RESEARCH REPORT 2006/20072010/2011 FOREWORD 04 The Helmholtz Centre for Infection Research – a portrait 07 Foreword 08 Orbituary – Jürgen Wehland

FOCUS 12 The German Centre for Infection Research (DZIF) – a great chance for the HZI 15 Highlights 2010-2011

RESEARCH REVIEWS 22 Frontier runners of Hepatitis C Virus 30 The aging of the immune system: challenges and perspectives 36 Novel nanoparticle-based technology platform for the delivery of vaccine antigens 42 Mycobacterial phagosomes and innate immunity

SPECIAL FEATURES 48 Where would we be without DNA sequences? 52 Nuclear magnetic resonance spectroscopy, a major player in the arsenal of platform technologies available in the HZI 60 International cooperation of Helmholtz Centre for Infection Research with India and China: a success story

SCIENTIFIC REPORTS 64 INFECTION AND IMMUNITY 67 Microbial Pathogenesis 69 Structural analysis of virulence factors 70 Virulence factors of streptococci and pneumococci 71 Molecular mechanisms of intracellular traffi cking,survival and persistence of streptococci 72 Analysis of protein networks induced by early host-pathogen interactions 73 Structural and mechanistical analysis of functional amyloids 74 Microtubule dynamics and bacterial pathogenesis 75 Function and regulation of Yersinia virulence factors 76 Structural characterisation of pathogen defence factors 77 Mycobacterial phagosomes and immunity 78 Molecular mechanisms of host-cell pathogen interactions 79 Signalling to actin dynamics 80 Generation and exploitation of DNA sequence data

81 Host Pathogen Interactions 84 Pathogenesis of chronic Pseudomonas aeruginosa infections 85 Unravelling mechanisms of host defence against Gram-positive pathogens in the mouse model 86 Microbial communication 87 Systems genetics of infection and immunity 88 The structural basis of mammalian prion transmission barriers 89 Biofi lm communities 90 Metabolic diversity SCIENTIFIC REPORTS 91 Infection and Immunity 92 Development and functional properties of Foxp3+ regulatory T cells 93 Mucosal immunity and infl ammation 94 Immuneffectors: molecules, cells and mechanisms 95 Interferons in viral defence and immunity 96 Cell-death mechanisms in immunity 97 Infl ammation and regeneration 98 Cellular models for infection

99 Strategies for Prevention and Therapy 102 Molecular mechanisms of infection and replication in the hepatitis C virus 103 Interaction between innate and adaptive immunity 104 Antigen delivery systems and vaccines 105 Chronic infection and cancer 106 Therapeutic cellular vaccines 107 Molecular diagnostics of mircobial pathogens

108 Pharmaceutical Research 110 Microbial diversity and natural product discovery 111 Medicinal chemistry of natural products 112 Chemical biology of infectious disease 113 Identifi cation of molecular targets of antiinfectives

114 New Project Groups 115 Regulation and herpesviral immune modulation of toll-like receptor signalling 116 Systems immunology 117 The National (“Helmholtz“) Cohort 118 Immune aging 119 Development of novel diagnostic and therapeutic tools for tuberculosis control and prevention 120 Exploring and exploiting microbial proteomes

121 POF II INDEPENDANT RESEARCH 122 Functional genomics and niche specifi ty 123 Structural biology of the cytoskeleton 124 Structural analysis of the innate immune system

125 TECHNOLOGICAL PLATFORMS 126 Central animal facility 127 Recombinant protein expression 128 Gene expression analysis 129 Peptide synthesis 130 Histo-pathology platform 131 Analytical instruments

132 The Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS)

138 TWINCORE, Centre for Experimental and Clinical Infection Research GmbH

148 Publications 2010-2011

FACTS AND FIGURES 172 Facts and Figures 4 PORTRAIT

The Helmholtz Centre for Infection Research

Our name refl ects the scientifi c programme: At the Infections are always caused by molecular interactions Helmholtz Centre for Infection Research in Braunschweig between humans and pathogens and between the pathogens approximately 350 scientists and 350 employees from themselves. Central element of this interplay are the technology and administration lay the foundations for new proteins involved in infections. These consequently play a prevention methods, diagnostic procedures, medicines and key role in the comprehension of infectious diseases. The active agents with which infectious diseases can be better microorganisms organise and catalyse their life with treated or more effectively prevented. The paths that lead to thousands of proteins. The goal of infection researchers is this goal are as varied as the paths by which the pathogens not only to describe these proteins but also to understand enter our bodies. Microbiologists investigate how bacteria their functions. The proteins on the cell surface, in particu- and viruses manage to enter our bodies, how bacteria lar, are responsible for the contact with the host – and the communicate with one another and how exactly they make question hanging over the research remains: how do bacteria us ill. Geneticists investigate our genetic make-up in search manage to infect us? The arsenal of these different pathogen- of reasons why one person falls ill with fl u, for example, ity factors, with which bacteria, viruses and other microbial whilst his neighbour does not. Immunologists investigate pathogens interfere with the process of human cells is a how organisms react to an intruder and resist it. Structural modest one – however, knowing where these are located and biologists research the molecular structures of key mole- being able to name these factors does not yet mean that they cules with all of their interactions. Chemists use this are understood. Proteins are very large molecules with knowledge to investigate and develop new agents that can complex structures and it is precisely in these structures that in turn be employed to combat pathogens. Vaccine research- the secret of their success lies. The scientists at the HZI take ers have their sights set on the best way to combat germs: a close look at their structure - atom for atom. They look for they aim to prevent these from making us ill at all. niches and hooks with which they brush alongside one another, cling to and release one another. New diagnostic approaches, vaccines or medicine can only be successfully developed when the mechanisms of Once the structural biologists have familiarised themselves infectious diseases have been properly understood. A with these decisive parts of the molecular structure the starting point from which researchers at the Helmholtz chemists can begin to put this knowledge to further use. Centre for Infection Research approach the complex Their goal is to develop tailored molecules that fi t this network of “infection” is the cell - both that of the host and structure exactly - and subsequently have the opportunity that of the bacteria. One example: opportunistic infections. to interrupt an infection. To this end chemists look for These are a problem in hospitals. In a place where patients natural inhibitors or create new synthetic ones, using these with weakened immune systems are treated, bacteria to block the functions of the pathogen proteins in a targeted transform themselves from unobtrusive companions to approach. dangerous aggressors. In extreme cases they ensconce themselves permanently in our bodies by forming a The basis for new active agents, which can then attack the so-called biofi lm, causing chronic illness under certain weak points of the bacterial proteins, are often natural circumstances. In biofi lms the bacteria are surrounded by a agents. These originate in traditional, recently rediscovered protective sheath that protects them very effectively against healing plants, from fungi or bacteria. A special role in the attacks from the immune system or antibiotics. But what HZI research is played by the myxobacteria. These live in has to occur in order for a seemingly harmless germ to the soil and defend themselves against bacterial competitors become an aggressor? How do bacteria communicate with with a range of chemical substances - substances that are one another and the host? Only when scientists have highly effective when precisely analysed by infection understood how the pathogens and their host cells interact researchers and then developed into medicines. Microbial and which are the mechanisms behind these interactions active agent principles for combating infectious diseases they can disrupt the communication between bacteria in a are therefore seen as the optimal initial substances for targeted manner. developing new therapies against bacteria for use in hospitals. PORTRAIT 5

The task of the chemists in infection research is easily imperviously to an infection. In this, the genes infl uence described but diffi cult to implement: chemists search for one another, with the consequence that a defective gene effective components that enable myxobacteria to defend may be compensated by another gene or defects in genetic themselves against other microorganisms or turn a plant material reinforce one another. The system-genetic investi- into a healing plant. And in these molecules they search for gation of such interrelations will lead to new therapeutic decisive structural characteristics and chemical groups in approaches. The instruments for this are not limited to the order to recreate these and even improve their effective- petri dish, microscope or mouse, but are above all computers. ness. Synthesising or modifying natural agents to create potent medicines is a discipline somewhere between pure This research bridge, which the Helmholtz Centre for research and medicinal application for the HZI researchers. Infection Research spans from the molecular interaction between pathogen and host to new active agents and An important step on the way for the application of natural prevention is aimed to lead directly to humans. Cooperation products is the further development of these compounds. with the Hannover Medical School has resulted in the New and often multi-resistant pathogens demand an TWINCORE in Hannover – a translation centre where effi cient development of new antiinfectives. The Helmholtz clinical research and basic science meet. Medical personnel Institute for Pharmaceutical Research Saarland (HIPS), a and pure scientists are working there together under one branch of the HZI and established in 2009, deals with that roof, drawing up joint solutions for the problem of infection task. Scientists at HIPS use genetic and genome-analytical in doctors’ surgeries and clinics. The involvement in the processes to optimize on one side the producers of natural new research centres enables the Helmholtz Centre for products and to further develop molecules, which are Infection Research to reinforce the health research potential candidates for an antibiotic treatment, for medical capabilities of the Braunschweig-Hannover district. And, and clinical purposes. With regard to this they are looking step by step, progress is being made towards resolving an for methods able to better transport such natural products old - and yet current - problem: protecting people against or their derivatives through biological barriers to the place infectious diseases. of their action and they are developing new and further optimized formulations for those natural products.

The vaccine researchers at the HZI also tread a fi ne line between basic and clinical research. Vaccines are held to be the most effective and economic method for protecting humans and animals against infectious pathogens. The Helmholtz Centre for Infection Research Although the scientists are obviously in search of new Inhoffenstraße 7 vaccines against illnesses such as AIDS or fl u, one of their 38124 Braunschweig, Germany specialities is the optimisation of vaccines. Adjuvants are Tel: +49 (0)531-61 81-0 the key. These agents have no effect on infectious pathogens Fax: +49 (0)531-61 81-2655 themselves, but help the vaccines to develop their full [email protected] effectiveness. www.helmholtz-hzi.de

An infection requires the pathogens, the organism con- cerned and environmental factors. As the environmental factors have not proven readily comprehensible thus far, the HZI researchers are focusing upon the interaction between pathogen and host. The research fi eld that aims to address the associated, highly-complex questions is in the fi rst stages of development: system genetics. A whole series of genes determine whether a host responds sensitively or 6 Guests at HZI events

Participants of the opening ceremony of the symposium “Networking and Technology Transfer”, organized by HZI, InWEnt, and Thai Red Cross Photo: QSMI/HZI

Photos: HZI FOREWORD | Prof. Dr. Dirk Heinz 7

Foreword

Prof. Dr. Dirk Heinz | Acting Scientifi c Director

The past two years have comprised a phase of change and transition for the Helmholtz Centre for Infection Research (HZI). After its successful scientifi c refocusing on infection research, the Scientifi c Director Rudi Balling left in 2009 - fi rst for a scientifi c sab- batical to the Broad Institute and shortly after permanently for a new position as Director of the Luxembourg Centre for Systems Biomedicine. With Jürgen Wehland, former head of the Cell Biology Department, an excellent successor for this crucial position was appointed in January 2010. Keenly aware of the research performed at the HZI, he was a natural choice for steering the centre and for shaping its profi le and scientifi c strategy. After less than one year in offi ce, his tragic death on 16th August 2010 saw the loss of a highly respected colleague and dear friend to many of us. In spite of this overshadowing event, and with the third Scien- tifi c Director in offi ce in less than three years, the HZI has continued to emerge as a centre of excellent infection research. In the spirit of the changes initiated by Rudi Balling, continued and realigned by Jürgen Wehland, a number of important projects have been launched and we are now successfully implementing these further, both on the HZI campus and beyond:

• In 2009, the Helmholtz Institute for Pharmaceutical Research Saarbrücken (HIPS) was founded in collaboration with the Uni- versity of Saarbrücken. The pharmaceutical know-how at the HIPS ideally complements the expertise in infectious disease and natural compound research at the HZI. • Together with the Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM), the Hannover Medical School (MHH), the Leibniz-University Hannover, the University of Veterinary Medicine Hannover and the University of Braunschweig, the HZI founded the “Translationsallianz in Niedersachsen” (TRAIN) in 2008 to promote translational drug and vaccine research. In May 2011, construction of the Hannover Centre for Translational Medicine (HCTM) will start. The HCTM is a joint project of the TRAIN partners ITEM, MHH and HZI and will serve as a platform for early clinical studies. • The HZI, together with its partners in the Braunschweig-Hannover region, successfully took part in the competitive selection of founding members of the German Centre for Infection Research (DZIF), following an initiative of the Bundesministerium für Bil- dung und Forschung. Together with 26 other research institutions from all over Germany, the HZI is now actively participating in the foundation process. The DZIF will join forces in infection research and places emphasis on the translation of basic research into medical application. The central DZIF administrative offi ce will be located at the HZI. • Working together with several partner institutions in northern Germany, the HZI took the lead in the development of a concept for the Centre for Structural Systems Biology (CSSB). The CSSB will bring together expertise in structural and systems biology to allow the analysis of the molecular processes taking place during infection and other diseases on a hitherto unattainable level of tempo-spatial resolution. The founding agreement for the CSSB was signed early in 2011. • A number of new buildings on the HZI campus have been completed or are about to be fi nished: · The new animal facility (Tierhaus II) commenced operation in 2010. · The administrative annex building was set-up at the end of 2009. · The novel BSL3-building is now complete and will be operational in 2011. · The offi ce-annex for building D was topped out in spring 2011 and will allow establishment of new lab space upon completion.

As a consequence of these successful projects and collaborations, the HZI is now on course for the next round of the Helmholtz Programme-oriented Funding (PoF III) in 2013.The HZI is well prepared for the challenges posed in infection research through its clear strategic focus on the elucidation of infection processes complemented by its anti-infectives research and its on-going efforts in the translation of basic research in collaboration with its academic and industrial partners. In future we will further strengthen our scientifi c profi le by fostering outstanding projects and recruiting additional scientifi c expertise.

Prof. Dr. Dirk Heinz 8 OBITUARY | Jürgen Wehland

… during a staff outing … a visit of politicians

… during the visit of Minister Prof. Schavan … during a PhD commemoration

… during a PhD commemoration … during the TRAIN event

… during the TRAIN event

All Photos: HZI OBITUARY | Jürgen Wehland 9

Jürgen Wehland †

Scientifi c Director 2009-2010 | Head of the Division of Cell and Immune Biology 1997-2009 | Scientist at the HZI since 1989

The Helmholtz Centre for Infection Research mourns Jürgen Wehland who suddenly and unexpectedly passed away on August 16, 2010, while on vacation in Scandinavia.

Jürgen Wehland was a key fi gure in the HZI for many years and in numerous aspects: As an excellent scientist with international standing, he made seminal contributions to the fi elds of cell and infection biology. Using Listeria monocytogenes as a model organism, he could show how the human pathogen utilizes the host’s actin cytoskeleton to promote infection. Together with other leading scientists in the fi eld, he laid the foundation for a new discipline, cellular microbiology, tackling host-pathogen interactions at various molecular levels, without which modern infection biology would simply be unthinkable.

As an outstanding scientifi c manager, he not only developed his own department into one of the most successful ones of the HZI but also represented the mission and vision of the entire centre by skilfully shaping the research programme, by fostering strategic alliances with our partner institutions and by promoting a number of crucial recruitments to Braunschweig and our subsidiary institutions. Even though only a short time in offi ce as the Scientifi c Director of the HZI, he was leading the centre towards exciting and important future developments.

As a caring supervisor and an inspirational mentor he actively supported the scientifi c careers of numerous young scientists within the HZI and beyond.

As a dear colleague and friend to so many of us his door was always open for scientifi c and personal advice. He just always took the time.

Beside his many great accomplishments, Jürgen Wehland remained a remarkably modest character, a great example to all of us.

We sadly miss him! FOCUS RESEARCH REVIEWS SPECIAL FEATURES

Photos from left to right: The representatives of the HZI PhD-student organization announcing the award for the best PhD-supervisor of the year 2010: Prof. Dr. Eva Medina. | Thomas Gazlig, the head of the HGF department of Public Relations, during a talk at the exhibition “Wunderkammer der Wissenschaft” at the Braunschweig “Schloss-Akarden” | Prof. Dr. Kurt Wütherich, Nobel Laureate, with Maxi Scheiter discussing her results of the poster presentation Photos: HZI, Hübner (le) | HZI (ce) | HZI, Hübner (ri) SCIENTIFIC REPORTS FACTS AND FIGURES

12 The German Centre for Infection Research (DZIF) – a Great Chance for the HZI – an Interview

15 Highlights 2010–2011 12 FOCUS | The German Centre for Infection Research (DZIF) – a Great Chance for the HZI – an Interview

The German Centre for Infection Research (DZIF) – a Great Chance for the HZI

INTERVIEW WITH | Prof. Dr. Dirk Heinz | Acting Scientifi c Director of the Helmholtz Centre for Infection Research – HZI

Professor Heinz, in November 2010 the HZI was selected as a partner institution for the German Centre for Infec- tion Research. What does this decision mean for the centre in Braunschweig?

Dirk Heinz: We are proud to be included in the small circle of leading research facilities that have been assigned to jointly established the German Centre for Infection Re- search (Deutsches Zentrum für Infektionsforschung, DZIF). The DZIF is part of a nation-wide initiative of the Federal Ministry of Education and Research. Several German cen- tres for health research will be launched in order to take a more targeted course of action against the major diseases. The aim is to consolidate research efforts, to improve ex- change between participating institutions and to accelerate the transfer of innovation from basic research into the clinic – the so-called translation.

Which diseases are the focus of attention for the Federal Government?

DH: In the context of this initiative, four German centres for health research are to be founded: the German consortium for translational cancer research as well as German centres for research on cardiovascular diseases, pulmonary di- seases and, of course, infection research. The prerequisites The participating institutes of the DZIF are located in the cities, for selection of the sites were proven scientifi c expertise, marked in the map. Further details see Interview. and the ability to expedite translation research. Both in- frastructures as well as personnel will be fi nanced for this purpose on a sustained basis. Which topics and which pathogens are to be investigated at How will the national health research centres be the DZIF? organized? DH: It is a primary goal to tackle important health issues DH: In most of the centres, and the DZIF as well, it is a relevant to Germany, which clearly not excludes major infec- matter of inter-site constructs. No new major research faci- tious diseases relevant to the rest of the world. Therefore, lities are being built, but leading institutions are brought chronic viral diseases such as HIV/AIDS and hepatitis are, together in order to facilitate networking and coordination among others, a major topic. Another focus are gastro- of work under one common organisational roof. This is also intestinal infections by Helicobacter, Yersinia and other the case for the DZIF with seven locations in Hannover- pathogenic bacteria. These are areas to which our consorti- Braunschweig, Hamburg-Lübeck-Borstel, Bonn-Cologne, um will also make considerable contributions. Furthermo- Tübingen, Munich, Heidelberg and Gießen-Marburg-Langen. re, tuberculosis and malaria are highly relevant topics in The central administrative offi ce, which coordinates the infection research to be addressed by the DZIF. One should work of the various research projects within the DZIF, will not forget hospital-acquired infections with persistently be located in Braunschweig on the HZI campus. reoccurring problems of antibiotic resistance and biofi lm formation. MRSA and pseudomonads, in particular, play a role here. Moreover, the researchers at the DZIF will be dealing with overriding issues of infection research. These will include control of pathogens by the immune system, improved vaccinations and, most importantly, new active compounds against infections. FOCUS | The German Centre for Infection Research (DZIF) – a Great Chance for the HZI – an Interview 13

What is the advantage of such a large alliance like the DZIF an important role. Together we are constantly expanding over the numerous cooperations that we already have – the pool of available natural substances from myxobacteria. nationally as well as internationally? They have proven to be a highly potent source for a diversity of bioactive compounds. Currently 80 percent of all antibio- DH: It is a different approach. In the future we will not carry tics found on the market originate from natural substances. out research and cooperate as before. Rather, we will initiate This shows that, in the search for new anti-infective agents, new approaches. Together the strong partner institutions we have to revert back to the existing, albeit insuffi ciently can, through consolidation in the DZIF, make use of common developed, pool of natural substances. Furthermore, novel infrastructures and expertise. This will allow a more con- compounds can be produced by means of targeted genetic sistent translation from fundamental research to clinical modifi cation of production strains, or previously known studies. The ultimate target is the development of new thera- structures can be produced in larger quantities. The existing peutic agents or preventative measures such as new vaccines bank of natural substances at the HZI will now be continu- and vaccination strategies. We have to work together and ously expanded. We will make it accessible to the various synergistically make our respective expertise available, so consortia for research on completely different pathogens. Our that optimal results can be achieved for public health. chemists enable the transition from the screening hit to lead structure candidates. Moreover, we are handling topics such What shall be the task – the special function – of the HZI as drug delivery procedures. It is in this context that our within the DZIF alliance? pharmaceutical research at HIPS comes into play. Without it, we could not make use of our bio-active compounds as phar- DH: On the basis of our special expertise, research on maceutical products that can then be used in clinical studies. natural bioactive compounds is the central theme for the HZI Academic basic research as a basis for the development of researchers in the DZIF. In this regard TWINCORE and the new drugs: this aspect of our DZIF application represents a Helmholtz Institute for Pharmaceutical Research Saarland unique characteristic. It shows great promise for the future. (HIPS), in addition to the activities on the HZI campus, play This is, in addition to our experience in translation research, a main criterion for our funding.

What about organisation? You mentioned that the admini- strative offi ce will be located in Braunschweig…

DH: For all of the German centres for health research there is a Helmholtz Centre, through which the funds from the Federal Ministry of Education and Research fl ow into the respective consortia. This means that we will take the responsibility for both distribution of funds and the controlling. That is why the subsidy management has to be residing within the HZI. The DZIF administrative offi ce will work closely with the department for subsidy manage- ment - another important reason for its location at the HZI. However, the offi ce itself will be independent of the HZI and our own projects.

What level of funding are we talking about?

DH: In the fi nal developmental stage from 2015 on, almost 40 M€ per year have been planned for the complete DZIF. The core estimated funding begins at developmental levels …Together the strong partner institutions can, through consoli- this autumn. The portion of our own consortium will dation in the DZIF, make use of common infrastructure and amount to several million euros per year in the fi nal stage. expertise…. Photo: HZI; Gramann 14 FOCUS | The German Centre for Infection Research (DZIF) – a Great Chance for the HZI – an Interview

The scientists whose studies have gone into the application process for the DZIF are already employed. They have been working on their scientifi c objectives for many years now. And today the objectives of the DZIF are added to this. Are resources being restocked? What will the fi nances from the DZIF be used for?

DH: First of all, money is being used to build up infra- structure and expertise. This will allow us to do justice to the claim that we work and live scientifi cally according to translation principles. For example we shall, by means of specifi c appointments, strengthen the research in bioactive compounds on the HZI campus. This means that colleagues will be specifi cally fi nanced through the DZIF. An appoint- ment in bio-informatics would be equally conceivable. We are investing new fi nancial means primarily into this. Of course, this does not forbid colleagues to incorporate their expertise into the major thematic topics. A few examples … I view the DZIF as a consistent continuation of our trans- are: Eva Medina in staphylococci, Thomas Pietschmann in lation activities…. Photo: HZI; Gramann hepatitis and Susanne Häußler in resistant gram-negative bacteria. And, by the way, for this purpose we are bringing a considerable portion of capital resources into the DZIF. What does the work at the DZIF specifi cally mean for the Let us return to the topic of translation, which represents a HZI? Will the employees here on campus notice that the primary concern of the DZIF. The HZI has already achieved DZIF exists? much in this regard. We already have the Translation Alli- ance in Lower Saxony, TRAIN, and there is the TWINCORE DH: Some employees will strongly notice, but certainly not Centre for Experimental and Clinical Infection Research. all. We did not involve all departments directly in our appli- There are partners here who shall become a part of the cation to the DZIF. We placed our emphasis on host-pathogen DZIF and who are already connected with one another in interactions and functional genome research, but most of all their translation activities. What will interaction with the on active compounds. Colleagues who are involved in these DZIF look like? areas will actively participate in the corresponding topics. They will become part of a translation pipeline. DH: Without TRAIN, we would conceivably not have TWIN- CORE either. It is possible that the expert evaluators would And when will it all begin? have perceived the basic research here in Braunschweig and the clinical research in Hannover as isolated site activities DH: Presumably in the summer of 2011, but in the autumn that only collaborate on small projects. Giving money to at the latest. After appraisal of the joint application in April, such facilities would probably not raise expectations that a decision will be made. We will then know whether all they would be doing research together in a superordinate specifi cally-announced research projects within the DZIF structure in fi ve years. TRAIN and TWINCORE thus were will be funded in their entirety*. When all these decisions the pre-conditions for the fact that we are forming, jointly have been made, we can start. We are already looking for- with the MHH, such a momentous double site within the ward to working with all of our partners. DZIF. I view the DZIF as a consistent continuation of our translation activities. We are currently expanding beyond local activities, with topics that are specifi ed within the DZIF, and make use of the strong local structures for this The interview was performed through the Public Relations purpose. One should realize that TRAIN and TWINCORE Department of the HZI in March 2011. are dealing with other projects as well, as opposed to those which are planned in the DZIF. FOCUS | Highlights 2010-2011 15

Highlights 2010-2011

AUTHORS | Manfred Braun | Head of the Department of Public Relations | [email protected] | Dr. Bastian Dornbach | Department of Public Relations | [email protected]

Helmholtz Touring Exhibition in Braunschweig The Herbert Waldmann 2010 awardee of the Inhoffen Medal exhibition “Wunderkammer Wissenschaft” (“Chamber Winner of the Inhoffen award for 2010 was Prof. Dr. Herbert of Scientifi c Wonders”) was a special attraction in the Waldmann from the Max-Planck-Institute for Molecular Braunschweig “Schloss-Arkaden” from 8 to 16 April 2010. Physiology in Dortmund. Herbert Waldmann received the More than 335,000 visitors attended the touring exhibi- award donated by the “Friends of the Helmholtz Centre” tion and gathered information regarding research at the (Förderverein des HZI) for his syntheses of new active Helmholtz-Association. About 500 images and multi-media compounds. The biochemist Herbert Waldmann sought offerings in huge opened trunks provided insights into after models, in nature, for new potential medications. miniscule nano-worlds and gigantic large-scale devices at These are biologically-active molecules, that are produced the Helmholtz Centres. The spectrum ran the gamut from a by plants, microorganisms and animals as, for example, close-up image of mould-fungus to photos of distant planets defence mechanisms or as messenger substances between and appealed to a broad audience from pupils to adults. The cells and organs. Many natural substances affect not only photos – in part animated, but always animating in their their originally-intended targets but also certain human effect – led to intensive discussions with the exhibit attend- metabolic processes. Almost half of the medications now ants from the HZI. available are based on natural substances or natural substance-like structures. Herbert Waldmann and his co-workers were successful through their efforts of many years in synthesizing a variety of new substances. A few of them have a fundamental effect on the growth of breast, ovary and stomach tumours, while others are able to regu- late the blood-sugar levels or participate in the conduction of nerve stimuli.

A view of the exhibition “Wunderkammer Wissenschaft” at the Schloss-Arkaden Braunschweig. Photo: HZI, Ritter

Science Shopping – Science late into the night Under the auspices of the “Moonlight Shopping” in the Braun- schweig city-centre, the HZI presented, as one of 13 regional research facilities, their “Science late into the night” exhibit (“Wissenschaft bis in die Nacht”). Visitors were able to gather information at four different learning stations regarding infectious diseases and carry out experiments with scientists in the afternoon until 11 p.m. The topics: How does vaccination actually work and how does our immune system function? How come the infl uenza virus does not know borders? What are prions? Information boards displayed the infl uence of infectious diseases upon contemporary art. Young researchers were able to fi nd out for themselves why it is that red cabbage is also called blue The HZI participating in the event “Moonlight Shopping” at cabbage. The event was organised by the local research in- Braunschweig city-centre within the part “Science late into the stitutions in cooperation with Braunschweig City Marketing night”. Photo: HZI, Morczinietz and business proprietors in the Braunschweig city-centre. 16 FOCUS | Highlights 2010-2011

HZI at NDR Info: Big fear of small viruses – Tracking down pathogens NDR Info, the Northern German news radio station, introduced in March 2010 in collaboration with the “Braunschweig Newspaper” and the House of Science (“Haus der Wissenschaft”) in Braunschweig, the new discus- sion and broadcast series “Logo – Science from Braunsch- weig”. Braunschweig has, according to an EU study, the most active research region in Europe with the highest concentra- tion of scientists. More than 15,000 individuals work in 26 scientifi c organisations and research facilities and there are about 250 light industries within the high-tech sector. For the second broadcast of this series in June, the Logo modera- Prof. Dr. Herbert Waldmann (middle) with the Inhoffen Medal tor Regina Methler discussed with experts from the HZI and 2010 together with Prof. Dr. Jürgen Wehland (†, at that time with the audience topics concerned with bacteria and viruses Scientifi c Director of HZI, left) and Prof. Dr. Joachim Klein as well as our fears and the opportunities and limitations (President of the HZI Förderverein, right) Photo: HZI, Gramann inherent in infection research. These included: “When will we experience the next pandemic?” and “Are animal experi- ments still necessary in infection research?”

Summer holiday course “Everything about Sugar” CSSB agreement Infection researchers and physicists in Learning about biochemical principles through experi- Northern Germany are blazing new trails together in their ments and result reportage was the objective of a four-day research of infectious diseases; the new “Centre for Struc- summer holiday course for pupils, which the School Lab tural Systems Biology” (CSSB) is being developed on the “BioS” and the Public Relations Department of the HZI campus of the German Electron-Synchrotron (DESY) in Ham- organized jointly. The topic: sugar in all its manifold forms. burg-Bahrenfeld with scientifi c coordination from the HZI. Pupils became acquainted with biochemical verifi cation The CSSB is building a bridge between structural biology methods for common sugar and got a fi rst impression of and systems biology. In this context, biologists, chemists, how laboratory studies are carried out. In the second part health and medical professionals, physicists and engineers of the course, pupils learned various journalistic formats are studying the interaction between pathogens and their and processed their knowledge of topics such as diabetes hosts. For this purpose, unique tools are being made avail- and lactose intolerance. This combination of biochemical able to them Germany-wide through DESY: the storage ring- studies and refl ection regarding scientifi cally-applied top- based X-ray source PETRA III, the free-electron laser FLASH ics was quite successful. Not only did it introduce pupils and the X-ray laser European XFEL, which is presently being the concept of having fun while working but they also built. A federal government and state agreement will make learned journalistic approaches to the technique of asking 50 million euros available for construction of the CSSB. suitable questions to fi nd relevant information.

A group of pupils that participated in the Summer holiday After signing of the CSSB agreement at the DESY facilities in course at the BioS-Laboratory. On the right side Dr. Iris Hamburg. From left to right: Prof. Dr. Helmut Dosch, President Eisenbeiser from the BioS-Laboratory. Photo: HZI, Fischer of the DESY Board of Directors; Prof. Dr. Johanna Wanka, Minister of Science and Culture of Niedersachsen; Christoph Ahlhaus, Lordmayor of the Freie und Hansestadt Hamburg; Prof. Dr. Annette Schavan, Minister of Education and Re- search, BMBF; Dr. Herlind Gundelach, Senator of Science and Research of Hamburg; Prof. Dr. Dirk Heinz, acting Scientifi c Director of the HZI. Photo: DESY, Reipka FOCUS | Highlights 2010-2011 17

Peter Seeberger receives the Inhoffen Award for 2011 For his fully automatic carbohydrate-syntheses, Prof. Dr. Peter Seeberger received the 2011 Inhoffen Award. The department head and managing director at the Max-Planck Institute for Colloids and Interfaces in Golm near Potsdam was successful in his attempts to produce completely synthetic complex carbohydrates, which play a signifi cant role in almost all physiological processes. Carbohydrates, or sugar molecules, fulfi l multifarious tasks in and on our cells. They deliver energy, form the foundation for surface structures such as phlegm, but are also used by patho- During the press conference of the State of Niedersachsen with gens. They play, therefore, a decisive role in infections and the participating institutions of the German Centres for Health immuno-reactions and are attractive target molecules for Care Research in Niedersachsen. 2nd from the left side: Prof. Dr. medical research. However, a chemical synthesis method Dirk Heinz, acting Scientifi c Director of HZI. Photo: MHH, Kaiser had been missing hitherto for producing biologically impor- tant sugars in large amounts – a gap that Peter Seeberger was able to fi ll. State Secretary Dr. Georg Schütte as a guest at the HZI On Monday, January 17, 2010, Dr. Georg Schütte State Secretary from the Federal Ministry for Education and Re- search (BMBF) was a guest at the HZI to become informed of the research at the HZI. In addition to information regarding the world of viruses and microbes, he had the op- portunity, through extensive discussions with the manage- ment board, to fi nd out about the challenges and research goals of the HZI. He then visited the often awarded School Lab “BioS”, where he was able to gain insights regarding the facility for promotion of a rising generation of young scientists in biotechnological research.

Prof. Dr. Peter Seeberger (middle) with the Inhoffen Medal 2011 together with Prof. Dr. Joachim Klein (President of the HZI Förderverein, left) and Prof. Dr. Dirk Heinz (acting Scientifi c Director, HZI, right) Photo: HZI, Hübner

HZI participating in the German Centre for Infection Re- search To investigate pathogens and to develop new strate- gies against them – these are the core tasks of the “Ger- man Centre for Infection Research” (DZIF), whose research programme will start in the summer of 2011. The initiative of the Federal Ministry for Education and Research (BMBF) comprises numerous partners from university and non- State Secretary Dr. Georg Schütte (BMBF) during his visit university research groups. The HZI is participating in at the HZI and BioS. Here talking with a pupil at the BioS DZIF as part of the Hannover-Braunschweig consortium. Laboratory. Prof. Dr. Dirk Heinz (acting Scientifi c Director The administrative centre of the DZIF will also be located of HZI, right) and Ulf Richter (Administrative Director, at the HZI. Partner facilities in the region Hannover-Braun- background) Photo: HZI, Hübner schweig are, in addition to the HZI, the Medical School of Hannover, the University of Vetenary Medicine Hannover (TiHo), the Technical University of Braunschweig (TU BS), the German Collection of Microorganisms and Cell-Cul- tures (DSMZ) and TWINCORE, the Centre for Experimen- tal and Clinical Infection Research. 18 FOCUS | Highlights 2010-2011

First “North-Regio-Day on Infection“ at the HZI On 1st and 2nd October 2010, scientists from universities and research facilities in Northern Germany met at the HZI on the occasion of the fi rst “North-Regio-Day on Infection“; the researchers presented up-to-date research results, became familiar with new technical developments and discussed important issues and future challenges in infec- tion research. The focus was on bacterial pathogens. The meeting was coordinated jointly by the HZI, the Technical University of Braunschweig and the Robert-Koch-Institute in Wernigerode (RKI) under the auspices of the HZI Gradu- ate School. The goal of the “NORDI” symposia in the future will be the promotion of exchanges between researchers in Prof. Les Baillie from the Welsh School of Pharmacy of the northern Germany and promotion of junior scientists. Cardiff University, UK, during his presentation. Photo: HZI, Oumard

Gene laboratory and school The workshop “Genlabor & Schule” was a guest at the HZI from 24 to 25 September 2010. The Society for Biochemistry and Molecular Biology (GBM) and the Braunschweig School Lab “BioS” invited participants to the fi fth workshop of the Network “Genlabor & Schule”, a unique event in the German-speaking coun- tries. These people responsible for almost 40 school labs in Germany, Austria and Switzerland have met annually or bi-annually since 2002. The workshop “Genlabor & Schule” offered an extensive programme consisting of technical lectures, poster presentations and panel discussions. It is The organizers of the symposium. From left to right: Antje a forum for everybody who deals with the subject of junior Flieger (Robert-Koch Institute, Berlin), Petra Dersch (HZI) and scientists in the natural sciences. Scientists, directors and Michael Steinert (TU Braunschweig). Photo: HZI, Dornbach employees of the school lab, experts from the fi eld of biol- ogy, as well as representatives from the Cultural Ministries of Hessen and Niedersachsen exchanged their experiences with the goal of developing the school lab as an extracur- “Day on deadly killers“ On the “Day on Deadly Killers“ ricular place of learning. on March 3, 2011, international experts discussed the cur- rent status of infection research. The pathogens for such dreaded epidemics as cholera, anthrax, rabies and AIDS represented the focus of the symposium at which experts from Europe, the USA and Asia presented their research results and discussed new therapeutic approaches against infectious diseases.

Among the speakers was the renowned US scientist Dr. Henry F. Chambers from General Hospital in San Francisco, specialist for antibiotic-resistant hospital infections.

Dr. Iris Eisenbeiser (left) giving some explanations during the workshop “Gene lab & School” Photo: HZI FOCUS | Highlights 2010-2011 19

Nobel Laureate Prof. Kurt Wüthrich guest at the HZI An international star of science came to visit Braunschweig on April 4, 2011. Prof. Dr. Kurt Wüthrich visited the HZI to discuss research with colleagues and to exchange fi ndings. The Swiss-born scientist is currently supervising a labora- tory at the Scripps Institute in La Jolla, USA, as well as a second one at the Federal Technical University (Eidgenös- sische Technische Hochschule) in Zürich. His research focus has a direct connection with research at the HZI – clarifi cation and examination of the spatial structure of biological molecules. Kurt Wüthrich developed, collectively with other colleagues, the technology of the so-called “mul- Prof. Dr. Rolf Müller, HZI Photo: HZI, Gramann ti-dimension NMR spectroscopy” as a powerful analytical tool for the structural elucidation of large biomolecules. For his pioneering work Kurt Wüthrich was awarded the Nobel Prize for Chemistry in 2002. Cells meet Surface In May 2010, scientists took a deeper look at cell surfaces: “Cells meet Surface”, a two-day sym- posium, organized by the German Association of Transfu- sion Medicine (“Berufsverband Deutscher Transfusions- mediziner”, BDT), Fhg IST, SFB 578, the InnoNet project ”InnoSurf“, the Helmholtz Centre for Infection Research (HZI) and the DECHEMA, took place at the HZI Forum. For a long time the interaction between cells and (artifi cial) surfaces was not taken into account. However, scientists learned that this interaction is essential for the well-being of a cell and that the cultivation of cells can be infl uenced positively or negatively by physical or chemical modifi cations of surfaces. The scope of this meeting was to present to clini- cians and clinical-oriented scientists the latest developments Visit of Nobel Laureate Prof. Wütherich at HZI. From left to right: in this fi eld. Main emphases of this symposium were put on Prof. Dr. Christiane Ritter, Dr. Torsten Lührs (both HZI), Prof. the modifi cation of surfaces and the technical possibilities Dr. Kurt Wüthrich (Scripps Institute La Jolla, USA), Prof. Dr. to prove these and to infl uence the characteristics of cells. Dirk Heinz (acting Scientifi c Director HZI). Photo: HZI, Hübner Furthermore, new ways of cell analysis based on magnetic signals, the successful use for the generation of cell products and initial attempts in clinical practice were presented.

Rolf Müller awarded the DECHEMA Prize of the Max- Buchner Research Foundation Prof. Rolf Müller received the 20,000 euro DECHEMA Prize of the Max-Buchner-Re- search Foundation on 26 November 2010 for his research in biologically active natural materials that are produced from bacteria living in the soil and used to develop new medica- tions. Rolf Müller is Managing Director of the Saarbrücken branch of the HZI, known as HIPS, and teaches at the University of Saarland. The department led by Rolf Müller concentrates on analysis and production of molecules de- rived from myxobacteria and actinomycetes with biological activity. An example for a microbial active ingredient made from myxobacteria is epothilone, which has been success- fully deployed in cancer therapy in the USA. FOCUS RESEARCH REVIEWS SPECIAL FEATURES

Photos from left to right: Niña S. Cortina, HIPS, feeding a Genetix clone picking robot. | Dr. Maximiliano Gutierrez acquiring confocal images of eukaryotic cells infected with mycobacteria | Wiebke Ginter, Twincore, during the preparation of a new experiment Photos: HIPS/HZI (le) | HZI, Bierstedt (ce) | Twincore/HZI (ri) SCIENTIFIC REPORTS FACTS AND FIGURES

22 Frontier Runners of Hepatitis C Virus

30 The Aging of the Immune System: Challenges and Perspectives

36 Novel Nanoparticle-based Technology Platform for the Delivery of Vaccine Antigens

42 Mycobacterial Phagosomes and Innate Immunity 22 RESEARCH REVIEWS | Frontier Runners of Hepatitis C Virus

Frontier Runners of Hepatitis C Virus

CORRESPONDING AUTHOR | Prof. Dr. Thomas Pietschmann | Department of Experimental Virology | TWINCORE | [email protected] | [email protected]

CO-AUTHORS | Dr. Sandra Ciesek | Dr. Eike Steinmann | both at Department of Experimental Virology | TWINCORE

Transmission of hepatitis C occurs via direct blood-to-blood contact: prior to 1990 primarily blood transfusions were the problem, however, today transmission by means of blood products, at least in Germany, no longer plays a role due to highly sensitive and accurate screening procedures. Risk factors to date are non-sterile handling of injection utensils in non-industrialised nations and intravenous drug consumption in industrialised nations. In the case of tattooing, piercing, acupuncture and medical inter- ventions, application of insuffi ciently sterilised instruments can lead to transmission of the virus. It is actually quite seldom that hepatitis C virus (HCV) infections are attributed to sexual intercourse with hepatitis-C-positive sexual partners. Transmission of the infection from an HCV-positive mother to her child prior to or during birth occurs in no more than 4% of cases. How ever, for approximately one-third of HCV-patients, the origin of the transmission is no longer traceable.

With currently approximately 130 million virus carriers, hepatitis C virus (HCV) infection is one of the most wide- spread infectious diseases worldwide. According to data from the Robert-Koch Institute, there are ca. one-half million virus carriers living in Germany. In the USA and Europe it is estimated that 1.5% of the population are infected, while in Egypt and Central Africa the ratio of up to 20% is signifi cantly higher. The virus is highly mutable and is, therefore, able to evade, time and time again, the immune system. On the basis of sequence analyses, the viruses are separated into seven genotypes, which deviate from one another by 30% and respond to medications with varying success.

The natural course of a HCV infection can be roughly devided into two stages: an acute and a chronic phase. During the fi rst six months after being infected, the patient will experience the acute, mostly symptom-free phase. A segment of the patients is able to control virus replication during this acute phase and the infection is spontaneously cleared. But for the majority of patients (50% to 90%), the virus is not eliminated and a chronic infection ensues. Occasionally, non-specifi c symptoms will emerge such as fatigue, depression, nausea, pains in the upper abdomen and indigestion – often times the infection is noticed and confi rmed many years after the initial contagion. Of these chronically-infected patients, up to 40% develop a progres- sive liver disease with formation of cirrhosis of the liver (Figure 1). HCV cirrhosis is, furthermore, one of the main risk factors for emergence of a heptocellular carcinoma (HCC). The effects of a chronic hepatitis C virus infection are considered among the most frequent indications for Fig. 1. Comparison of a cirrhotic liver (above) with a healthy liver transplantation3. In light of the world-wide dispersal liver (beneath). Photo: Twincore of the HCV infection, considerable clinical signifi cance is attributed to this pathogen on the grounds of its frequency and morbidity. RESEARCH REVIEWS | Frontier Runners of Hepatitis C Virus 23

Currently, world-wide there are only insuffi cient options The basis for our collaborative efforts is a cell-culture available for treatment of hepatitis C. Since the beginning system developed by us for HCV. With this groundwork, of the 90’s chronic HCV infection has been treated with we can examine the mechanisms of virus-replication in interferon-alpha, a cytokine of our immune system, which human liver cells in the Petri dish, identify new targets for triggers mechanisms in the cell to control a virus infec- therapy, and search for new active compounds that are able tion. In recent years this therapy has been substantially to inhibit propagation of the virus. We also use this model optimised. The application of interferon alpha (IFN-alpha) in order to be able to, on the one hand, better estimate derivatives with an increased half-life time (pegylated HCV transmission risks within the clinical environment interferon) and in combination with ribavirin, a nucleoside or amongst the drug milieu, for example, and, on the other analogue, the response rates have improved considerably. hand, to defi ne disinfection agents and hygienic procedures With this combination it is possible to achieve a long-term that can safely inactivate the virus. virological response with 50% of the patients with the HCV genotype 1, while patients with genotype 2 and 3 viruses The development of HCV cell-culture models As opposed are even able to attain long-term therapeutical success in to bacteria or fungi, viruses are obligatory intra-cellular more than 80% of the cases12. New and directly effective parasites. They have no self suffi cient metabolism of their antiviral medications will increase the options for therapy own and are therefore dependent upon suitable host cells. by the end of 2011. However, these novel therapeutics are HCV-replication can therefore only be examined in cell unfortunately not suitable for the treatment of all viral cultures – however, for many years scientists had been genotypes. Since the virus is able to become resistant very unsuccessful in their attempts to develop such systems for quickly, following application of these combinations without the propagation of HCV. further medications (mono-therapy), these preparations supplement standard therapy, but are not able to replace the It was not until ten years after the discovery of HCV that IFN-ribavirin treatment with its multiple side-effects. This Lohmann and Bartenschlager were able to achieve some leaves us with continued demand for the development of new success on the way to establishing a cell-culture model suit- forms of therapy with preferably diminished side-effects, able for HCV propagation11. They constructed HCV “mini- genotype-overlapping effectiveness and a high barrier genomes”, so-called sub-genomic HCV replicons. These rep- against viral resistance. licons possess all viral proteins that are necessary for the propagation of the virus genome. However, in place of the In this tug-of-war between fundamental questions gener- viral structural proteins that are required for the formation ated by the virus and the clinical challenges of therapy, we of new virus particles, they carry a resistance gene. This at TWINCORE are focusing on HCV. Our team is composed resistance gene is required to separate cells that propagate of natural-science scientists and physicians. We work the replicons from other cells that do not: after transfection closely with the Department of Chemical Biology of the HZI of the replicons into the human hepatoma-cell-line Huh-7, (Helmholtz Centre for Infection Research - Dr. Ronald Frank these cells are treated with an antibiotic that is inactivated und Dr. Florenz Sasse), and with the Clinic for Gastroen- by the replicon-encoded resistance gene. Consequently, terology, Hepatology and Endocrinology (Prof. Dr. Michael only those cells survive that have assumed the replicons Manns) of the MHH (Hannover Medical School). This close and effectively propagate them (Figure 2). This is a system collaboration between TWINCORE scientists, HCV experts with two essential limitations: The formation and release of in the clinic and natural scientists at the HZI fosters the viruses as well as the infection process itself could not be successful operation of a translational research programme. examined, since the sub-genomic replicons were missing Convenient proximity to the MHH is quite helpful since the structural proteins. it facilitates pragmatic implementation of the vision of translational research – for example, when young assistant The discovery that retroviruses, from which the cognate physicians oversee research projects within their respec- envelope protein had been genetically removed, are able tive medical environment at TWINCORE. to incorporate the HCV envelope proteins E1 and E2 in a functional fashion in their virus envelope placed one more important milestone on the pathway to establishing a cell- culture system for HCV. By using this technique Bartosch and her colleagues were able to show in 2003 that these 24 RESEARCH REVIEWS | Frontier Runners of Hepatitis C Virus

Viral pseudoparticle (1) DNA with cloned HCV Genome (2) In vitro Transcription (3) HCV RNA Transcripts (HCVpp) Structural Non-structural proteins proteins T7-RNA Polymerase E2 E1 HCV Manipulation of HCV Genome Retroviral Genome HCV Complete genome Retroviral Capsid Resistence IRES Non-structural gene proteins (5) Defection infected cells (4) Transfection (IFM, qRT-PCF)

96 h 72 h HCV Replicon Huh-7 Hepatoma cells Infection Transfection HCV Replicon cells IFM Selection Manipulation of Analysis of the host cell Phenotypes (e.g. RNAi) α-NS3 Model for the Model for the

HCV HCV Mutant Infection process RNA Replication Wild type

Fig. 3. Experimental procedure for examination of HCV replica- Fig. 2. Important HCV cell-model systems prior to development tion with the aid of the JFH1 infection system. IFM, immuno- of the JFH1 infection system. IRES, internal ribosome entry fl uorescence; qRT-PCR, quantitative Reverse-Transcriptase Poly- point. Graphic: Twincore merase Chain Reaction; RNAi, RNA-interference. Graphic: Twincore

mixed-viruses (“HCV-pseudo-particles” = HCVpp), with a With the aid of molecular-biological techniques we can now retroviral core and an HCV-typical virus envelope, prefer- manipulate, in a targeted manner, either the virus genome entially infect liver cells1. Since the early stages of infec- or the host cell and subsequently characterise the infl uence tion are conveyed essentially by the proteins in the virus of these modifi cations on virus propagation (Figure 3). In envelope (the HCV E1 and E2 proteins, for the HCVpp case), this fashion we gain essential knowledge concerning the a system was made available that permitted analysis of the virus’ essential interactions with the host cell, its method of HCV infection process by means of molecular-biological propagation within the cell, and we can identify new targets technology (Figure 2). It was the developments of 2005 to for development of a direct antiviral therapy. Moreover, new 2006 that ultimately rendered the methods and approaches therapy procedures can be evaluated regarding their of classic virology accessible to HCV. Together with Wakita effectiveness. Our HCV cell-culture model represents and other colleagues, we have been able to show that a thereby the central key to solving clinically relevant issues new HCV-isolate taken from a Japanese patient with a case that are current in HCV research. of fulminant hepatitis (“Japanese fulminant hepatitis 1” = JFH1) could not only effi ciently replicate in Huh-7 cells, but The pathway of the virus through the clinic An important it could also yield virus particles in the culture medium15. aspect in the management of hepatitis C is prevention, More over, we were also able to show that these particles since the mode of transmission from blood to blood should (“cell culture grown HCV” = HCVcc) are infectious not only be controllable with suitable hygienic measures. Particu- in cell cultures, but in vivo as well. By constructing JFH1 larly, transmissions within the hospital environment, variations with structural proteins from other virus isolates, the stability of the virus under variable environmental we substantially enhanced the effi ciency level of the infec- conditions and its sensitivity to chemical disinfection tion system13. Further HCV chimeras have been constructed agents are in the focus of our efforts. Examinations and in the mean time by us and other groups. In this manner, experiences heretofore have been based, due to a lack of comparative examinations between different isolates are HCV in vitro models, almost exclusively on studies with the now possible as regards the route of transmission and the bovine diarrhoea Virus (BVDV), which is a relative of the mechanisms of virus formation and virus release. HCV and which has been cultivated for a fairly long time to date. However, these studies with the surrogate virus for HCV allow only a semi-dependable estimation of the infectiousness of the HCV. RESEARCH REVIEWS | Frontier Runners of Hepatitis C Virus 25

With the aid of our HCV infection model, we were able to The infl uence of immuno-suppressive drugs on HCV show that HCV still remains infectious after 28 days at Once HCV has been transmitted, the infection process room temperature, and at 4°C the virus is still infectious proceeds through the stages described above, frequently even after 150 days5. Since HCV in vivo is associated with leading to chronic liver disease. HCV-associated liver human serum, the infl uence of human serum from healthy failure is one of the most frequent reasons for liver patients on HCV stability was analysed. The presence of transplantation3. Since in practice neither virus-neutra- serum, however, had no infl uence upon the stability of HCV lising antibodies nor potent antivirals have been made particles. Incubation of HCV on various surfaces such as available that could hinder re-entry of the virus into the plastic, steel and gloves indicated a comparable half life of liver, re-infection of the transplanted liver can currently not the virus between these materials. be avoided. Consequently, the graft liver is almost univer- sally re-infected by HCV. Unfortunately, disease progression It is a limiting factor of these studies that it is currently after transplantation is usually more rapid than before, unknown whether cell-culture viruses and naturally often leading to severe liver disease. In fact, the time span occurring viruses possess precisely the same characteris- between transplantation and development of liver cirrhosis tics. However, since in vivo animal studies based on has unfortunately become shorter in the last ten years2. chimpanzees are controversial due to high costs and ethical This means that patients who underwent transplantations concerns and as small animal models for HCV replication in 1988/89 experienced deterioration of liver function by are still not broadly available, cell culture studies with cell one fi brosis grade on average within approximately 5 years. culture derived HCV represent the optimal model to address For patients who underwent transplantation in 1996, this these important questions. time span amounted to only one year.

One important fi nding of our study was that detection of In the meantime several factors that may account for this HCV-RNA does not necessarily correlate with the viral development have been identifi ed2,8,9. It must also be infectiousness of HCV. Several different published studies mentioned that patients with a chronic hepatitis C virus are based nevertheless on a verifi cation of HCV-RNA by infection after liver transplantation experience a worse means of a PCR (polymerase chain reaction). Knowledge development than patients with other liver diseases. They concerning the missing correlation between detection of have a worse prognosis in comparison to patients with HCV-RNA and infectiousness must therefore be taken into alcohol- or hepatitis B-associated liver cirrhosis, or primary consideration in the interpretation of such studies. More- sclerosing cholangitis (PSC = a liver disease which involves over, to evaluate the effi cacy of HCV inactivation procedures, destruction of the bile ducts), or autoimmune hepatitis. In various alcohols and seven commercial disinfection agents this context the particular kind of immuno-suppressive were tested regarding their virucidal characteristics. We drug administered after the transplantation plays a role. An were able to show in this context that certain disinfection HCV-specifi c and optimised immuno-suppression could agents led to complete virus deactivation only with contribute to an improvement in the disease process for undiluted application14. These fi ndings provide a valuable these patients. It was a goal in this study to systematically frame work to defi ne safe hygiene procedures for handling examine all currently and routinely administered immuno- of HCV-positive materials in the clinical daily routine. We suppressive drugs as regards their infl uence on the overall are currently working on other studies that concern life-cycle of HCV. We thus examined the entry of the virus, stability of HCV in body fl uids such as breast milk and the RNA replication and the release in the presence of these semen, since these could also potentially contain either drugs utilizing our new HCV in-vitro-system6. virucidal substances or factors that may facilitate virus transmission. Furthermore, it has been planned to examine, With regard to RNA replication, both cyclosporine A and by means of special carrier experiments, the behaviour and mycophenolic acid as well as FTY720 act in an inhibitive stability of HCV on various surface areas after drying. manner in the infection model6,7. Other frequently used This knowledge will contribute to the assessment of immuno-suppressive drugs such as tacrolimus, everolimus, transmission risks and in turn help to prevent new HCV basiliximab and prednisolone had no infl uence on HCV- infections. RNA replication. When we examined the complete viral lifecycle, we discovered important correlations: incubation 26 RESEARCH REVIEWS | Frontier Runners of Hepatitis C Virus

with higher doses of prednisolone and other steroids identifi ed the human peripheral neuroblastoma cell-line increased HCV infectivity. By means of time-kinetic SKNMC, which expresses all four essential HCV-entry experiments, we were able to prove that HCV under factors and was susceptible to cell entry by our HCV treatment of steroids can penetrate the liver cells more pseudo-particles (HCVpp). Our investigations did however quickly. With the aid of retroviral pseudotypes, we could show as well that the virus cannot effi ciently propagate in show that the increase in infectiousness was specifi c for these cells. Nevertheless, our results point to the fact that HCV. Further mechanistic studies revealed that prednisolo- HCV can for all intents and purposes penetrate into ne treatment increased the mRNA and the protein expres- non-hepatic cell types. These interactions may in turn sion of SR-BI and occludin, two proteins that are essential infl uence the development of the disease and particularly components of the HCV receptor complex. extra-hepatic disease manifestations.

It was possible to ablate this steroid effect through the use Small molecules up against an unsolved problem The of the glucocorticoid receptor inhibitor RU-486, which options available for therapy against HCV are currently still indicates that this signalling cascade is responsible for the insuffi cient. Although a series of direct antiviral substances infl uence on the receptor expression and the HCV cell entry. are in clinical development, undesirable side-effects, resistance development and genotype-specifi c antiviral Moreover, we noted that prednisolone not only facilitated activity still pose substantial challenges. For this reason, the virus cell entry and spread in the human hepatoma-cell-line on-going search for appropriate therapy measures continues. Huh-7.5: the incubation with prednisolone also increased The focus is now on combinations of well-tolerated active viral dissemination in dimethyl-sulphoxide-differentiated compounds that act at different steps within the HCV liver cells and primary human hepatocytes. This result replication cycle. In this manner, a combination therapy correlates with the clinical experience in the patient: with drugs acting via distinct mode of action should prevent Various clinical studies had reported that administration of rapid development of viral resistance. high dosages of steroids, for example in the context of acute organ rejection treatment, was associated with increased In order to identify substances that disturb the various viral load and augmented liver infl ammation8. Correspond- steps in the HCV replication cycle, we have developed a new ingly, avoidance of highly-dosed steroids could contribute to dual-reporter-gene assay, and adapted it in collaboration an improvement in clinical course after HCV-induced liver with scientists at the HZI to a high-through-put-screening transplant. procedure (Figure 4). This system encompasses the entire HCV life-cycle and, therefore, enables identifi cation of Hepatitis C – not only in the liver? To date research on inhibitors against each individual step in viral propagation10. HCV has been primarily focussed on the liver – an obvious The assay is based on an HCV reporter virus that carries a focus, since the symptoms can be attributed predominantly gene for a luciferase-enzyme of the fi refl y (F-Luc; fi refl y to damage of this organ. Upon more precise investigation luciferase). The host cells, which are used for propagation of however, one can see that infection with HCV generates a HCV, were equally furnished by us with a luciferase gene series of other symptoms as well, and these symptoms are from the deep-sea crab gaussia (G-Luc; gaussia luciferase). not necessarily in direct relation to the liver infection. Since the two luciferase enzymes use different substrates Patients with a chronic HCV infection often times present and do not infl uence one another, propagation of the HCV with various nervous system-associated symptoms. Chronic and the vitality of the cells are simultaneously determined. fatigue syndrome, depression and cognitive disorders In this manner we are able to differentiate between frequently appear. It is however unclear whether these antiviral molecules and inhibitors of cell growth and symptoms can be attributed to a direct HCV replication in cytotoxic compounds, i.e. separate the wheat from the chaff. the brain and the peripheral neuronal cells, or to indirect Subsequently we validated the system with the aid of effects upon the central and peripheral nervous system. For known HCV inhibitors for cell entry, replication and virus this reason we investigated whether host cells from these tissues – among them human neuroblastoma and glioblas- toma cell-lines and microglial cells – are susceptible to HCV infection and can propagate the virus4. In this context we RESEARCH REVIEWS | Frontier Runners of Hepatitis C Virus 27

production. Finally, we tested compounds of myxobacteria taken from the HZI compound library. A series of candi- dates emerged from this screening that inhibit either the virus’ entry into the liver cells or the respective propaga- tion and exit from the cell. With these molecules it is our goal, in collaboration with our partners at the HZI and the myxobacteriologists working with Rolf Müller at HIPS, to develop lead molecules for new HCV inhibitors. Parallel to this, we want to use these compounds to better understand how HCV propagates in liver cells (Figure 5). In this regard the myxobacterial natural materials provide us with a unique gateway to facilitate manipulation of cell-biological processes, and to be able to observe how HCV is infl uenced thereby. For these purposes, also those compounds that aug- ment propagation of HCV are by all means quite important. Once we have come to understand precisely which processes in the cell are responsible for these processes, we will be able to fi nd ways in the future to use this interaction Fig. 5. Infl uence of small molecules with antiviral activity between virus and cell for therapeutic purposes. against HCV on the formation of cellular lipid droplets in liver cells. HCV infected cells were incubated with solvent or an inhibitor. Lipid droplets were coloured green with an anti- A Firefl y HCV Reportervirus Virus production ADRP antibody (left) or a lipid colouring agent (small picture). +/- Substance HCV core was detected with a core-specifi c antibody (on the ”F-Luc” right and marked with red in the small picture). Incubation with the inhibitor leads to expansion and aggregation of lipid droplets, as well as to the enrichment of HCV core on the Deep sea crab Liver cell with Reportergene surface. Graphic: Twincore

”G-Luc” Measurement of reporter activity B

Fig. 4. HZI robot in the S3* safety laboratory for execution of high-throughput screenings. Graphic & Photo: Twincore 28 RESEARCH REVIEWS | Frontier Runners of Hepatitis C Virus

Sandra Ciesek born 1978 in Goslar, studied medicine in Göttingen and Hannover and received her M.D. degree with honours under Prof. M. P. Manns and Dr. H. Wedemeyer at the Clinic for Gastroenterology, Hepatology and Endo- crinology at the MHH (Hannover Medical School). Sandra Prof. Pietschmann and his work group. Photo: Twincore/HZI Ciesek received numerous prizes for her doctoral thesis and has been working since then as Assistant Physician in the Department Gastroenterology, Hepatology and Endocrinology and as a scholarship-funded scientist at TWINCORE with Prof. Thomas Pietschmann. Under the auspices of an indivi- dually contracted position funded by the German Research Foundation (DFG), she is now doing research at TWINCORE on the infl uence of cyclosporine A on the hepatitis C non- structural protein NS2.

Thomas Pietschmann born in 1971 in Würzburg, studied biology with emphasis on biochemistry, animal physiology, virology and immunobiology at the University of Würzburg and the Duke University (Durham, NC, USA). After com- pleting his studies in 1996, he received his Ph.D. degree in biology at the Institute for Virology of the University of Würzburg and subsequently worked as postdoc at the Institute for Virology in Mainz and in the Department for Molecular Virology in the University Clinic of Heidelberg. Eike Steinmann born 1978 in Bremen, studied biology at Thomas Pietschmann established there an independent Leibniz University Hannover, with emphasis on Virology, research group that investigated the mechanisms of mor- Microbiology and Molecular Biology. After a DAAD scholar- phogenesis and cell entry of the hepatitis C virus. From the ship for study at Northeastern University in Boston, he year 2006 his group was supported by an Emmy Noether completed his diploma dissertation under Prof. Herrler at fellowship from the German Research Community (Deutsche the Institute for Virology of the Veterinary University of Forschungsgemeinschaft). In the spring of 2007 he was Hannover. For his Ph.D. thesis Eike Steinmann changed to appointed with his work group to TWINCORE. He now leads the Department for Molecular Virology at the University the Department for Experimental Virology there. Clinic of Heidelberg and did research in the group of Prof. Bartenschlager regarding the function of p7 protein in the HCV replication cycle. With his advisor, Prof. Thomas Pietsch mann, he then was appointed to TWINCORE. His research is now concentrated on various aspects of the HCV assembly process and its release, as well as a search for new antiviral targets. Furthermore, he is examining the environmental stability and susceptibility of HCV to disin- fection agents. RESEARCH REVIEWS | Frontier Runners of Hepatitis C Virus 29

Literature 9. Gane, E. J., B. C. Portmann, N. V. Naoumov, H. M. Smith, J. A. Underhill, P. T. Donaldson, G. Maertens, and R. Williams. 1. Bartosch, B., Dubuisson, J., & Cosset, F. L. (2003) Infec- 1996. Long-term outcome of hepatitis C infection after liver tious hepatitis C virus pseudo-particles containing functional transplantation. New England Journal of Medicine 334:815- E1-E2 envelope protein complexes. Journal of Experimental 820. Medicine 197, 633-642. 10. Gentzsch, J., Hinkelmann, B., Kaderali, L., Irschik, H., 2. Berenguer, M., Prieto, M., San Juan, F., Rayon, J. M., Jansen, R., Sasse, F., Frank, R., & Pietschmann, T. (2011). Martinez, F., Carrasco, D., Moya, A., Orbis, F., Mir, J., & Hepatitis C virus complete life cycle screen for identifi cation Berenguer, J. (2002) Contribution of donor age to the recent of small molecules with pro- or antiviral activity. Antiviral decrease in patient survival among HCV-infected liver trans- Research 89(2), 136-148. plant recipients. Hepatology 36, 202-210.

3. Brown, R. S. (2005) Hepatitis C and liver transplantation. 11. Lohmann, V., Körner, F., Koch, J. O., Herian, U., Theil- Nature 436, 973-978. mann, L., & Bartenschlager, R. (1999) Replication of sub- genomic hepatitis C virus RNAs in a hepatoma cell line. 4. Bürgel B, Friesland M, Koch A, Manns MP, Wedemeyer H, Science 285,110-113. Weissenborn K, Schulz-Schaeffer WJ, Pietschmann T, Stein- mann E & Ciesek S (2010) Hepatitis C virus enters human 12. Manns, M. P., Wedemeyer, H., & Cornberg, M. (2006). peripheral neuroblastoma cells - evidence for extra-hepatic Treating viral hepatitis C: effi cacy, side effects, and compli- cells sustaining hepatitis C virus penetration. Journal of Viral cations. Gut 55, 1350-1359. Hepatology Jun 23 [Epub ahead of print]. 13. Pietschmann, T., Kaul, A., Koutsoudakis, G., Shavinskaya, 5. Ciesek, S., Friesland, M., Steinmann, J., Becker, B., Wede- A., Kallis, S., Steinmann, E., Abid, K., Negro, F., Dreux, M., meyer, H., Manns, M. P., Steinmann, J., Pietschmann, T., Cosset, F. L., & Bartenschlager, R. (2006) Construction and & Steinmann, E. (2010) How stable is the hepatitis C virus characterization of infectious intragenotypic and intergeno- (HCV)? Environmental stability of HCV and its susceptibility typic hepatitis C virus chimeras. Proceedings of the National to chemical biocides. Journal of Infectious Diseases 201, 1859- Acdademy of Sciences USA 103, 7408-7413. 1866. 14. Steinmann, J., Becker, B., Bischoff, B., Paulmann, D., 6. Ciesek, S., Steinmann, E., Iken, M., Ott, M., Helfritz, F. A., Friesland, M., Pietschmann, T., Steinmann, J., & Steinmann, Wappler, I., Manns, M. P., Wedemeyer, H., & Pietschmann, T. E. (2010) Virucidal activity of 2 alcohol-based formulations (2010) Glucocorticosteroids increase cell entry by hepatitis C proposed as hand rubs by the World Health Organization. virus. Gastroenterology 138, 1875-1884. American Journal of Infection Control 38, 66-68.

7. Ciesek, S., Steinmann, E., Wedemeyer, H., Manns, M. P., 15. Wakita, T., Pietschmann, T., Kato, T., Date, T., Miyamoto, Neyts, J., Tautz, N., Madan, V., Bartenschlager, R., von Hahn, M., Zhao, Z., Murthy, K., Habermann, A., Krausslich, H. G., T., & Pietschmann, T. (2009) Cyclosporine A inhibits hepa- Mizokami, M., Bartenschlager, R., & Liang, T. J. (2005) Pro- titis C virus nonstructural protein 2 through cyclophilin A. duction of infectious hepatitis C virus in tissue culture from Hepatology 50, 1638-1645. a cloned viral genome. Nature Medicine 11, 791-796.

8. Forman, L. M., Lewis, J. D., Berlin, J. A., Feldman, H. I., & Lucey, M. R. (2002) The association between hepatitis C infection and survival after orthotopic liver transplantation. Gastroenterology 122, 889-896.

30 RESEARCH REVIEWS | The Aging of the Immune System: Challenges and Perspectives

The Aging of the Immune System: Challenges and Perspectives

AUTHORS | Priv.-Doz. Dr. Eva Medina | Research Group Infection Immunology | [email protected] | Dr. Dr. Luka Cicin-Sain | Junior Research Group Immune Aging and Chronic Infections | [email protected]

How old is old? Looking back through human history, the average life expectancy during the Stone Age was 20 to 35 years, just enough time to allow procreation and ensure the continuation of the human race. In the late 19th and the 20th centuries, however, life expectancy in industrialized nations exploded, nearly doubling over the last 100 years. The average life expectancy is now ≈75 years for men and somewhat over 80 years for women, with a still upwardly trend and nobody is able to predict where it may end (Figure 1). Although the news that we are living longer is positive, this fact presents new challenges to both individuals and to society. The increase in the number of people with advanced age is accompanied by an epidemic of chronic diseases, including Alzheimer disease, arthritis, atherosclerosis or diabetes. A common denominator of all these conditions is their infl ammatory pathogenesis, indicating that aging results in a dysbalance of the immune function referred to as “immunosenescence”. This age-associated deterioration of the immune system is responsible for the increased prevalence and severity of infectious diseases, as well as the low effi cacy of vaccination in the elderly. Indeed, the elderly population is particularly susceptible to infection and vulnerable in case of disease (Gavazzi and Krause, 2002). For example, deaths resulting from infl uenza and pneumonia represent the sixth leading cause of death among persons aged 65 or older in developed countries (Stupka et al., 2009). Vaccinations could help to overcome this increased risk of infectious death in elderly persons. However, the protective effect of vaccination is also partially lost in the elderly population (Chen et al., 2009). Understanding the basic mechanisms of immune dysfunction that occur with age will help to develop interventions to delay or even reverse the detrimental effects of immunosenescence, and thereby ensure a better protection of the vulnerable elderly population and a “healthy aging”.

Development of pathogen diagnostics: Mechanisms of tious agent and rapidly recognize them upon reinfection. immunosenescence The aging process can be viewed Lymphocytes constitute a population of millions of clonally as a progressive breakdown of the homeostatic balance in diverse cells, which can each recognize a different molecu- an individual. As we age, it becomes increasingly diffi cult lar moiety that is not cognate to the host (antigen). Once a to react to external stress and restore the homeostasis. naïve lymphocyte clone recognizes a novel antigen, it pro- Therefore, the aging host requires increasingly more time liferates to form a large population of cells recognizing and and energy to recover its homeostatic footing, which makes neutralizing pathogens carrying this moiety. A number of them comparatively less fi t than younger individuals. Sys- these lymphocyte clones persists after infection resolution tems that allow an individual to adapt to its environment – as memory cells in the host, conferring immunity against such as the nervous system, which allows the adaptation to recurrent infections with the same pathogen. the immediate physical environment or the immune system, which allows the host to adapt to its microbiological envi- Generally, the aging process can affect both branches of the ronment – are particularly affected by the aging process, immune system, yet the functional losses of the adaptive because their rapid response to external stimuli is essential branch of the immune system have been exhaustively docu- for host survival. mented, while the documented age-related changes of the innate immune system are less exhaustive and sometimes The immune system can be divided into an innate part, contradictory (Nikolich-Zugich and Cicin-Sain, 2010). While consisting mainly of monocytes, neutrophils, natural a number of studies showed an increase in the activity of killer and dendritic cells, as well as into an adaptive part, the innate immune system and the infl ammation associated represented by B and T lymphocytes. The innate immune with aging has been prominently termed “infl ammaging” system allows prompt reactions to pathogen-associated (Franceschi and Bonafè, 2003), it remains unclear if this is molecular patterns (PAMP) but responds indiscriminately due to a compensation of the progressively weaker function to an infectious threat. The adaptive immune system is able of the adaptive immune branch or an intrinsic increase in to respond in a more pathogen-specifi c manner and has the the activity of the innate immune system. ability to remember novel moieties associated with an infec- RESEARCH REVIEWS | The Aging of the Immune System: Challenges and Perspectives 31

Fig 2. Loss of naive cytotoxic T-lymphocytes in aged individuals (adapted from Cicin-sain et al. PNAS 2007). The population of naive lymphocytes, characterized by low CD95 and high CD28 expression, is typically abundant in young adult rhesus monkeys, but severely diminished in old ones. This results in relative increases of memory subsets. Graphic: HZI

to immune aging are the thymic involution and the conse- quent loss of newly generated naïve T cells (Figure 2) as well as metabolic stress, in particular oxidative stress (Alt- meyer and Hottiger, 2009). It remains less clear if infl amma- tory conditions contribute to immune aging or are merely a consequence of immunosenescence. Similarly, the role Fig. 1. The male (blue histogrammes) or female (red histo- of chronically persisting infections in immunosenescence grammes) general population has been traditionally is not entirely understood (Virgin et al. 2009) and it is not distributed across age categories to form a pyramid, where clear if persistent infections contribute to immune aging or the most numerous individuals were present in the youngest if immunosenescence results in chronic infections. These age cohorts. In Germany, the demographic changes resulted questions can be answered by experimental modelling of in an altered, mushroom shaped, distribution. If current the immune processes in aging animals. trends persist, in 25 years the population between 65 and 75 years of age will be the most frequent one. Graphic: HZI Immunosenescence has multiple clinical consequences. The elderly show a signifi cant increase in the susceptibility to infections that are controlled by naïve lymphocyte populations, such as emerging infl uenza strains, West Nile On the other hand, there is a clear consensus that aging Virus infections or pneumococcal infections. This problem adversely affects the adaptive branch of the immune sys- is complicated by the inability of naïve cell populations to tem, and in particular the response to emerging infections generate protective immunity upon vaccination in elderly occurring in individuals that have reached an advanced age individuals. On the other hand, the elderly are also more (Nikolich-Zugich and Rudd, 2010). In contrast, the memory vulnerable to a variety of other conditions, particularly responses to pathogens and vaccines encountered earlier in chronic infl ammatory conditions including arthritis, life are maintained even in very advanced age (Ammana et infl ammatory bowel disorders, atherosclerosis or conditions al., 2007). Consequently, clonally diverse naïve lymphocytes with a strong infl ammatory component like diabetes or are more profoundly affected by aging than clonally restricted Alzheimer disease. memory subsets. Therefore, the loss of clonal diversity in the lymphocyte pool is a prominent change associated with Our understanding of the cellular and molecular changes immunosenescence. that underlie the decline in immune function with age has increased signifi cantly in recent years, and clinical trials to The causes of immunosenescence are multiple and not en- evaluate methods by which to augment immunity in elderly tirely understood. Among the well characterized contributors individuals are now underway. 32 RESEARCH REVIEWS | The Aging of the Immune System: Challenges and Perspectives

Centenarians as a model for healthy aging Centenarians, which are by defi nition very old individuals, are the best model in which to study human longevity. The centenarians are persons not only older than 100 years, but also in good mental and physical condition. Centenarians were once considered to be exceedingly rare; however this is no longer the case as their numbers are dramatically increasing. Thus, according to published predictions, those surviving longer than 95 or 100 years will soon represent a considerable population (Olshansky et al., 1990; Barinaga M, 1991). Cen- tenarians are the best example of successful healthy aging since they have avoided and/or survived the most impor- tant pathologies that are responsible for the morbidity and mortality of aged people. In order to be capable of avoiding or postponing major age-related diseases, centenarians should be equipped with well preserved and effi cient immune and defence mechanisms and have optimal combinations of ap- Fig 3. The mouse model has proved to be a very useful tool propriate lifestyle and genetic background (Franceschi and for studying the ageing process as well as for elucidating Bonafè, 2003). Studies of their immune system have revealed the basis for the increased susceptibility to infection that parameters that follow the deteriorating trend often present accompanies advance age. Photos: HZI in aged people (e.g. reduction of B and T lymphocytes), whereas other parameters are well preserved (e.g. chemo- taxis, phagocytosis). Whether these components, rather than environmental and genetic determinants, are responsible for The mouse models (Figure 3), on the other hand, have reaching such as an advanced age still remains to be deter- provided signifi cant data on the changes in the adaptive im- mined. Thus, the study of centenarians, and particularly that mune response with advancing age (Maue et al., 2009). The of healthy centenarians, is not only of broad biological and immunosenescence models include mice with alterations in medical interest, but can also help in identifying genes that telomerase activity, tumour suppressor function, oxidative prevent the above-mentioned age-related diseases. stress, hormone expression and various other molecules associated with immune development and differentiation as Animal models for the study of immunosenescence well as longevity. A number of unique fi ndings have been Ethical concerns pose considerable limitations on our made in these models regarding basic immune function and ability to perform experiments in humans. Hence, much of the relationship between cellular networking and signalling what we have learned is owed to the utility of experimental pathways associated with longevity and immune function. cellular or animal models of aging. A number of animal Furthermore, the mouse model represents a very useful tool models have been established to examine the age-associated for evaluating strategies to prevent or reverse the immune effects in the immune function and specifi c pathways that aging process, such as those aimed at improving the ability have been shown to be altered by aging. Invertebrates offer of aged animals to successfully respond to vaccination or to pragmatic benefi ts for such studies as they generally have combat pathogen challenge. short generation times, short life spans and can be raised in large numbers. However, the translation of the knowledge One major limitation of extrapolating the fi ndings obtained gained in invertebrate organisms is complicated by the fact with the mouse model of immune aging to immunosenes- that invertebrates rely solely on an innate immune response cence in human medicine has been the relatively short and lack the components of the adaptive immune system lifespan of mice (≈2.5 years). A more suitable alternative is found in vertebrates. Nevertheless, the lack of components offered by studies performed on aging rhesus non-human of the adaptive immune response has the advantage that it primates (NHP), which are closer to humans both from the allows the study of effects of age on the innate immune sys- phylogenetic perspective and also in terms of their lifespan tem while excluding the confounding interactions between clock (average life expectancy in rhesus monkeys ≈25 years). the innate and adaptive immune mechanisms. The most On the other hand, research in primate models is limited by extensively used invertebrates for aging studies have ethical concerns and by technical restrictions. One can only been Drosophila melanogaster and Caenorhabditis elegans house and use a limited number of NHP in an experiment (Kurz and Tan, 2004). and the advanced genetic models which are freely available in experimental mice cannot be practically implemented in RESEARCH REVIEWS | The Aging of the Immune System: Challenges and Perspectives 33

long-lived hosts. To this end, different models offer varying Using a mouse model of aging, the researchers from the benefi ts in the study of immunosenescence. Identifying the Group Infection Immunology have investigated specifi c comparative advantages of individual experimental models defi ciencies in the immune function that lead to age-related and their use in the appropriate context is key to successful increased susceptibility to infection with the bacterium research. Streptococcus pyogenes (Goldmann et al., 2010). This patho- gen is an important cause of severe, life-threatening infec- Immune rejuvenation: more than just a dream? As we tions among the elderly population. They found that, similar learn more about the cellular and molecular changes that to humans, young animals (aged two to three months) are underlie aging of the immune system, it is likely that new ap- able to combat the infection successfully while aged mice proaches to reverse this process will be discovered. As immu- (older than 20 months) died even if they were infected with nosenescence is associated with a decline in naïve B and T fewer bacteria (Figure 4). cell production, the repopulation of the immune system by B and T cells derived from pluripotent stem cells might provide The immune mechanisms affected by immunosenescence a means to revert immunosenescence in elderly patients, that make aged mice more susceptible to bacterial infections provided that adult somatic cells may be reprogrammed into are also a focus of research in this Group. They centre their pluripotent stem cells to raise naïve lymphocytes. Another studies on macrophages because these cells are the fi rst line potential strategy that provided encouraging results in ani- of defence for combating bacterial infections. They have found, mal models is based on the restoration of the thymic epithe- for example, that the amount of macrophages is highly re- lium function and the consequent delay of thymic involution duced in the tissue of aged mice compared to young animals after treatment with keratinocyte growth factor (Min et al., (Figure 5). As the amount of macrophages in the organs de- 2007). An additional promising strategy for rejuvenation of pends on the production of a specifi c growth factor (M-CSF), the aged immune system is to target lymphocyte production the researchers have investigated whether treatment with to increase the number of naïve B and T cells that migrate this growth factor could induce repopulation of resident tis- to secondary lymphoid organs. Increases in the numbers of sue macrophages in aged mice and increase their resistance new B and T cells in the circulating pool may improve the during infection. Indeed, the treatment with M-CSF made ability of elderly individuals to mount a response to new or aged mice much more resistant and they were able to fi ght recall antigens. Several interventions, including exercise, the infection much better. The outcome of these investigations have also been proposed to restore immune function in older indicates that repeated prophylactic administration of M- populations. The fi ndings from some, but not all studies, sup- CSF can help to maintain the macrophage compartment in port the possibility that exercise may attenuate immunose- the elderly and the fi tness of the immune system. nescence and, therefore, may be an effi cacious therapy for restoring immune function in the elderly. Another possible strategy to counteract immunosenescence is to reduce the antigenic load represented by pathogens, such as infl uenza virus and cytomegalovirus (CMV). From this point of view, a systematic search for chronic viral infections in the elderly and the establishment of safe procedures to eradicate them would be likely to have a benefi cial impact on longevity. Because the aging process varies widely between individu- als, it will be important to develop ways of identifying those patients who would benefi t most from immunomodulatory treatments. Therefore, it would be useful to have simple biomarkers with which to accurately measure the effects of aging on the immune system. Progress in this area is already underway. Fig 4. Mice exhibit the age-related loss of resistance to Streptococcus pyogenes seen in humans. This fi gure shows Aging research at the HZI Aging research is currently that young mice can recover and survive when they are conducted at the HZI by two Research Groups: Infection infected with S. pyogenes (open symbols). On the other Immunology (INI) and Immune Aging and Chronic Infections hand, aged mice (closed symbols) are very susceptible (IMCI). The research efforts of these groups are focused on to streptococcal infection and all mice died shortly after identifying age-related changes in the immune function in bacterial inoculation. Graphic: HZI the hope of developing intervention strategies to delay or pre- vent the decline in the functionality of the immune system. 34 RESEARCH REVIEWS | The Aging of the Immune System: Challenges and Perspectives

Fig 5. Tissue macrophages are very important cells for combating S. pyogenes infection. This fi gure shows that aged mice had a signifi cantly lower number of resident tissue macrophages (indicated by an arrow) than young animals. Fig 6. Vaccination with an attenuated poxvirus (Modifi ed This can be one of the reasons for the elevated susceptibility vaccinia Ankara strain) results in detectable immune of aged mice to S. pyogenes infection. Graphic: HZI responses of tissue resident cytotoxic T-lymphocytes in adult rhesus monkeys, but not in aged ones. This loss of immune responses correlates directly to the loss of naive cells in aged hosts (see fi gure 2). Graphic: HZI The newly established Immune Aging and Chronic Infec- tions Group focuses on the mechanisms and effects of im- mune aging. In a recent collaborative study, the Group was able to show that aging NHP (≈18-25 yrs old) show signifi - cantly weaker immune responses to vaccination than adults (≈7-10 yrs old), and that this response could be predicted by the relative and absolute count of naïve cells in the blood of monkeys (Cicin-Sain et al., 2010) (Figure 6). The vaccination was performed with an attenuated virus (modifi ed vaccinia Ankara – MVA) that may be realistically considered for vac- cinations in elderly populations. Interestingly, the naïve cell counts and vaccine responses correlated only if the fraction Eva Medina is the head of the Infection Immunology of naïve cells as a pool of total lymphocytes dropped below Research Group at the HZI. Eva Medina graduated in Bio- a threshold of approximately 20%. Therefore, the clonal logical Sciences at the University of Seville (Spain) in 1987 diversity of naïve T cells appeared to defi ne the intensity of and obtained her PhD at the Medical School of Seville in responses to the vaccine only if the clonality was a limiting 1990. She worked as a postdoctoral researcher from 1991 to factor. In a follow up study submitted for publication the 1993 in the Immunology Department at the Royal Free Hos- authors show that the same population of NHP can mount pital School of Medicine in London. In 1993, she undertook a vigorous memory responses to a pathogen to which they postdoctoral fellow position at The Trudeau Institute in New were exposed early during their life (Rhesus cytomegalovi- York studying several aspects of the host response to infection rus – RhCMV). The Group now focuses on the effects that with Mycobacterium tuberculosis. From 1997 to 2004 she the persistent infections exert in a chronically infected host worked as a postdoc at the Department of Microbial Pathoge- and their contribution to the immune aging process. nesis and Vaccine Research in the HZI. On 2004 Eva Medina was appointed as Head of the Infection Immunology Conclusions To conclude, at present aging must be con- Research Group in the same institution. sidered an unavoidable end point of the life history of each individual. Nevertheless, our increasing knowledge about the mechanisms regulating aging and immunosenescence allows us to envision many different strategies to cope with and delay the processes in order to endow everybody with a long and healthy life. RESEARCH REVIEWS | The Aging of the Immune System: Challenges and Perspectives 35

Franceschi, C., & Bonafè, M. (2003) Centenarians as a model for healthy aging. Biochemical Society Transactions 31(2), 457-61.

Gavazzi, G., Krause, K. H. (2002) Ageing and infection. Lan- cet Infectious Diseases 2(11), 659-66.

Goldmann, O., Lehne, S., & Medina, E. (2010) Age-related Luca Cicin-Sain born in 1972, is head of the junior research susceptibility to Streptococcus pyogenes infection in mice: group Immune Aging and Chronic Infections at the Helm- underlying immune dysfunction and strategy to enhance im- holtz Centre for Infection Research (HZI). He studied at the munity. Journal of Pathology 220(5), 521-9. University of Rijeka, Croatia, and obtained his MD in 1996 and his PhD in 2006. From 2001 to 2004 he worked at the Kurz, C. L., & Tan, M. W. (2004) Regulation of aging and in- Max von Pettenkofer Institute in Munich and then moved to nate immunity in C. elegans. Aging Cell 3(4), 85-93. the Oregon Health and Science University in Portland, OR, USA, where he worked as a postdoctoral DFG fellow until Maue, A. C., Yager, E.J., Swain, S. L., Woodland, D. L., Black- 2007 and as a Research Assistant Professor until 2009. In man, M. A., & Haynes, L. (2009) T-cell immunosenescence: 2009 he moved back to Germany and joined the HZI in his lessons learned from mouse models of aging. Trends in Im- current position. In 2011 he was awarded the European Re- munology 30(7), 301-5. search Council Starting Grant to expand his research focus on the molecular and cellular mechanisms of herpesviral Min, D., Panoskaltsis-Mortari, A., Kuro-O., M., Holländer, G. antigenic induction and on the effects of persistent infec- A., Blazar, B. R., & Weinberg, K. I. (2007) Sustained thy- tions on immune homeostasis and aging. mopoiesis and improvement in functional immunity induced by exogenous KGF administration in murine models of aging. Blood 109(6), 2529-37.

Literature Nikolich-Zugich, J. & Cicin-Sain, L. (2010) Aging of the Im- mune System Across Different Species. In: Comparative Biol- Altmeyer, M., & Hottiger, M. O. (2009) Poly(ADP-ribose) ogy of Aging (Wolf, N., ed.), Springer Science and Business polymerase 1 at the crossroad of metabolic stress and infl am- Media, New York, pp 232-249. mation in aging. Aging (Albany NY) 1(5), 458-69. Nikolich-Zugich, J., & Rudd, B. D. (2010) Immune memory Amanna, I. J., Carlson, N. E., & Slifka, M. K. (2007) Duration and aging: an infi nite or fi nite resource? Current Opinion in of humoral immunity to common viral and vaccine antigens. Immunology 22, 535-40. New England Journal of Medicine 357(19), 1903-15. Olshansky, S. J., Carnes, B. A., & Cassel, C. (1990) In search Barinaga, M. (1991) How long is the human life-span? Science of Methuselah: estimating the upper limits to human longev- 254(5034), 936-8. ity. Science 250(4981), 634-40.

Chen, W. H., Kozlovsky, B. F., Effros, R. B., Grubeck-Loeben- Stupka, J. E., Mortensen, E. M., Anzueto, A., & Restrepo, M. I. stein, B., Edelman, R., & Sztein, M. B. (2009) Vaccination in (2009) Community-acquired pneumonia in elderly patients. the elderly: an immunological perspective. Trends in Immu- Aging Health 5(6), 763-774. nology 30(7), 351-9. Virgin, H. W., Wherry, E. J., & Ahmed, R. (2009) Redefi ning Cicin-Sain, L., Smyk-Paerson, S., Currier, N., Byrd, L., Koudel- chronic viral infection. Cell 813 (1), 30-50. ka, C., Robinson, T., Swarbrick, G., Tackitt, S., Legasse, A., Fischer, M., Nikolich-Zugich, D., Park, B., Hobbs, T., Doane, C. J., Mori, M., Axthelm, M. T., Lewinsohn, D. A., & Nikolich- Zugich, J. (2010) Loss of naive T cells and repertoire constric- tion predict poor response to vaccination in old primates. Journal of Immunology 184(12), 6739-45. 36 RESEARCH REVIEWS | Novel Nanoparticle-based Technology Platform for the Delivery of Vaccine Antigens

Novel Nanoparticle-based Technology Platform for the Delivery of Vaccine Antigens

CORRESPONDING AUTHOR | Prof. Dr. Claus-Michael Lehr | Department of Drug Delivery, Helmholtz-Institute for Pharmaceutical Research Saarland – HIPS | [email protected] | Chair for Biopharmaceutics and Pharmaceutical Technology, Saarland University, Saarbruecken | [email protected]

CO-AUTHORS | Dr. Steffi Hansen | Research Group Transdermal Drug Delivery – HIPS | Prof. Dr. Ulrich F. Schäfer | Chair for Biopharmaceutics and Pharmaceutical Technology, Saarland University, Saarbruecken | Prof. Dr. Dr. Carlos A. Guzmán | Department of Vaccinology and Applied Microbiology – Helmholtz Centre for Infection Research – HZI

Infections are responsible for approximately one third of all deaths occurring each year in the world. In addition infectious agents are also directly involved in the pathogenesis of many cancers and chronic diseases. Furthermore, infections are often the fi nal cause of death in patients affected by other primary diseases (e.g. trauma, cardiovascular or respiratory syndromes). Thus, it is of the upmost importance to develop strategies to prevent and treat infectious diseases. In this context vaccines are the most cost-effi cient tool to prevent infections. Moreover, their therapeutic application for both infectious and non-infectious diseases is attracting general interest.

Classical vaccines were based on live attenuated or in- in the muscles. In contrast, dendritic cells are present in activated pathogens. However, due to their complex and high densities in the dermis, and Langerhans cells are ill-defi ned nature, such vaccines can vary in quality from located in the epidermis accumulating around the hair batch to batch and can induce adverse events. In recent follicles. Therefore, it is highly attractive to target vaccines years we have seen the advent of subunit vaccines. These to the skin immune system by chemical, mechanical or preparations are based on the use of well-defi ned subcel- nano-technological means and devices thereby promoting lular components of the corresponding pathogen which are access of vaccine antigens to these APCs. In fact, intrader- in turn critical for the stimulation of a protective response. mal immunization can induce strong mucosal and systemic Subunit vaccines can be prepared with native components immune responses, as well as protection against infections. derived from the pathogen or obtained by DNA-recombinant technologies or in vitro synthesis (e.g. recombinant proteins, synthetic peptides, capsular carbohydrates, etc.). Classical vaccines were traditionally very immunogenic, due to the complex nature of these formulations and the presence of pathogen-derived components with built-in adjuvant pro- perties. In contrast, purifi ed components are usually very poor immunogens rendering essential the incorporation of adjuvants in the formulation. Adjuvants do not only allow to improve the overall strength of the elicited responses but also to reduce the amount of antigen needed and the time required to achieve a threshold of protective immunity as well as to modulate the quality and expand the breadth of the elicited response. Finally, adjuvants enable the stimula- tion of long lasting memory responses, thereby reducing the need for frequent boost vaccinations.

Most of the traditional vaccines have been administered by subcutaneous or intramuscular injection. However, the use of this route is associated to lack of acceptance by the public and safety issues (e.g. risk of contamination). It also requires skilled health personnel which in turn represents a logistic constraint. In addition, an injection (e.g. intra- muscular) does not deliver the vaccine optimally to antigen presenting cells (APCs) which are the relevant target to Fig. 1. Penetration of methylene blue into porcine skin. prime naïve T cells to initiate an effi cient adaptive immune The dye penetrates deep into the hair follicle. Photo: HIPS/HZI response. In fact, only a limited number of APC are present RESEARCH REVIEWS | Novel Nanoparticle-based Technology Platform for the Delivery of Vaccine Antigens 37

At the same time the skin and especially the horny layer of the stratum corneum provides nearly perfect protection not only against the invasion of chemicals or pathogens but also against therapeutics topically applied to the skin. Thus, the highly compact structure of the stratum corneum with alter- nating hydrophilic and lipophilic areas presents, indeed, a very effective fi rst line defense, in particular against large and hydrophilic molecules. Furthermore, purifi ed antigens are highly unstable when applied in their native state to the skin. Innovative delivery strategies are urgently needed (i.e. suitable carrier devices or formulations), which enable antigen stabilization and facilitate permeation. Fig 2. Penetration of methylene blue into mouse skin. The dye penetrates deep into the living skin layers and the hair follicle Pathways for passive diffusion of small molecules have been (arrow). Photo: HIPS/HZI identifi ed, among them the inter- and intra-cellular paths, as well as permeation via the appendages, such as hair fol- licles and pores. The trans-appendage pathway is especially interesting for the permeation of larger molecules since comparable. Transcutaneously immunized volunteers it allows sidestepping the tight stratum corneum. Corni- exhibited both CD4+ and CD8+ T-cell responses, whereas the fi ed cells are present only in the upper thirds of the hair latter was missing in conventionally immunized volunteers.9 follicles, whereas the lower part is lined with an epithelium containing tight junctions. However, Langerhans cells accu- The common principle of these permeability enhancing mulating around the follicles build up a second immunologic methods is that the mechanical action helps to overcome the line of defense in places where the mechanical barrier is very effective stratum corneum barrier to facilitate the access weakened. Although Langerhans cells represent only about of the antigen to the APCs. It was further hypothesized that 1% of the total cell population in the skin, they cover a large the mechanic action leads to unspecifi c pre-activation of surface area of approximately 20% through their horizontal immune cells, thereby improving antigen-specifi c immune orientation and long interdigitating protrusions.2 responses. A historical example where this concept was successfully used is the immunization against smallpox. Transdermal Vaccination – Innovative Approaches An effective and strong immune response was induced Various approaches have been pursued to deliver antigens subsequent to the degradation of the skin barrier as a result across the skin. New technologies are in progress to ensure of scarifi cation. the targeting of vaccine compounds to the APCs present in the epidermis. Membrane permeabilization agents such as Several such physical permeability enhancing methods cholera toxin used on patches that were combined with high have been combined with innovative formulations of vac- doses of antigens induced signifi cant immune responses.5 cines in order to achieve an improved immune response, New approaches which include micro-needles6, Gene gun or reduce the risk of severe side effects and lower the required PowderJect technologies7, electroporation, iontophoresis and dose. To this end different delivery vectors such as lipo- dermal abrasion have been proposed to facilitate DNA and somes, virosomes, transferosomes, nano- or micro-beads protein vaccine delivery by the percutaneous route.8 and viral vectors have already been tested in animal models.2 It has been demonstrated both in vitro and in vivo In a clinical phase 1 trial including 11 healthy volunteers, that microparticles with a size below 5 μm are ingested by a the normal seasonal fl u vaccine was applied either by wide variety of phagocytic cells.10 For submicron particles, conventional intramuscular injection or transcutaneously to there are contradictory reports in the literature with regard an area of 16 or 32 cm2, respectively, on the upper arm after to the differential uptake of nanoparticles by APCs, as weakening the horny layer barrier. Previously the horny compared to microparticles, depending on the nature of the layer at the application site had been pretreated with super- particle, the involved antigen, the route of delivery and the glue and adhesive tape. The procedure was generally well study endpoint. There is indeed evidence that nanoparticles tolerated leading to a slight erythema in a single volunteer. show increased uptake by APCs, which suggests that they The immune response to both immunization methods was may be better inducers of immune responses than micro- 38 RESEARCH REVIEWS | Novel Nanoparticle-based Technology Platform for the Delivery of Vaccine Antigens

particles.11 In addition, it was shown that carrier size could Development of pollen-mimetic vaccine particles play a role in determining the type of response induced. Scientists from HIPS and Saarland University, in colla- Virus-like particles induced IFN-γ and cell-mediated type boration with the Charité Universitätsmedizin Berlin¸ 1 responses, whereas larger beads mostly induced type 2 recently showed that nanoparticles accumulate selectively responses.12 in the openings of hair follicles.13 These nanoparticles were made of the biodegradable and biocompatible polymer However, strategies for transdermal vaccination that reduce polylactic-co-glycolic acid (PLGA) using polyvinyl alcohol the protective barrier function of the stratum corneum for a as a stabilizer. signifi cant period of time are intrinsically problematic due to the risk of invasion of pathogens. This imposes a limit to A plain aqueous nanoparticle suspension as well as a such methods for mass vaccination campaigns in countries hydrogel formulation that was massaged into pig ear skin with critical hygienic conditions and calls for alternatives in vitro, permeated deeper and to a greater extent into hair that leave the stratum corneum intact. Therefore, the trans- follicles than an aqueous solution applied with similar appendage pathway seems to be an appealing alternative to physical force. It was hypothesized that this is due to a pas- circumvent the stratum corneum barrier and directly target sive targeting effect, elicited by the nano-scale size of the Langerhans cells via the hair follicles. delivery device. We could further show that such particles may release different encapsulated compounds that then enter the stratum corneum.

As outlined above, the trans-follicular route appeals as a very attractive and safe strategy for the delivery of vaccine antigens to APCs. Hair bulbs are also an excellent reservoir, being only slowly cleared by hair growth and sebum pro- duction.16 An activation of the APCs via this route is a com- monly occurring phenomenon in allergic contact dermatitis in people allergic to pollen antigens. In fact, pollen can be considered as a micrometer sized carrier that accumulates in the follicle opening, sebaceous gland or dermatoglyphs (skin folds). Antigen release from the pollen is triggered by a moist atmosphere with suffi cient humidity, such as occurs on the skin by sweating. The idea is now to mimic pollen delivery in order to vaccinate across the intact skin barrier. Therefore, we are currently working to create pollen-mime- tic carriers allowing antigens to be stably encapsulated in order to target the hair follicles. This would allow releasing the antigen at the site of action in a controlled fashion in Fig 3. Overlay of transmission and fl uorescence image of order to activate skin APCs, thereby eliciting an effective a longitudinal section of skin from a porcine ear. Green humoral and cellular immune responses. Co-encapsulation fl uorescent nanoparticles (ca. 500 nm) applied to the ear of different adjuvants would enable to strengthen the accumulate in the opening of the hair follicle. Photo: HZI overall responses, as well as to modulate them according to the specifi c needs. First experiments show that the model antigens can be encapsulated into PLGA particles. These particles can be freeze-dried to be stored in a stable form. They can then be re-dispersed in water directly before the experiment to be tested in vitro or in vivo. The particles shall later be formulated as a gel or lotion making them easy to manufacture, acceptable to patients and safe to use. RESEARCH REVIEWS | Novel Nanoparticle-based Technology Platform for the Delivery of Vaccine Antigens 39

The research group at the Department of Prof. Dr. Claus-Michael Lehr at the University of Saarbrücken / HIPS Photo: HZI

Claus-Michael Lehr studied pharmacy at the universities of Mainz and Hamburg. After that, he wrote his PhD thesis at the Leiden University in the Netherlands. In 1991, he started as a postdoc at the University of Southern California in Los Angeles, USA, and returned to the Netherlands in the follow- ing year, where he worked at the Leiden/Amsterdam Centre The research group of Prof. Dr. Dr. Carlos Guzmán at HZI for Drug Research. In 1993, Claus-Michael Lehr became pro- Photo: HZI fessor for pharmaceutical technology at the Phillips Univer- sity Marburg and, since 1995, is the head of the department Biopharmaceutics and Pharmaceutical Technology at the Saarland University in Saarbrücken. He was engaged in the founding of the Across Barriers GmbH Saarbrücken in 1998 and co-founded the Centre for Bioinformatics Saarbrücken in 2000, where he still is the deputy director. His successful re- search was rewarded with numerous awards, for example the International Prize of the Belgian Society of Pharmaceutical Sciences (2009) and the APV Research Award for Outstan- ding Achievements in the Pharmaceutical Sciences (2006). In 2010 he was became „Fellow“ of the American Association of Pharmaceutical Scientists (AAPS). So far only 5 scientists from Germany have received this recognition.

Since 2009 Claus-Michael Lehr leads the department Drug Delivery of the newly founded Helmholtz Institute for Phar- maceutical Research Saarland (HIPS) and is co-founder of the PharmaBioTec GmbH in Saarbrücken. 40 RESEARCH REVIEWS | Novel Nanoparticle-based Technology Platform for the Delivery of Vaccine Antigens

In 2005 he was appointed as Head of the new HZI Depart- ment of Vaccinology. He has been working in the fi eld of Vaccinology since 1989. His work has been instrumental for the development of new adjuvants and the establishment of Salmonella spp. as a delivery system for DNA vaccines and therapeutic molecules.

Steffi Hansen studied pharmacy at Ernst-Moritz-Arndt Universität, Greifswald and graduated 2004 with a diploma in pharmacy. 2004 she also received her approbation as a pharmacist. From 2005 to 2009 she joined the department of Prof. Claus-Michael Lehr at Saarland University to prepare her doctoral thesis entitled „Development of a physiology based diffusion model to predict dermal absorption using experimental input parameters“. Subsequently Steffi Hansen started a postdoc at University of Cincinnati, USA sponsored by the German Academic Exchange Service (DAAD). She Ulrich Schäfer studied Pharmacy at Kiel University came back to Saarland to the newly founded Helmholtz-Insti- from 1971 to 1974 and qualifi ed as pharmacist in 1974. He tute for Pharmaceutical Research in 2010 where she works as obtained his Ph.D. at the Saarland University in 1980 for his a project leader on new strategies for transdermal vaccina- thesis work on dissolution processes of poorly soluble tions on immune homeostasis and aging. substances. In 1994, he was nominated as a so-called ‚Fachapotheker‘ for Pharmaceutical Technology. In 1995, he was permitted to further educate Pharmaceutical Technolo- gy as a professional training. Dr. Schäfer is member of the CRS, the APV, and the DPhG. His research interests are the biopharmaceutical investigations of semisolid dosage forms for dermatological applications, the development of ex vivo models for the determination of penetration of drugs and preservatives into human skin, the optimization of (trans) dermal dosage forms, and the solubility improvement of poorly soluble drugs. In December 2010, he was appointed APL Professor from the President of Saarland University as Carlos A. Guzmán born in 1959, is Head of the Department appreciation for his commitment and achievements in the of Vaccinology and Applied Microbiology at the Helmholtz Pharmaceutical Sciences. Centre for Infection Research (Braunschweig, Germany) and APL-Professor at the Medical School of Hannover. He is also external Lecturer of the Doctorate in Biotechnology from the University of Catania (Italy). He graduated in Medicine at the National University of Rosario (1981) and obtained his Board Certifi cation in Medical Bacteriology (1986) in Argen- tina. In 1987, he moved to the Institute of Microbiology from the University of Genoa (Italy), as Research Fellow from the Italian Foreign Offi ce Ministry. In Italy he also graduated as Doctor of Medicine and Surgery and obtained his Doctorate of Research in Microbiological Sciences. In 1994 he moved to Germany, where he became Head of the Vaccine Research Group at the German Research Centre for Biotechnology. RESEARCH REVIEWS | Novel Nanoparticle-based Technology Platform for the Delivery of Vaccine Antigens 41

Literature 10. O’Hagan, D. T., Singh, M., & Ulmer, J. B. (2004) Micro- particles for the delivery of DNA vaccines. Immunological 1. Martin Mdel, P., Seth, S., Koutsonanos, D. G., Jacob, J., Reviews 199, 191-200. Compans, R. W., & Skountzou, I. (2010) Adjuvanted infl uenza vaccine administered intradermally elicits robust long-term 11. Foged, C., Brodin, B., Frokjaer, S., & Sundblad, A. (2005) immune responses that confer protection from lethal chal- Particle size and surface charge affect particle uptake by hu- lenge. PLoS ONE 5(5), e10897. man dendritic cells in an in vitro model. International Journal of Pharmacy 298(2), 315-322. 2. Babiuk, S., Baca-Estrada, M., Babiuk, L. A., Ewen, C., & Foldvari, M. (2000) Cutaneous vaccination: the skin as an 12. Fifi s, T., Gamvrellis, A., Crimeen-Irwin, B., Pietersz, G. A., immunologically active tissue and the challenge of antigen Li, J., Mottram, P. L., McKenzie, I. F., & Plebanski, M. (2004) delivery. Journal of Controlled Release 66(2-3), 199-214. Size-dependent immunogenicity: therapeutic and protective properties of nano-vaccines against tumors. Journal of Im- 3. Hansen, S., Henning, A., Naegel, A., Heisig, M., Wittum, munology 173(5), 3148-3154. G., Neumann, D., Kostka, K. H., Zbytovska, J., Lehr, C. M., & Schaefer, U. F. (2008) In-silico model of skin penetration 13. Lademann, J., Richter, H., Teichmann, A., Otberg, N., based on experimentally determined input parameters. Part Blume-Peytavi, U., Luengo, J., Weiß, B., Schaefer, U. F., Lehr, I: Experimental determination of partition and diffusion coef- C.-M., Wepf, R., & Sterry, W. (2007) Nanoparticles - An effi - fi cients. European Journal of Pharmaceutics and Biopharma- cient carrier for drug delivery into the hair follicles. Euro- ceutics 68(2), 352-367. pean Journal of Pharmaceutics and Biopharmaceutics 66(2), 159-164. 4. Mitragotri, S. (2003) Modeling skin permeability to hy- drophilic and hydrophobic solutes based on four permeation 14. Luengo, J., Weiss, B., Schneider, M., Ehlers, A., Stracke, F., pathways. Journal of Controlled Release 86, 69-92. König, K., Kostka, K.-H., Lehr, C.-M., & Schaefer, U. F. (2006) Infl uence of nanoencapsulation on human skin transport 5. Glenn, G. M., Rao, M., Matyas, G. R., & Alving, C. R. (1998) of fl ufenamic acid. Skin Pharmacology and Physiology 19(4), Skin immunization made possible by cholera toxin. Nature 190-197. 391(6670), 851. 15. Stracke, F., Weiss, B., Lehr, C.-M., König, K., Schaefer, U. 6. Koutsonanos, D. G., Martin, M. d. P., Zarnitsyn, V. G., Sul- F., & Schneider, M. (2006) Multiphoton microscopy for the livan, S. P., Compans, R. W., Prausnitz, M. R., & Skountzou, investigation of dermal penetration of nanoparticle-borne I. (2009) Transdermal infl uenza immunization with vaccine- drugs. Journal of Investigative Dermatology 126(10), 2224- coated microneedle arrays. PLoS ONE 4(3). 2233.

7. Arora, A., Prausnitz, M. R., & Mitragotri, S. (2008) Micro- 16. Lademann, J., Richter, H., Schaefer, U. F., Blume-Peytavi, scale devices for transdermal drug delivery. International U., Teichmann, A., Otberg, N., & Sterry, W. (2006) Hair fol- Journal of Pharmaceutics 364(2), 227-236. licles - A long-term reservoir for drug delivery. Skin Pharma- cology and Physiology 19(4), 232-236. 8. Combadière, B., & Mahé, B. (2008) Particle-based vaccines for transcutaneous vaccination. Comparative Immunology, Microbiology and Infectious Diseases 31(2-3), 293-315.

9. Vogt, A., Mahé, B., Costagliola, D., Bonduelle, O., Hadam, S., Schaefer, G., Schaefer, H., Katlama, C., Sterry, W., Autran, B., Blume-Peytavi, U., & Combadiere, B. (2008) Transcutane- ous anti-infl uenza vaccination promotes both CD4 and CD8 T cell immune responses in humans. Journal of Immunology 180(3), 1482-1489. 42 RESEARCH REVIEWS | Mycobacterial Phagosomes and Innate Immunity

Mycobacterial Phagosomes and Innate Immunity

AUTHOR | Dr. Maximiliano G. Gutierrez | Junior Research Group Phagosome Biology | [email protected]

Although potential pathogens are encountered routinely, only on rare occasion they do cause diseases. The vast majority of pathogenic agents are eliminated rapidly by innate defenses operating in our bodies.

In 1882, Ilya Metchnikoff in a revealing experiment observed phagocytes surrounding and attempting to avidly eat a splinter he had introduced into a transparent starfi sh larva. At that time, he postulated that phagocytes are critical part of the host immune defense and elaborate the theory of phagocytosis and the evolutionary perspective from eating for nutrition to eating for defense. In 1908, in recognition of his work on immunity, he was awarded the Nobel Prize in Physiology or Medicine, jointly with Paul Ehrlich. Metchnikoff’s theory identifi ed and established the basis of our current knowledge of phagocytosis and the innate immune response (reviewed in Tauber, 2003).

Phagocytosis Phagocytosis is the process by which profes- Second, phagocytic cells make use of phagocytic pathway sional and non-professional phagocytes and other cells to direct processed protein and lipid antigens to the Major ingest particles whose size exceeds 1 μm. The resulting Histocompatibility Complex (MHC) class I, MHC class II intracellular vacuoles, termed phagosomes, go through and CD1 positive compartments. This loading and exposure dynamic changes that modify the composition of both their of antigens on plasma membrane allows the development limiting membrane and their contents, by a sequence that of a more customised adaptative immune response. Thus, resembles the progression of the endocytic pathway. This phagosomes play a dual role: as an innate immune effector process is referred to as phagosome maturation, and confers and as a bridge between the innate and acquired immune the vacuole with degradative properties, which are central system (Figure 3). Actually, phagosome maturation allows to its microbicidal function, representing the fi rst line of de- the shaping of a cellular compartment where killing, fense against infection that vertebrates possess (Haas, 2007). degradation and antigen processing take place in a very organised manner (Jutras & Desjardins, 2005). The study of Phagosome maturation and immunity Advances in the phagosome biology is evolving rapidly and refl ects recent understanding of phagosome biology have been possible by advances in a broad range of disciplines including cell bio- the use of the latex bead system. This model was originally logy, proteomics, and immunology. A better understanding introduced by Weisman and Korn in the late 1960s and re- of the mechanisms of phagosome formation and maturation discovered in the early 1990s (Desjardins et al, 1994). Latex and its interactions with intracellular compartments will beads are a versatile system for both in vitro and in vivo analyses of many phagosomal functions. This system allows an easy approach to understand the very complex process of phagocytosis (Figure 1). Because the latex fl oats in sucrose gradients, latex bead phagosomes are much simpler to isolate in pure fractions in order to analyse their content (Figure 2). Much of what we understand today about phago- cytosis and phagosome biology in cellular systems has been obtained from studies from many groups that use the latex-bead phagosome system (Desjardins & Griffi ths, 2003). Moreover, it is possible to coat these beads with different proteins or with specifi c ligands, which selectively bind to cellular receptors to study the impact during internalisation. Recently, it has been shown that particular substrates can also be coupled to the beads to monitor enzymatic activity Fig. 1. Phagocytosis of latex beds. RAW 264.7 macrophages in real-time (Yates et al, 2005). expressing Rab34-GFP (green) ingesting avidly IgG-coated latex beads (red). Nucleus is shown in blue. This process By ingesting microbial pathogens, phagocytic cells achieve mimics the IgG-opsonised bacteria that also enter macro- two essential immune functions orchestrated by the phago- phages via Fcγ receptors (FcγRs). A. Beads at different stages some. First, phagosomes initiate microbial elimination, in of internalisation. B. 3D diagram of the localisation of Rab34 part by directing ingested microbial pathogens to lysosomes on the phagosomal membrane. Photo: HZI, Kasmapour in a process known as phagosome maturation pathway. RESEARCH REVIEWS | Mycobacterial Phagosomes and Innate Immunity 43

allow the development of novel strategies to modulate the innate and adaptive immune response.

Mycobacterial phagosomes: the subverted compartment More than 1.5 million people die every year from tubercu- losis (WHO, TB report 2008) and the appearance of multi- and extensive-drug-resistant strains in eastern Europe is now moving swiftly towards western European countries (www.eurotb.org). Although 3.2 billion people are infected with Mycobacterium tuberculosis, only in 10% of those people the bacteria do cause disease. The other 90% control the dis- ease and rely on innate and acquired immune processes of Fig. 2. Latex bead phagosome purifi cation. The scheme shows the body that contain and restrict bacterial growth (Korbel et the purifi cation of latex bead phagosomes from macrophages al, 2008). Therefore, in most of the cases the innate immune for biochemical and proteomics analysis. Briefl y, latex beads system is able to effectively eradicate the infection with M. are internalised and then isolated in a sucrose gradient using tuberculosis. The aim of our research is to give insights into its property of fl otation. Electron microscopy pictures show the host factors that facilitate this. The exact mechanisms the different steps during the process of purifi cation. Isolated that govern this initial sterilising reaction are not com- LBP are used for different biochemical assays and proteomics. pletely understood. Therefore, understanding how host cells Photo & Graphic: HZI eliminate mycobacteria is important to fi nd possible novel therapeutic strategies that enhance this natural response.

To live within eukaryotic cells, through evolution Myco- bacterium tuberculosis has developed an impressive set of mechanisms to subvert phagosome maturation. Many clinical manifestations and problems during treatment of tuberculosis are a direct consequence of a population of intracellular bacilli (Russell, 2001). The key event during M. tuberculosis infection is the ability of this pathogen to survive within phagosomes in host cells (Figure 4). This

Fig 3. The phagosome links the innate and adaptative immune response. The phagosome of M. tuberculosis plays two func- tions. The fi rst one is the continuous acquisition of bacteri- cidal properties. This important step in the development of the innate immune response is achieved mainly by the interaction of the phagosome with late endosomes and lysosomes (left Fig. 4. Visualising mycobacterial phagosomes in macrophages. side). On the other hand, in the phagosome bacteria are RAW264.7 macrophages infected with mycobacteria. degraded and lipids and peptides are presented in the context Cells were visualised using confocal microscopy (Green: of MHC class I, class II and CD1 molecules. This activates mycobacteria; Blue: nucleus; Red: actin cytoskeleton). Same different populations of lymphocytes that participate in the infected macrophages can be observed by electron microscopy. onset of the adaptive immunity. Some of these pathways are In this case the lysosomes are loaded with BSA-Gold. As actually impaired by M. tuberculosis at different levels but shown, lysosomes are not fusing with the phagosomes are omitted for clarity. containing mycobacteria. Photo: HZI, Gutierrez 44 RESEARCH REVIEWS | Mycobacterial Phagosomes and Innate Immunity

capability is linked to the aptitude of the live pathogen to of a sub-class of lysosomal proteins from the Golgi to the manipulate phagosome maturation. The strategy by which phagosome (Wähe et al, 2010). Therefore, our observations M. tuberculosis manipulates the cellular traffi cking machin- raise the question of the regulation of the sortilin-pathway ery is likely to be both multifactorial and complex. In spite functions in the context of phagosomes containing intracel- of the signifi cant progress in the last few years, a mechanis- lular pathogens. tic understanding of how M. tuberculosis infection proceeds at the cellular level and how the fi rst steps of host defenses Perspectives Although in vitro and in vivo studies have are organised against this pathogen is still elusive. shed light on some aspects of tuberculosis pathogenesis, we still do not completely understand how M. tuberculosis Our research focuses on the mechanisms whereby M. tuber- manages to survive in eukaryotic cells and why host cells culosis arrests phagosome maturation and avoids killing are in some cases able to eradicate this pathogen. Therefore, by macrophages. Towards this goal, we are studying the a deep understanding of the very fundamental biology of intracellular transport of non-pathogenic and pathogenic M. tuberculosis that considers the intracellular lifestyle is mycobacteria in macrophages. We have identifi ed novel crucial for developing new therapies towards tuberculosis factors involved in vesicular traffi cking and protein sorting, control. particularly phago-lysosome fusion, during mycobacterium infection. These proteins are promising candidates for being The study of the cell biology and cellular immune response involved in the lysosomal-mediated killing of mycobacteria, of macrophages to mycobacteria is a very productive area of as well as in the molecular events linking innate and adap- research and its understanding is critical for the under- tive immune responses (Gutierrez et al., 2009; Gutierrez et standing of mycobacterial pathogenesis. These studies will al., 2008). shed light on fundamental questions of the host to pathogen response and will also provide knowledge of basic charac- The most promising candidate proteins are rab proteins and teristics of cellular and innate immune functions. With our lysosomal receptors. The fi rst ones are the molecular switches studies we expect to contribute to a better understanding that regulate membrane traffi cking and spatio-temporal of how macrophages kill mycobacteria and other infectious organisation of vesicular traffi cking (Stenmark, 2009). The intracellular pathogens. second ones are receptors for some important lysosomal com - ponents and enzymes in charge of pathogen degradation.

Rab proteins and phagosomes Rab proteins are proteins that have a key role in organising membranous compart- ments facilitating transport and regulating vesicular traffi cking. A complex network of Rab GTPases controls the dynamic changes of phagosome maturation. We have identi- fi ed some Rabs potentially implicated in response to myco- bacterial infection previously not related to infection with intracellular pathogens (Gutierrez et al, 2008). However, the Maximiliano Gabriel Gutierrez born in 1976 in Mendoza, exact role of many of these proteins in phagosome matura- Argentina, is head of the Junior Research Group Phagosome tion is unknown. We are now defi ning the function of those Biology at the Helmholtz Centre for Infection Research, proteins during phagocytosis and evaluating the impact on Braunschweig. He studied at the University of San Luis, the innate immune response to mycobacterial infection. Argentina and obtained a biochemistry and molecular biology degree from the university in 2001. In 2005, he was also Lysosomal protein receptors and phagosomes For one awarded a PhD from the University of San Luis, Argentina. of the identifi ed proteins, sortilin, we showed its role dur- During his PhD studies he spent some time as a research ing phagosome maturation. Up to now, the possibility of a visitor in the US and Germany. After that, he spent almost direct delivery pathway for some lysosomal enzymes from three years as a postdoctoral scientist at EMBL, Heidelberg, the Trans Golgi Network (TGN) to the phagosome has been Germany, as a fellow of the von Humboldt Foundation and suggested. However, the pathways involved in this process EMBO. Since November 2008 he has been the head of the and the identity of the enzymes delivered is unknown. Our Junior Research Group Phagosome Biology. The main area fi ndings with sortilin in macrophages identifi ed a novel of research in his group is the cell biology of host-pathogen and important contribution of this protein to the delivery interactions with a focus on mycobacteria. RESEARCH REVIEWS | Mycobacterial Phagosomes and Innate Immunity 45

Literature

Desjardins, M., Celis, J. E., van Meer, G., Dieplinger, H., Jahraus, A., Griffi ths, G., & Huber, L. A. (1994) Molecular characterization of phagosomes. The Journal of Biological Chemistry 269, 32194-32200.

Desjardins, M., & Griffi ths, G. (2003) Phagocytosis: latex leads the way. Current Opinion in Cell Biology 15, 498-503.

Gutierrez, M. G., Gonzalez, A. P., Anes, E., & Griffi ths, G. (2009) Role of lipids in killing mycobacteria by macrophages: evidence for NF-kappaB-dependent and -independent killing induced by different lipids. Cellular Microbiology 11, 406-420.

Gutierrez, M. G., Mishra, B. B., Jordao, L., Elliott, E., Anes, E., & Griffi ths, G. (2008) NF-kappaB activation controls phagolysosome fusion-mediated killing of mycobacteria by macrophages. Journal of Immunology 181, 2651-2663.

Haas, A. (2007) The phagosome: compartment with a license to kill. Traffi c (Copenhagen, Denmark) 8, 311-330.

Jutras, I., & Desjardins, M. (2005) Phagocytosis: at the cross- roads of innate and adaptive immunity. Annual Review of Cell and Developmental Biology 21, 511-527.

Korbel, D. S., Schneider, B. E., & Schaible, U. E. (2008) Innate immunity in tuberculosis: myths and truth. Microbes and Infection / Institut Pasteur 10, 995-1004.

Russell, D. G. (2001) Mycobacterium tuberculosis: here today, and here tomorrow. Nature Reviews 2, 569-577.

Stenmark, H. (2009) Rab GTPases as coordinators of vesicle traffi c. Nature Reviews 10, 513-525.

Tauber, A. I. (2003) Metchnikoff and the phagocytosis theory. Nature Reviews 4, 897-901.

Wähe, A., Kasmapour, B., Schmaderer, C., Liebl, D., Sand- hoff, K., Nykjaer, A., Griffi ths, G., & Gutierrez, M. G. (2010) Golgi-to-phagosome transport of acid sphingomyelinase and prosaposin is mediated by sortilin. Journal of Cell Science 123, 2502-2511.

Yates, R. M., Hermetter, A., & Russell, D. G. (2005) The kinet- ics of phagosome maturation as a function of phagosome/ lysosome fusion and acquisition of hydrolytic activity. Traffi c (Copenhagen, Denmark) 6, 413-420. FOCUS RESEARCH REVIEWS SPECIAL FEATURES

Photos from left to right: During the offi cial retirement of Dr. Helmut Blöcker. Dr. Siggi Weiss giving some refl ections on the work of Helmut Blöcker at the GBF/HZI | Dr. Kathrin Dinkla, Prof. Dr. G. Singh Chhatwal, Katja Mummenbrauer, PD Dr. Manfred Rohde, Dr. Susanne Talay (from left to right) during a discussion on streptococci | Prof. Dr. Christiane Ritter preparing for a new NMR experiment Photos: HZI, Dornbach (le) | HZI, Ammerpohl (ce) | HZI, Gramann (ri) SCIENTIFIC REPORTS FACTS AND FIGURES

48 Where Would We Be without DNA Sequences?

52 Nuclear Magnetic Resonance Spectroscopy, a Major Player in the Arsenal of Platform Technologies available in the HZI

60 International Cooperation of Helmholtz Centre for Infection Research with India and China: A Success Story 48 SPECIAL FEATURES | Where Would We Be without DNA Sequences?

Where Would We Be without DNA Sequences?

AUTHOR | Dr. Helmut Blöcker | Department of Genome Analysis | [email protected]

Three weeks of hard work in the radioactivity laboratory, in the darkroom, in the biology lab and having snacks always impatiently in between. Then it was done. Just when my colleagues returned from the cafeteria, I had developed the crucial X-ray fi lm, rinsed it under running water and had a fi rst glance at it. Together we were able to actually read a sequence of 33 nucleotides! A team of undergraduates, graduate students and postdocs had worked together in the early seventies to produce chemically and enzymati- cally the world’s fi rst structural gene coding for a polypeptide. Around three years work of the team, and I was fi rst to hold the la- boriously produced evidence for the existence of the target compound in my hands: 33 bands on X-ray fi lm.

How can it be that today one is able to sequence complete human genomes with billions of DNA building blocks, whereas, within a single researcher’s life, at that time happiness was in a short sequence of 33 nucleotides?

The development of sequencing technology (1) During gels, but performed on capillaries fi lled with separating my doctoral work, in the years before the 33-base success, material. In addition, the capillaries were loaded automati- sequencing procedure of choice was the “wandering spot cally with samples from microtiter plates. This generation method”. Maximum read length was about 13 nucleotides. of devices formed the technical basis for the fi rst complete Just good enough to check the chemically synthesized sequence of the human genome as it was developed by the oligonucleotides for the construction of the said 33mer international public human genome consortium including duplex. After a working visit at MIT, Hans Gross had the our department. method established at the MPI for Biochemistry in Martins- ried/München for RNA analysis. Together we tested it there The sequencers and the procedures around them were so successfully for DNA sequences. sophisticated that one could quite get read lengths of 1,300 nucleotides per separation capillary at up to 384 capillaries To check longer sequences, the development of base-specifi c per device. For aesthetes, the gray boxes were not a chemical cleavage of DNA, the method of Maxam and revelation - that has not changed until today. Our many Gilbert, proved advantageous. But how to get the details of visitors were impressed at the most with the cost (several the tricky and not yet published method? Without the hundred thousand Euros per unit). At the beginning of this existence of the internet, e-mail, pdf attachment, or even a decade hardly any of the laboratory staff could imagine that fax machine? Six weeks later, an airmail letter to Allan the entire Sanger-based technology would become hope- Maxam at MIT brought permission to take a copy of a still lessly inferior in a few years and would be ready for the secret protocol of Heinz Schaller’s at DKFZ. So that’s the museum. 33-base success. Already at the heyday of the Sanger technology experts Even a hard-working chemist does not like to work with were informed at congresses and in particular from toxic and volatile substances such as in the Maxam/ Gilbert international review documents that soon a whole new protocol. It is, therefore, not surprising that this method generation of devices and procedures would be on the was soon replaced completely by the dideoxy method of market (“next generation sequencing”). Today, the three Sanger (“chain termination synthesis”). Soon you could main types of techniques and equipment of this generation achieve read lengths of several hundred nucleotides and are marketed by the companies Roche, Life Technologies analyse together several tens of samples over slab gel and Illumina. In 2007, we opted for the Illumina devices electrophoresis devices. We had about a dozen of such that are now dominant. It is surprising that within about devices, so we had established a room especially for the two years the sequencing productivity of our equipment preparation of electrophoresis cassettes (pouring gels, clean could be raised to about 1,500 percent through comprehen- glass plates, etc.) including a special dishwasher. sive optimization. Today one can generate on a single sequencer, the raw sequence of a complete human genome, This drudgery was over when commercial sequencers were at a moderate cost of several thousand Euros. Remember available, in which the separation of sequencing reaction that the fi rst estimates for the cost of the public Human mixtures were no longer done next to each other on slab Genome Project was around two billion U.S. dollars! SPECIAL FEATURES | Where Would We Be without DNA Sequences? 49

In fact, there are announcements of the fi rst devices that are smaller, highly integrated, very much lower in acquisi- tion cost and can operate at extremely low cost. In future, it will be possible to routinely sequence single molecules. The DNA material will be used free of artifi cial markers, and read lengths of tens of thousands of nucleotides are only a few years (if not months) ahead. A focus will be on develop- ments in nanotechnology and microfl uidics. The fi rst sequencers for around 50 kEuros are on the market. A British company announced a disposable sequencer the size of a USB stick they want to develop in the coming years. Fig. 1. Automated cluster analysis from Illumina sequencer. Simply breathtaking! Within about two years overall sequencing productivity could be raised to about 1,500 percent through comprehensive No wonder that with such a price perspective, DNA optimization. Right-hand side: blow-ups. Photo: HZI sequencing will soon fi nd its way into daily routine analysis, in hospitals, the pharmaceutical industry, food industry, breeding research, and health and food inspection agencies, and perhaps on farms and in doctors’ offi ces. An amazing journey from “handmade” 33 base pairs to the machines of today. However, no deep knowledge is neces- Compared to the period before the present devices, almost sary to imagine that this extremely rapid development will nothing remained as it used to be. This is true for effi ciency continue. You can fi nd a graphic explanation of general and also the vastly greater versatility of new devices and lines of technology development in Clayton Christensen’s procedures developed for them. It is no longer merely about book “The Innovator’s Dilemma” (2). the control of cloning reaction or de novo sequences of genomes, but also, for example, the analysis of transcripts, metagenomics, genome-wide methylation patterns, gene duplications, and SNPs. It is this methodical expansion of sequencing technology, which not only cannibalizes existing chip technology and reduces it to pure niche technology, but makes sequencers become an indispensable part of any molecular biology laboratory for a very long time. In future, a PS (“Personal Sequencer”) in the labora- tory will have come up to standard. Just like a PCR machine.

It would be an undue exaggeration to argue that the current and next sequencers could be operated by well-trained apes. But at least the next devices will demand less from the technical personnel and will nevertheless produce almost unimaginable amounts of data. We should strongly and immediately adjust to the situation that we need intelligent bioinformaticians and experts with a broad molecular- Fig. 2. Clayton Christensen. How low-end disruption occurs over biological and medical knowledge that can deal with mass time. (http://en.wikipedia.org/wiki/Disruptive_technology) data. Despite enthusiasm for the rapid development of pure data generation, this has been our permanent reminder – not least in my HZI internal paper of 2006 on an assess- ment around sequencing (“Some Remarks Relating to Next Generation Genetic Analyzers”). 50 SPECIAL FEATURES | Where Would We Be without DNA Sequences?

Excursus funding policy Without any massive public and With the above chapter on the development of sequencing it private investment, the current international genome-based is clear that one could only compete on an international top research would simply not exist. After our interest in level, if one would accept the challenge of technological genome research aroused in the mid-1980s, it was very innovation. We tried this by developing the “Shortmer” clear that substantial progress could only be reached with sequencing primer technology, by ongoing developments in strong funding. My paper “Technology Development and robotics, bioinformatics and biochemistry that still give us, Genomics” became the basis of a funding programme of the as in the work of Gerhard Kauer (4) and Igor Deyneko, a German Ministry of Research. It was run over a number of competitive advantage. As a consequence, since the years and mobilized a total of about 150 million DM for the beginning of the nineties the various activities around scientifi c landscape in Germany. Part of the logic behind sequencing were the focus of the GBF/HZI group later the initial funding period and the following ones today known as the Department of Genome Analysis. Fortunately, appears, however, questionable. The logic goes something we had an early chance to prove ourselves in international like this: fi rst, we develop technology, we generate data, we sequencing projects such as those supported by the EU - the interpret the data, we develop new approaches for diagnosis yeast and Arabidopsis genome projects. and new therapies, and fi nally, we market the fi nal results. Nice thought, but unrealistic. To date, most top countries act The ultimate project was certainly the public Human differently. The best examples are the USA and China. Genome Programme (5), where we took part as one of 16 There is continuous, substantial funding for each link in genome centres worldwide. Often referred to as a moon the chain, from technology development to the medical use. landing project of bio-research this project caused a global Specifi cally, the technology sector in the United States may echo in the media and society like no other in the biological have made billions of revenue. Unfortunately, early and sciences. A few years ago there were rumours from multiple pioneering developments in Germany did not encounter sources, that our international consortium was proposed as stable funders in this country. In a modifi cation of the title a candidate for a Nobel Prize. Well, fi rst a rumour is a of this paper we might ask: Where could we be in Germany rumour, and secondly, Helmut Kohl has been rumoured to if more DNA sequences had been made?! be proposed several times for a Nobel Prize – he has not been awarded the prize to this day. I found the Nobel From a nucleic acid chemist to a sequencing guy rumour interesting. I was in the USA when I learnt from a After completing my graduate work at the former GMBF in British offi cial about the Nobel story. My immediate Braunschweig I joined a young and dynamic team working reaction was: “Brilliant! I would love to become 0.2 percent on gene synthesis in Hamburg. It was mainly concerned of a Nobel laureate!” After all, a few hundred scientists were with the aforementioned DNA double strand of 33 bases in involved in the project! However, unlike in physics, there length, which carries the genetic information for the are rules for the life sciences that the prize may be awarded peptide hormone angiotensin II (3). only to a maximum of three people.

In 1980, with all our experience and developments from One may view genomics with its core element sequence Hamburg University in mind, Ronald Frank and I together analysis as drudgery for research, development and set up a new research group at the GBF. Within three years, application in the life sciences and medicine. For these we had reached our fi ve-year goal. We synthesized oligonu- areas it will be indispensable. As early as 2000 the journal cleotides and then produced longer genes by chemical-enzy- Science entered the sequencing of whole genomes in their matical procedures. Having produced DNA double strands, annals of interdisciplinary breakthroughs of the year (6). our curiosity was aroused for optimal expression systems. Francis Collins, an advisor to President Obama, director of Expressed synthetic genes made us dream of artifi cial NIH, and previously speaker of our Human Genome proteins. Working with the genes of protease inhibitors and Programme has recently put it this way: “Genome research genes of other rather small proteins, however, taught us that is really just beginning and it can be dizzying to live on an even in these protein families there was too little information exponential curve”. So it was and it is. available about the relationship of specifi c gene variants to the respective biologically active proteins. It seemed reasonable to work towards an acceptable algorithm. This could be attempted at that time only via mass DNA sequenc- ing since it was (and still is) superior to peptide sequencing. SPECIAL FEATURES | Where Would We Be without DNA Sequences? 51

Back to the headline Where would we be without DNA Literature sequences? This is what I ask in the title of this essay. The simplest answer would be (correct, but a discussion killer): (1) for various sequencing details look here: without DNA sequences no life form would exist, including http://en.wikipedia.org/wiki/DNA_sequencing#cite_note-6 ourselves. But research and development have enabled us to undertake all kinds of travel in the universe of our interior (2) Christensen, Clayton M. (1997). The innovator‘s dilemma: by working with nucleic acids sequences. As a fan of space when new technologies cause great fi rms to fail. Harvard exploration I am interested in this inner universe much Business Press. ISBN 9780875845852. more than in the universe around us. Perhaps a self-cen- tered thought, but certainly closer to discoveries that give (3) Köster, H., Blöcker, H., & Frank R., Geussenhainer S., us more knowledge about ourselves and permits us to make & Kaiser W. (1975) Total Synthesis of a Structural Gene for life a little better - if used responsibly. This is what I the Human Peptide Hormone Angiotensin II. Hoppe-Seyler´s secretly hope for myself. Against the background of Zeitschrift für physiologische Chemie 356 (2), 1585–1594. thousands of human genomes, which are currently being analysed in the world, my own genome sequence should tell (4) Kauer, G. & Blöcker, H. (2003) Applying signal theory to some interesting secrets. I also expect this for the genome the analysis of biomolecules. Bioinformatics 19, 2016-2021. sequences of my three colleagues in Braunschweig, whose genomes are being analysed together with my sequence. (5) International Human Genome Sequencing Consortium (2001) Initial sequencing and analysis of the human genome. Profound knowledge of DNA sequences is the key to Nature 409, 860-921. knowing more about ourselves and our living environment, today and tomorrow. As a nucleic acid person, what else can (6) Pennisi, E. (2000) Breakthrough of the year. Genomics you expect from your professional life? Comes of Age. Science 290, 2220–2221.

Fig. 3. Four genomes under investigation: Michael Jarek, Tschong-Hun Im, Maren Scharfe, Helmut Blöcker, all next to hand-crafted DNA (farewell present to H.B.) Photo: HZI

Helmut Blöcker born in 1945, obtained his diploma in chemistry at the Technical University of Braunschweig (1972). He did his doctoral thesis (Dr. rer. nat.) at the Uni- versity of Hamburg (1972-1975) and worked for 5 years as a postdoctoral scientist in the same group. In 1980 he set up the DNA Synthesis Group at the GBF. He was head of the department of Genome Analysis at the GBF/HZI (1994-2010). 52 SPECIAL FEATURES | Nuclear Magnetic Resonance Spectroscopy, a Major Player in the Arsenal of Platform Technologies

Nuclear Magnetic Resonance Spectroscopy, a Major Player in the Arsenal of Platform Technologies available in the HZI

AUTHOR | Dr. Victor Wray | Research Group Biophysical Analysis | [email protected]

The analytical instruments platform is the oldest core facility in the HZI with well established modern instrumental techniques and expertise for structural investigations of all types of biomolecule. It provides detailed structural data in numerous forms that are crucial for understanding the underlying molecular mechanisms and interactions governing the cellular processes involved in host- pathogen interactions.

The platform provides large instrumental facilities for determining the three dimensional structure of all types of natural product and fi nds application in most, if not all, of the main areas of research within the “Infection and Immunity” programme of the HZI.

Recent reports in this series have featured the use of electron microscopy and X-ray crystallography. Electron microscopy can visualise a wide range of super macromolecules, organelles and bacteria, as well as providing intimate details of the adherence to, and invasion of, host cells by a wide range of pathogens. The complementary technique of X-ray crystallography provides structural details at the atomic level of proteins and their molecular complexes with ligands and other proteins. It has provided considerable detail of both the mechanisms of bacterial adhesion, invasion and propagation, as well as human receptor signalling. Here we will consider, in some detail, the use of nuclear magnetic resonance spectroscopy for studies of small molecular weight natural products in solution. It plays a major role in the discovery and development of novel anti-infectives, as well as affording complementary information to that gained by X-ray crystallography.

Introduction Early in the development of the institute it The power of the nucleus In the late 1940s it was shown was recognised that instrumentation capable of investigat- that the nucleus of certain atoms absorb electromagnetic ing biological structures was necessary for an understand- radiation in a magnetic fi eld and the frequency of the ing of basic natural processes. Enormous advances have radiation was dependent on the strength of the magnetic been witnessed over the last four decades in various instru- fi eld, the nucleus involved and, very importantly, on the mental areas with the rapid development of fundamental particular chemical environment of that nucleus. Of the physical techniques and the integration of the potential of common atoms only 1H, 13C, 15N and 31P showed this computer technologies. Of particular importance for struc- phenomenon and clearly had great potential for delving tural biology has been the rapid evolution of nuclear mag- into the structures of organic compounds, and hence most netic resonance (NMR) spectroscopy, mass spectrometry natural products. Subsequently, the development of power- (MS), X-ray crystallography (X-ray) and electron microscopy ful, stable magnetic fi elds led to the high resolution of the (EM). These techniques are grouped together as one of the signals arising from the same nuclei in different chemical core facilities in the HZI and have the ability of provid- environments. In tandem, a decisive step forward was the ing intricate details of the whole spectrum of biological introduction of pulsed NMR spectroscopy, the development structures from the smallest of natural products (NMR and of which was associated with the rapid advancements in MS), through macromolecules and their complexes (X-ray) computer technology, allowing enormous gains in sensi- to the largest of structures that include organelles and bac- tivity. However, the development of two-dimensional (2D) teria (EM) (Fig. 1). In a previous report 2008/9 the elegant NMR methodology in the 1970s, particularly by R. R. Ernst, application of X-ray crystallography was shown to yield for which he received a Nobel Prize in 1991, heralded the intimate details of microbial pathogen interactions with start of a new era in NMR spectroscopy. The use of these the host organism at atomic resolution. Such details allow techniques now allowed the ready identifi cation of bonding the development of new strategies and tools to broaden our patterns in molecules via either the through-bond interac- understanding of bacterial infections. Here we will concen- tions of similar nuclei (homonuclear 2D NMR) or dissimilar trate on the complementary technique of NMR spectroscopy nuclei (heteronuclear 2D NMR), as well as conformational, and its application to the investigation of biologically active sequential and folding properties of units in a molecule by natural products. the observation of through-space interactions. SPECIAL FEATURES | Nuclear Magnetic Resonance Spectroscopy, a Major Player in the Arsenal of Platform Technologies 53

Small Natural Products

Macromolecules: Proteins

NMR Spectroscopy Mass Spectrometry Molecular Complexes: Protein-ligand Protein-Protein

Super Macromolecules: Organelles Bacteria X-ray Crystallography Electron Microscopy

Fig. 1. Analytical Instruments Platform Photos: HZI

Since the early 1970s NMR instrumentation in our institute Infection and Immunity programme of the HZI. The has closely followed these technical breakthroughs as it structure of such compounds is a pre-requisite for their has become the most powerful technique for the routine development and is most easily achieved by a combination structure elucidation at the atomic level of biologically of NMR spectroscopy and mass spectrometry. active natural products in solution. For pharmacologically relevant substances with molecular weights under 2000 Da In contrast to chemical synthesis there is generally no prior it has the advantage over X-ray of not requiring crystals of knowledge of the structure of a compound isolated from the material and allowing rapid access to structural details. plants, bacteria or fungi, all potential sources of new active As a consequence, over the years NMR spectroscopy has compounds. Consequently, after isolation, a small amount of been used to solve the structures of an extremely large material is dissolved in an organic solvent and a simple variety of natural products emanating from a diverse proton (1H) spectrum is recorded (Fig. 2). Such a spectrum number of research groups both within the HZI and from already contains a wealth of information regarding the external sources. nature of units present in the molecule. The position of signals in the spectrum refl ects the local environment of The quest for novel anti-infectives: Structures of the observed proton, allowing distinction of aromatic, sugar biologically active substances The emergence of new and and aliphatic groups. While the signal integral indicates the reoccurring pathogens continues to pose a serious threat to number of protons present, their splittings provide details human health even in developed countries. As a conse- of the number and disposition of protons in the immediate quence the search for, identifi cation, characterisation and vicinity. Additional information is afforded by a carbon (13C) clinical development of novel anti-infectives from natural spectrum which again shows a dispersion of signals that sources is, and will continue to be, a major goal of the allow the number and types of carbon atom to be readily determined. 54 SPECIAL FEATURES | Nuclear Magnetic Resonance Spectroscopy, a Major Player in the Arsenal of Platform Technologies

Aromatic region Sugar region Aliphatic region

1H spectrum

2D NMR experiments Yield molecular fragments

13C spectrum

Fig. 2. 1D NMR spectra of an unknown compound in methanol-d4 Graphic: HZI

AB NMR Data OR Me O OH MeO OH

C OH Me O OH

Me O O

O OMe Me O OH Me O OH

Fig. 3. Analytical procedure (A), 2D 1H- 1H spectrum showing sugar unit (B) and complete structure (C) Graphic: HZI SPECIAL FEATURES | Nuclear Magnetic Resonance Spectroscopy, a Major Player in the Arsenal of Platform Technologies 55

The use of 2D NMR spectroscopy now allows the defi nition amounts of material to be investigated (0.2 to 1 mg) without of fragments in the molecule (pieces of the jigsaw, Fig. 3). the need for chemical derivation. As is evident from Thus homonuclear 2D 1H-1H spectra shows off-diagonal previous features in these reports, NMR structure elucida- signals corresponding to through-bond interactions tion has played a major role in the discovery and evaluation between hydrogen atoms two and three bonds apart. The of new anti-infectives, particularly in unravelling the identifi cation of such interactions allows the tracing of structures of the complex number of biologically active series of these interactions that identify the various compounds produced by the myxobacteria. This is well fragments in the molecule. Further use of heteronuclear 2D illustrated by the large number of basic structures of 1H-13C spectra confi rms the types of fragment present and secondary metabolites from the single bacterium Sorangium allows the pieces of the jigsaw to be pieced together to give cellulosum, Fig. 4. To date over 600 unique myxobacterial a fi nal three-dimensional structure. The exact conforma- metabolites have been identifi ed and their structures tional details of this structure are then available from elucidated. Over the years a very promising number of inspection of the magnitude of the splittings in the 1D 1H small molecules with anti-tumour, antiviral (proteasome spectrum and from through-space interactions of the inhibitors), anti-infective (interferon regulators) and protons in the different fragments. Finally, confi rmatory anti-bacterial (antibiotics, cytolysin inhibitors, biofi lm data regarding the molecular weight is available from mass inhibitors) activities have been discovered and character- spectrometry and other data from ultra-violet and infra-red ised. The most important of these was the discovery of spectroscopies, as well as chemical information. epothilone, a compound that has led to a treatment of aggressive metastatic or locally advanced breast cancer. This approach has been widely applied over the last three decades to elucidate the structure of numerous natural NMR also provides the main means of establishing the products and it continues to be the method of choice. The structure of synthetic derivatives of natural products improvements in instrumentation particularly in terms of prepared for structure-activity studies, a pre-requisite for sensitivity and signal resolution provided by modern drug development, as well as a priori chemical syntheses for superconducting magnet systems now allows small groups active in chemical biology and medicinal chemistry.

MeO HO OH OH OMe O S OH OH OH O HO N N O O O O OH OH OH OH N O O O OM e O N CO OH O O N O O OH H OH S O O O OH O O O O O MeO OH O OH O OM e O O HO OH O H O O O O OH OH N O O MeO OH O O O HO S H H N OH O O OMe O O O MeO HO HO HO O O N O OH OH HO OH HO H O O OH

OH O O OH O O H HOOC O HN OH COO H H NH H H OH OH O OH Cl O HN O O Cl H O

N HO O O HO O - Me Cl O H O O COO H H O B O O O N O + O NH HN O O Na O H N O O O O O O OH HO O OH OH O N H N OH O OH N O O O O O NH O OH O O N O OH O O O HO HO N O O OMe O OH OH O O HO OH O O OMe O O OH O OH OH OH O O OH H H OH HO OH HO O O O OH OH HO O S O OM e HO O OH OH Me N OH OH O OH OH N O OMe OH O OH O O O CO OH OH COO H MeO OH N H O H HO OH N HO O OMe OM e O OH O O O O O N OMe H S O MeO O N OH OM e O H OMe HO O OH OH O N O OH O O MeO O O O OH OH OH H OH O OMe NH2 O N H3C CH 3 OM e N O Me N N S H MeS O HO OM e Me O N O HO OC O H3CO CH3 O O O N Cl O O O O O O OH O O OH N H3C OH O OH OH NH H HO OH OMe OH OH O O Cl

Fig. 4. Secondary metabolites from the myxobacterium Sorangium cellulosum Graphic: HZI, Gerhard Höfl e 56 SPECIAL FEATURES | Nuclear Magnetic Resonance Spectroscopy, a Major Player in the Arsenal of Platform Technologies

In the past signifi cant collaborations with universities and Biosynthetic pathways and monitoring of biological other institutes have also been forged that have involved systems Information of the biosynthesis of secondary investigations of natural products from bacteria, fungi, metabolites can also be furnished by monitoring the plants and marine sources. products after “feeding” the producer bacterium with 13C-enriched precursor molecules. Under these conditions the precursors are incorporated into the product that shows enhanced signals in the 13C NMR spectrum only for those OH carbons derived from the enriched material. An example is O shown in Fig. 5 for apicularen B biosynthesised from O N acetate, glycine and methionine. O H O The use of NMR spectroscopy for monitoring changes in biological systems was recognised very early and was used Apicularen B to investigate non-invasively the cytoplasm-vacuole differential acidity and phosphate metabolites in plant cell O 1,2-13C-acetate suspension cultures of Nicotiana tabacum and potato by NH in vivo 31P NMR spectroscopy. More recently the pathways O 1-13C-glycine involved in the biodegradation of substituted aromatics O 13 C-methyl-methionine have been followed by adding specifi c enzymes from HO particular bacteria in situ to key intermediates and OH OH identifying the resulting compounds by 1H NMR spectros- copy. This provides direct information for the reconstruc- tion of the metabolic networks found in aromatic degrada- Fig. 5. Biosynthetic precursors of apicularen B Graphic: HZI tion and permits a rational exploitation of the capabilities of the microorganisms involved.

FROM THE GENOME TO FUNCTIONAL GENOMICS

Functional genomics

Genome Proteome Cellular Function & Regulation

Proteomics Protein 3D Structure 2D gel elecrophoresis MALDI / ESI MS Protein sequencing Protein Production Structural Methods

Recombinant X-ray Crystallography Post-translational Solid State Methods NMR spectroscopy modifi cation peptide Cloning synthesis Expression Flexible non-crystalline 1D Sructure Protein Fermentation proteines

Fig. 6. Instrumental techniques used in functional genomics Graphic: HZI SPECIAL FEATURES | Nuclear Magnetic Resonance Spectroscopy, a Major Player in the Arsenal of Platform Technologies 57

Structural biology: Application of NMR in protein studies The impetus to protein research generated by the decoding A of the both human and subsequent genomes at the begin- ning of the century has been a result of the enormous progress made in many areas of instrumentation. This was refl ected in the 2002 Nobel prizes in chemistry awarded to Fenn and Tanaka for the development of mass spectrometric analyses of biological macromolecules and to Wüthrich for his development of NMR spectroscopy for determining the three-dimensional structure of biological macromolecules in solution. Soft desorption ionisation MS methods (ESI and MALDI MS, fi gure 6) provide the one-dimensional sequence of amino acids of proteins and indentify post-translational modifi cations. This information is a prerequisite for the determination of their three-dimensional structures and Ion channel Interaction subsequent correlations with cellular function and activity with CD4 regulation (fi gure 6). The importance of X-ray crystallogra- phy for structural investigations of crystalline proteins has B been stressed in an earlier feature of these reports. Here an alternative approach is considered that exemplifi es the use of NMR spectroscopy to elucidate the solution structure of small fl exible proteins, molecules that do not form crystals and are not readily produced by recombinant methods.

The HIV genome encodes a number of enzymes and structural proteins as well as six accessory proteins. Of these the HIV-1 specifi c virus protein U (Vpu) is an 81 amino acid class I integral membrane phosphoprotein that induces degradation of the virus receptor CD4 in the Fig. 7. Model of the membrane-bound structure of monomeric endoplasmic reticulum and enhances the release of virus Vpu (A) and its dynamic forms (B) Graphic: HZI particles from infected cells. Although this fl exible protein does not form crystals, suffi cient quantities of the protein and related fragments were available from solid state peptide synthesis that allowed studies to be undertaken by bilayers using 15N solid-state NMR spectroscopy. These data circular dichroism and NMR spectroscopy. Both methods allowed a model of the monomeric structure of the molecule indicated that the structure of the molecule was dependent to be constructed, fi gure 7, in which the fi rst helix (α1) of on the solution conditions and only in a hydrophobic the N-terminal domain forms a trans-membrane helix membrane-like environment did the molecule assume a attached to the fi rst helix (α2) of the cytoplasmic domain stable secondary structure, a phenomenon characteristic of which binds parallel to the membrane surface. This is then many small proteins in solution. Detailed 2D 1H NMR attached to the fi nal helix (α3), which shows only weak studies of the 50 amino acid cytoplasmic C-terminal domain interactions with the membrane. Such a structure is in its most structured state possessed a well defi ned compatible with the ion channel activity of the N-terminus helix-interconnection-helix-turn structure in which the two and the interaction of the C-terminus with the cytoplasmic serine residues undergoing post-translational phosphoryla- domain of CD4, as well as the requirement of a phosphor- tion were positioned in the interconnection between the ylated Vpu for this function. Similar studies provided helices. The position of these structures and the helix of the details of the structure of a second HIV-1 accessory protein, N-terminal anchor domain in a bilayer membrane-like Vpr, and more recently indicated its unusual interaction environment were established in oriented phospholipid with a host protein, cyclophilin A. 58 SPECIAL FEATURES | Nuclear Magnetic Resonance Spectroscopy, a Major Player in the Arsenal of Platform Technologies

Large proteins and the compatibility with X-ray sickness and Chagas disease) and leishmaniasis. The global The question now arises as to whether NMR spectroscopy folding of the structure determined by three-dimensional can also provide structural details for larger proteins and NMR spectroscopy was very similar to that of the crystal are the structures determined in solution compatible with structure although regions near the active site were less those in the solid phase from X-ray crystallography. In well defi ned in solution (fi gure 8, A and B). Clearly, if the general, the answer to both questions is yes. Laboratories structure was the only aim of the work, X-ray crystallogra- such as the HZI Protein Sample Production Facility can now phy would have been the method of choice. However, NMR produce 15N,13C-doubly-labelled protein using recombinant allowed the binding of inhibitory substrate analogues to be methods suitable for multi-dimensional heteronuclear NMR readily investigated and disclosed regions on the protein investigations in solution. A salient example is the elucida- surface that are functionally relevant (fi gure 8, C and D), tion of tryparedoxin, a virulence determinant in trypano- which set a framework for the design of more rigid and somatids, the causative agent of trypanosmiasis (sleeping active ligands.

Fig. 8. Comparison of NMR and X-ray structures of tryparedoxin (A & B), surface model showing ligand-binding positions (C & D) SPECIAL FEATURES | Nuclear Magnetic Resonance Spectroscopy, a Major Player in the Arsenal of Platform Technologies 59

Future perspectives There will be a continued intensive Literature use of the platform technologies in the foreseeable future that is underscored by recent major investments in new 1. Frank, R. (2007) The chemical pipeline – A research pro- instrumentation. These will play a signifi cant role in the gramme and infra-structure for the discovery and evaluation research undertaken in the new Drug Discovery Centre that of new anti-infectives. Research Report 2006-2007, 32-43. is currently in the planning stage. All aspects of structure elucidation of old and new natural products of all types will 2. Reichenbach, H. (2007) Natural products: An indispensible continue to be a key area in infection research as envisaged source of new drugs. Research Report 2006-2007, 54-59. in the current research initiative of the HZI. The continued advances in technology, particularly the imminent avail- 3. Wray, V., Schiel O., Berlin, J. & Witte, L. (1985) Phospho- ability of ultra-high magnetic fi elds (≥ 1 GHz), opens up the rus-31 nuclear magnetic resonance investigation of the in possibility of studying even larger systems such as vivo regulation of intracellular pH in cell suspension biomacromolecular assemblies of highest complexity and culture of Nicotiana tabacum: The effect of oxygen supply, biomedical signifi cance by solution NMR techniques that nitrogen, and external pH change. Archive of Biochemistry will complement those studied by X-ray. Clearly this is a and Biophysics 236, 731-740. further important area of research that will make signifi - cant contributions to modern biology and biomedicine by 4. Pieper, D. H., Pollmann, K., Nikodem, P. Gonzalez, B. & increasing our knowledge of the structures of macromol- Wray, V. (2002) Monitoring key reactions in degradation of ecules and their interactions within the cellular context. chloroaromatics by in situ 1H nuclear magnetic resonance: Solution structures of metabolites formed from cis-dienelac- tone, Journal of Bacteriology. 184, 1466-1470.

5. Wray, V., Kinder, R., Federau, T., Henklein, P., Bechinger, B. & Schubert, U. (1999) Solution structure and orientation of the transmembrane anchor domain of the HIV-1-encoded virus protein U by high-resolution and solid-state NMR spec- troscopy. Biochemistry 38, 5272-5282.

6. Krumme, D., Budde, H., Hecht, H-J., Mange, U., Ohlenschlä- ger, O. Ross, A., Wissing, J., Wray, V. & Flohé, L. (2003) NMR studies of the interaction of tryparedoxin with redox-inactive substrate homologues. Biochemistry 42, 14720-14728.

Victor Wray born in 1945, obtained his B.Sc. (1966) and 7. Solbak, S. M. Ø., Reksten, T. R., Wray, V., Bruns, K., Horvli, Ph.D. (1969) in Chemistry at the University of Hull. Postdoc- O., Raae, A. J., Henklein, P., Henklein, P., Röder, R., Mitzner, toral scientist in the Department of Organic Chemistry at D., Schubert, U., Fossen, T. (2010) The intriguing cyclophilin Imperial College, London (1969-1973). Joined the Physical A-HIV-1 Vpr interaction: Prolyl cis/trans isomerisation cata- Measurements Group of the Centre for Molecular Biological lysis and specifi c binding. BMC Structural Biology 10, 31. Research, a forerunner of the GBF and HZI (1973). Head of the NMR spectroscopy group since 1987 and head of the Bio- physical Analytics Research Group (including the Analytical Instruments Platform) since 2003. 60 SPECIAL FEATURES | International Cooperation of Helmholtz Centre for Infection Research with India and China

International Cooperation of Helmholtz Centre for Infection Research with India and China: A Success Story

AUTHOR | Prof. Dr. G. Singh Chhatwal | Department of Medical Microbiology | [email protected]

Infectious diseases, which are responsible for a quarter of all deaths, are a serious global health problem. One reason is that the infectious agents do not require passport, visa or fl ight ticket to travel from one country to another. A few years ago it took the SARS virus just 24 hours to travel from Hong Kong to Toronto. To add to this mobility problem, pathogenic microorganisms differ considerably in their antigen repertoire in different geographical regions. It is well known that strains belonging to the same species have different types in China, India, Europe or North America. For an effective control of infectious diseases a global effort is required, not only to predict and manage the epidemics, but also to design and develop region-specifi c intervention strategies. Helmholtz Association, therefore, aims to strengthen international cooperation in the area of health research. Motivated by this initiative, HZI has strengthened its international cooperation in the last few years, illustrated by the following examples.

Indo-German Science Centre for Infectious Diseases (IG- in a rapid review process. Besides funding the projects, SCID): a role model for international cooperation the Centre sponsors joint workshops on particular areas of In 2006 HZI, Medical University Hannover (MHH), and In- infectious diseases in India and Germany. The Centre puts dian Council of Medical Research (ICMR) presented a vision great emphasis on the training of young investigators from to establish a Virtual Centre for Indo-German cooperation both countries. German investigators who travelled to India in the area of infectious diseases, especially the develop- benefi ted from on-hand experience with clinical infection ment of new diagnostics, vaccines and antiinfectives. The research as well as epidemiological surveys and Indian rationale behind this vision was strong clinical research in investigators gained expertise in Braunschweig and Han- the fi eld in India and state-of-the-art modern technologies nover in modern technologies and new animal experimental and experimental animal facilities at HZI and MHH. This techniques. The Centre recently started a new fellowship high degree of complementarity made the concept into a win- programme where scientists from India and Germany can win situation. This vision became reality in 2006, when Prof. undertake exchange visits and do research for a period of N.K. Ganguly, Director General of ICMR, and Prof. Jürgen up to 3 months. Since both German partners come from Mlynek, President of Helmholtz Association, signed a Memo- Niedersachsen, its activities were presented during the randum of Understanding in the presence of the Prime Min- visit of the Honourable Chief Minister, Mr. David McAllister, ister of India, Dr. Manmohan Singh, and the Chancellor of in New Delhi in September 2010. The Centre is a useful Germany, Dr. Angela Merkel, in Hannover. After this signing addition to the joint activities of Niedersachsen with India the events took place fast and both Helmholtz Association in science and technology. and ICMR ensured the fi nancing of the centre. In April 2007 the Centre was inaugurated in New Delhi in the presence of There is a desire from both sides to continue this coopera- dignitaries from Helmholtz Association, HZI, MHH, ICMR tion and to look for possibilities to transform this Virtual and a number of scientists working in the area of infectious Centre into a physical one, most probably in India. diseases. An offi ce of IG-SCID was set up at the headquarters of ICMR in New Delhi. Six months after inauguration IG- SCID was expanded to include Jawaharlal Nehru University (JNU) and a Memorandum of Understanding in this regard was signed by Dr. Rajendra Prasad, Rector JNU, Dr. N.K. Gan- guly, and Prof. Rudi Balling (Director HZI) in the presence of the Honorable Ministers Dr. Annette Schavan and Mr. Kapil Sibal in New Delhi.

A centre with such elite partners was deemed to be a success, but it has now become clear that this Centre has become a role model for international cooperation. A unique concept of this Centre has contributed towards this achieve- ment. The Centre funds joint research projects based on excellent science and high relevance for both countries. The Fig. 1. Signing of MoU by Prof. Ganguly (left) and Prof. Mly- projects are peer-reviewed by a joint steering committee nek in April 2006 in Hannover Photo: HZI SPECIAL FEATURES | International Cooperation of Helmholtz Centre for Infection Research with India and China 61

Fig. 2. Inauguration of IG-SCID in May 2007 in New Delhi Fig. 5. Sino-German meeting in Guangzhou, China coordi- (from left: Prof. Balling, Prof. Chhatwal, Prof. Mlynek, nated by Prof. Chhatwal from German side and Prof. George Prof. Ganguly and Prof. Hasnain) Photo: ICMR Fu Gao, Vice President of Beijing Institutes of Life Sciences, Chinese Academy of Sciences, from Chinese side in December 2010. Prof. Han Jianguo, Director of the Sino-German Center, also participated in this meeting. Photo: HZI

Cooperation with China: an important initiative Infectious diseases represent a huge health hazard in China. In the last decade the research activity in this fi eld in China has substantially increased. The results fl owing in from this research indicate that the strains in China differ not only in their virulence repertoire but also cause different clinical manifestations. To develop global control strate- gies against infectious diseases, a strong cooperation with China is extremely important. Therefore, HZI has started an Fig. 3. Expansion of IG-SCID to include Jawaharlal-Nehru Uni- initiative with Chinese Academy of Sciences for intensive versity as a partner during the visit of Dr. Annette Schavan, collaboration. This initiative has resulted into two Sino- German Federal Minister of Education and Research, in New German workshops in Goslar in 2009 and in Guangzhou in Delhi in October 2007 Photo: HZI 2010. These activities were fully sponsored by the Chinese- German Centre for Science Promotion in Beijing. This centre clearly identifi ed the need for such cooperation. From German side, beside HZI, Justus Liebig University Giessen, RWTH Aachen, TiHo Hannover and MHH participated in workshops. From Chinese side, different institutes of Chi- nese Academy of Sciences were partners. These workshops resulted in a number of bilateral cooperations, and an appli- cation for a bigger Sino-German project is being submitted. We hope that the collaboration with China will be a useful supplement to our other international cooperation.

Fig. 4. An event to present the scientifi c activities of IG-SCID during the visit of Mr. David McAllister, Chief Minister of Niedersachsen, in New Delhi in September 2010 (Prof. Heinz, Mr. McAllister, Prof. Chhatwal) Photo: Henning Noske FOCUS RESEARCH REVIEWS SPECIAL FEATURES

Photos from left to right: Lisa Schreiber performing magnetic cell sorting | The assistance to the head of HIPS: administrative manager David Hofmann, personal assistant Birgitta Lelarge, and scientifi c assistant Dr. Markus Ehses (from left to right) | Dr. Leonor Gama-Norton watches a crucial analysis step performed by the PhD student Marcin Cebula. Photos: HZI, Krämer (li) | HIPS/HZI (ce) | HZI, Scheibe (ri) SCIENTIFIC REPORTS FACTS AND FIGURES

64 Infection and Immunity

114 New Project Groups

121 PoFII Independent Research

125 Technological Platforms

132 The Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS)

138 TWINCORE, Centre for Experimental and Clinical Infection Research GmbH

148 Publications 2010-2011 64 SCIENTIFIC REPORTS | Infection and Immunity

INFECTION AND IMMUNITY

PROGRAMME SPEAKER | Prof. Dr. Dirk Heinz | Department of Molecular Structural Biology | [email protected]

Infections remain the third most frequent cause of deaths worldwide and hence pose a serious threat to human societies in all hemispheres. Despite the discovery and development of antibiotics, vaccines and improved hygiene conditions, we are confronted with the fact that many infectious diseases are re-emerging. The increasing prevalence of antimicrobial resistance added to globalization, climate change and growing aging populations in industrialized countries poses a continuous challenge in combating and controlling infectious diseases of humans and domesticated animals. The infl uence of these anthropogenic factors is best- illustrated by epidemic outbreaks of previously unknown diseases, such as novel zoonotic infections like HIV, SARS, and avian or swine infl uenza. Moreover, infectious diseases such as tuberculosis, that used to steadily decline in the past, are currently re- emerging as global threats.

In addition to the increasing occurrence of epidemic viral diseases, industrial countries are facing novel challenges in the combating and containment of bacterial infections. With the advances in modern medicine, the number of immunosuppressed patients highly susceptible to opportunistic infections (e.g. transplantation patients or those under intensive care) is growing, and a high suscepti- bility to infection is observed in the ageing population as a result of an age-related decline in the body’s immune defences. Further- more, resistance against virtually all antibiotics presently on the market poses an immense burden on health systems. Novel strategies for diagnosis, prevention and therapy of infectious diseases are therefore essential for controlling these threats to public health.

The HZI, its subsidiary institute, the Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), and its associated institute TWINCORE (Centre for Experimental and Clinical Infection Research) together tackle a number of the most important questions in infection research by building a strong basis in fundamental research and applying this knowledge to develop new strategies for prevention and therapy of microbial infections for translation into public health benefi ts. They have set up the Helmholtz programme “Infection and Immunity”, which comprises fi ve areas of infectious disease research refl ected in the topics

• Microbial Pathogenesis • Host-Pathogen Interactions • Infl ammation and Immunity • Strategies for Prevention and Therapy • Pharmaceutical Research

The former topic “Translational Infection Research” has become a cross-topic activity since it is highly relevant to all fi ve topics. The complete infrastructure and expertise necessary to plan, prepare and conduct clinical trials has been developed together with the Hannover Medical School (MHH). Currently a clinical trial centre for early clinical trials is being set up as a collaborative effort by the Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM), MHH and HZI. SCIENTIFIC REPORTS | Infection and Immunity 65

Microbial Pathogenesis tackles the fundamental question of how bacterial pathogens cause disease. Adherence to host cells, invasion, intracellular survival, dissemination and immune evasion are strategies used by microorganisms to establish infections in a host. Understanding these complex processes is achieved by investigating the mechanisms of pathogenicity, the role of specifi c virulence factors and pathogen diversity. Bacterial pathogens under investigation in this topic include Streptococci as causative agent for respiratory and invasive diseases, Yersiniae and Listeriae as both enteropathogens and model organisms, and Myco- bacterium tuberculosis, the causative agent of tuberculosis. State-of-the-art technologies including structural biology, electron microscopy and proteomics are utilized for the in-depth investigation of the molecular processes involved.

Studies in Host-Pathogen Interactions focus on understanding the interplay between the infected host and bacterial or viral pathogens, where genetic and cellular factors controlling the infection process are studied in complex model systems like the mouse. This also includes the complexity of microbial communities within the host involved in the formation of persistent biofi lms on medical implants or in cystic fi brosis lungs that are refractory to antimicrobial treatment. Other research activities deal with the transmission of zoonotic pathogens like infl uenza and prion protein diseases. Large-scale epidemiological studies, such as the newly developed National Cohort, that help to further understand the complexity of host factors responsible for susceptibility or resistance against pathogens are also part of this topic.

Infl ammation and Immunity addresses the basic processes involved in eliciting innate and adaptive immune responses against infectious agents. A strong emphasis is placed on understanding the mechanism and regulation of the interferon (IFN) system as an important constituent of the early immune response and mediator of infl ammation. T cells as key players in the adaptive im- munity are studied with respect to their delicate balance between host protection, tolerance and autoimmunity. New technologies developed in this research area include the development of novel cell lines and humanized mice as models for infection and host response. Systems biology approaches are used to model molecular pathways involved in immunity.

The central aim of Strategies for Prevention and Therapy is the development of innovative tools and strategies for the preven- tion, management and control of infections and infection-related diseases. One major research focus is the development of new antigen delivery systems and adjuvants, in particular those amenable for the development of mucosal vaccines. This is linked to activities where preclinical validation models based on humanized mice for screening, selection and prioritization of candidate in- terventions are established. New drug targets relevant for hepatitis C virus (HCV) replication and propagation are investigated us- ing a cell-based HCV model. Furthermore, virus and host associated factors affecting the quality and quantity of anti-viral cytokine responses and the protective mechanism of IFN are being studied.

The topic Pharmaceutical Research aims at integrating anti-infective research to bridge the gap between basic biological and biochemical research and clinical applications, and consequently initiate an early interaction with the pharmaceutical industry. The central goal is to identify and develop natural products and chemically synthesized compounds as bioactive lead structures, which can be further developed using medicinal chemistry, total synthesis and biosynthetic approaches. Finally, further studies involve optimisation of formulation and drug delivery technology to facilitate transport of promising compounds to their targets. 66 SCIENTIFIC REPORTS | Infection and Immunity

With the programme “Infection and Immunity”, the HZI addresses current and future fundamental problems of infection research not only through the existing areas of research and expertise but also by implementation of a comprehensive set of novel and/ or complimentary initiatives to the current lines of research. These are concentrated in the provision of a pipeline for the develop- ment of novel anti-infectives and their rapid translation into clinical applications.

Currently, expertise and infrastructure in functional genomics and proteomics are further strengthened to provide new approaches for unravelling the mechanisms and pathways involved in host-pathogen interactions during infection. These will be exploited for the identifi cation and characterization of targets as a prerequisite for anti-infective intervention. The discovery and development of anti-infectives is being intensifi ed and signifi cantly expanded by exploiting the enormous genetic potential of microorganisms for yielding novel bioactive compounds. This will accelerate the development of therapeutic tools with the ultimate goal of translation into the clinic. Within the newly developed German Centre for Infection Research (Deutsches Zentrum für Infektionsforschung (DZIF)) it is planned that the HZI will mainly contribute its strengths in natural product research for the development of novel anti- infectives and its expertise in basic research on bacterial and viral infections. Together with the MHH, the German Collection of Microorganisms and Cell Cultures (DSMZ) and the Technische Universität Braunschweig, the HZI will signifi cantly expand its activi- ties in promoting and developing translational infection research in the Braunschweig-Hannover area and beyond to foster effi cient exploitation of basic research results in the DZIF.

Most important ifectious diseases No. of deaths - cases/year Year AIDS/HIV Lower Respiratory 2.8 Mill. Infections 3.9 Mill. Lower Respiratory Infections 3.900.000 2002 HIV/AIDS 2.400.000 -3.300.000 2001-2009 Diarrhea / Gastroenteritis 1.800.000 -2.600.000 2002-2009 Tuberculosis 1.600.000 -3.000.000 2001-2009 Meningitis Malaria 1.300.000 -2.700.000 2002-2009 170.000 Diarrhea/ Tetanus Measles 700.000 2001 Gastroenteritis Pertussis 210.000 2.6 Mill. 290.000 Hepatitis 520.000 -700.000 2005

Hepatitis Pertussis 290.000 2002 Measles 600.000 700.000 Tuberculosis Tetanus 210.000 2002 1.8 Mill. Malaria 1.3 Mill. Meningitis 170.000 2002

Infectious diseaes worldwide: Death cases per year Source: WHO (2001-2009) SCIENTIFIC REPORTS | Infection and Immunity | Microbial Pathogenesis 67

01 Microbial Pathogenesis

TOPIC SPEAKER | Prof. Dr. G. Singh Chhatwal | Department of Medical Microbiology | [email protected]

In spite of the availability of a large number of different antibiotic and antiviral agents, the disease burden caused by infections is continuously increasing and markedly impairs the achievements of medical care. Due to advanced medical practises chronic per- sistent infections are becoming more and more apparent and constitute a major challenge for the medical profession. Furthermore, the increasing emergence of antimicrobial resistant bacterial isolates is a major concern and multi- and pan-resistant pathogens are frequently identifi ed especially in the clinical setting. The decreasing therapeutic options make it necessary to develop new treatment strategies. To meet these challenges, a detailed understanding of the mechanisms of pathogenicity is of utmost impor- tance. Adherence to and invasion of the host cells, intracellular survival, dissemination, immune evasion – especially evasion of intracellular defences – and persistence are just a few of the strategies used by microorganisms to establish an infection in the host. In addition, the carrier stage of the pathogen, which has been neglected in the past, has become an important research theme because of its role in latent infections. Another area of concern is the diversity of many infectious agents. Many bacterial and viral species have hundreds of different serotypes with strong antigen variation which impedes development of effective therapies. Therefore, a multi-disciplinary approach has to be applied in order to fully understand the pathogenic mechanisms of microorganisms.

The main objective of this topic is the in-depth study of the biology of pathogenic microorganisms and their interaction with the host cell. The topic deals with the following research themes:

Study of the host cell-pathogen interactions at the molecular and cellular level In the course of their co-evolution with the host, bacterial pathogens have developed sophisticated strategies to exploit the host cell and immune system for their own benefi t. Intracellular survival, dissemination in the host, and pathogen persistence require a complex series of interactions within the host cell. A prime target is the cytoskeleton that is involved in numerous cellular functions and processes that depend on the intrinsic dynamics of its constituents, in particular the actin system. A large number of actin binding proteins have been described which regulate the dynamic reorganisation of the cytoskeleton. This research area deals with the elucidation of exact signalling pathways and principles of the control of actin assembly. Whereas the subversion of the actin system by a pathogen has been a topic of interest for a long time, the contribution of the dynamic microtubule system to bacterial pathogenicity is emerging as an important new area of infection research and has been included in this topic. Listeria monocytogenes and Yersinia enterocolitica are the focus of this research area.

Identifi cation of novel virulence factors from pathogenic bacteria Pathogenic microorganisms produce a variety of virulence factors with diverse functions in processes such as adherence, invasion, intracellular survival, evasion of the host immune response and bacterial communication. Their functional characterisation not only contributes towards our understanding of pathogenicity, but also helps in identifying promising candidates for the development of vaccines, diagnostics and novel therapeutics. Fibronectin, collagen and plasminogen are important constituents of the extra- cellular matrix shown to be directly involved in host-pathogen interactions. Fibronectin binding proteins of pathogenic bacteria are important factors in adherence, invasion and also intracellular survival. Collagen binding to bacteria plays a role in certain autoimmune manifestations and the activation of plasminogen induced by bacterial proteins is crucial for tissue invasion. The main focus of this research area is the virulence factors of different streptococci and Yersinia enterocolitica. 68 SCIENTIFIC REPORTS | Infection and Immunity | Microbial Pathogenesis

Analysis of bacterial virulence using proteomics In this theme, the model organism L. monocytogenes is used to unravel the spatial and temporal dynamics of host-pathogen interactions at the protein level. In particular effector mediated host cell signalling is studied using a qualitative phosphokinome analysis. Since the progression from fi rst P. aeruginosa acquisition until sustained colonisation of the lung of CF patients correlates directly with general life expectancy, a highly sensitive immunoproteome workfl ow for the identifi cation of diagnostic and prog- nostic antigenic markers is being established together with the outpatient clinic of the Hannover Medical School. Furthermore, the project investigates signal transduction processes specifi c both for the bacterial pathogen P. aeruginosa and for lung epithelial cells derived from CF patients.

Structural analysis of proteins involved in host-pathogen interactions The elucidation of host-pathogen interactions at the atomic level provides mechanistic insights for the development of new approaches to specifi cally interfere with infection processes. The 3D-structures of microbial virulence factors and, if known, their complexes with host cell interaction partners and receptors are determined at high resolution, using the well-established techniques of X-ray crystallography and NMR spectroscopy. The main emphasis is placed on virulence factors that contribute to microbial adherence and invasion, remodelling of the host cell cytoskeleton, components and effectors of type III secretion systems, key gene regulators of microbial virulence and amyloids involved in neurodegenerative diseases. We have also included in this topic the protein production in eukaryotic cells for structural analysis.

The above-mentioned research areas are being handled at project level by a multi-disciplinary team of scientists that belong to different departments and research groups at HZI and bring with them a variety of different expertise. The highlights of the results obtained at project level are given in the following project reports.

Genome map of a highly infectious clinical Mycobacterium tuberculosis strain from Latin America. Genes coding various functional categories are depicted in different colours. (see page 80) SCIENTIFIC REPORTS | Infection and Immunity | Microbial Pathogenesis 69

01.1 Structural Analysis of Virulence Factors

PROJECT LEADER | Prof. Dr. Dirk Heinz | Department of Molecular Structural Biology | [email protected]

PROJECT MEMBERS | Davide Ferraris | Thomas Heidler | Dr. Christian Kambach | Dr. Björn Klink | Dr. Jörn Krauße | Dr. Anja Menzel | Nick Quade | Dr. Stefan Schmelz | Ulrich Wiesand

Pathogenic bacteria use an arsenal of virulence factors to A new class of bacterial guanine nucleotide exchange breach natural host barriers, circumvent or block defense factors for human GTPases Actin cytoskeleton remodelling mechanisms or reprogramme host molecular processes is essential for cell division, motility or cell-cell communica- for their own benefi t. As most of these bacterial factors are tion. It is tightly regulated by multiple signalling pathways missing in the host cell, they represent potential targets for employing many different actin-binding proteins. Most if not novel antibiotics. The aim of this project is to gain a precise all pathways converge on small Rho family GTPases which understanding of pathogen-host interactions during infection act as switches inducing different actin-fi lament structures. by X-ray crystallographic structural analysis of microbial GTPase signalling requires control by guanine nucleotide ex- virulence proteins in complex with their host target proteins. change factors (GEFs). Rho-GTPases are key targets of patho- gens modifying cell signalling or actin remodelling for their Listeria InlB activates the human receptor tyrosine kina- own needs. Pathogens frequently bypass or activate Rho- se Met through dimerization L. monocytogenes is a harmful GTPase pathways by producing virulence factors mimicking food pathogen which can cause listeriosis, a systemic disease Rho-interacting proteins. This allows them to induce tight with high mortality rates in neonates, the elderly and other adherence to or entry into host cells. Shigella fl exneri IpgB1 immuno-compromized individuals. The unique capability of and IpgB2 and Map from pathogenic E. coli are prototypic the bacterium to breach several important host-cell barriers members of an intriguing novel family of bacterial effectors. critically depends on two invasins, internalin A and B (InlB), These were originally classifi ed as GTPase mimics, despite located at the bacterial surface. InlB interacts with the re- the lack of sequence homology or GTP binding. They induce ceptor tyrosine kinase Met, leading to bacterial uptake. Met fi lopodia (Map) or lamellipodia and stress fi bers indicative is the receptor for hepatocyte growth factor (HGF), making of Rac1 and RhoA activity for IpgB1 and IpgB2, respectively. it a key player in cell growth, cell migration, wound healing We solved the crystal structures of IpgB2 and its complex and cancer metastasis. Growth factor-mediated receptor with human RhoA (Fig. 2), unambiguously identifying IpgB2 activation typically involves dimerization of the receptor by as a bacterial RhoA-GEF not as a functional mimic. Struc- the ligand, in this case as InlB2/Met2. In 2007, we deter- tures of the complex in various nucleotide bound states re- mined the crystal structure of the InlB/Met complex. Like in vealed the molecular mechanism of GDP release, an essential the InlB structure, the complex displays a head-to-tail InlB prerequisite for GTP binding. dimer interface leading to a Met/InlB/InlB/Met-arrangement compatible with Met function, tyrosine kinase cross-phos- phorylation and receptor activation. To confi rm the putative role of InlB-mediated dimerization for Met activation, we crosslinked InlB by two symmetrical inter-InlB disulfi de bridges like in the InlB dimers (Fig. 1). Strikingly, this cova- lent InlB dimer leads to strong Met activation outperforming even HGF. Conversely, weakening the InlB dimer interface by introducing opposing positively charged amino acids completely abrogates Met signalling.

Fig. 2. Structure of the complex between IpgB2 (blue) and RhoA (brown). GDP is shown in orange and the GDP-coordina- ting Mg2+ is a yellow sphere. Red spheres are water molecules coordinating Mg2+. The switch I region of RhoA is shown as a yellow loop, switch II in green. The „catalytic loop“ of IpgB2 is shown in red.

Ferraris, D. M., Gherardi, E., Di, Y., Heinz, D. W. & Niemann, H. N. (2010). Ligand-medi- ated dimerization of the Met receptor tyrosine kinase by the bacterial invasion protein Fig. 1. Structure of the InlB dimer (blue) covalently cross-lin- InlB. Journal of Molecular Biology 395, 522-532. ked by the introduction of two disulfi de bridges (yellow sticks, Klink, B. U., Barden, S., Heidler, T. V., Borchers, C., Ladwein, M., Stradal, T. E., Rottner, with electron density). K. & Heinz, D. W. (2010). Structure of Shigella IpgB2 in complex with human RhoA: Im- plications for the mechanism of bacterial GEF-mimicry. Journal of Biological Chemistry 285, 17197-17208. 70 SCIENTIFIC REPORTS | Infection and Immunity | Microbial Pathogenesis

01.2 Virulence Factors of Streptococci and Pneumococci

PROJECT LEADER | Prof. Dr. G. Singh Chhatwal | Department of Medical Microbiology | [email protected]

PROJECT MEMBERS | Silva Amelung | Dr. René Bergmann | Dr. Simone Bergmann | Dr. Marcus Fulde | Angela Hitzmann | Melanie Lüttge | Dr. Andreas Nerlich | Dr. Patric Nitsche-Schmitz | Priv.-Doz. Dr. Manfred Rohde | Dr. Vivek Sagar | Dr. Susanne Talay

Streptococcus pyogenes and S. pneumoniae (pneumococci) are The correlation of SDSE with rheumatic heart disease is a well known human pathogenic bacteria capable of causing a cause of concern. SDSE strains from South India can bind wide spectrum of diseases. Group C and group G streptococci, collagen through PARF peptide leading to autoimmune such as S. dysgalactiae ssp equisimilis (SDSE), S. anginosus or reaction. High prevalence of SDSE indicates that this S. canis, which have been considered as animal pathogenic, neglected streptococcal species must be taken seriously are emerging as human pathogenic bacteria. For example and should be eradicated. SDSE has recently been related to rheumatic heart disease. In addition, oral streptococci which can cause dental caries Interaction of streptococci with endothelial cells are also capable of causing serious invasive diseases in hu- Prerequisite for streptococcal invasive disease is the dissemi- mans. In spite of the availability of antibiotics the morbidity nation of bacteria via blood circulation. To evade the immune and mortality due to streptococcal infections remains very system the streptococci invade endothelial cells through a high. It is necessary to elucidate the pathogenicity mecha- phagocytosis-like process. We could show that a streptococcal nisms in order to develop novel control strategies. This is the protein SpyCep, which can degrade IL-8, is involved in the major objective of the project. The highlights are: immune evasion process leading to invasive infection.

Neglected streptococcal diseases Our results showed that oral streptococci possess a pathogenicity mechanism that has until now been described only for S. pyogenes and S. pneumoniae. This mechanism involves binding and acti- vation of plasminogen by oral streptococci leading to tissue destruction and dissemination in the host. The major bacte- rial factors involved in this process are M-proteins, which are expressed by S. pyogenes, and, as we described recently, also by SDSE and S. canis. Invasion of SpyCep-expressing S. pyogenes (left) and SpyCep- coated latex beads (right) into human endothelial cells Photo: HZI, Rohde

Pneumococci are the major organism involved in human pneumonia. Using a human genome microarray we could show that pneumococci can infl uence the gene expression profi le of lung endothelium. In addition, we could identify phosphoglyceratkinase, a cytoplasmic enzyme of Pneumo- coccus that is found on bacterial surface and can also bind plasminogen thereby facilitating tissue invasion.

In summary, the elucidation of the mechanisms involving interaction of streptococci with human endothelial cells and Activated plasmin(ogen) on the surface of S. canis can destroy plasminogen offers new control strategies for invasive strep- fi brin, leading to bacterial dissemination in the host Photo: HZI, Rohde tococcal diseases. The study on neglected streptococcal species allows us to understand the common pathogenicity mechanisms among different streptococci that may lead to The role of group C and group G streptococci in human novel diagnostic and therapeutic approaches. diseases is not well investigated. Our results showed that in South India, where such infections are very common, 80 Bergmann, R, Dinkla, K, Nitsche-Schmitz, DP, Graham, RM, Lüttge, M, Sanderson- Smith, M, Nerlich, A, Rohde, M, Chhatwal, GS (2010) Biological functions of GCS3, a percent of the diseases were caused by SDSE. Surprisingly, novel plasminogen binding protein of Streptococcus dysgalactiae ssp. equisimilis. S. anginosus was a causative organism in 20 percent of the International Journal of Medical Microbiology. doi:10,1016/j.ijmm.2010.06.007 infections. We have recently developed a test which can Itzek A, Gillen CM, Fulde M, Friedrichs C, Rodloff AC, Chhatwal GS, Nitsche-Schmitz DP (2010) Contribution of plasminogen activation towards the pathogenic potential of oral identify S. anginosus with a high degree of specifi city. streptococci. PLoS One 5: e13826 SCIENTIFIC REPORTS | Infection and Immunity | Microbial Pathogenesis 71

01.3 Molecular Mechanisms of Intracellular Traffi cking, Survival and Persistence of Streptococci

PROJECT LEADER | Priv-Doz. Dr. Manfred Rohde | Dept. Medical Microbiology | [email protected]

PROJECT MEMBERS | Katja Branitzki-Heinemann

Streptococcus pyogenes, the group A Streptococcus (GAS), is With the help of SfbIGaro coated gold-nanoparticles we could the major cause of human streptococcal infections that range show that fi bronectin binding does not result in integrin from uncomplicated to severe and life-threatening infections clustering (D) as seen for GfbA. Only GfbApro mediated like necrotizing fasciitis. Streptococci are also able to cause fi bronectin binding results in integrin clustering (H). Thus, recurrent infections such as erysipel and tonsillitis. This pheno- we demonstrate for the fi rst time a biological function of the menon is described as carrier stage of streptococcal infections. aromatic domain of a fi bronectin binding protein of a patho- It allows GAS not only to persist but also to resist prolonged genic bacterium. In addition, fi rst evidence is coming up that antibiotic treatment. The carrier stage of streptococci has streptococci invading via membrane ruffl ing always seem to been more or less neglected in the past. Only scant informati- fuse with lysosomes. on is available on the mechanisms and the factors involved.

Invasion and survival mechanisms of streptococci Fibronectin binding proteins of streptococci have been demonstrated to play an important role in adherence and invasion. The streptococcal fi bronectin-binding protein (SfbI) from group A streptococci mediates adherence and inva- sion through invaginations (A) and is also involved in the intracellular survival of streptococci by formation of a novel compartment termed caveosome. SfbI utilizes fi bronectin as a bridging molecule to bind to α5ß1 integrins. By co-opting this caveolae-mediated pathway SfbI-carrying streptococci bypass the degradation mechanism of the host cell since no fusion with lysosomes is detectable.

GfbA-expressing Group G streptococci exhibit GfbA on their surface which also binds fi bronectin and interacts with

α5ß1 integrins. But GfbA-mediated invasion leads to the formation of membrane ruffl es (E), rearrangements of the host-cell cytoskeleton, despite the fact that similar amounts of fi bronectin are bound compared to the SfbI-carrying strains. GfbA-expressing streptococci follow the classical endocytic pathway: fusion of lysosomes and phagosomes to form phagolysosomes. By heterologous surface expression of A)SfbI-expressing streptococci invade host cells via the formati- GfbA in the non-pathogenic S. gordonii it was demonstrated on of invaginations. E) GfbA-expressing streptococci invade via that GfbA alone is responsible for the morphological distinct membrane ruffl es. B) heterologous expression of SfbI contai- invasion mechanism compared to the SfbI-mediated mecha- ning the aromatic domain of GfbA (SfbIGaro) on S. gordonii nism. Sequencing of the GfbA gene demonstrated that only leads to invasion via membrane ruffl es and fusion with lysoso- the C-terminal part shows a high similarity with SfbI and the mes (C); streptococci in red, lysosomes (green) are labeled for N-terminal part, including the aromatic domain of GfbA and the Lamp-1 protein. F) deletion of the aromatic domain in GfbA SfbI, shows signifi cant differences. (GfbApro) results in an invasion via invaginations and no fusi- on with lysosomes (G). D) binding of SfbIGaro with fi bronektin Expression of chimaric proteins from SfbI and GfbA onto integrins results in no integrin-clustering since only single Based on the sequence data the N-terminal aromatic domain gold-nanoparticles are detectable (D, arrows), whereas binding was assumed to be responsible for the different invasion me- of fi bronectin to GfbApro leads to integrin-clustering, large chanism. Therefore, a GfbA protein was constructed without gold-nanoparticles aggregation (H, arrows). Photos: HZI, Rohde the aromatic domain (GfbApro) and the aromatic domain in SfbI was replaced by the aromatic domain of GfbA (SfbGaro). Nerlich, A., Rohde, M., Talay, S., Genth, H., Just, I., Chhatwal, G.S. (2009) Invasion of Both proteins were expressed on the surface of S. gordonii. endothelial cells by tissue invasive M3 type Group A streptococci requires Src kinase and activation of Rac1 by a PI3-kinase independent mechanism. J. Biol.Chem., 284, GfbApro mediated invasion showed the typical caveolae- 20319-20328 mediated invasion pattern (F) without fusion with lysosomes Rohde, M., Graham, R.M., Branitzki-Heinemann, K., Borchers, P., Preuss, C., Schleicher, (G). On the other hand SfbIGaro was now accompanied by I., Zähner, D., Talay, S.R., Fulde, M., Dinkla, K., Chhatwal, G.S. (2011) Differences in the aromatic domain of homologous streptococcal fi bronectin-binding proteins trigger membrane ruffl ing (B) with lysosomal fusion events (C). different cell invasion mechanisms and survival rates. Cell. Microbiol. 13, 450-468 72 SCIENTIFIC REPORTS | Infection and Immunity | Microbial Pathogenesis

01.4 Analysis of Protein Networks Induced by Early Host-pathogen Interactions

PROJECT LEADER | Dr. Lothar Jänsch | Research Group Cellular Proteomics | [email protected]

PROJECT MEMBERS | Dr. Uwe Kärst | Dr. Sebastian König | Dr. Manfred Nimtz | Dr. Tobias Reinl | Dr. Josef Wissing | Evelin Berger | Susanne Freund | Christoph Gernert | Alexander Iphöfer | Thorsten Johl | Kirstin Jurrat | Zofi a Magnowska | Maxi Scheiter

The Research Group Cellular Proteomics focuses on the analysis of fundamental events during signal-transduction processes which are important for infectious processes in humans and the host’s adaptive immune responses towards pathogens. Modules for cell biological, biochemical, mass spectrometric and bioinformatic approaches have been de- veloped and combined for this purpose. Our aim: a quantita- tive and time-resolved analysis of protein expression, locali- sation, interaction and their post-translational modifi cations (PTMs) in primary and immortalised human cells.

Methods Quantitative chemical proteome analyses facili- tate the identifi cation of cellular “target proteins” and the understanding of signalling networks. Therefore, we opti- mise small molecules used in therapeutics and use them after immobilisation as “baits” for interacting proteins. In combination with chromatographic and mass spectrometric methods these tools enable the analysis of transient struc- “Kinase tree” of all known human kinases; identifi ed human tural modifi cations on signalling components. In the case kinases are labelled red, the corresponding orthologs from of phosphorylations this refers to protein kinases, i.e. their mouse in green. Graphic: HZI activities and their molecular interactions with substrate molecules. iTRAQ™-technology is used for quantitative peptide sequencing (Orbitrap Velos) and iTRAQassist for Characterisation of signal transduction pathways in acti- statistical evaluations, both matching the quality criteria vated NK and T cells T lymphocytes are essential for regu- for clinical studies. lating the immune system. Their actual cellular processes and effector functions depend directly on the activation sta- Signalling pathways depending on phosphorylations tus of such pathways. Quantitative phosphokinome analyses during invasion by Listeria monocytogenes The bacterial with different regulatory T cells detected novel components pathogen L. monocytogenes causes severe illnesses and of the CD3/CD28-dependent activation pathways. As part prenatal infections. The virulence factors InlA and InlB of a comparative analysis of CD3/CD28-activated signal induce the invasion of host cells by interacting with the networks of effectory and regulatory T cells we analysed adhesin E-Cadherin (InlA) and the receptor tyrosine kinase the expression and phosphorylation status of about 150 c-Met (InlB). The signalling pathways involved are control- kinases. We identifi ed novel signalling components and led through protein phosphorylations by kinases. A direct phosphorylation sites that are currently being investigated analysis of time-resolved phosphorylation events on kinases to clarify their roles in the formation and function of sup- was demonstrated for the InlB-activated c-Met pathway for pressor T cells in humans and mice. The methods developed the fi rst time worldwide. This work led to the identifi cation for analysing T cells are now also being applied for the fi rst of signalling components involved in listerial invasion that time for the characterisation of activated NK-(natural killer) were not described so far for the c-Met pathways activated cells that are part of the innate immune response. by the physiological ligand HGF. Functional investigations of these kinases thus analyse their contribution to the invasion as well as to motogenic and mitogenic processes. Hemmen, K. Reinl, T.; Buttler, K. Behler, F., Dieken, H. Jaensch, L., Wilting, J., Weich, H.A. (2010) High-resolution mass spectrometric analysis of the secretome from mouse Chemical proteomics at the HZI already can access about lung endothelial progenitor cells. Angiogenesis, in press 70% of the total kinome from humans and mice and in addi- Reinl T., Nimtz, M., Hundertmark, C., Johl, T., Kéri, G., Wehland, J., Daub, H. and Jänsch, tion has devised novel tools, which are now complementing L. (2009) Quantitative phosphokinome analysis of the Met pathway activated by the information from the ubiquitin-signal network. invasin InlB from Listeria monocytogenes. Molecular Cell Proteomics 8(12):2778-95. Hundertmark C., Fischer R., Reinl T., Klawonn F. and Jänsch L. (2009) MS-specifi c noise model reveals the potential of iTRAQ™ in quantitative proteomics. Bioinformatics 25(8):1004-11. SCIENTIFIC REPORTS | Infection and Immunity | Microbial Pathogenesis 73

01.5 Structural and Mechanistical Analysis of Functional Amyloids

PROJECT LEADER | Prof. Dr. Christiane Ritter | Junior Research Group Macromolecular Infections | [email protected]

PROJECT MEMBERS | Agnes Zimmer | Madhu Nagaraj | Johannes Spehr | Tobias Schubeis

Bacterial fi brillar adhesins with an amyloid-like fold have recently been identifi ed as a novel class of adhesins that are expressed by many bacteria, both gram-positive and gram-negative. A study on biofi lms from different habitats es- tablished that up to 40% of the bacteria present within these communities carry amyloid-like fi brils on their surface. They extracellular space have been identifi ed as virulence traits important for biofi lm formation, interaction with host proteins and promoting survival in a wide range of conditions. Amyloid-like fi brils are ordered aggregates that have a high content of specifi c b-sheet secondary structure. Long associated with human diseases such as Alzheimer’s disease, Parkinson’s disease or Creutzfeld-Jacob disease the amyloid fold is now known to be formed natively by a large set of diverse proteins without Model of curli biogenesis. Curli expression is controlled by toxic side-effects. the positive transcriptional regulator CsgD. CsgA is the major structural subunit which forms a fi brillar structure upon nu- The major focus of our group lies on the determination of cleation by the homologous protein CsgB. CsgG is a membran the structural and mechanistical basis of these functions for chanel required for export of the structural subunits, and selected bacterial and fungal amyloids. CsgE and CsgF a chaperone-like molecules crucial for effi cient curli assembly. The insert depicts a model of a single CsgA Tools for the structure determination of amyloid fi brils molecule within the fi brillar structure derived from quenched One major bottleneck in amyloid research is the sparseness hydrogen exchange NMR and sequence alignments. of high-resolution structural information on naturally occur- ring amyloids, because the size and non-crystalline nature of the fi brils drastically restrict the use of established structural proteins. In addition, we analyse the structure and function techniques. We have, therefore, introduced a new, gene- of chaperones that are essential for curli biogenesis in vivo. rally applicable approach that derives the fold of amyloid A mechanistical understanding of curli biogenesis will allow fi brils from a combination of quenched hydrogen exchange us to identify new ways to interfere with fi bril formation in measured by nuclear magnetic resonance (NMR), solid state order to reduce bacterial virulence. NMR and other spectroscopic techniques. Solid-state NMR is a new method to determine high-resolution structures of HET-s: a functional prion found in fi lamentous fungi proteins. It is particularly interesting for fi brous proteins. We Only subsets of amyloids are self-propagating prions. To un- are therefore in the process of establishing this technique at derstand the molecular determinants that govern the infecti- the HZI, and we develop biochemical tools to generate prote- vity of an amyloid we investigate the biophysical properties in samples with selective isotope labelling patterns. of the functional prion protein HET-s from the fi lamentous fungus Podospora anserina and of a recently identifi ed homo- Curli: an amyloid coat that increases bacterial virulence log from Fusarium graminearum. Despite limited sequence Curli is the major proteinaceous component of the extracel- identity the amyloid fi brils formed by both proteins can lular matrix produced by Enterobacteriaceae such as E. coli cross-seed each other, i.e. they are able to breech the species and Salmonella typhimurium. Curli fi brils are involved in the barrier. Using quenched hydrogen exchange NMR we could adhesion to biotic and abiotic surfaces and the promotion explain this observation with a high structural similarity of of biofi lm formation. They also interact with several host the amyloid fi brils. proteins resulting in increased tissue penetration, infl am- mation and sepsis. In vivo curli biogenesis is dependent on the nucleation of the major curli component, CsgA, by Wasmer, C., Zimmer, A., Sabate, R., Soragni, A., Saupe, S.J., Ritter, C., and Meier, B.H. (2010). Structural Similarity between the Prion Domain of HET-s and a Homologue the homologous protein CsgB. In order to understand the Can Explain Amyloid Cross-Seeding in Spite of Limited Sequence Identity. Journal of features responsible for the different functionalities of CsgA Molecular Biology 402(2): 311-325. and CsgB we analyse the structures, aggregation kinetics Greenwald, J., Buhtz, C., Ritter, C., Kwiatkowski, W., Choe, S., Maddelein, M.L., Ness, F., Cescau, S., Soragni, A., Leitz, D., Saupe, S.J., and Riek, R. (2010). The Mechanism of and thermodynamic stabilities of the fi brils formed by both prion inhibition by HET-S. Molecular Cell 38: 889-899. 74 SCIENTIFIC REPORTS | Infection and Immunity | Microbial Pathogenesis

01.6 Microtubule Dynamics and Bacterial Pathogenesis

PROJECT LEADER | Prof. Dr. Jürgen Wehland (†) | Research Group Cell Biology

PROJECT MEMBERS | Dr. Marco van Ham ([email protected]) | Dr. Andreas Fischer | Dr. Marcin Ura | Ramona Baier

Whereas the subversion of the actin system by pathogens and ataxia and died within 24 hours after birth due to an has been studied in great detail, much less is known about intricate combination of brain defects involving anomalies the contribution of the dynamic microtubule system in in neuronal proliferation/differentiation balance and in bacterial pathogenicity. Microtubules are essential for a defective control of neurite outgrowth and differentiation. variety of cellular functions such as cell division, intra- On the cellular level, our results indicate that the tubulin cellular organelle/vesicle transport and different forms tyrosination cycle regulates microtubule interactions with of cell motility. During the past few years we have been CAP-Gly domain-containing microtubule plus-end tracking focusing on a specifi c post-translational modifi cation of proteins, which provide explanations for the involvement of tubulin to study the contribution of microtubule sub-sets TTL in the establishment of cell polarity. to these various functions. A unique enzyme respon sible for tubulin-tyrosination, the tubulin-tyrosine-ligase (TTL), Conditional TTL knockout in mice Since the constitutive is well characterized due to substantial contributions from TTL knockout leads to perinatal death, the question arises our laboratory. The tyrosination cycle is highly conserved as to whether TTL defi ciency per turbs other physiological among eukaryotes. To test the physiological importance of activities outside the brain and in addition affects adult the tubulin tyrosination cycle, we recently generated TTL mice. To explore this, we recently have generated a condi- defi cient mice. Analysis of these TTL null mice showed se- tional TTL knockout mouse using the cre/lox system. Pre- vere disorganization of the brain, involving loss of cells and liminary data showed cell type specifi c deletion of the TTL abnormal neuronal extensions leading to perinatal death gene resulting in enriched levels of detyrosinated tubulin. demonstrating the vital role of this highly specifi c tubulin We are now in the phase of expanding TTL defi cient mouse modifi cation. lines that will be used to analyze the aforementioned brain phenotype in more detail. Furthermore, we will analyse the TTL null mice Constitutive TTL null mice were generated involvement of the TTL cycle in cells of different hematopoi- by conventional gene knockout and newborn TTL null mice etic lineages. were undistinguishable from their wild type littermates. However, TTL null mice displayed defective breathing Perspective We are aiming at a detailed understanding of the tubulin tyrosination cycle. This includes the elucidation of the function of specifi c microtubule-binding proteins (e.g. CLIPs) in the regulation of microtubule dynamics in the background of TTL defi ciency. This will be pursued both in vivo and in vitro by the means of conditional TTL knockout mice and using tissue culture systems focusing on cell adhesion and polarity, respectively. In addition, a central question of particular interest is in the cytoskeleton fi eld: how microtubules interact with the actin cytoskeleton and how does the communication between the two cytoskeletal systems work. Furthermore, of utmost interest are the ques- tions, (i) how bacterial and viral pathogens can exploit the microtubule system for their own advantage especially by usurping specifi c host cell pathways, and (ii) how pathogens interfere with the crosstalk between both the actin and microtubule system.

Schwan, C., Stecher, B., Tzivelekidis, T., van Ham, M., Rohde, M., Hardt, W.D., Wehland, J., Aktories, K. (2009) Clostridium diffi cile toxin CDT induces formation of microtubule- A migrating mouse embryonic fi broblast immunostained for the based protrusions and increases adherence of bacteria. PLoS Pathogens 5, e1000626. microtubule network (red) and actin-rich structures in the lamel- Erck, C., Peris, L., Andrieux, A., Meissirel, C., Gruber, A.D., Vernet, M., Schweitzer, A., lipodium (green). Photo: HZI Saoudi, Y., Pointu, H., Bosc, C., Salin, P.A., Job, D., Wehland, J. (2005) A vital role of tubulin-tyrosine-ligase for neuronal organization. Proceedings of the National Academy of Sciences USA 102, 7853-7858. SCIENTIFIC REPORTS | Infection and Immunity | Microbial Pathogenesis 75

01.7 Function and Regulation of Yersinia Virulence Factors

PROJECT LEADER | Prof. Dr. Petra Dersch | Department of Molecular Infection Biology | [email protected]

PROJECT MEMBERS | Katja Böhme | Katharina Herbst | Dr. AnnKathrin Heroven | Dr. Annika Kochhut | Wiebke Opitz | Dr. Fabio Pisano | Rebekka Steinmann | Tatjana Stolz | Tanja Thiermann | Frank Uliczka

Enteropathogenic Yersinia species cause a large range of studies and kinase activation assays, we could demonstrate different gut-associated diseases, including enteritis, colitis, that the protein kinase B (Akt), the phospholipase C-γ and mesenteric lymphadenitis which are commonly called yersi- variants of the protein kinase C are recruited to bacteria- niosis. In rare cases they can also induce certain autoimmu- bound membranes and are activated in a time-dependent ne diseases such as reactive arthritis. Yersiniae are widely manner. Application of inhibitors and use of knock-out cell distributed and contaminated porc is the main source of lines and RNA interference demonstrated that activation of infection for men. They express a set of special surface struc- these factors occurs after activation of the integrin-bound tures which allow them to bind and internalize into host cells. focal adhesion kinase (FAK), c-Src and the PI3 kinase and These outer membrane proteins (adhesins and invasins) are required for the bacterial entry process. Furthermo- promote effi cient attachment to cell receptors which results re, we obtained evidence that, besides the small GTPase in the induction of certain signal transduction pathways Rac-1, also other GTPases of the Rho family and different within the cell. Recruitment and activation of certain signal actin-associated proteins, such as N-WASP and the Arp2/3 molecules to bacteria bound cell receptors induce rearran- complex, are implicated in the uptake process. Furthermore, gements of the actin cytoskeleton and lead to the formation we characterized two newly identifi ed adhesins of Y. pseudo- of special membrane protrusions which migrate around the tuberculosis with high homology to the invasin protein, and bacteria and enclose it into a membrane-bound phagosome. found that both promote tight binding to human enterocytes. Cell invasion allows transcytosis of the bacteria through the First analyses in the mouse infection model revealed that gut epithelium into the underlying lymphatic tissues and loss of the adhesins prolonged the life time of infected mice deeper organs. We focus on the characterization of the func- signifi cantly. tion and expression of Yersinia invasion factors to gain more information about how enteric bacteria colonize host tissues Temperature-dependent expression of Yersinia viru- and become resistant against the host immune system. lence factors Another important goal is to understand the molecular control mechanism of Yersinia virulence genes Molecular analysis of the Yersinia-induced signal trans- during the infection process. Temperature is one of the most duction in epithelial cells In order to identify signalling pa- crucial factors sensed by the pathogens to adjust expression thways which are essential for the internalization of Yersinia of their virulence factors after entry from a cold external pseudotuberculosis, we studied the cell uptake promoted by environment into a warm-blooded host. We found that the the Yersinia invasion factors InvA and YadA which inter- regulator protein RovA controlling expression of the invasin act directly or indirectly via extracellular matrix proteins protein undergoes a reversible conformational change upon with β1-integrin receptors of the host cell. By colocalization a temperature shift from 30°C to 37°C. This reduces the DNA-binding affi nity of the regulator and renders it more susceptible for proteolysis by the bacterial protease Lon. In addition, other post-transcriptional regulatory mechanisms were analysed. These control systems are used to adjust virulence gene expression during the infection process to availability of nutrients and stress superimposed by the host immune system.

Herbst, K., Bujara, M., Heroven, A.K., Opitz, W., Weichert, M., Zimmermann, A., & Dersch, P. (2009) Intrinsic thermal sensing controls proteolysis of Yersinia virulence regulator RovA PLoS Pathogens 5(5):e1000435.

Uliczka, F., Kornprobst, T., Eitel, J., Schneider, D., & Dersch P. (2009) Cell invasion of Yersinia pseudotuberculosis by invasin and YadA requires protein kinase C, PLC-γ 1 and Adherent Y. enterocolitica on human epithelial cells. Akt kinase Cellular Microbiology 11, 1782–1801. Photo: HZI, Rohde Heroven, A.K., Böhme, K., Rohde, M., & Dersch, P. (2008) A Csr regulatory system, inclu- ding small non-coding RNAs, regulates the global virulence regulator RovA of Yersinia pseudotuberculosis through RovM Molecular Microbiology 68,1179-1195. 76 SCIENTIFIC REPORTS | Infection and Immunity | Microbial Pathogenesis

01.8 Structural Characterisation of Pathogen Defence Factors

PROJECT LEADER | Dr. Konrad Büssow | Research Group Recombinant Protein Expression | [email protected]

PROJECT MEMBERS | Sonja Wilke | Sarah Tokarski

Many different proteins are involved in the defence against pathogens. The elucidation of the three dimensional struc- ture of proteins via X-ray structural analysis provides im- portant information about the way in which these function. Structural investigations of many human proteins have not proved possible to date, due to the fact that they were un- able to be produced in a pure state in suffi cient quantities. This is the prerequisite in order for protein crystals to be cultivated and X-ray diffraction data recorded.

The majority of proteins for use in X-ray structural analysis are produced in bacteria. The bacteria are genetically modifi ed to enable them to produce the respective target protein in large quantities. This procedure is both rapid and economical. However, many human proteins involved in the defence against pathogens are unable to be produced in this manner. The necessary processing stages required to transfer these proteins to their biologically active form are not implemented.

Proteins from animal cell cultures Cultivated animal cells are an alternative to bacteria in the production of proteins for X-ray structural analysis. In the majority of cases insect Cloning of a stable GFP cell line through fl uorescence-activated cells are infected with genetically modifi ed baculoviruses. cell sorting Graphic: HZI However, mammalian cell lines are also highly useful, in particular in the case of proteins discharged from the cells in their natural environment that are found in extracellular ducing cell lines. RMCE uses site-directed recombination fl uid or on the exterior of cells. to replace a marker gene such as GFP with another gene of choice. This process takes just a few weeks to be completed. These discharged proteins, for example antibodies or We have combined the cloning of a GFP cell line via cell cytokines, are often stabilized via disulphide bridges and sorting with RMCE and have already been able to success- carry carbohydrate chains on their surface. The hamster cell fully demonstrate the cassette exchange in CHO-Lec cells. line CHO-Lec 3.2.8.1 is suitable for producing these for X-ray structural analysis purposes. Mutations here mean that the We have established production cell lines for a number carbohydrate chains are small and uniform, with the conse- of lysosomal associated membrane proteins (LAMP) with quence that the proteins produced are easily crystallized. this technique. These cell lines secret engineered LAMP proteins that lack the membrane spanning region. LAMPs Accelerated cell line generation A disadvantage here is play an important role in the lysosomes and are required the long time, approximately one year, that is required for for elimination of pathogens by phagocytosis. A number of the creation of a genetically modifi ed CHO-Lec cell line using LAMPs were produced in larger amounts and crystallized. standard methods. Introducing a new procedure we have The spatial structure of a LAMP was determined for the now been able to signifi cantly reduce this time. This pro- fi rst time. However, the quality of the structure still has to cedure is based on a fl uorescent reporter gene, GFP, which be improved for a detailed understanding of the molecular enables the cloning of genetically modifi ed CHO-Lec cells details of these important proteins. with particularly good production properties with fl uores- cence activated cell sorting (FACS).

Wilke,S., Krausze,J., Gossen,M., Gröbe,L., Jager,V., Gherardi,E., van den,H.J., & Bussow,K. Recombinase-mediated cassette exchange technology (2010) Glycoprotein production for structure analysis with stable, glycosylation mutant CHO cell lines established by fl uorescence-activated cell sorting. Protein Science 19, (RMCE) enables a further acceleration of the process of pro- 1264-1271. SCIENTIFIC REPORTS | Infection and Immunity | Microbial Pathogenesis 77

01.9 Mycobacterial Phagosomes and Immunity

PROJECT LEADER | Dr. Maximiliano G. Gutierrez | Junior Research Group Phagosome Biology | [email protected]

PROJECT MEMBERS | Achim Gronow | Bahram Kasmapour | Gang Pei | Dr. Cristina Vazquez

M. tuberculosis owes its success to its ability to survive Rab GTPases and phagosome maturation For Rab34, we within macrophage phagosomes. M. tuberculosis blocks have performed the in-deep characterization of a polyclonal phagosome maturation, specifi cally the stage of phagosome- antibody we generated and determined that the main loca- lysosome fusion, and as a consequence grows within macro- lization of Rab34 in macrophages is the Golgi complex. We phages, contributing to disease. The mechanisms whereby also found, using live cell imaging, western blot of purifi ed M. tuberculosis arrests the phagosome maturation are still phagosomes and electron microscopy, that Rab34 is asso- elusive. The machinery by which mycobacteria manipulate ciated to phagosomes transiently. As this rab protein likely intracellular defenses is likely multifactorial and dynamic. has a role in phago-lysosomal fusion, we tested the effect Many intracellular targets have been invoked to explain the of Rab34 mutants in the acquisition of lysosomal markers block in bactericidal performance of the infected macrophage by phagosomes. Our data indicate that expression of the such as rab proteins. From these studies, it becomes clear constitutively active mutant increased the fusion between that interference of intracellular traffi cking is a key event in phagosomes and lysosomes. the survival strategy of this pathogen. In the case of Rab20, we cloned Rab20 in pEGFP-C1 and ge- We have already shown that NF-kB activation is an event nerated a negative mutant (unable to bind GTP) for studies in required for the killing of intracellular mycobacteria by macrophages. Preliminary studies revealed that Rab20 was macrophages (Gutierrez et al., 2009). More important, block associated to the Golgi and large vesicular structures that of NF-kB activation leads to inhibition of mycobacterial remain to be characterized. Our preliminary data point out phagosomes fusion with lysosomes. In this study, a whole for a role during macropinosome formation. Interestingly, we genome microarray analysis also identifi ed some interesting also have found associated to latex bead phagosomes during candidates on which the projects in our laboratory are based. early times of internalization. In the context of mycobacterial infection, Rab20-GFP was heavily recruited into mycobacteri- The most promising candidates are Rab34, Rab20 and sorti- al phagosomes (Figure 1). We are currently testing whether lin. Since the role of these proteins in phagosome maturation this recruitment could have an impact in phagosome matu- is not known, the fi rst part of the project consisted in the ration. characterization and elucidation of protein function in the context of phagosome maturation. This knowledge will allow Sorting of lysosomal enzymes to phagosomes We found us to understand the intracellular behavior of these proteins that sortilin mediates the direct traffi cking of selected during mycobacterial infection of macrophages. lysosomal enzymes from Golgi to the latex bead phagosome. This represents a novel pathway by which selected proteins are transported to the phagosome bypassing lysosomes during maturation of phagosomes (Wähe et al., 2010). We plan to expand these studies to mycobacterial traffi cking and killing using a sortilin KO mouse (in collaboration with Dr. A. Nykjaer, Aarhus, Denmark).We are also investigating the role of sortilin in mycobacterial phagosome maturation using different mutants of sortilin that are not able to signal for adaptor proteins, endocytosis or retromer interaction.

Fabrino, D. L., Bleck, C. K., Anes, E., Hasilik, A., Melo, R. C., Niederweis, M., Griffi ths, G., & Gutierrez, M. G. (2009) Porins facilitate nitric oxide-mediated killing of mycobacteria. Microbes and Infection 11, 868-875.

Gutierrez, M. G. & Griffi ths, G. (2009) Phagosome-cytoskeleton interactions. In: Intracel- Fig. 1. RAW 264.7 macrophage expressing Rab20-GFP (green) lular Niches of Pathogens A Pathogens Guide through the Host Cell, (Schaible, U.; Haas, A.) Wiley-VCH, Weinheim, Germany. and heavily infected with mycobacteria (red). Some of the bac- teria are localized in large vacuoles positive for Rab20-GFP. Wähe, A., Kasmapour, B. , Schmaderer, C., Liebl, D., Sandhoff, K., Nykjaer, A., Griffi ths, G., & Gutierrez, M. G. (2010) Sortilin mediates the transport of Acid Sphingomyelinase Photo: HZI, Gang Pei and Prosaposin from the Golgi to phagosomes. Journal of Cell Science 123, 2502-11 (Cover). 78 SCIENTIFIC REPORTS | Infection and Immunity | Microbial Pathogenesis

01.10 Molecular Mechanisms of Host-cell Pathogen Interactions

PROJECT LEADER | Prof. Dr. Klemens Rottner | Former Research Group Cytoskeleton Dynamics | [email protected]

PROJECT MEMBERS | Dr. Jennifer Block | Dr. Stefan Köstler | Markus Ladwein | Margit Oelkers | Dr. Malgorzata Szczodrak

This project aims at characterising the precise molecular mechanisms driving actin reorganisation during different types of motility processes and the interaction of different pathogens with their hosts.

Actin polymerisation is catalysed by protein complexes enhancing actin fi lament nucleation, such as the Arp2/3- complex or formins. Prominent important actin regula- tors include activators of Arp2/3-complex, e.g. WASP and WAVE family members, which can operate downstream of the Rho-GTPases Cdc42 and Rac1. Novel members of this Arp2/3-complex activators include proteins of the WHAMM/JMY subfamily and WASH (Rottner et al., 2010), the latter of which we could recently identify to contribute to Salmonella invasion (Hänisch et al., 2010).

Another prominent Arp2/3-complex binding protein that Fig. 1. Cortactin is essential for InlB-mediated Listeria invasion. had previously been implicated in dynamic actin reorgani- Quantifi cation of Listeria entry in the presence (Control) and zation events such as lamellipodium protrusion or differ- absence (Cortactin KO) of cortactin, as indicated. Listeria ent types of host-pathogen interaction is the Src-substrate genetically defi cient for both Internalin (A) and InlB (grey) were cortactin. We have generated cells and mice harbouring a used as non-specifi c invasion control. Graphic: HZI conditional mutation of the cortactin gene. To our surprise, genetic removal of cortactin in fi broblasts did not abol- ish Arp2/3-complex activation and actin polymerization stimulated by constitutively active Rac1. However, cortac- Finally, in more recent efforts, we explored the role of tin deletion abrogated signal transduction to activation of cortactin in different types of host-pathogen interaction. Rac or Cdc42 small GTPases, for instance downstream of Interestingly, cortactin appears essential for InlB-mediated growth factor activation, thus affecting migration and actin Listeria invasion (Fig. 1), but neither for entry triggered by reorganization more indirectly than previously thought (Lai Shigella fl exneri nor for actin pedestal formation induced et al., 2009). at the cell surface by different types of pathogenic E. coli (Oelkers/Lai et al., unpublished). These results are consistent with more recent data obtained by employing a novel actin assembly assay that we could recently develop. We demonstrated that cortactin is incapa- ble of driving Arp2/3-complex-dependent actin assembly at ectopic sites in living cytoplasm (Oelkers et al., submit- ted). They also confi rmed our previous data on the relative turnover of cortactin and Arp2/3-complex in the lamellipo- Rottner, K., Hänisch, J., & Campellone, K. (2010) WASH, WHAMM and JMY: Regulation dium, which had already indicated that the majority of the of Arp2/3 complex and beyond. Trends in Cell Biology 20(11), 650-61. cortactin molecules associating with the structure cannot Hänisch, J., Ehinger, J., Ladwein, M., Rohde, M., Derivery, E., Bosse, T., Steffen, A., Bumann, D., Misselwitz, B., Hardt, W.-D., Gautreau, A., Stradal, T.E.B., & Rottner, K. engage in Arp2/3-complex activation at the lamellipodium (2010) Molecular dissection of Salmonella-induced membrane ruffl ing versus invasion. tip (Lai et al., 2008). Cellular Microbiology 12(1), 84-98. Lai, F.P.L., Szczodrak, M., Oelkers, J.M., Ladwein, M., Acconcia, F., Benesch, S., Auinger, S., Faix, J., Small, J.V., Polo, S., Stradal, T.E.B., & Rottner, K. (2009) Cortactin promotes migration and platelet-derived growth factor-induced actin reorganization by signaling to Rho-GTPases. Molecular Biology of the Cell 20(14), 3209-23.

Lai, F.P.L., Szczodrak, M., Block, J., Faix, J., Breitsprecher, D., Mannherz, H.G., Stradal, T.E.B., Dunn, G.A., Small, J.V., & Rottner, K. (2008) Arp2/3-complex interactions and actin network turnover in lamellipodia. EMBO Journal 27(7), 982-92. SCIENTIFIC REPORTS | Infection and Immunity | Microbial Pathogenesis 79

01.11 Signalling to Actin Dynamics

PROJECT LEADER | Prof. Dr. Theresia E.B. Stradal | Former Research Group Signalling and Motility | [email protected]

PROJECT MEMBERS | Stefan Arens | Jan Hänisch | Kathrin Schloen | Kai Schlüter

The manipulation of actin cytoskeletal turnover is a One example of a more direct manipulation of actin common theme in bacterial pathogenesis. Bacteria do this assembly constitutes pathogenic E. coli of the types EPEC to establish a niche for replication, to evade the immune (enteropathogenic E. coli) and EHEC (enterohaemorrhagic E. system by hiding inside non-phagocytic cells or by prohi- coli): Both of them fi rmly attach to the host cell surface and biting phagocytosis, to name only some. Manipulation of induce actin polymerisation directly beneath the bacteri- actin dynamics is achieved directly by modifying actin, or um. EPEC induce actin assembly by mimicking receptor indirectly via regulators controlling actin dynamics such as tyrosine kinase signalling, a mechanism that is also used members of the Rho family of GTPases. This preference can by pox viruses in a strikingly similar fashion [3]. EHEC- be explained by the crucial function of Rho-protein regula- induced-actin assembly requires translocation of two bacte- ted and actin dependent processes for innate and adaptive rial proteins: the translocated intimin receptor Tir, which immunity. Rho-GTPases serve as molecular switches that serves as attachment anchor traversing the host cell plasma

- while cycling - turn actin remodelling on and off. Bacterial membrane, and EspFU /TccP, activator of N-WASP, and thus cytotoxins affect all stages of this cycle, thereby activating the host cell actin polymerization machinery is essential for

or inactivating Rho GTPases. For instance, reversible this process. Since Tir does not directly bind to EspFU , ad- shut-down comes from bacterial effectors with GAP-like ditional factors must bridge these components. In order to activities (e.g. SptP from Salmonella) and activation can elucidate this pathway, we analysed the composition of Es- be mediated by factors with GEF-activity (e.g. SopE from pFu-based protein complexes using mass spectrometry. We Salmonella; [1]). A novel family of bacterial virulence could identify host cell IRSp53 as direct interactor of both

factors termed WxxxE-family exerts bacterial GEF-activity, Tir and EspFU. Moreover, IRSp53 co-localized with EspFU in similar to SopE, with which they also share structural actin pedestals, and loss of IRSp53-function abrogated actin similarity. We were recently able to pinpoint residues assembly into pedestals. Thus, we identifi ed IRSp53 as the

of the interaction surface between the Shigella fl exneri missing factor linking bacterial Tir and EspFU [4]. Current WxxxE-factor IpgB2 and human RhoA that are crucial for work is focussed on elucidating the precise molecular bacterial GEF activity [2]. We currently identify interac- interactions between host cell and bacterial factors driving tion networks around bacterial and cellular GEFs, GAPs pedestal formation, which will also include analyses of the and their regulated GTPases. binding surfaces at atomic resolution (collaboration SB).

Murine embryonic fi broblasts expressing a constitutively active mutant of human RhoA, wildtype IpgB2 or its attenuated mutant Q116A (adapted from Klink et al.,2010). Note that the virulence factor IpgB2 phenotypically induces RhoA activity . Photos: HZI

Hänisch, J., Ehinger, J., Ladwein, M., Rohde, M., Derivery, E., Bosse, T., Steffen, A., Bumann, D., Misselwitz, B., Hardt, W.-D., Gautreau, A., Stradal, T.E.B., & Rottner, K. (2010) Molecular dissection of Salmonella-induced membrane ruffl ing versus invasion. Cellular Microbiology 12(1), 84-98.

Klink,B.U., Barden,S., Heidler,T.V., Borchers,C., Ladwein,M., Stradal,T.B.*., Rottner,K., & Heinz,D.W.*. (2010) Structure of Shigella IpgB2 in complex with human RhoA: implicati- ons for the mechanism of bacterial guanine nucleotide exchange factor mimicry. Journal of Biological Chemistry 285, 17197-17208.

Rottner,K. & Stradal,T.E.*. (2009) Poxviruses taking a ride on actin: new users of known hardware. Cell Host and Microbe 6, 497-499. 80 SCIENTIFIC REPORTS | Infection and Immunity | Microbial Pathogenesis

01.12 Generation and Exploitation of DNA Sequence Data

PROJECT LEADER | Dr. Helmut Blöcker | Department of Genome Analysis | [email protected]

PROJECT MEMBERS | Cyril Alozieuwa | Dr. Sabin Bhuju | Dr. Igor Deyneko | Ayssar Elamin | Michael Jarek | Yulia Kalybaeva | Dr. Gabriele Nordsiek | Ibrahim Sabra | Maren Scharfe | Prof. Dr. Mahavir Singh | Dr. Matthias Stehr | Hanaa Wanas

Sequence analysis projects Sequencing and analysis of primary DNA is one of the principal basic techniques in modern biological research. Our work includes the compara- tive sequence analysis of clinical isolates from pathogenic organisms such as M. tuberculosis with special emphasis on genes involved in virulence, persistence, antibiotic resist- ance and host preference.

We analysed selected regions from the horse, pig and cattle genomes, most of which are suspected to be disease-related. Deep annotation of a number of bacterial genomes is com- Fig. 1. Distribution of DNA clusters to be sequenced. About two plete. Recently, we sequenced three more of the big myxo- years of technology development increased the yield of mega- bacterial genomes. bases per sequencer to 1,500 percent. Photos: HZI

The implementation of two so-called next generation DNA sequencers (Illumina/Solexa) enables us to considerably expand the genomic analyses, quantitatively as well as quali- 100 new candidate proteins that are currently evaluated. tatively (Fig. 1.). Accordingly, several expression analyses The enzyme Antigen85A synthesizes the most abundant gly- have been run on sequencers with samples from cow, badger, colipid in the cell wall, TDM (trehalose 6,6’-Dimycolate; Cord mouse and man. To allow for RNAi screening with complex factor). Antigen85A is an important component in the cell libraries, especially negative selection screening, we estab- wall but can also be used as a vaccine. We have produced lished deep sequencing and successfully analysed a rather the recombinant protein for a detailed study of the immuno- large amount of samples. A further focus was on ChIP-Seq logical properties of the enzyme. We were fi rst to report the which is about interaction of proteins (for example transcrip- development of a non-radioactive assay, which was used to tion factors) with DNA. We mostly investigated disease-rele- determine the enzyme´s catalytic parameters. The new as- vant human samples. say is also suitable for high-throughput experiments and for the screening of substance libraries in order to explore new Mycogenomics Our goal is the development of new methods lead structures. for tuberculosis diagnosis and treatment. The focus is primarily on the identifi cation of clinical multiple-resistant Novel bioinformatics technology We are exploring applica- Mycobacterium tuberculosis strains and on the characteriza- tions of signal theory for the function-oriented analysis of tion of virulence-associated proteins. biomolecules. Our intent is to reveal similarities, homologies and analogies based on considerations of their physico-chem- Tuberculosis kills approximately two million human beings ical properties and to confi rm these fi ndings by wet lab data. worldwide per year. A big problem is the growing issues of The software “FeatureScan” is publicly available (http://ge- multiple-resistant bacteria (MDR) or extremely resistant patho- nome.helmholtz-hzi/featurescan). Our system is able to spot gens (XDR) which can only be treated with so called Second- property-dependent similarities where letter code-based sys- Line Drugs. We develop new diagnostic tools for MDR-TB. tems (A, C, T, G) fail. Thus, comparing promoters from man and chimpanzee, our novel technology revealed functional In the EU project Fast XDR-Detect we develop a colour-based regions which otherwise would have been undiscovered. assay for a 96-well plate format to detect MDR tuberculosis. For the development of this assay, the genomes of numerous clinical Mycobacterium tuberculosis strains were sequenced to identify resistance-associated mutations (Fig. 2, see page Deyneko,I.V., Kalybaeva,Y.M., Kel,A.E., & Blöcker,H. (2010) Human-chimpanzee promo- 68). The identifi ed mutations are used as molecular markers ter comparisons: Property-conserved evolution? Genomics 96, 129-133. for the assay. Von Groll A., Martin A., Stehr M., Singh M., Portaels F., da Silva P.E. & Palomino JC. (2010) Fitness of Mycobacterium tuberculosis strains of the W-Beijing and Non-W- Beijing genotype. PLoS One 5, e10191.

In the EU project FASTEST-TB we are looking for new my- Adhikary,T., Kaddatz,K., Finkernagel,F., Schonbauer,A., Meissner,W., Scharfe,M., cobacterial antigens (diagnostic protein markers) for rapid Jarek,M., Blöcker,H., Muller-Brusselbach,S., & Müller,R. (2011) Genomewide Analyses Defi ne Different Modes of Transcriptional Regulation by Peroxisome Proliferator-Activa- tests in a 96-well plate format. So far, we have found about ted Receptor-beta/delta (PPARbeta/delta). PLoS ONE 6, e16344. SCIENTIFIC REPORTS | Infection and Immunity | Host Pathogen Interactions 81

02 Host Pathogen Interactions

TOPIC SPEAKER | Prof. Dr. Klaus Schughart | Department of Infection Genetics | [email protected]

The severity of an infectious disease is determined by the intricate interactions between pathogen and host. Many studies have been performed to identify specifi c virulence factors in individual pathogenic microbe species but our knowledge about important factors in the host is still very limited. For example, genetic and environmental factors are crucially involved in the host response and determine favorable or unfavorable disease outcomes. Also very little is known about bacterial communities in the host and how they may infl uence the activities of pathogenic microbes or the host response. Furthermore, pathogens that are able to cross the species barrier represent a constant threat to the human population. Here, not much is known about the molecular mecha- nisms of host adaptation.

Therefore, the objective of this topic is to obtain a better understanding about the complexity of the interactions between the host and and infectious pathogens. The different research projects aim to identify host factors which infl uence its susceptibility and resistance to infections, to unravel mechanisms of cross-species transmission, and to characterize microbial communities and how they may modulate the host response to pathogens. To achieve these goals, the topic will perform research projects in the following areas.

Microbial communities in the host and their relevance for infectious diseases In the oral cavity approximately 500 bacterial species have been identifi ed so far. Many of these bacteria are able to form biofi l m s , so called dental plaque. In order to form biofi lms, bacterial species must communicate with each other. We are, therefore, studying the molecular basis of bacterial communication pathways and investigate possible ways to disrupt them.

In cystic fi brosis (CF) patients, Pseudomonas aeruginosa is the most dominant bacterial pathogen causing chronic lung disease. However, P. aeruginosa isolates from patients revealed that they do not represent a single variant but rather different phenotypes. And the presence of different phenotypes has been correlated with poor prognosis in CF patients. To better understand the under- lying biological and molecular mechanisms of phenotype changes, we will study the diversity of bacterial communities and isolates in CF patients.

Biofi lms play an important role in the establishment of pathogenic bacterial colonistaion in the host. Very little is known so far about pathogenic communities in biofi lms and the relationship between the biodiversity and pathogenicity in presumed sterile environments of the host. We are thus determining the biodiversity in biofi lms from patient material like pacemakers, bone and dental implants. With the knowledge which bacterial communities live at the different niches in the human body new approaches to control or prevent biofi lms and to optimize their biodiversity with regards to the control of pathogenic bacteria are being developed.

The nasal cavity and the gastro-intestinal tract harbor a huge number of bacterial organisms which may be advantageous or, in some cases, pathogenic to the host. We are, therefore, aiming to defi ne the conditions which favor or exclude the colonization with pathogenic bacteria. A special focus is the colonization of the nose with multi-resistant Staphylococcus aureus and its interaction with other community members. 82 SCIENTIFIC REPORTS | Infection and Immunity | Host Pathogen Interactives

The host response to viral and bacterial pathogens in experimental models of infection Group A streptococci are prevalent human pathogens capable of causing a variety of diseases ranging from simple pharyngitis to very severe infections, such as necrotizing fascitiis and streptococcal toxic shock syndrome. We have previously shown that strains of mice from various genetic backgrounds differ markedly in their susceptibility to Streptococcus pyogenes. Using these resistant and susceptible mouse strains, we will identify the immunological and molecular mechanisms controlling the resistance or susceptibility to S. pyogenes and design new strategies to enhance resistance in susceptible hosts. Staphylococcus aureus is one of the most important causes of life-threatening bacterial infections in Western countries.

The incidence of S. aureus infections has dramatically increased in the last decade, occurring in both, community-acquired and hospital-acquired cases. Despite the substantial progress made in understanding the mechanism employed by S. aureus to persist within the host, few or none studies have addressed the contribution of host factors to the establishment of S. aureus infection. Research in this area has been hampered by the lack of feasible animal models that recapitulate the different aspects of S. aureus infection. We have established an experimental mouse model that sustains a long-term S. aureus infection and, therefore, enables us to dissect the mechanisms underlying the divergent functionality of the host immune response during the different phases of infection.

Infl uenza A virus represents one of the most important threats to human health and economics. Thus far, very little is known about host resistance or susceptibility to infl uenza infections. At the HZI, we have established an infection model for infl uenza A H1N1 virus in the mouse and identifi ed signifi cant strain differences in the LD50 response. We perform comparative studies on the patho- genicity, gene expression patterns in the lung, viral load, and immune response in infected mice from different strains. This infor- mation will be used to map quantitative trait loci (QTL) that contribute to infection susceptibility. Potential candidate genes will then be tested by phenotyping combinations of single gene mouse mutants (knock-out, gene trap, classical or ENU-induced) in various genetic backgrounds.

Molecular specifi city of interspecies and zoonotic pathogen transmissions The mammalian prion protein, PrPC, is able to switch its conformation from a monomeric soluble form into an aggregated trans- missible prion state, PrPSc. Once a prion is introduced into a susceptible host, it triggers a PrP conversion cascade which leads to prion disease. Individual prion strains may lead to distinct prion disease pathologies in one host species. Interspecies transmission will be strongly infl uenced by the extent to which a particular PrPSc conformation is compatible with the primary PrP amino acid sequences of both species. The zoonotic transmission of BSE to humans, and the resistance of humans towards sheep scrapie are well known examples of this process. However, the underlying molecular mechanism of conformational conversion and prion propagation is not at all understood. A major obstacle in the research on prions was the limited availability of samples for prion 3D structure investigations. At the HZI we have established a novel technology to overcome this bottleneck. In particular NMR spectro scopic techniques and other biophysical techniques are used to unravel the structural origin of prion transmission barriers SCIENTIFIC REPORTS | Infection and Immunity | Host Pathogen Interactives 83

and prion adaptations to new hosts. The 3D structures of mammalian prion associated PrP will be revealed and should provide detailed insight into biophysical mechanisms of the PrPC to PrPSc conformation conversion.On a broader scope, we have a general interest in the molecular structural investigation of specifi c host:pathogen protein interactions that modulate the interspecies or zoonotic transmissibility of viruses and bacteria.

Epidemiological studies in humans to determine environmental and genetic risk factors The causes of human diseases are multi-factorial and consist of environmental, life style, and genetic risk factors. To better assess these factors, we will study the incidence of infectious diseases in humans and determine their genetic and environmental risk factors. To this end, the HZI and other centres of the Helmholtz Association as well as many university partners have initiated a nationwide population-based prospective cohort study, the National (Helmholtz) Cohort. This cohort will contain about 200,000 healthy volunteers from all over Germany. We will administer a questionnaire on acute and chronic infections, vaccination status, and exposure to pet and farm animals. In addition, swabs from the nasal cavity, saliva and feces will be collected and analysed for their microbial communities. The participants of the cohort will be followed for 10-20 years, and we will seek to determine the risk factors which contribute to infectious diseases and to secondary diseases that may be directly or indirectly associated with infections. 84 SCIENTIFIC REPORTS | Infection and Immunity | Host Pathogen Interactions

02.1 Pathogenesis of Chronic Pseudomonas aeruginosa Infections

PROJECT LEADER | Prof. Dr. Susanne Häußler | Research Group Chronic Pseudomonas Infections | [email protected]

PROJECT MEMBERS | Dr. Andreas Dötsch | Dr. Mathias Müsken | Juliane Schmidt | Sebastian Schulz | Dr. Piotr Bielecki

The diagnostic and therapeutic strategies that have served Cooperation is supported by communication Apart from us well in the treatment of acute bacterial diseases have two homoserine lactones, P. aeruginosa produces a third not yielded favourable outcomes when applied to chronic intercellular signal that is referred to as the Pseudomonas infections where bacteria grow in matrix-enclosed sessile quinolone signal (PQS). It is involved in cell density- biofi lm communities. Although every single cell is able to dependent virulence factor regulation – also known as induce a stress response with a characteristic change in quorum sensing (QS) – and the establishment of biofi lms. the gene expression pattern, living in populations provi- However, the molecular mechanism underlying down- des a species with additional mechanisms of survival, the stream PQS signalling is largely unknown. PqsE, encoded most obvious being heterogeneity and cooperation. on the last gene of the PQS biosynthetic operon, seems to play a key role in the translation of the presence of the Diversity facilitates survival Pseudomonas aeruginosa is PQS signal and bacterial behaviour at the single cell level. the most dominant bacterial pathogen causing chronic The elucidation of the function of this important enzyme is lung infection in cystic fi brosis (CF) patients. Although a major focus of the group. most patients are colonized with only one or few P. aeru- ginosa clones, the isolation of various morphotypes is a very characteristic microbiological fi nding. This diversity seems to play a key role in the persistence of chronic lung infections. Our research focuses on the elucidation of the molecular mechanisms responsible for this diversity and the characterization of particularly well adapted P. aerugi- nosa biofi lm phenotypes.

Small Colony Variants We have previously demonstra- ted that an adherent subgroup of so-called small colony variants (SCVs) is selected in the CF lung. The switch to an autoaggregative biofi lm-forming SCV phenotype of P. aeruginosa seems to be linked to the expression of the “Chaperone Usher Pathway” (CupA) gene cluster. This gene Susanne Häußler preparing for a new experiment in the lab. cluster encodes for bacterial fi mbriae and is regulated via Photo: Twincore/HZI the modulation of the newly identifi ed bacterial signal molecule, cyclic di-GMP, in an antagonizing way. With the aim to identify those mutations that are responsible for the switch to a biofi lm forming P. aeruginosa phenotype as well as the acquisition of antibiotic resistances, we will apply a whole genome sequencing approach. Therefore, the genomes of pools of clinical P. aeruginosa strains will be sequenced using next generation sequencing technologies Pommerenke C, Müsken M, Becker T, Dötsch A, Klawonn F, Häussler S. (2010). Genotype- and we will search for consistent nucleotide exchanges Phenotype correlation in Pseudomonas aeruginosa. PLoS Pathogens. 6(8): e1001074.

in sets of strains which express a similar phenotype. The Müsken M, Di Fiore S, Römling U, Häussler S. (2010) 96-well plate based optical method for next step will be to verify whether the respective mutation the quantitative and qualitative evaluation of Pseudomonas aeruginosa biofi lm formation and its application for susceptibility testing. Nature Protocols 5(8):1460-9. Epub 2010 Jul 29. is responsible for the biofi lm or the resistance phenotype. Dötsch, A., F. Klawonn, M. Jarek, M. Scharfe, H. Blöcker, S. Häussler. (2010). Evolutionary The knowledge about the genotypes that are specifi cally conservation of essential and highly expressed genes in Pseudomonas aeruginosa. BMC selected at different stages of the disease should signifi - Genomics. 11(1):234. cantly advance our knowledge on the evolution and Müsken M, Di Fiore S, Dötsch A, Fischer R, Häussler S. (2010) Genetic determinants of Pseudomnoas aeruginosa Biofi lm establishment. Microbiology. 156:431-41 adaptation mechanisms of P. aeruginosa to its habitat. It Häussler S. (2010) Multicellular signalling and grwoth of Pseudomonas aeruginosa. Interna- might aid in the development of new antimicrobial treat- tional Journal of Medical Microbiology. 300(8):544-8. ment regimes for the clinical management of chronically Häussler S, Parsek MR. Biofi lms 2009: (2010) new perspectives at the heart of surface- infected CF patients. associated microbial communities. Journal of Bacteriology. 192(12):2941-9.

SCIENTIFIC REPORTS | Infection and Immunity | Host Pathogen Interactions 85

02.2 Unravelling Mechanisms of Host Defence against Gram-positive Pathogens in the Mouse Model

PROJECT LEADER | Priv.-Doz. Dr. Eva Medina | Research Group Infection Immunology | [email protected]

PROJECT MEMBERS | Dr. Oliver Goldmann | Dr. Jens Abel | Christina Ziegler | Daniela Bruhn | Alva Rosendahl

Staphylococcus aureus and Streptococcus pyogenes are major human pathogens. The focus of the Infection Immunology Research group is to unravel the immune mechanisms involved in host defense against these pathogens.

The mouse model of genetic predisposition to severe Staphylococcus aureus infection A prominent feature of S. aureus infections is their inter-individual phenotypic va- riability, ranging from mild to lethal clinical manifestations. Common genetically determined variations in the immune system may contribute to the observed heterogeneity in the host response to this pathogen. We have examined the Bacterial loads in resistant C57BL/6 (mouse 2) and suscepti- immune mechanisms underlying the host genetic predis- ble A/J mice (mouse 3) infected for 24 h with the biolumines- position to severe S. aureus infection in the murine system. cent S. aureus strain SH1000 ALC2906. Uninfected C57BL/6 Our results confi rmed a signifi cant inter-strain variation in (mouse 1) and A/J (mouse 4) are included for comparison. resistance to this pathogen. Thus, whereas C57BL/6 mice were the most resistant and survived infection, A/J, DBA/2 and BALB/c mice were highly susceptible and succumbed (macrophages and neutrophils) to the infection site. Further- to infection shortly after bacterial inoculation. Susceptibi- more, MyD88-/- but not C57BL/6 mice exhibited a massive lity of mice to S. aureus was associated with an inability to infi ltration of eosinophils in infected organs (Figure 2) which limit bacterial growth in the kidneys and development of can be explained by an impaired production of the regula- pathology (Figure 1). tory chemokines, MIG/CXCL9 and IP-10/CXCL10, shown to be able to inhibit transmigration of eosinophils. Our results Depletion of neutrophils rendered resistant C57BL/6 mice indicate that MyD88 signalling targets effector cells to the completely susceptible to S. aureus indicating that neu- site of streptococcal infection and prevents extravasation trophils are essential for the observed resistance. We also of cells that can induce tissue damage. Therefore, MyD88 found that a delay in neutrophil recruitment to the site of signalling may be important for shaping the quality of the infection may underlie the increased susceptibility of A/J infl ammatory response elicited during infection to ensure mice to S. aureus. Therefore, the superior resistance of optimal effector-functions. C57BL/6 mice is likely to be the result of a fast recruitment of effector cells to the site of infection that bring straight away under control the infectious pathogen.

Role of TLR/MyD88 signalling in the pathophysiology of experimental Streptococcus pyogenes infection Streptococcus pyogenes represents one of the most frequent causes of mild infection of the upper respiratory tract and the skin with the potential to cause life-threatening invasive diseases such as necrotizing fasciitis and septicemia. Based H&E stained lung tissue sections obtained from S. pyogenes- on our previous in vitro studies, the myeloid differentiation infected MyD88-/- mice. Left, low magnifi cation photograph factor 88 (MyD88) was found to be involved in the response showing a massive cellular infi ltration. Right, a higher of innate immune cells to S. pyogenes. To determine the magnifi cation photograph showing that the infi ltrating cells relevance of these in vitro observations to the pathogenesis are eosinophils. of S. pyogenes in vivo, we have investigated the impact of MyD88 defi ciency on host resistance to S. pyogenes in a von Köckritz-Blickwede M., Rohde M., Oehmcke S., Miller L.S., Cheung A.L., Herwald H., mouse model of infection. Our results show that MyD88-/- Foster S., and Medina E. (2008). Immunological mechanisms underlying the genetic predis- position to severe Staphylococcus aureus infection in the mouse model. American Journal of mice harbored signifi cantly more bacteria in the organs and Pathology. 173:1657-68. succumbed to infection much earlier than MyD88+/+ animals. von Köckritz-Blickwede M., Konrad S., Foster S., Gessner J.E., and Medina E. (2009). Protec- Absence of MyD88 resulted in diminished production of in- tive role of complement C5a in an experimental model of Staphylococcus aureus bacteremia. Journal of Innate Immunity. 2:87 – 92. fl ammatory cytokines such as IL-12, IFN-γ, and TNF-α as well Loof T., Goldmann O., Gessner A., Herwald H., and Medina E. (2010). Aberrant Infl ammatory as chemoattractants such as MIP-2 and KC and hampered Response to Streptococcus pyogenes in Mice Lacking Myeloid Differentiation Factor 88. recruitment of effector cells involved in bacterial clearance American Journal of Pathology. 176: 754-63. 86 SCIENTIFIC REPORTS | Infection and Immunity | Host Pathogen Interactions

02.3 Microbial Communication

PROJECT LEADER | Prof. Dr. Irene Wagner-Döbler | Research Group Microbial Comunication | [email protected]

PROJECT MEMBERS | Dr. Helena Sztajer | Dr. Brigitte Kunze | Dr. Michael Reck | Andre Lemme | Xiaoli Xue | Ina Buchholz | Jürgen Tomasch | Thomas Riedel | Diana Patzelt | Szymon Safranski | Hui Wang

Bacteria synthesize signalling molecules, so-called auto- SDSF (Streptococcus diffusible signal factor). It suppresses inducers, which are excreted into the medium and control the formation of hyphae of the fungus, which are crucial important physiological traits in a concentration dependent for virulence and biofi lm formation. SDSF is structurally way. Cells sense autoinducer concentrations by specifi c related to farnesol, the quorum sensing signal of C. albicans, receptors in the membrane or within the cytoplasm. Above so this is a case of quorum sensing mimicry across huge a threshold concentration, the so-called quorum, the signal- phylogenetic distance, e.g. between the kingdom of bacteria ling molecules induce, for example, genetic competence and the kingdom of fungi. in the caries bacterium Streptococcus mutans, resulting in increased genetic exchange and biofi lm formation. There- Biofi lm inhibitors We have identifi ed small chemical fore, this type of cell-cell communication is termed quorum molecules which interfere with microbial communication. sensing. It plays an important role for infections and sym- Carolacton, which is produced by the soil bacterium Soran- bioses. Understanding the mechanisms of quorum sensing gium cellulosum, is particularly interesting because it has opens up new possibilities for infl uencing infections and no antibacterial or cytotoxic activity, but inhibits selectively developing alternatives for antibiotics. biofi lm formation of Streptococcus mutans. Carolacton causes membrane damage and cell death and thus disturbs Interkingdom signalling: The caries bacterium Strepto- biofi lm formation. Microarray investigations suggest that coccus mutans inhibits hyphae formation of the human several of the two component systems of S. mutans, which pathogenic fungus Candida albicans Streptococcus are its main sensory systems for environmental adaptation mutans lives in the polymicrobial biofi lm on our teeth and and quorum sensing, are impaired by carolacton. Details of is strongly associated with caries. Among hundreds of other this mechanism and possible applications of carolacton for species of bacteria also fungi occur in the oral cavity, e.g. caries prevention are currently studied in a systems biology the human pathogen Candida albicans, which can cause bad joint project. infections in immuno-compromised patients, after anti- biotic therapy or in the elderly. When studying signalling Quorum sensing in the Roseobacter group Bacteria of molecules of S. mutans we discovered a fatty acid compound, the Roseobacter group are important for biogeochemical cycles in the ocean, because they comprise up to 25 % of all bacterial cells in marine habitats. Dinoroseobacter shibae, one of the model organisms in the Roseobacter Transregio, lives in symbiosis with unicellular marine algae. D. shibae provides the algae with the essential vitamins B12 (coba- lamin) and B1 (thiamine), and receives sugar compounds for its heterotrophic metabolism in turn. Currently, we are studying the role of quorum sensing for this symbiosis, as well as the contribution of photosynthesis to the metabo- lism of the bacterium, which in the light gains energy by photophosphorylation, allowing it to burn less carbon and

thus “save” CO2.

Dinoroseobacter shibae on its host algae, the marine Xue, X., Tomasch, J., Sztajer, H., and I. Wagner-Döbler (2010). The delta subunit of RNA polymerase, RpoE, is a global modulator of Streptococcus mutans environmental adaptation. dinofl agellate Prorocentrum lima. The bacteria have been Journal of Bacteriology. 192:5081-92. specifi cally stained by CARD-FISH (catalyzed reporter Kunze B, Reck M, Dötsch A, Lemme A, Schummer D, Irschik H, Steinmetz H, Wagner-Döb- deposition fl uorescent in situ hybridisation). The remaining ler I. (2010) Damage of Streptococcus mutans biofi lms by carolacton, a secondary metabolite from the myxobacterium Sorangium cellulosum. BMC Microbiology. 10:199. bacterial community is invisible under these conditions. The Wagner-Döbler, I., Ballhausen B, Baumgart M, Brinkhoff T, Buchholz I, Bunk B, Cypionka orange spot appearing in the left dinofl agellate is the so- H, Daniel R, Drepper T, Gerdts G, Hahnke S, Han C, Jahn D, Kalhoefer D, Kiss H, Klenk H-P, Kyrpides N, Liebl W, Liesegang H, Meincke L, Pati A, Petersen J, Piekarski T, Pommerenke called pyrenoid, a region of the plastid which contains very C, Pradella S, Pukall R, Rabus R, Stackebrandt E, Thole S, Thompson L, Tielen P, Tomasch high concentrations of the rubisco enzyme. Photo: HZI J, von Jan M, Wanphrut N, Wichels A, Zech H, Simon M. (2010) The complete genome sequence of the algal symbiont Dinoroseobacter shibae – a hitchhiker´s guide to life in the sea. ISME Journal. 4(1):61-77. SCIENTIFIC REPORTS | Infection and Immunity | Host Pathogen Interactions 87

02.4 Systems Genetics of Infection and Immunity

PROJECT LEADER | Prof. Dr. Klaus Schughart | Department of Infection Genetics | [email protected]

PROJECT MEMBERS | Dr. Rudi Alberts | Dr. Mahmoud Bahgat | Leonie Dengler | Dr. Feng He | Dr. Bastian Hatesuer | Dr. Berenike Henneberg | Dr. Heike Kollmus | Dr. Tatiana Nedelko | Dr. Bastian Pasche | Dr. Claudia Pommerenke | Dr. Nuno Viegas | Dr. Esther Wilk | Silke Bergmann | Barkha Bhatnagar | Paulina Blazéjewska | Hairong Chen | Haiya Wu

Our research group is identifying genetic factors which contribute to the susceptibility or resistance of the host to infl uenza A infections. For these studies, we are using mouse genetic reference populations consisting of mouse fa- milies with different genetic backgrounds as well as mouse mutants which carry deletions in specifi c gene loci.

Every year, about 10,000 to 30,000 people die from infl u- enza infections in Germany. The most severe pandemic in recent human history, in 1918, has claimed about 30-50 Million deaths world-wide. The infl uenza virus is able to change its antigenic properties very rapidly. In addition, the viral genome is segmented and these segments can be ex- changed between different virus subtypes in other species. Such re-assorted viruses may then adapt to the human host and cause new pandemics. (A) Weight loss and late death in mice carrying a mutation in the Rag2 gene (A). The Rag2 knock-out mutation results in It has long been suspected that genetic factors may play an the absence of T and B cells of the adaptive immune response. important role for resistance or susceptibility to infl uenza in- Mice survive the early phase of infection because the innate fections in humans. However, such studies are very diffi cult immune system can control virus replication to a certain ex- to perform in humans and are often blurred by environmen- tent, but infected mice succumb later because the virus cannot tal factors, e.g. other serious health conditions, nutrition or be fi nally cleared from their lungs. (B) Histological sections medication. Therefore, we have established an experimental of mouse lung tissue infected with infl uenza A virus H1N1. model in which we infect mice with different mouse-adapted The brown staining detects the presence of the infl uenza A infl uenza A viruses. In particular, we are studying mouse virus Nuclear Protein in Rag2 mutant mice at 10 days after families derived from laboratory-inbred mouse strains, fami- infection whereas wild type mice (C57BL/6J) have cleared the lies in which two or more parental genomes are segregating virus (reproduced from Wu et al., 2010). and families in which parts of the genome were derived from wild mice and introduced into the genomic background of a laboratory strain. In addition, we are analysing the role of individual genes for the host defense to infl uenza infection the host-pathogen interactions at the molecular level and to by studying mouse lines in which a single gene locus has relate changes in gene expression with cellular responses of been mutated by genetic engineering. the immune system and pathologies in infected lungs. Our research activities are integrated into several national and So far, we demonstrated a very strong infl uence of the gene- international networks. We co-ordinate GeNeSys (German tic background on host susceptibility in mice. One mouse Network for Systems Genetics) and SYSGENET (European family, the DBA/2J inbred strain, was highly susceptible to network for systems genetics to understand human diseases), the PR8 H1N1 virus whereas another family, the C57BL/6J and we participate in FluResearchNet (German infl uenza inbred strain, was very resistant. In the susceptible mouse research network), Infrafrontier (European network to strain the infl uenza virus replicated much faster and the establish mouse phenotyping and archiving infrastructures), susceptible strain also exhibited a very strong innate im- CASIMIR (European network for mouse database integration) mune response. Our results suggest that the high viral load and the CTC (world-wide Complex Trait Consortium). in the lungs and the hyper-infl ammatory response of the immune system are responsible for the fatal outcome of the infection in the susceptible strain. In addition, we showed in P. Blazejewska, L. Koscinski, N. Viegas; D. Anhlan; S. Ludwig; K. Schughart, (2011). Patho- genicity of different PR8 infl uenza A virus variants in mice is determined by both viral and mouse mutants that the Rag2 and the Irf7 genes are impor- host factors. Virology Journal, 412(1):36-45 tant for the host to survive an infection with infl uenza virus. M. Bahgat, P. Błazejewska, and K. Schughart. Inhibition of lung serine proteases in mice: a new approach to control infl uenza infection. Virology Journal, 8:27 We are now performing genome-wide gene expression B. Srivastava, P. Blazejewska, M. Heßmann, D. Bruder, R. Geffers, S. Mauel, A. D. Gruber, K. Schughart. (2009) Host genetic background strongly infl uences the response to infl uenza A analyses over the course of an infection to better understand virus infections. PLoSONE, 4, e4857. 88 SCIENTIFIC REPORTS | Infection and Immunity | Host Pathogen Interactions

02.5 The Structural Basis of Mammalian Prion Trans- mission Barriers

PROJECT LEADER | Dr. Thorsten Lührs | Junior Research Group of Structure-Based Infection Biology | [email protected]

PROJECT MEMBERS | Dr. Felix Deluweit | Dr. Vandana Gupta

The mammalian prion protein, PrPC, is able to switch its con- Transmission barriers Between different mammalian spe- formation from a monomeric soluble form into an aggregated cies usually exists a so-called ‘species barrier’: mice do not transmissible prion state, PrPSc. Once a prion is introduced develop prion disease after challenge with hamster prions. into a susceptible host, it triggers a PrP conversion cascade, Transgenic mice that express additionally hamster PrP can which leads to prion disease (Fig. 1A). Interspecies transmis- be infected with hamster prions and then selectively incor- sion barriers have been observed to correlate with the amino porate hamster PrP into prion particles. The exclusive ex- acid sequence of PrP. On the other hand, multiple prion pression of a mouse/hamster chimeric PrP, termed MH2M, strains of the same PrP amino acid sequence have been makes mice susceptible to both hamster and mouse prions, identifi ed that cause characteristic pathologies in one host and the resulting prions transmit disease to both wild-type species. We aim to investigate prion 3D structures in order to hamsters and wild-type mice. Thus, the species barrier is in understand the transmission barrier between mice and ham- part determined by the primary amino acid sequence of the sters at atomic resolution, and to understand the structural prion protein, and the substitution of only a few amino acid basis of prion strains in relation to prion host range. There- residues can completely alter prion transmission patterns. fore, we have established a novel technology for the in vitro This notion has gained signifi cant importance because generation of specifi c aggregated prion conformations. We human variant Creutzfeld-Jacob Disease has been causally employ a combination of solution NMR, solid state NMR, and linked to the exposure to BSE prions of cattle. other biophysical or chemical techniques in order to obtain meaningful structural information of these aggregates. Prion sample preparation and structural analysis A key technique in achieving our research goals is the conversion of isotopically labelled (2H, 13C and 15N) recombinant PrP into PrPSc. In order to obtain homogeneous particle prepara- tions, we have invented a novel technique in 2010 (Fig.1B/C). Such aggregates are further characterized by asymmetric fl ow-fi eld-fl ow fractionation (AF4), which has recently been introduced into amyloid research. This chromatography-like technique is capable of separating particles by size without the need for a stationary matrix. At the same time the abso- lute hydrodynamic properties of individual particle fractions are determined by multi-angle and dynamic light scattering. Fig. 1: Progress on Prion in vitro Amplifi cation. (A) The This technique is also routinely used to characterise the benign cellular prion protein, PrPC, is an abundant oligomerisation status of other proteins. The core techniques mammalian protein, which may undergo conformational for the protein structure determinations are NMR in solution conversion and aggregation into infectious prions, PrPSc. (B) and in solids, but also other biophysical techniques like We have developed a novel method, that employs specifi c fl uorescence, FTIR and CD-spectroscopy. The strength of shear forces (left) for the effi cient in vitro amplifi cation this approach lies in its robustness towards subtle structural of prions. A device, the Shear Induced Fragmentation / heterogeneity of the investigated aggregates. Thus, with this Aggregation (ShIF/A) array, has been developed by our group a “consensus structure” can be obtained to provide essential to effi ciently optimize many conversion reaction in parallel. structural insights. (C) Using ShIF/A we can convert recombinant PrPC into patially proteinase K (PK) resistant PrPSc aggregates, using natural hamtser Sc237 prions as seeds (top). In unseeded reactions no PK resistance is observed (bottom).For optimal prion amplifi cation, the rotation frequency (Hz) of the rotor has to be controlled within narrow boundaries. SCIENTIFIC REPORTS | Infection and Immunity | Host Pathogen Interactions 89

02.6 Biofi lm Communities

PROJECT LEADER | Dr. Wolf-Rainer Abraham | Research Group Chemical Microbiology | [email protected]

PROJECT MEMBERS | Andreía B. Estrela | Ahmed S. Gebreil | Dr. Marcela G. Heck | Rolf Kramer | Dr. Heinrich Lünsdorf | Maira Peres de Cavalho | Esther Surges

This project understands microbial communities in biofi lms as functional units and pursues the goal of fi nding new ways to their control by investigation of the diversity of mi- crobes and their interactions. The focus is on the fact that in nature bacteria mostly live not as pure strains but in com- munities. It is of special interest how pathogenic bacteria live in their hosts and how they deal with non- or facultative pathogenic bacteria in biofi lms in the human body.

Formation of dental biofi lms One of the main complica- tions with implants in the hospital is the infection which is mostly accompanied by biofi lm formation on the implant. Such biofi lms are diffi cult to treat since the bacteria in biofi lms exhibit special protective mechanisms against anti- biotics and the immune system. Problems arise in particu- lar in the oral cavity which harbours by nature a multitude of different bacteria. We wanted to know which bacteria settle fi rst on implants in the oral cavity and, therefore, we examined two weeks old biofi lms of tooth implants from Biofi lm community recorded by confocal laser microscopy. different patients. We always found complex communities The cells were stained with Nile Red and slightly hydrophobic of bacteria which strongly varied from patient to patient. By bacteria cells (red) can be discerned from highly hydrophobic statistic analysis we could, however, show that there are ones (green) Photo: Ahmed Gebreil/ Maximilliano Gutierrez some characteristic species of bacteria which were found at certain sites of the implant again and again and thus are characteristic for these sites. Interestingly enough, we found at the implants of the same patient more frequently Natural products to control biofi lms The aim of all of Streptococci than on the natural teeth, however, no amass- these investigations is a better understanding of biofi lm ment of pathogenic bacteria was observed. communities with the goal of modulating them and of displacing pathogenic bacteria. We use well-known natural Metabolic activities in biofi lms The knowledge which substances but we also look for new ones in mushrooms. A bacteria in which biofi lms occur at which places is, how- fi rst investigation of fungal isolates revealed that many of ever, only the fi rst step in order to understand the complex them produce a number of quite diverse natural products phenomenon biofi lm. We must also examine the role of the which are able to dissolve biofi lms of pathogenic bacteria individual bacteria in the biofi lm community. One approach without, however, being antibiotic. These investigations are is the question about substrate use. In order to clarify how still at the beginning; however we are already able to state quickly and how strongly a substrate is incorporated by that mushrooms are a rich source for substances which can bacteria we use substrates which are labelled with stable modulate biofi lms. isotopes, thus are not radioactive, and follow their incor- poration into the individual types of bacteria. In this way, we can determine kinetics and model the fl ow of material in the community. Since we are not only interested in the interactions of the bacteria among themselves but also in host-bacteria interactions we also began to accomplish such studies in cell cultures and animal models. As the materials A. B. Estrela, M. G. Heck, W.-R. Abraham (2009) Novel approaches to control biofi lm infec- are not radioactive no special safety precautions are neces- tions. Curr. Med. Chem., 16, 1512-1530. sary. With such models we can show that the same bacteria A. B. Estrela, W.-R. Abraham (2010) Combining biofi lm controlling compounds and antibio- have different metabolic pathways in different organs, and tics as a promising new way to control biofi lm infections. Pharmaceuticals, 3, 1374-1393. in one organ e.g. glucose is directly taken up while in a W. Heuer, M. Stiesch, W. R. Abraham (2010) Microbial diversity of supra- and tumour bacteria nourish themselves preferentially of its subgingival biofi lms on freshly colonized titanium implant abutments in the human mouth. European Journal of Clinical and Microbiological Infection Diseases, metabolites. http://dx.doi.org/10.1007/s10096-010-1068-y 90 SCIENTIFIC REPORTS | Infection and Immunity | Host Pathogen Interactions

02.7 Metabolic Diversity

PROJECT LEADER | Prof. Dr. Dietmar Pieper | Research Group Microbial Interactions and Processes | [email protected]

PROJECT MEMBERS | Dr. Ramiro Vilchez | Dr. Melissa Wos | Macarena Marin | Daiana Morales | Amelia Silva

Microorganisms in their natural habitats typically live in Intestinal communities: The structure of the human gut complex communities where the composition and functio- microbial community (microbiome) is determined by host ning is strongly infl uenced by the environmental conditions. genetics and environmental factors, where alterations in The understanding of complex community functioning with its structure have been associated with the onset of many the goal of their rational manipulation and exploitation different diseases. Establishing a defi ned human gut is not only important in environmental habitats such as microbiome within inbred rodent models provides a means marine or terrestrial ecosystems but also in other habitats to study microbiome-related pathologies. We now compared like the human body which is also colonised by bacterial the effi cacy, quality and stability of the human microbiome communities in scales that outnumber human cells. While established within germ-free (GF) rats, GF mice, and anti- these host-associated microbes may be benefi cial for human biotic-treated mice. Remarkable differences were observed health, the human body can also be a reservoir for opportu- between the different rodent models. While the majority of nistic pathogens. abundant human-donor bacteria were established in the GF rats, only a subset was present in the GF mice. Specifi cally Nasal communities As an example, the human anterior members of Clostridia cluster IV, which comprise important nares are the major source and risk factor for invasive butyrate producers contributing to processes important for infections by Staphylococcus aureus, an increasingly multi- colonic health, failed to colonise the mouse gut. Our results resistant pathogen causing a large spectrum of infectious indicate that the genetic background of the recipient system diseases. However, very little is known about the composi- strongly infl uences the effi cacy and quality of the popula- tion of the nasal microbiota and possible community inter- ting human gut microbiota and clarifi es the suitability of actions. We have now elucidated the community composi- the models. tion of 40 individuals using advanced culture-independent approaches and have shown the presence of previously not Environmental communities and new metabolic pathways expected dominant community members such as Finegol- We have also improved our analytical tools to study micro- dia magna, Dolosigranulum pigrum, Anaerococcus sp. or bial communities in contaminated environments whereby uncultured actinomycetes. We also evaluated interactions using a novel validated catabolic gene array dedicated to between S. aureus and other microbial species present in achieve fast monitoring of catabolic gene diversity and the anterior nares and, as an example, observed a statisti- abundance and thus the catabolic landscape in combination cally signifi cant converse association between S. aureus and with metagenomic screening approaches, structure (on the F. magna, indicating that these species have a substantially DNA level) and function (by analyzing mRNA) could reliab- different distribution and abundance pattern refl ecting ly be described. These culture-independent analyses were their inability to share the same niche. The previously complemented by the elucidation of novel metabolic routes undiscovered co-colonization patterns and natural vari- and enzymes. ations in species composition provide insights for future intervention strategies for the control of health care- and community-associated S. aureus infections.

Scheithauer BK, Wos-Oxley ML, Ferslev B, Jablonowski H, Pieper DH. (2009). Characteriza- tion of the complex bacterial communities colonizing biliary stents reveals a host-dependent diversity. ISME Journal 3:797-807.

Wos-Oxley M, Plumeier I, von Eiff C, Taudien S, Platzer M, Vilchez-Vargas R, Becker K and Pieper DH. (2010). A poke into the diversity and associations within human anterior nare microbial communities. ISME Journal 4:839-851.

Brennerova MV, Josefi ova J, Brenner V, Pieper DH, Junca H. (2009). Metagenomics reveals diversity and abundance of meta-cleavage pathways in microbial communities from soil highly contaminated with jet fuel under air sparging bioremediation. Environmental Microbiology 11:2216-2227.

Vilchez-Vargas R, Junca H, Pieper DH. (2010). Metabolic networks, microbial ecology and ‚omics‘ technologies: towards understanding in situ biodegradation processes. Environmental Microbiology 12:3089-3110. SCIENTIFIC REPORTS | Infection and Immunity | Infl ammation and Immunity 91

03 Infl ammation and Immunity

TOPIC SPEAKER | Prof. Dr. Hansjörg Hauser | Department of Gene Regulation and Differentiation | [email protected]

The hosts’ immune response against invading pathogens can lead to massive infl ammatory reactions which might result in immunopathology, thereby causing severe problems to the host. Thus, understanding the mechanisms how pathogens’ actions on the one hand induce protective immune responses and on the other hand cause tissue damage is the key for the development of protective and therapeutic measures.

The topic “Infl ammation and Immunity” deals with pathogen-induced reactions, including innate immune responses, infl ammatory responses, and induction of long-term immunity. While these aspects are studied with the aim of understanding basic principles, the topic also aims at defi ning novel drug targets and therapeutic intervention strategies.

Induction of interferons and their activities The recognition of pathogen-associated molecular patterns by various receptors leads to signaling events, which fi nally result in the induction of interferon (IFN) genes. Upon secretion, IFNs have diverse activities on their target cells. The topic aims at identifying the involved molecular players, understanding their interaction with viral proteins and describing the dynamics of IFN-induced signal transduction in different cell types.

Consequences of infl ammation In most cases, infl ammatory events have benefi cial outcomes to the organism. Post-infl ammatory actions involve apoptosis and autophagy, which are of central importance to eliminate infected or damaged cells. In this topic, the involved basic mechanisms are studied as such, but also with regard to pathogen-mediated modulation of these processes. In cases of chronic infl ammatory reactions, e.g. in rheumatoid arthritis (RA), the dysregulated immune responses have negative outcomes. An important translational aspect within this topic concerns the suppression of chronic infl ammation in murine models of RA by inhibition of a central signalling molecule in proinfl ammatory T cells.

T cells in autoimmunity and tolerance Effector T cells play a critical role for the elimination of different types of pathogens from the infected host. However, if directed against self tissues, they also can drive autoimmune reactions, thereby causing severe damage to the host. To prevent such self- destructive immune reactions, the immune system has evolved a peculiar T cell subpopulation, the reglatory T cells, which play an important role in the maintenance of self tolerance. Within this topic, the generation of diverse T cell subsets and their functional properties are studied in detail in infection and infl ammatory mouse models.

Systems Biology: Theoretical models for complex immune responses In the past, researchers have been mainly focussing on the elucidation of linear pathways of biological events. With the develop- ment of high-throughput techniques and with the acquisition of complex data sets it has become more and more diffi cult to trans- late this huge amount of information into comprehensive concepts. In this topic, we use mathematic modeling approaches to describe dynamic and complex immunological processes such as IFN signalling and B and T cell homeostasis. 92 SCIENTIFIC REPORTS | Infection and Immunity | Infl ammation and Immunity

03.1 Development and Functional Properties of Foxp3+ Regulatory T Cells

PROJECT LEADER | Prof. Dr. Jochen Hühn | Department of Experimental Immunology | [email protected]

PROJECT MEMBERS | Pedro Almeida | Devesha Bhagwat | Sascha Cording | Dr. Stefan Flöß | Larissa Fournier | Yi-Ju Huang | Dr. Katjana Klages | Dr. Julia Polansky | Lisa Schreiber | Dr. René Teich | Aras Toker

Regulatory T cells (Tregs) play a key role for the control of numerous immune reactions and the maintenance of immunological tolerance. CD4+ Tregs are characterized by the expression of the transcription factor Foxp3 which is central for both the development of Tregs and their sup- pressive characteristics. The majority of Foxp3+ Tregs is already formed during the development of T cells within the thymus and shows features of a stable T cell line. We could recently show that epigenetic modifi cations within an evolutionarily conserved region of the foxp3 gene are decisive for the stability of Foxp3 expression (Huehn et al., 2009). These epigenetic modifi cations are already initiated during the thymic maturation of Tregs. The epigenetic modifi cations of this evolutionarily conserved region are essential for its activation, allowing the binding of specifi c transcription factors (Polansky et al., 2010). Thus, these Foxp3+ cells within the spleen: The cells were stained with epigenetic modifi cations can be regarded as a kind of mo- fl uorescently labelled antibodies. B cells, T cells und Foxp3+ lecular switch. cells are stained in blue, red and green, respectively. Picture: Dr. Eberl, Pasteur Institute, Paris, France. Foxp3+ Tregs are not exclusively generated in the thymus but also in the periphery, where antigen recognition under tolerogenic conditions leads to de novo induction of Foxp3+ Tregs from conventional, naïve T cells. In particular, we conditions leads to a reactivation of the tumour-specifi c observed a very effi cient de novo induction in mucosa- immune response. The most effi cient regression of tu- associated lymphoid tissues with the lymph node stroma mour growth was observed when we vaccinated against cells playing a key role. Currently, we are studying the tumour-associated antigens simultaneously to the transient mechanisms leading to the ‘tolerization’ of stroma cells depletion of Foxp3+ Tregs. In future projects we aim to fi nd within mucosa-associated lymphoid tissues. Moreover, we ways to selectively eliminate Foxp3+ Tregs or to transiently are investigating whether Foxp3+ Tregs which were de novo inhibit their suppressive capacity. These studies are also of induced within mucosa-associated lymphoid tissues have a major interest for the development of vaccination strategies superior suppressive capacity to prevent the development of against chronic infectious diseases. chronic-infl ammatory diseases of the gut. All in all, we expect that our investigations will result in Foxp3+ Tregs also can very effi ciently suppress wanted both, a better understanding in the molecular mechanisms immune responses; in a large number of tumour patients of Treg development and function in vivo. This will provide and in various in vivo tumour models a preferential ac- us new strategies for the induction and modulation of Tregs cumulation of Foxp3+ Tregs within the tumour tissue has in the scope of a therapeutic approach. been observed. Currently we are studying to which extend this accumulation can be explained by specifi c recruitment, local expansion or de novo induction of Foxp3+ Tregs within the tumour tissue. In any case, the large number of Foxp3+ Klages, K., C.T. Mayer, K. Lahl, C. Loddenkemper, M.W. Teng, S.F. Ngiow, M.J. Smyth, A. Tregs within tumour tissues very effi ciently prevents the Hamann, J. Huehn*, and T. Sparwasser*. (2010) Selective depletion of Foxp3+ regulatory T cells improves effective therapeutic vaccination against established melanoma. Cancer generation of tumour-specifi c immune responses. With Research 70:7788-7799. *equally contributed the aid of a transgenic mouse that allows the selective Polansky, J.K., L. Schreiber, C. Thelemann, L. Ludwig, M. Krüger, R. Baumgrass, S. Cor- + depletion of Foxp3 Tregs we were able to demonstrate that ding, S. Floess, A. Hamann, and J. Huehn. (2010) Methylation matters: Binding of Ets-1 + to the demethylated Foxp3 gene contributes to the stabilization of Foxp3 expression in the transient depletion of Foxp3 Tregs under therapeutic regulatory T cells. Journal of Molecular Medicine 88:1029-1040.

Huehn, J., J.K. Polansky, and A. Hamann. (2009) Epigenetic control of FOXP3 expression: the key to a stable regulatory T-cell lineage? Nature Reviews in Immunology 9:83-89. SCIENTIFIC REPORTS | Infection and Immunity | Infl ammation and Immunity 93

03.2 Mucosal Immunity and Infl ammation

PROJECT LEADER | Priv.-Doz. Dr. Dunja Bruder | Research Group Immune Regulation | [email protected]

PROJECT MEMBERS | Andrea Autengruber | Harro Frauendorf | Dr. Marcus Gereke | Dr. Andreas Jeron | Dr. Sabine Stegemann | Dr. Milena Tosiek | Julia Volckmar

We focus on the basic mechanisms underlying T cell-media- ted infl ammation in mucosal tissues, mechanisms involved in peripheral tolerance induction and the impact of infec- tions on the balance between tolerance and autoimmunity. Further activities concentrate on pathogen-induced immune modulation following bacterial and viral infections of the re- spiratory tract as well as a therapeutic vaccine to reactivate cellular immunity to chronic HCV infection.

Tolerance, autoimmunity and infection T cell reactivity to self antigen in the lung is studied in a transgenic mouse model based on the expression of infl uenza hemagglutinin (HA) in alveolar type II epithelial cells (AECII). Self-antigen Fig. 2. Identifi cation of alveolar type II epithelial cells (AECII) recognition in the lung by CD4+ T cells results in chronic in the alveoli of a murine lung section by staining for surfac- obstructive pulmonary disease, followed by the induction tant protein-C (SP-C) (in red) as a typical AECII marker. of Foxp3+ regulatory CD4+ T cells (Tregs) that suppress Foto: MHH, Christian Hennig&Jana Bergmann infl ammatory immune reactions. These cells release immu- nosuppressive mediators such as TGF-ß and IL-10 which directly contribute to Treg induction. Moreover, they also express a variety of surface molecules complementary to function dependent on listeriolysin O. Ongoing studies shall receptors on Tregs, suggesting a broader function of AECII in help to clarify whether break down of self-tolerance is a peripheral tolerance induction. In addition, AECII from the result of the infection itself. infl amed lung express elevated levels of antiviral genes. Immune modulation in the context of respiratory infec- Whereas HA antigen recognition by CD4+ T cells results in tions Pathogens such as Bordetella bronchiseptica modulate the induction of Tregs, self-reactive CD8+ T cells from the host immune responses thereby facilitating persistence. infl amed lung are neither activated, nor regulatory. However, Together with C. Guzmán, we study the impact of chronic some of HA-specifi c CD8+ T cells represent memory cells. B. bronchiseptica infection on molecular plasticity of Tregs. Foxp3+ Tregs appear to be dispensable for regulation of CD8+ We found that Tregs from chronically infected mice express T cell-mediated lung infl ammation since ablation of Tregs elevated levels of neuropilin-1, an activation-independent does not result in gain of effector function in CD8+ T cells. marker for Tregs. Currently, we are analyzing the coherence This suggests that tolerance mechanisms being active in CD8 between the observed changes in Treg phenotype and sup- and CD4 T cell-mediated lung infl ammation may differ. pressive function.

Studies in mice predisposed to the development of type Using a mouse model for infl uenza A virus / Streptococcus 1diabetes revealed that infection with Listeria monocytogenes pneumoniae superinfection we study the mechanisms leads to rapid onset of the disease. Infection impairs Treg underlying the increased lethality of mice by S. pneumo- niae induced sepsis following primary infection with the infl uenza virus. Increased susceptibility toward S. pneumo- niae infection is not the result of toll-like receptor (TLR)-7 induced lymphopenia. However, TLR-7 signaling contributes to the fi ne tuning of antiviral immune responses. Using TLR- 7 defi cient mice we could demonstrate that signalling by this receptor that senses viral RNA infl uences the outcome of secondary pneumococcal infection.

Chronic HCV infection represents the major cause for the development of hepatocellular carcinoma. In frame of the HGF fi nanced alliance “Immunotherapy of Cancers” we are aiming at develop a therapeutic vaccine against HCV. The ap- proach used is based on in vivo targeting of HCV antigen to Fig. 1. Alveolus of a murine lung. blue: DAPI; red: tubulin maturing Dendritic Cells (DC). We observed a very effi cient Photo: HZI&SySy GmbH, Christian Erck CD4+ and CD8+ T cell response as well as comparably high antibody titers. 94 SCIENTIFIC REPORTS | Infection and Immunity | Infl ammation and Immunity

03.3 Immuneffectors: Molecules, Cells and Mechanisms

PROJECT LEADER | Dr. Siegfried Weiss | Research Group Molecular Immunology | [email protected]

PROJECT MEMBERS | Imke Bargen | Nicole Dietrich | Dr. Sandra Düber | Dr. Nelson Gekara | Dr. Jadwiga Jablonska | Katja Kochrübe | Dr. Sara Leschner | Dr. Stefan Lienenklaus | Dr. Bishnudeo Roy | Swati Shukla | Evgeniya Solodova | Christian Stern | Dr. Nuno Viegas | Kathrin Wolf | Natalia Zietara

Our immune system consists of several lines of defense. One of the fi rst of such lines are type I interferons (IFN) with their most important members IFN-α and IFN-β. Originally, IFNs have antiviral activity but are also induced by all kinds of other infections and play an important role under normal physiological conditions. In our experiments, we could show that in macrophages IFNs are also induced by ligands of the toll like receptor 2 (TLR2). TLR2 ligands are found in the cell wall of Gram-positive bacteria and mycoplasms. Interestingly, to be able to trigger IFN production, the TLR2-ligand complex has to be endocytosed by the macrophages. Inhibition of endocytosis leaving the receptor-ligand complex at the cell surface allows only production of pro-infl ammatory cytokines like TNF-α. In accordance, the ability of a cell to endocytose surface TLRs decides whether the cell can produce IFNs after TLR-ligand binding or not. Along this line, we could show that mast cells are not able to endocytose such TLRs or to phagocytose bacteria that might have engaged TLRs. Therefore, mast cells are not able to produce IFN during an infection by bacteria although they are perfectly able to do so in case of a virus infection. Anatomically, mast cells are Enhanced tumour angiogenesis and growth is mediated by positioned underneath the various epithelia. Most likely, they neutrophilic granulocytes unresponsive to type I IFN. Mice were represent the fi rst immune cells to encounter pathogens that subcutaneously injected with B16F10 melanoma cells that were invade the body. During bacterial infection production of IFN mixed either with neutrophils obtained from tumor bearing is often detrimental. Thus, the lack of IFN production and Ifnar1-/- mice (IFNAR-/-+B16) or from tumour bearing wild type the restriction of mast cells to the secretion of pro-infl amma- mice (C57BL/6+B16). The tumours mixed with IFN-unresponsive tory cytokines might represent a well-balanced evolutionary neutrophils grew much faster. Blood vessels in the tumors were adapted defense mechanism. investigated by immune histology using anti-laminin to stain endothelial cells (red) and anti-actin to stain smooth muscle cells We noticed that mice that lacked the gene for IFN-β or of one (green). The presence of high amounts of actin in the IFNAR-/- of the receptor chains (IFNAR) exhibited an altered angio- +B16 tumours is indicative for the enhanced maturity of the genesis i.e. formation of blood vessels. This was especially blood vessels and thus of a better blood supply of the tumour. obvious in fast growing transplantable tumours: In IFN-β ko- Photo: HZI or IFNAR ko-mice the tumours grew much faster. This was due to an enhanced angiogenesis resulting in more and more mature blood vessels in tumours of the recombinant mice. Essential for the specifi c expression of such substances is We could show that neutrophilic granulocytes were involved the defi nition of bacterial promoters that restrict expression in this process (Figure 1). Without a functional IFN system to the tumour. To this end we have screened a so-called the tumours contained more of such granulocytes and they promoter-trap-library with our Salmonella and could detect produced more pro-angiogenic factors. By isolating granu- several promoters with the appropriate specifi city. Presently, locytes from tumours of the ko-mice we could show that we characterize such promoters using bioinformatics as well already small amounts of IFN-β down-regulate the produc- as experimental approaches. In parallel, we investigated tion of the pro-angiogenic factors. Thus, it becomes clear that tumour-specifi c bacterial gene expression using micro ex- IFNs are a natural component of the tumour surveillance pression arrays. We were able to defi ne more than 20 genes system of our body. that were specifi cally expressed in the tumour and not in the spleen. First of all this will help to understand the physiologi- Many bacteria like Salmonella typhimurium migrate to solid cal status of the bacteria residing in the tumour. In addition, tumours after systemic application and proliferate there. this will also lead to additional control elements that will Thus, they can be used as shuttle to express therapeutic genes enable us to express therapeutic genes specifi cally in the directly in the neoplastic tissue sparing the healthy organs. cancerous tissue. SCIENTIFIC REPORTS | Infection and Immunity | Infl ammation and Immunity 95

03.4 Interferons in Viral Defence and Immunity

PROJECT LEADER | Dr. Andrea Kröger | Department of Gene Regulation and Differentiation | [email protected]

PROJECT MEMBERS | Katja Finsterbusch | Lucas Kemper | Dr. Mario Köster | Antje Ksienzyk | Ramya Nandakumar | Berit Neumann | Dr. Ulfert Rand

Interferons (IFNs) are central signalling molecules in the defense against pathogens and have essential functions in the interplay of immune cells. The IFN system is the fi rst line of defense against infections. The effect is based on the transcriptional activation of multiple antiviral and immune modulatory genes. The aim of the project is to characterize the molecular and cellular context of host defense against infections. The detailed understanding of the involved me- chanisms and the spatio- and temporal dynamics will open new opportunities for the prevention of infectious diseases.

The IFN network The network of IFN production and action is strictly regulated, and its deregulation can lead to a Signal progression during viral infection Time-lapse pathologic condition in the host. Spatio-temporally resolved microscopy of IFN induction (green) and response (red) single cell data on viral infections have revealed IFN induc- resolves spatio-temporal dynamics in IFN signal propagation. tion mechanisms to be a highly stochastic process while the Photo: HZI response to IFN is bimodal. Based on these data dynamics and quantitative mathematical models describing the un- derlying signaling circuits are developed. This should allow the prediction of the outcome of an infection. To investigate IRF-1 induces innate and adaptive immune responses the coordinated functions of the IFN network in vivo we Apart from the antiviral response the IFN system is also generated reporter mice which allow the investigation of the essential for the induction and stimulation of immune IFN signal distribution in the whole body. With these tools responses. Enhanced immune cell activation is critical for we found that viruses with different pathogenicities lead to anti-tumour responses. The expression of IRF-1 in a lung me- IFN responses which differ in quality, quantity and temporal tastases model inhibits the development of metastases. This activity. effect is mediated by the attraction and activation of natural killer cells. Concurrently, the induction of IRF-1 results in Fail-safe pathway for antiviral activity Many viruses have tumour specifi c CD8+ T cell response which protects against developed strategies to circumvent the IFN system and there- secondary tumour development. The infl ammatory tumour by avoid the defense of the host. We identifi ed an alternative microenvironment plays an important role in the outcome of mechanism of antiviral defense which is IFN-independent. the immune response. Detailed analysis of the microenviron- The transcription factor IRF-1 plays an important role in ment and the signalling cascades of the infi ltrating immune this IFN-independent mechanism. Mice which lack IRF-1 cells will elucidate the mechanism of immune response are more susceptible against viral infections. We identifi ed induction in future. viperin as a gene which plays a major role in this effect. On encountering viral infections the IRF-1 gene usually regulates the IFN response, but if the virus has developed strategies to circumvent this response there is an alternative interferon independent mechanism of antiviral defense. The impact of the IFN-independent mechanism in host defense will be investigated in other infection models like HCV.

Stirnweiss, A., Ksienzyk, A., Klages, K., Rand, U., Grashoff, M., Hauser, H., Kröger, A. (2010) IFN regulatory factor-1 bypasses IFN-mediated antiviral effects through viperin gene induction. Journal of Immunology 184(9), 5179-5185.

Pulverer, J.E., Rand, U., Lienenklaus, S., Kugel, D., Zietara, N., Kochs, G., Naumann, R., Weiss, S., Staeheli, P., Hauser, H., Köster, M. (2010) Temporal and spatial resolution of type I and III interferon responses in vivo. Journal of Virology 84(17), 8626-8638.

Dietrich, N., Rohde, M., Geffers, R., Kröger, A., Hauser, H., Weiss, S., Gekara, N.O. (2010) Mast cells elicit proinfl ammatory but not type I interferon responses upon activation of TLRs by bacteria. Proceedings of National Academy of the Science USA 107(19):8748-8753. 96 SCIENTIFIC REPORTS | Infection and Immunity | Infl ammation and Immunity

03.5 Cell-death Mechanisms in Immunity

PROJECT LEADER | Prof. Dr. Ingo Schmitz | Research Group Systems-oriented Immunology and Infl ammation Research | [email protected]

PROJECT MEMBERS | Michaela Annemann | Frida Ewald | Ralf Höcker | Dr. Yvonne Rauter | Marc Schuster | Tanja Telieps

Types of cell death Multicellular organisms have evolved Apoptotic signal transduction Two major apoptosis path- multiple cell-death mechanisms which are activated upon ways exist referred to as the extrinsic and intrinsic pathway, different stimuli and cell type-dependent to allow a fi ne- respectively. The intrinsic pathway acts on the mitochon- tuning of cellular responses to environmental conditions. drium and is regulated by proteins of the Bcl-2 family. The Necrosis is characterized by swelling and rupture of the cells extrinsic signalling cascade is initiated at the cell surface so that intracellular contents leak out of the cell and initiate by so-called death receptors with CD95 being a prototypical an infl ammatory immune response. During apoptosis, on family member. CD95 plays a crucial role in a number of the other hand, cells shrink and the integrity of the plasma immunological processes such as T cell-dependent immune membrane stays intact. Therefore, leakage of intracellular responses but also in tumour development. Not much is components does not take place and activation of the im- known about the apoptosis signalling mechanisms which mune system is inhibited. Apoptosis plays an important role operate during acute and chronic infections in vivo. The aim in the interplay between pathogens and the host cell. In of our project is to analyse these signalling pathways using this regard, immune cells may kill infected tissue cells by different transgenic mice strains and apoptosis-modulating the induction of apoptosis. On the other hand, viruses and substances (e.g. caspase inhibitors). One family of proteins bacteria are able to inhibit host cell apoptosis to ensure their we may focus on are the FLICE-inhibitory proteins (FLIP), own survival and further spreading. Deregulation of apopto- important regulators of CD95 signaling. FLIP proteins have sis may lead to various diseases such as viral hepatitis (too been identifi ed in mammalian cells (cellular FLIP = c-FLIP) much apoptosis) or cancer (too little apoptosis). as well as in γ-herpesviruses (v-FLIP). We could recently show that, though structurally very similar, viral and cellular FLIP proteins act by different biochemical mechanisms. The functional consequences and potential therapeutic windows opened by these differences will be analysed in the future.

Autophagy Autophagy is a lysosomal degradation pathway that is important for cellular homeostasis, mammalian devel- opment and immunity. On the one hand, autophagy is able to eliminate intracellular pathogens. However, some pathogens use the autophagic pathway to escape immune recognition. Though the process of autophagy has been elucidated on the molecular level in recent years, little is known about signaling mechanisms regulating the effector functions of these molecules. We have recently uncovered a novel regula- tory mechanism involving the stress kinase p38 and Atg5, The intrinsic apoptosis pathway is activated by different stimuli the latter being an essential component of the autophagic such as UV irradiation or growth factor deprivation. Subsequent- pathway. We will address the role of autophagy regulation for ly, pro-apoptotic BH3-only proteins are activated to inhibit the the intracellular survival of Staphylococcus aureus in mam- anti-apoptotic Bcl-2 family members. This event allows Bax and malian cells. Bak to form pores in the outer mitochondrial membrane so that apoptogenic factors such as cytochrome c are released. In the cytosol, cytochrome c binds to Apaf-1 to form a multiprotein com- plex, called the apoptosome, at which pro-caspase-9 is activated. Caspase-9 then cleaves caspase-3 to start a proteolytical cascade ultimately leading to cell death. The extrinsic apoptosis pathway Ueffi ng N, Keil E, Freund C, Kühne R, Schulze-Osthoff K, Schmitz I (2008) “Structural and mutational analyses of c-FLIPR, the only murine short FLIP isoform, reveal require- is activated by death receptors. Upon binding of trimeric ligands, ments for DISC recruitment”. Cell Death and Differ 15(4):773-82. the receptor oligomerizes and recruits the adapter molecule Ueffi ng N, Schuster M, Keil E, Schulze-Osthoff K, Schmitz I (2008) “Upregulation of c-FLIPshort by NFAT contributes to apoptosis resistance of short-term activated T cells”. FADD and pro-caspase-8 to form a death inducing signaling com- Blood 112(3):690-8. plex (DISC). Caspase-8 is activated at the DISC and can directly Ueffi ng N, Singh KK, Christians A, Thorns C, Feller AC, Nagl F, Fend F, Heikaus S, Marx activate caspase-3 or it cleaves the BH3-only protein Bid to tBid A, Zotz RB, Brade J, Schulz WA, Schulze-Osthoff K, Schmitz I*, Schwerk C (2009) “A SNP in the cellular FLICE-inhibitory protein (c-FLIP) gene determines protein isoform leading to activation of the intrinsic pathway. c-FLIP proteins production and is associated with risk of follicular lymphoma in humans”. Blood can inhibit the activation of caspase-8 at the DISC. 114(3):572-9. * corresponding author SCIENTIFIC REPORTS | Infection and Immunity | Infl ammation and Immunity 97

03.6 Infl ammation and Regeneration

PROJECT LEADER | Priv.-Doz. Dr. Gerhard Gross | Department of Gene Regulation and Differentiation | [email protected]

PROJECT MEMBERS | Dr. Thomas Böldicke | Markus Heine | Sandra Laggies | Dr. Virginia Seiffart | Stefan Somplatzki | Lijing Sun | Dr. Herbert Weich | Dr. Manfred Wirth

Infection of tissues and trauma, shock and sepsis induce a septic challenge with Bacillus subtilis. We will now focus severe infl ammatory response, the primary role of which is on the investigation whether or not TLR2- and the recently to eliminate pathogens, promote tissue repair and return the constructed TLR9-intrabodies are able to interfere with a host to optimal normal function. In contrast to acute infl am- chronic infl ammatory disease such as rheumatoid arthritis. mation, chronic infl ammation leads to a progressive shift in the type of cells which are present at the site of infl ammation Rheumatoid arthritis is a severe chronic, systemic, infl amm- and, in general, leads to the destruction of the tissue in or atory, autoimmune disorder that may affect many tissues near the infl ammatory process often resisting regeneration. and organs but principally attacks and destroys synovial joints. About 1% of the world‘s population is affected. This We follow the hypothesis that inhibition of the infl ammato- disorder may lead to a substantial loss of mobility and, in ry signaling cascades will allow the design of new anti- general, patients suffer from serious pain and are affl icted infl ammatory and therapeutic modalities. In this regard, we by an increased mortality. Current therapies for rheumatoid investigate the role of caveolin-1 in infl ammation in general arthritis are often unsatisfactory and an ongoing search and in virus infection in particular. Caveolin-1 represents a for new therapeutic modalities is, therefore, required. We multifaceted membrane protein involved in cellular transport recently showed that a novel systemic targeting strategy for a and signaling. It interacts with the human infl uenza virus central infl ammatory signaling mediator (TGF-beta-activated matrix protein M2 and, moreover, modulates virus produc- kinase-1; TAK1) using the RNAi technology dramatically alle- tion. Further investigation of M2/Caveolin-1 interaction and viates rheumatoid arthritis symptoms in a murine collagen- its signal transduction pathways may, therefore, result in a induced arthritis model. Down-regulation of TAK1 protects novel target for therapeutic intervention. the synovial joints from degradation and causes a signifi cant reduction of infl ammatory Th1 and Th17 cells. TAK1 is, As another anti-infl ammatory strategy we are developing there fore, a novel and attractive therapeutic target to inter- specifi c intrabodies which are able to retain the Toll-like fere with rheumatoid arthritis. At present, we aim to transfer receptors 2 and 9 (TLR2 / TLR9) in the endoplasmic reticu- these results onto other chronic infl ammatory disorders. lum (ER). We could already demonstrate that an adenovirus vector-dependent, systemic expression of an anti-TLR2 Angiogenesis and the activation of the blood vascular system intrabody has the capacity to provide resistance to a lethal play important roles in sustaining a chronic infl ammation. In contrast, however, the role of the lymphatic vasculature in chronic infl ammation has remained unclear. We have esta- blished novel potent isolation techniques which allowed for the fi rst time the isolation of human lymph endothelial cells from normal skin and from diseased tissue (lymphangiomas).

In addition, we were successful in isolating mouse endothe- lial progenitor cells from adult lungs which showed a bipo- tential capacity to differentiate in vitro and in vivo into blood endothelial cells and lymph endothelial cells. These primary cells have been extensively compared and also subjected to expression profi ling to fi nd genes as possible targets for the therapy of infl ammatory disorders.

Becker, J., Pavlakovic, H., Ludewig, F., Wilting, F., Weich, H.A., Albuquerque, R., Ambati, J., & Wilting,J. (2010) Neuroblastoma progression correlates with downregulation of the In vitro differentiation of endothelial progenitor cells (EPCs) lymphangiogenesis inhibitor sVEGFR-2. Clinical Cancer Research 16, 1431-1441. from mouse lung: Bipotential capacity for the generation Charbord, P., Livne, E., Gross, G., Haupl, T., Neves, N. M., Marie, P., Bianco, P., & Jorgen- sen, C. (2010) Human bone marrow mesenchymal stem cells: A systematic reappraisal of blood- and lymph-endothelial cells. Prox1, marker of via the genostem experience. Stem Cell Review, in press lymph-endothelial cells (green); marker of blood- and lymph Courties, G., Seiffart, V., Presumey, J., Escriou, V., Scherman, D., Zwerina, J., Riuz, G., endothelial cell (red). Photo: HZI Zietara, N., Jablonska-Koch, J., Weiss, S., Hoffmann, A., Jorgensen, C., Apparailly, F., & Gross, G. (2010) In vivo RNAi-mediated silencing of TAK1 decreases infl ammatory Th1 and Th17 cells through targeting of myeloid cells. Blood, in press. 98 SCIENTIFIC REPORTS | Infection and Immunity | Infl ammation and Immunity

03.7 Cellular Models for Infection

PROJECT LEADER | Dr. Dagmar Wirth | Research Group Model Systems for Infection and Immunity | [email protected]

PROJECT MEMBERS | Muhammad Badar | Lacramioara Botezatu | Milada Butueva | Leonor Gama-Norton | Natascha Kruse | Daniel Maeda | Prof. Dr. Peter P. Müller | Dr. Kristina Nehlsen | Dr. Upneet Sandhu | Stephanie Sievers

Within this project we develop cellular models as well as A toxin/antitoxin system To achieve long term expression transgenic mouse model systems based on previously devel- of transgenes selection systems are usually used that allow oped strategies for controlled transgene expression. These enriching transgene-expressing cells in presence of selection systems are employed to address specifi c questions within drugs. Since this regimen is not feasible in in-vivo systems, infection research. we have developed an alternative strategy to stabilize trans- gene expression without selection. For this purpose a cell Targeted integration of expression cassettes In the past, system was developed in which toxin- induced cell death is we developed highly effi cient tools to predictably express overcome by expression of an antitoxin. Upon coupling ex- transgenes in cells and mice. For this purpose we used pression of the anti-toxin to the transgene we could stabili ze recombinase-mediated targeted integration of expression transgene expression over time in absence of selection drugs. cassettes into previously tagged chromosomal loci (hot-spots). This enabled us to investigate and optimize expression Prevention of microbial biofi lms on medical implants cassettes at defi ned chromosomal loci. We could show that To prevent the formation of bacterial biofi lms on implants optimal expression features depend on the interplay of chro- slow release coatings were developed with the aim to provide mosomal site and the regulatory elements of the incoming bactericidal activity for a critical period after implantation. vector. This allowed identifying optimal cassette design for a Two different strategies were employed. A surface-modifi ed panel of different integration sites. mesoporous silica layer containing drug deposits could release an antibiotic over an extended period of time. Alter- Tightly controlled and tissue-specifi c transgene natively, a coating derived from a biologically synthesized expression in the mouse: a model for induced hepatitis secondary metabolite provided antibacterial in vitro activity Based on the strategies for predictable transgene expression, against P. aeruginosa for approximately one week. we have established a platform of mouse-embryonic stem cells for targeted integration of transgene cassettes. This strategy was employed to induce liver-specifi c expression of viral antigens from HBV and HCV. We could show that induc- tion of antigens in the liver results in a massive activation of CD8 T cells resulting in pronounced hepatitis. This mouse model is currently being characterized to elucidate the inter- action of T cells with liver antigens.

Sandhu, U., Cebula, M., Behme, S., Riemer, P., Wodarczyk, C., Reimann, J., Schirmbeck, R., Hauser, H. and Wirth, D. (2011) Strict control of transgene expression expression in a mouse model for sensitive biological applications. Nucleic Acids Research (in press)

Gama-Norton, L., Herrmann, S., Schucht, R., Coroadinha, A., Löw, R., Bartholomae C, Schmidt M, Alves, P., Baum, C., Schambach, A., Hauser, H., and Wirth, D. (2010) Retro- viral vector performance upon integration into defi ned chromosomal loci of modular packaging cell lines. Human Gene Therapy 21(8):979-91.

Nehlsen, K., Herrmann, S., Zauers, J., Hauser, H. and Wirth, D. (2010) Toxin-antitoxin based transgene expression in mammalian cells. Nucleic Acids Research 38(5), e32.

Mouse models with induced liver-specifi c activation of antigens May, T., Butueva, M., Bantner, S., Markusic, D., Seppen, J., Weich, H., Hauser, H., and Wirth, D (2010). Synthetic gene regulation circuits for control of cell expansion. Tissue A. Proof of the activated luciferase through bioluminescence Engineering Part A. 6(2): 441-452 B. The activation of ovalbumin results in a damage of hepato- Nehlsen, K., Schucht, R., Gama-Norton, L., Kromer, W., Baer, A., Cayli, A., Hauser, H. and cytes and the liberation of specifi c enzymes (ALT) Wirth, D. (2009) Recombinant protein expression by targeting pre-selected chromoso- mal loci. BMC Biotechnology, 9, 100.

Badar M., K. Hemmen, M. Nimtz, H. Hauser, M. Stieve, M. Stiesch, T. Lenarz, U. Möll- mann, S. Vogt, M. Schnabelrauch, P.P. Mueller. (2011) Evaluation of madurahydroxylac- tone as a slow release antibacterial implant coating. TOBEJ, in press SCIENTIFIC REPORTS | Infection and Immunity | Strategies for Prevention and Therapy 99

04 Strategies for Prevention and Therapy

TOPIC SPEAKER | Prof. Carlos A. Guzmán, MD, PhD | Department of Vaccinology and Applied Microbiology | [email protected]

One third of all deaths occurring each year worldwide are directly caused by infectious agents. Microorganisms are also responsible for at least 15% of new cancers, and they are involved in the pathogenesis of many chronic non-infectious diseases. Furthermore, a recent report suggested that the incidence of lung cancer is signifi cantly increased in case of chronic tuberculosis. In addition, the major public health problem represented by infections is rendered even more dramatic by the global emergence of multiple drug-resistant strains. It is, therefore, critical to establish new approaches to fi ght microbial pathogens. Thus, the main objective of this topic is to develop innovative tools and strategies for the prevention, management and control of infections and infection- associated diseases. This is achieved by identifying novel intervention targets and bioactive molecules, designing new immune interventions, and establishing novel diagnostics and biomarkers.

In the project “Molecular Mechanisms of Hepatitis C Virus Infection and Replication” virus-host interactions crucial for hepatitis C virus (HCV) replication are investigated. These efforts should help to both defi ne new drug targets conferring inhibition of HCV replication and implement screening assays for the identifi cation of molecules preventing interactions crucially involved in HCV replication. To this end, cell based assays are exploited to identify cellular co-factors that HCV usurps for its propagation. Cell- based high-throughput screening systems are also being developed to identify HCV-specifi c inhibitors using HZI natural compound libraries. In this context, a novel luciferase-based screening system to monitor HCV replication was developed which was adapted to 384-well format in collaboration with the Department of Chemical Biology. This allowed the screening of large compound libraries for small molecules that may interfere with HCV replication. At the same time, the direct infl uence of immunosuppressive drugs on HCV propagation was assessed. This is of interest, because chronic HCV infection is one of the key indicators for liver transplantation, and immunosuppression is mandatory after transplantation to prevent graft rejection. Thus, these studies can help to eventually improve the management of HCV patients post transplantation. A particular focus of the work lays in the characterization of determinants defi ning the narrow species-tropism of HCV which should support the development of immuno- competent small animal models for this virus.

In the project “Senescence Surveillance in Chronic Hepatitis and Hepatocellular Carcinome” innovative mouse models are developed to study the role of the cellular senescence programme for immune surveillance during chronic liver infl ammation and liver cancer. This is linked with studies addressing the oncogenic potential of HBV and HCV structural proteins in hepatocarcino- genesis. Beyond this, functional genomics driven approaches are exploited to study the molecular mechanisms of liver damage, liver regeneration and liver cancer. To this end, microRNA based shRNA technology enabling stable or reversible RNAi-mediated inhibition of endogenous genes in cultured cells or in experimental mouse models is used. Genome-scale retro- and lentiviral shRNA libraries targeting the murine and the human genome are harnessed to identify new therapeutic targets in liver cancer and liver regeneration.

The project “Immune Aging and Chronic Infection” aims to defi ne the effects of persistent infections in immune homeostasis and immune senescence. In this context, there is a consensus that the adaptive immune response to emerging infections is compro- mised in the elderly. Aging results in a loss of both naïve T-cells and T-cell clonal diversity, thereby increasing the susceptibility to novel infections and decreasing the ability to generate protective immunity upon vaccination. However, the underlying mechanism 100 SCIENTIFIC REPORTS | Infection and Immunity | Strategies for Prevention and Therapy

has not been completely elucidated. Cytomegalovirus (CMV) is a ubiquitous pathogen, infecting the majority of the human population worldwide. CMV establishes a lifelong latent infection which dominates the repertoire of the memory T-cell compart- ment. Thus, the cellular and molecular mechanism of induction of the adaptive immune system during CMV infection is being elucidated, as well as the broader biological implications in terms of evolution of T-cell repertoires and immune function against CMV and other chronic infections. The knowledge emerging from this project is expected to facilitate the development of inter- vention strategies specifi cally tailored for the elderly.

The project “Interface between Innate and Adaptive Immunity” is dissecting the role played by type I interferon (IFN-I) for limiting viral spread, in periphery as well as at the level of the central nervous system. This is a critical issue since IFN-I is induced a few hours after infection, warranting initial host survival, since an adequate virus specifi c adaptive immune response able to eradicate the pathogen is activated after days. More specifi cally, the activities address how DNA (human CMV and vaccinia virus) or RNA (HCV and vesicular stomatitis virus) encoded viruses induce IFN-I responses and which type of IFN-inhibiting factors they have evolved. In this context anti-viral responses of human dendritic cell (DC) subsets such as conventional DC and plasmacytoid DC are studied in vitro, whereas the role of murine DC subsets is assessed exploiting in in vivo models. Gene stimulation signatures of virus-stimulated human DC subsets are of particular interest and may eventually yield new biomarkers. Interestingly, all examined viruses have developed evasion strategies to inhibit either the induction of IFN-I responses or the IFN-I effector function. Dedicated experiments are tackling the differential IFN-I induction in different anatomic niches, as well as the impact of virus induced IFN-I on the induction of virus specifi c CD8+ T cell responses, virus neutralizing antibody responses, and NK cell activity. The emerging knowledge will be exploited to improve pegylated IFN-α/Ribavirin therapy of chronic HCV infection and to establish new approaches for immunotherapies with antiviral cytokines.

The “Antigen Delivery Systems and Vaccines” project is focused on the development of tools and strategies to optimize the delivery of vaccine antigens, particularly by the mucosal route, and their subsequent exploitation for the generation of vaccines against specifi c diseases. This is linked to a programme aimed at the establishment of cost-effi cient preclinical validation models based on humanized mice for the screening, selection, and prioritization of candidate interventions. In the context of vaccination, it is critical to optimize antigen design. This is particularly true when pathogens which have evolved immune escape mechanisms are considered. In the case of Trypanosoma cruzi, despite strong immune responses elicited after natural infection or vaccination, parasite survival suggests that these responses are inadequate. Studies performed with recombinant proteins spanning different domains of cruzipain, a major cystein proteinase of T. cruzi, suggested that immunization with full-length cruzipain resulted in poor recognition of the N-terminal catalytic domain. In contrast, “masking” of protective determinants can be reverted by using the N-terminal domain as a tailored immunogen. This approach thus allows redirection of the host response, thereby providing enhanced protection against acute and chronic phase infection. Other activities were focused on optimizing antigen delivery by the mucosal route. In this context, the stimulation of specifi c T helper (Th) subsets is critical to promote effi cient immunity without side effects. However, there is an incomplete knowledge on the effector mechanisms triggered by mucosal vaccination. Thus, it was evaluated which Th subsets are stimulated following intranasal immunization. The results demonstrated that there is a preferential induction of Th17 cells. SCIENTIFIC REPORTS | Infection and Immunity | Strategies for Prevention and Therapy 101

The “Therapeutic Cellular Vaccines” project is geared towards the development of strategies to break the immune escape mechanisms operating in persistent infections. This can be achieved by specifi c stimulation of professional antigen presenting cells, especially dendritic cells (DC). However, a strong immune activation can also lead to immune pathogenic processes which may be controlled by immunosuppressive cells, such as mesenchymal stromal cells. Thus, antigen presenting cells were modifi ed using adenoviral vectors encoding antigens and immunomodulatory molecules to improve their antigen presentation capacities. It was also investigated if the stimulatory capacity of DC can be enhanced by infection with Mycobacterium bovis BCG. To facilitate translation of basic research into cell therapies, fl exible and robust good manufacturing praxis (GMP)-compliant processes for the cultivation and modifi cation of different cell types, including DC, were established using a closed integrated bag system. The use of dielectric barrier discharge (plasma) treatment in presence of suitable precursors allowed modifying the surface properties of the bag-system, thereby enabling the cultivation of adherent cells (e.g. mesenchymal stem cells).

The “Molecular Diagnostics of Microbial Pathogens” project is focused on the development and application of high resolution molecular diagnostic tools for the study of food- and waterborne bacterial pathogens. These tools aim at the identifi cation of single strains which are responsible for infections of individual patients or cause outbreaks of infectious diseases. They allow assessing the presence as well as the virulence and degree of metabolic activity of pathogens present in human, animal, and environmental samples. By implementing methods such as the Multi-Loci Variable Number of Tandem Repeats Analysis (MLVA), the project aims at understanding the infection routes of bacterial pathogens from food and water as well as to obtain insights into the evolution and epidemiology of pathogenic bacteria in clinical and natural environments. Validation of a MLVA method developed for Vibrio parahaemolyticus with a set of Chilean isolates indicated that, despite pandemic occurrence, local evolution is relevant for infectivity. Adaptation of the approved MLVA method for Legionella pneumophila to fi nished drinking water also allows detection and identifi - cation of specifi c MLVA-genotypes without cultivation.

Fig. 2. Infection of mouse cells with retroviral HCV pseudoparticles. These pseudoparticles carry either the wild-type envelope proteins (left side, upper left) or the envelope proteins adapted to the mouse CD81 (left side, upper right). Utilizing these pseudoparticles we in- oculated mouse cells that carry either all four human factors or the corresponding mouse factors (left side, bottom). The pseudo-particles transfer an eGFP reporter gene so that infected cells can be identifi ed on the basis of their eGFP fl uorescence. The quantity of infected cells is indicated (right side). (see next page) 102 SCIENTIFIC REPORTS | Infection and Immunity | Strategies for Prevention and Therapy

04.1 Molecular Mechanisms of Infection and Replication in the Hepatitis C Virus

PROJECT LEADER | Prof. Dr. Thomas Pietschmann | Department of Experimental Virology | Twincore | [email protected]

PROJECT MEMBERS | Dorothea Bankwitz | Dr. Julia Bitzegeio | Dr. Christiane Brohm | Dr. Sandra Ciesek | Martina Friesland | Dr. Sibylle Haid | Kathrin Hüging | Dr. Eike Steinmann

The Hepatitis C Virus (HCV) infects only humans and HCV chimpanzees. Therefore, besides chimpanzees, which can serial virus passage hardly be used for experimentation due to high costs and ethical concerns, the only animal model currently available human cells with are partially “humanised” mice. These animals lack an im- mouse CD81 mune system, permitting xenotransplantation with primary sequencing human liver cells, which then sustain HCV propagation. before after

108 108

C E1 E2p7 NS2 NS3 NS4B NS5A NS5B Although certainly very useful for drug development, this NS4A 107 107 /ml) /ml)

model is, however, unsuitable for the development of vac- 50 50 106 106 adapted virus genome cines, since no immunological studies can be carried out 105 105 due to the immune defect. Given these circumstances, we 104 104 103 103 are examining why HCV does not propagate in murine infectivity (TCID infectivity (TCID liver cells. By analysing the mechanisms that restrict HCV propagation in mouse cells we learn how HCV makes use of CD81 CD81 essential co-factors and which murine antiviral factors may limit HCV replication in mice. Collectively, these studies Fig. 1. Adaptation of HCV to murine CD81. HCV was transferred should help to defi ne what hurdles have to be surmounted to human liver cells that carry murine CD81 instead of human in order to develop an immune-competent small-animal CD81. The cell-culture supernatant of these cells was serially model for HCV. passaged to the human cells with mouse CD81. The success of this adaptation was evaluated (below) by inoculation of cells Entry of HCV into mouse cells cannot occur without human that carry either human or murine CD81. After the adaptation, cell-entry factors, since the proteins on the surface of the the viral genome was isolated and sequenced. Three mutations, virus are for the most part incompatible with the decisive in the area of the envelope proteins and responsible for the mouse proteins on the mouse liver cells. To enable HCV adaptation to CD81, were identifi ed; they have been indicated to enter into the liver cells, the virus must interact with with a star in the virus genome (below right). four different molecules on the surface of the target cell. These cellular proteins are SR-BI, CD81, Claudin-1 (CLDN1) and Occludin (OCLN). Among these, primarily CD81 and adaptation has exposed particularly sensitive areas on the OCLN are used by the virus in a species-specifi c fashion as surface of the virus, thus rendering it more susceptible to murine orthologs sustain HCV cell entry only poorly. Our neutralization by antibodies. It is possible that this “opened” latest results show that murine CLDN1 supports infection structure, which facilitates conformational changes of viral with limited effi ciency, indicating that CLDN1 also contri- proteins required for cell entry, is decisive for the usage of butes to HCV species specifi city. non-human entry factors.

Adaptation of HCV to murine cell-entry factors We at- Outlook Given these fi ndings, it should be theoretically tempted in this context to adapt the virus to the mouse. For possible to adapt the virus to further non-human cellular this purpose, a human liver cell-line was produced, which replication factors. In this manner we can fi nd out how HCV does indeed possess human SR-BI, Claudin-1 and Occludin; makes use of the respective host proteins, and why this is however, the human CD81 protein is missing. Subsequently, not possible in, for example, mouse cells. The obstacles that we artifi cially introduced the mouse version of CD81, after inhibit development of an immune-competent small-animal which the cells were again susceptible to infection; however, model can thus be identifi ed and in the long run strategies the level of effi ciency was approximately 100 times lower can be devised to ultimately overcome these. when compared to the human CD81 (Fig. 1). Through serial passage of the virus in these cells, a virus variant was se- lected that was able to utilize the mouse version of CD81 as Bitzegeio J, Bankwitz D, Hueging K, Haid S, Brohm C, Zeisel MB, Herrmann E, Iken M, Ott M, Baumert TF & Pietschmann T. (2010) Adaptation of hepatitis C virus to mouse well as the human protein (Fig. 2, see page before). This al- CD81 permits infection of mouse cells in the absence of human entry factors. PLoS teration produced a disadvantage for the virus; the adapted Pathogens 6:e1000978. virus has now become considerably more susceptible to Haid S, Windisch MP, Bartenschlager R & Pietschmann T. (2010) Mouse-specifi c resi- dues of claudin-1 limit hepatitis C virus genotype 2a infection in a human hepatocyte neutralization by antibodies from patients. Presumably, this cell line. Journal of Virology 4(2):964-75. SCIENTIFIC REPORTS | Infection and Immunity | Strategies for Prevention and Therapy 103

04.2 Interaction between Innate and Adaptive Immunity

PROJECT LEADER | Prof. Dr. Ulrich Kalinke | Research Group Experimental Infection Research | Twincore | [email protected]

PROJECT MEMBERS | Dr. Claudia Detje | Dr. Theresa Frenz | Elena Grabski | Sabrina Heindorf | Julia Heinrich | Christian Mers | Claudia Soldner

Following viral infection, type I interferon responses that strategies they have developed to undermine these responses secure the initial survival of the host are usually induced and how interferons secure the survival of the host. within hours. It is approximately a week later that adaptive immunity is activated to an extent that it is able to eradicate Mechanisms of type I interferon induction and its protec- the infecting pathogen. In earlier studies we showed that tive function The molecular mechanism of type I interferon upon viral infection a small number of highly specialised induction may vary depending on the inducing agent and the immune cells, also addressed as plasmacytoid dendritic cells analysed tissue of cell type. We study in the mouse model (pDC), produce large quantities of protective type I interfer- how the entry of the vesicular stomatitis virus (VSV) into the on. Practically all viruses examined more closely developed central nervous system via the olfactory system is inhibited countermeasures that inhibit the induction or the function interferon-dependently (see also Figure 1). Furthermore, we of such type I interferon responses. We are investigating how analyse the infl uence type I interferon responses have on different viruses induce type I interferon responses, which immune cell functions. In this context, we have found that a vaccinia virus-derived attenuated pathogen variant, modifi ed vaccinia virus (MVA), which triggers strong type I interferon responses, also induces a type I interferon-dependent expan- sion of virus-specifi c T cells. In this context type I interferon stimulation of both the antigen-presenting dendritic cells as well as T cells plays a central role. Furthermore, we investi- gate the infl uence type I interferon has on the induction of long-lasting antibody responses. This work plays an impor- tant role for the development of new vaccination strategies.

Virus-mediated activation of human pDC Some patients with chronic hepatitis C virus (HCV) infection can be suc- cessfully treated with an interferon-alpha/ribavirin combina- tion therapy. However, so far little is known about whether the body’s own interferon system is suffi ciently activated in HCV infection. We investigate which HCV and/or host encoded factors infl uence the strength and composition of HCV-induced cytokine response of human pDC. This work is carried out in vitro with primary human pDC isolated from blood samples from healthy donors. In the future also experi- ments with pDC from chronically HCV infected patients will be carried out.

Perspective An improved understanding of the multiple functions of type I interferon can help to optimize type I interferon-based therapies of tumors, autoimmune diseases and viral infections. Furthermore deeper knowledge of viral mechanisms of immune stimulation and evasion will provide new approaches for vaccine development.

After intranasal infection of mice, VSV infects olfactory nerves, travels over the axons to the olfactory bulb and is arrested in Frenz, T., Waibler, Z., Hofmann, J., Hamdorf, M., Lantermann, M., Reizis, B., Tovey, M,G., Aichele, P., Sutter, G., & Kalinke, U. (2010) Concomitant IFNAR-triggering of T cells and peripheral areas of the olfactory bulb by a type I interferon of DC is required to promote maximal MVA-induced T-Cell expansion. European Journal dependent mechanism. The image shows the histological ana- of Immunology 40, 2769-2777. lysis of the olfactory bulb of a mouse that was infected with Waibler, Z., Anzaghe, M., Frenz, T., Schwantes, A., Pöhlmann, C., Ludwig, H., Palomo- Otero, M., Alcamí, A., Sutter, G., & Kalinke, U. (2009) Vaccinia virus-mediated inhibition an eGFP-expressing VSV (upper panel). It is clearly visible that of type I interferon responses is a multifactorial process involving the soluble type I the virus is stopped in the glomerular layer of the olfactory interferon receptor B18 and intracellular components. Journal of Virology 83, 1563-1571. bulb (lower panel). Photos: Twincore Detje, C.N., Meyer, T., Schmidt, H., Kreuz, D., Rose, J.K., Bechmann, I., Prinz, M., & Ka- linke, U. (2009) Local type I IFN receptor signaling protects against virus spread within the central nervous system. Journal of Immunology 182, 2297-2304. 104 SCIENTIFIC REPORTS | Infection and Immunity | Strategies for Prevention and Therapy

04.3 Antigen Delivery Systems and Vaccines

PROJECT LEADER | Prof. Carlos A. Guzmán, M.D., Ph.D. | Department of Vaccinology and Applied Microbiology | [email protected]

PROJECT MEMBERS | Dr. Pablo D. Becker | Dr. Jennifer Debarry | Dr. Thomas Ebensen | Dr. Miriam Nörder | Dr. Peggy Riese | Rimma Libanova | Kirsten Scholz | Dr. Kai Schulze | Sebastian Weissmann | Dr. Beata Zygmunt

The main aim of this project is the development of tools and IgG titers were observed in mice vaccinated with either the strategies to optimize the delivery of vaccine antigens, parti- C-terminal domain or the full-length protein. In contrast, low cularly by the mucosal route, and their subsequent exploita- antibody titers were stimulated in mice immunized with the tion for the generation of vaccines against specifi c diseases. N-terminal domain, thereby suggesting a parasite immune escape mechanism by avoiding antibody production against Improving antigen design Despite the strong immune the catalytic N-terminal domain. Similarly, cellular responses responses elicited after natural infection with Trypanosoma were poorly restimulated with the N-terminal domain in cruzi or vaccination against it, parasite survival in the host mice immunized with full-length rCz, whereas most of the suggests that these responses are insuffi cient or inherently response was evoked by the C-terminal domain. However, the inadequate. T. cruzi contains a major cystein proteinase, cellular responses were much stronger when the N-terminal cruzipain, which is an attractive candidate vaccine antigen. domain was used for immunization, suggesting that cellular We produced full-length recombinant cruzipain (rCz), as responses can be down-regulated by full-length Cz to avoid well as truncated forms encompassing its N- and C-terminal recognition of the N-terminal domain. Masking of the essential domains. Groups of mice were then immunized with the domain can be reverted by using the N-terminal domain as recombinant proteins combined with CpG-ODN and Cz- a tailored immunogen to redirect host responses to provide specifi c antibody responses were measured. The highest enhanced protection. In fact, mice immunized with the N- terminal domain were able to better control infection, result- ing in signifi cant lower parasite loads throughout the acute phase and limited tissue injury during the chronic phase.

Optimizing vaccination strategies Mucosal vaccination is an attractive strategy for antigen administration, since this approach is not associated with pain or stress, has an extremely easy and cost-effi cient administration logistics and does not require highly trained personnel. However, although many features of the mucosal immune system have been dissected, there is an incomplete knowledge on the effector mechanisms triggered by mucosal vaccination. In this context, the stimulation of specifi c T helper (Th) subsets is critical to promote effi cient immunity without side effects. Thus, we investigated which Th subsets are preferentially stimulated following intranasal immuniza- tion. In comparison to parenteral immunization we detected an up-regulation of CCR-6 on CD4+ cells which correlated with an enhanced production of IL-17. This preferential induction of Th17 immune response is independent from the kind of adjuvant used.

It is known that Th17 cells play a critical role in immune (A) Antibody response in mice immunized with full-length rCz responses against important respiratory pathogens. Thus, or its domains. . (B) Parasite loads during the acute phase of our observation that a Th17 response pattern is specifi cally T. cruzi infection in vaccinated mice. (C) Micrographs of skel- promoted by intranasal immunization has important impli- etal muscle from control mice show severe infl ammation (×40), cations in the context of optimizing rational vaccine design. whereas animals immunized with rCz (D), the C-terminal (E) and the N-terminal (F) domain show moderate infl ammation with edema (lower and upper arrows, ×10), moderate infl am- Cazorla, S. I., Frank, F. M. , Becker, P. D., Arnaiz, M., Mirkin G. A., Corral R. S., Guzmán C. A*. and Malchiodi E. L.* (2010). Redirection of the immune response to the functional mation and collagen deposition (upper and lower arrows, ×40) catalytic domain of the cystein proteinase cruzipain improves protective immunity against Trypanosoma cruzi infection. Journal of Infectious Diseases. 202:136-144. and mild infl ammation (×20), respectively; *P < 0.05, **P < *Corresponding authors. 0.01, ***P < 0.001. Zygmunt, B. M., Rharbaoui F., Groebe L., and Guzman C. A. (2009). Intranasal immuni- zation promotes Th17 immune responses. Journal of Immunology. 183:6933-6938. SCIENTIFIC REPORTS | Infection and Immunity | Strategies for Prevention and Therapy 105

04.4 Chronic Infection and Cancer

PROJECT LEADER | Prof. Dr. Lars Zender | Junior Research Group Chronic Infection and Cancer | [email protected]

PROJECT MEMBERS | Dr. Tae-Won Kang | Dr. Michelle Stange | Dr. Torsten Wüstefeld | Dr. Tetyana Yevsa | Daniel Dauch | Florian Heinzmann | Lisa Hönicke | Anja Hohmeyer | Nils Jedicke | Marina Pesic | Ramona Rudalska

Liver Cancer (Hepatocellular Carcinoma) represents one 2. Translational Oncology, mosaic cancer mouse mod- of the most frequent and most deadly cancers worldwide. eling, in vivo RNAi, identifi cation of new, innovative The incidence of the disease is increasing, with more than therapeutic targets in liver cancer Tumour genetics can 700,000 new cases per year, 50,000 of which occur in guide the application of particular therapeutic strategies Europe. and can predict treatment outcome. By combining in vivo RNAi technology with powerful mosaic liver cancer mouse We are taking functional genomic approaches to study models, we have made substantial progress in pinpointing molecular mechanisms of liver damage, liver regeneration new cancer genes and tumour suppressor networks in hepa- and liver cancer. As our major tool, we are harnessing the tocellular carcinoma (HCC). In addition to probing tumour naturally occurring process of RNA interference (RNAi). suppressor genes in vivo one at a time, we also performed Over the past couple of years we have developed and refi ned in vivo screens to multiplex this approach. We recently per- microRNA based shRNA technology, which allows stable formed the fi rst in vivo RNAi screen, which identifi ed more or reversible RNAi-mediated inhibition of any endogenous than ten new tumour suppressor genes in HCC. gene in cultured cells or in mouse models of liver regenera- tion and liver cancer. The use of inducible RNAi allows us Current projects in the laboratory are aiming to identify to interfere with gene products within regenerating livers genetic vulnerabilities in hepatobiliary malignancies. These or growing tumours in mice, either at a single gene level or approaches are based on the concept of “synthetic lethality”, through pooled multi-gene screening. Genome-scale retro- which assumes that by virtue of their accumulated genetic and lentiviral shRNA libraries targeting the murine and alterations, tumour cells may acquire vulnerabilities that the human genome are used to conduct screens in order to create opportunities for new therapeutic interventions. identify new therapeutic targets in regenerative medicine Several high confi dence targets have been identifi ed and we and hepatobiliary oncology. are currently trying to translate these into new treatment strategies. 1. Translational Regenerative medicine and targeting of liver stem cells The liver has tremendous potential to regenerate following tissue damage caused by toxins or infection. It is unique that differentiated hepatocytes, which normally reside in the Go phase of the cell cycle, can re-enter the cell cycle subsequent to liver damage and give rise to new hepatocytes. However, when chronic liver dam- age occurs, there is an exhaustion of the regenerative capac- ity of hepatocytes and only partial compensation by a stem cell compartment. The consequence is chronic liver failure, which represents a major health problem worldwide.

We have established a unique system which allows conduct- ing in vivo RNAi screens to identify positive and negative Dr. Torsten Wüstefeld (le) und Florian Heinzmann (ri) discuss- regulators of hepatocyte proliferation during chronic liver ing results from an experiment. On the left side: Dr. Tetyana damage. A recently completed in vivo RNAi screen identi- Yevsa, on the right side: Ramona Rudalska. Photo: HZI fi ed a dual specifi city protein kinase as a promising thera- peutic target for hepatic regenerative medicine. Functional characterization showed that knockdown of the target by Zender, L., Xue, W., Zuber, J., Semighini, C.P., Krasnitz, A., Ma, B., Zender, P., Kubicka, S., Luk, J.M., Schirmacher, P., McCombie, W.R., Wigler, M., Hicks, J., Hannon, G.J., Powers, different shRNAs led to a more than 1000-fold increased S. & Lowe, S.W. (2008) An oncogenomics-based in vivo RNAi screen identifi es tumor proliferative capacity of hepatocytes in a mouse model of suppressors in liver cancer. Cell 135(5), 852-64. chronic liver damage and greatly improved survival. We Xue, W., Zender, L., Miething, C., Dickins, R.A., Hernando, E., Krizhanovsky, V., Cordon-Cardo, C. & Lowe, S.W. (2007) Senescence and tumour clearance is triggered by are currently working to translate the obtained genetic p53 restoration in murine liver carcinomas. Nature 445(7128), 656-60. information into new pharmacological strategies for hepatic Zender, L., Spector, M.S., Xue, W., Flemming, P., Cordon-Cardo, C., Silke, J., Fan, S.T., regenerative medicine. Luk, J.M., Wigler, M., Hannon, G.J., Mu, D., Lucito, R., Powers, S. & Lowe, S.W. (2007) Identifi cation and validation of oncogenes in liver cancer using an integrative oncogeno- mic approach. Cell 125(7), 1253-67. 106 SCIENTIFIC REPORTS | Infection and Immunity | Strategies for Prevention and Therapy

04.5 Therapeutic Cellular Vaccines

PROJECT LEADER | Dr. Werner Lindenmaier | Department of Gene Regulation and Differentiation | [email protected]

PROJECT MEMBERS | Dr. Kurt E. J. Dittmar | Dr. Wilhelm Meyring | Dr. Oliver Schön | Dr. Nadia Zghoul | Claudia Preuß | Ellen Kuppe

Cell-based immunotherapies have great potential for the As a tool to overcome this hurdle we have developed com- treatment of tumours and persistent infections. Activation pletely closed bag systems for cGMP compliant dendritic cell of the cellular immune response can eliminate the cancer generations. Using sterile docking, all steps starting from cell cells and the cells which are persistently infected. However, isolation up to the fi nal formulation and cryo-conservation tumour cells and persistent pathogens have evolved mecha- of adenovirally modifi ed DC can be realized without opening nisms to escape the normal immune response. Therefore, the system. In addition to the development of the cultivation specifi c stimulation by professional antigen presenting cells, system we investigated whether the stimulatory capacity of especially dendritic cells (DC), is necessary to overcome the DC vaccine is enhanced by infection with M. bovis BCG these obstacles. On the other hand, a strong induction of using fl ow cytometry, expression profi ling and confocal and the immune response, especially when directed against self electron microscopy and how the balance between anti-tu- tumour antigens, bears the danger of inducing pathogenic mour immunity and autoimmunity after DC vaccination can autoimmunity. In this case control of the overwhelming be controlled in a transgenic murine model system. response by immunesuppressive cells like mesenchymal stromal cells is an attractive option. So far, the bag system only allowed the cultivation of suspen- sion cells. Adherently growing cells like mesenchymal stem Sterile docking for cellular therapies However, the biggest cells require adherence-compatible modifi ed surfaces. This obstacle for broader application of cellular therapies is the modifi cation can be achieved using dielectric barrier dischar- requirement of autologous primary cells which have to ge (plasma). Plasma treatment in the presence of suitable be prepared under cGMP conditions in accordance to EU precursors such as silanes or amino-functionalized com- legislation. pounds allows selective modifi cation of surface properties. We have identifi ed suitable surface coatings, which support adherence and survival of MSC whereas cells in unmodifi ed bags aggregated and eventually died.

Modifi ed bag MSC in comparison to conventional fl ask MSC MSC grown on modifi ed bag surfaces were extensively analysed in comparison to conventional fl ask MSC. Analysis of cell specifi c surface markers, gene transfer effi ciency, adipogenic and osteogenic differentiation and global expres- sion profi les did not reveal signifi cant differences between bag- and fl ask-MSC. Bag modifi cation using dielectric barrier discharge therefore opens up new possibilities for the culti- vation of therapeutic cells in a closed system. Furthermore, secondary surface modifi cation (e.g. by coupling of cell-specifi c antibodies) has been used to isolate CD14+ monocytes from peripheral blood. Leukocytes and structured modifi cation of the surface allowed localized adherence and genetic modifi cation providing a tool for the analysis of interactions between different cell types.

Garritsen,H.S., Macke,L., Meyring,W., Hannig,H., Pägelow,U., Wörmann,B., Geffers,R., Dittmar,K.E., and Lindenmaier,W. (2010). Effi cient generation of clinical-grade gene- tically modifi ed dendritic cells for presentation of multiple tumor-associated proteins. Transfusion. 50, 831-842.

Macke,L., Garritsen,H.S., Meyring,W., Hannig,H., Pagelow,U., Wörmann,B., Adherent growth on modifi ed surface and karyotype stability Piechaczek,C., Geffers,R., Rohde,M., Lindenmaier,W., and Dittmar,K.E. (2010). Evalua- ting maturation and genetic modifi cation of human dendritic cells in a new polyolefi n of MSC A) Electron micrograph of MSC growing adherently on cell culture bag system. Transfusion. 50, 843-855. modifi ed bag surface (Coop.: M. Rohde, HZI). Dittmar,K.E.J., Simann,M., Zghoul,N., Schön,O., Meyring,W., Hannig,H., Macke,L., B) Even after passage 22 a normal diploid karyotype is Dirks,W., Miller,K., Garritsen,H.S.P., and Lindenmaier,W. (2010). Quality of Cell Products: Authenticity, Identity, Genomic Stability and Status of Differentiation. Trans- maintained. (Coop.: K. Miller, MHH). fusion Medicine and Hemotherapy 37, 57-64. SCIENTIFIC REPORTS | Infection and Immunity | Strategies for Prevention and Therapy 107

04.6 Molecular Diagnostics of Microbial Pathogens

PROJECT LEADER | Priv.-Doz. Dr. Manfred Höfl e | Department of Vaccinology and Applied Microbiology | [email protected]

PROJECT MEMBERS | Dr. Ingrid Brettar | Dr. Erika Harth-Chu | Karsten Henne | Leila Kalisch | Rolf Kramer

Molecular diagnostics of microbial pathogens focused on the development and application of high resolution molecular diagnostic tools for the study of foodborne and waterborne bacterial pathogens. These tools aim at the identifi cation of single strains which are responsible for infections of individual patients or cause outbreaks of infectious diseases. They are based on the genome-wide analysis of a set of Vari- able Number of Tandem Repeats (VNTR) which are used as highly specifi c and sensitive taxonomic markers. A set of these VNTR markers from across the bacterial genome is selected and can be used to identify the targeted bacterial species at the clonal level. This method, called Multi-Loci Variable Number of Tandem Repeats Analysis (MLVA), is a new tool to understand the infection routes of bacterial Epifl uorescence microscopy of drinking water bacteria (blue pathogens from food and water. Additionally, this genomic cells) and immunofl uorescence of Legionella pneumophila fi ngerprinting method provides insights into the evolution serogroup 1 cells (green) in biofi lm from a cooling tower. and epidemiology of pathogenic bacteria in clinical and Photo: Leila Kalisch, HZI natural environments.

Foodborne bacterial pathogens We have developed a MLVA method for Vibrio parahaemolyticus based on com- resolution electrophoresis to obtain sequences of single parative genome analysis of the two sequenced genomes. V. VNTRs. This approach allowed the detection and identifi ca- parahaemolyticus is a marine bacterium causing cholera-like tion of specifi c MLVA-genotypes in drinking water without diarrhea if contaminated mussels are eaten raw or semi- cultivation of the respective strains. We demonstrated the cooked. It causes outbreaks around the Pacifi c and in the applicability of this approach to common drinking water USA; recently, even occurrences in European countries have with low levels of L. pneumophila (< 1 cell/liter). Using this been reported, probably due to global warming. We validated culture-independent method we were able to identify several the method with a set of Chilean isolates in collabora- new clones in addition to isolated strains from the same tion with the University of Santiago de Chile. Clinical and drinking water. This novel tool for in-situ diagnostics of envi- environmental strains from the South and North of Chile had ronmental bacterial pathogens provides a new avenue for the a common evolutionary history and only Northern Chilean surveillance of waterborne pathogens after outbreaks have strains showed an evolutionary relationship to Asian strains. occurred and advances molecular epidemiology of bacterial These fi ndings indicate that, despite the pandemic occur- pathogens with high relevance to public health. rence of V. parahaemolyticus,the local evolution of a pathogen is of relevance for its infectivity.

Waterborne bacterial pathogens Legionella pneumophila is a freshwater bacterium abundant in man-made systems such as cooling towers, air conditions and drinking water supply systems (DWSS). It can cause atypical pneumonia in humans with high mortality stemming from the inhala- tion of contaminated aerosols. Detection and identifi cation of L. pneumophila is hampered by its fastidious growth on Harth-Chu, E. Especio R. T., Christen, R., Guzman, C.A. & M.G. Höfl e (2009) Multiple- locus variable-number of tandem-repeats analysis for clonal identifi cation of Vibrio agar plates and its tendency to go into a so-called viable but parahaemolyticus isolates using capillary electrophoresis. Applied and Environmental non-culturable (VBNC) state. We adapted the approved MLVA Microbiology 75, 4079-4088 method specifi c for L. pneumophila to fi nished drinking Kahlisch, L. K. Henne, L. Groebe, J. Draheim, M.G. Höfl e & I. Brettar (2010) Molecular analysis of the bacterial drinking water community with respect to live/dead status. water. Critical was the development and application of high Water Science & Technology-WST 61.1, 9-14

Kahlisch, L. K. Henne, J. Draheim, I. Brettar & M.G. Höfl e (2010) High-resolution in situ genotyping of Legionella pneumophila populations in drinking water by Multiple-Locus Variable-Number of Tandem Repeat Analysis (MLVA) using environmental DNA. Applied and Environmental Microbiology 76, 6186-6195 108 SCIENTIFIC REPORTS | Infection and Immunity | Pharmaceutical Research

05 Pharmaceutical Research

TOPIC SPEAKER | Prof. Dr. Rolf Müller | Research Group Microbial Drugs | [email protected] | Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS) | Department of Microbial Natural Products

Infectious diseases are one of the most important causes of death worldwide and contribute considerably to the need for clini- cal treatment. Strong interdisciplinary collaboration in anti-infective research is necessary to translate hits from initial stages of the drug development process to drugs in the clinic (“bench to bedside”) and to cope with the increasing antibiotic resistance of pathogens. Isolation, identifi cation and optimisation of small molecules interfering with processes involved in infectious diseas- es and modelling of the molecular interaction is crucial for understanding the infection process. The knowledge of the transport mechanism and the drug-target interaction mechanism is required to optimise the impact of the drug while reducing unwanted side effects. Most of the commercial drugs currently in use are derived from natural products or small molecules mimicking their pharmacophore. The topic “Pharmaceutical Research” investigates these compounds with chemical biology approaches in order to provide new classes of sustainable anti-infectives to the drug development pipeline.

The research topic was newly created after the establishment of the “Helmholtz-Institute for Pharmaceutical Research Saarland” (HIPS) in late summer 2009. It was founded to harvest the potential of a fostered interaction between the HZI and the Department of Pharmacy at Saarland University (UdS). The incorporation of HIPS adds pharmaceutical sciences to the Health research pro- gramme of the Helmholtz Association. As translational research was identifi ed as one of the most important tasks for future health research, the foundation of HIPS is a signifi cant contribution to close the translational gap in natural product based anti-infective research.

The topic is composed of seven groups working at both sites, in Braunschweig (HZI) and Saarbrücken (HIPS). The three depart- ments at HIPS, namely “Microbial Natural Products” (MINS), “Drug Development and Optimization” (DDOP) and “Drug Delivery” (DDEL) now commit their respective pharmaceutical expertise to the fi eld of infectious diseases. While MINS has traditionally strong ties with the group “Microbial Diversity and Natural Product Discovery” (MWIS) at HZI, DDOP and DDEL have built up colla- borations with HZI groups, which already lead to the attraction of third party funding and exchange of scientists. The HIPS depart- ments are presented separately within this report.

The group “Chemical Biology of Infectious Diseases” (CBIO) as well as the groups “Medicinal Chemistry” (MCH) and “Identifi cation of Molecular Targets of Anti-infectives”(BISA) were integrated into the topic “Pharmaceutical Research” because they engage in highly overlapping activities: CBIO uses various chemical biology approaches to characterise the activity and the mode of action of natural products. MCH uses medicinal chemistry and synthetic approaches to optimise these natural products as lead structures and make them available in signifi cant quantities. BISA synergistically works on the identifi cation of antifungal compounds and on deciphering their mode of action.

The group “Chemical Biology of Infectious Diseases” (CBIO) at HZI focuses on the elucidation of molecular mechanisms of infec- tious processes by low-molecular drugs. They develop new, miniaturize and run existing activity assays to identify lead struc- tures and elucidate the mode of action of hits from compound libraries. These libraries consist of approx. 90,000 compounds with unique collections of natural products from myxobacteria, compounds provided by cooperation partners, commercially available libraries as well as substances synthesized in-house. CBIO has developed a semi-automated microscopic assay that provides hints on the mode of action from the changes in the phenotype, coupled with statistical analysis of huge data sets. By software-based photo analysis of the cells, a biological profi le of the compounds is generated. In a hierarchical cluster analysis, new substances group with known compounds and similar profi les. The procedure could be validated and already gave valuable hints on new modes of action and potential new drugs, which will be further investigated. SCIENTIFIC REPORTS | Infection and Immunity | Pharmaceutical Research 109

In another project, a high-throughput microscopic adhesion assay was established to investigate the interaction of the pathogenic bacterium Staphylococcus aureus with human epithelia cells. After screening of 4,000 substances and mixtures, new promising compounds could be identifi ed which will now be tested in further studies: with human nasale primary cells on their effi ciency and in cell-based assays on their toxicity.

The group “Identifi cation of Molecular Targets of Anti-Infectives” (BISA) at HZI deals with commensal and persistent microorgan- isms. These microorganisms like Candida albicans and Mycobacterium bovis (BCG) are adopted to survive in presence of human cells and are only causing infections if the immune defence of the human is weakened. To identify new sites of action for anti-in- fectives, BISA simulates the conditions found in humans to screen potential sources for new drugs like libraries of chemical com- pounds, extracts from myxobacteria and commercially available inhibitors. One project investigates, if the elimination of C. albi- cans by phagocytes can be infl uenced by the treatment with kinase inhibitors. It could indeed be shown that all steps relevant for the course of the infection can be controlled by chemical substances that in part are formed even by the organism itself, like Gen- istein from soya. Another project shows that the histidine kinase CaNik1 of C. albicans is the site of action for fungicides. Unex- pectedly, CaNik1 causes a change in the metabolism but not in growth inhibition. The deletion of several HAMP domains in CaNik1 affects the sensitivity of the fungicides depending on the specifi c compound.

The group “Medicinal Chemistry” (MCH) at HZI deals with syntheses of anti-infectives as well as derivatives and analogues thereof to optimise the biological profi le and to get insights into the structure-activity relationships. Recently, the absolute confi guration of the natural product Chondramid C could be confi rmed. On the route to Corallopyronin the closely related natural product Myxopy- ronin could be synthesised by a stereoselective and convergent strategy. The successful synthesis of Chivosazol confi rms the pro- posal for the assignment of 10 stereo centres. The key step is an unusual Stille coupling for the formation of the macrocycle.

The research topic “Pharmaceutical Research” further develops the close interaction of the group “Microbial Diversity and Natural Product Discovery” (MWIS) at HZI and the department “Microbial Natural Products” (MINS) at HIPS to further utilise the potential of gliding bacteria for the production of natural products. Methods of classical microbiology, functional genomics, secondary me- tabolomics, advanced screening, and structure elucidation are being employed and further developed. In addition, novel assays for interference with bacterial cell-cell communication are developed. MWIS collaborates with CBIO, MCH and BISA to extend the chemical and biological profi le of strains from their rich collection of myxobacteria.

By staining the nuclei of the epithelial cell line A549 with DAPI (blue) and the bacteria with fl uorescent antibodies (green), the adhesion can be visualized. After taking pictures with the automatic microscope (A + B), a software module allows the quantifi cation of the nuclei and the adherent bacteria. If a substance reduces the bacterial adhesion, the effect can be quantifi ed. Picture B shows the result after incubation of the cell line with the bacteria in the presence of compound HZI-MW195 at 5 μM concentration in comparison to the control A without added substance. The concentration dependence of the effect is shown in fi gure C (see page 112). 110 SCIENTIFIC REPORTS | Infection and Immunity | Pharmaceutical Research

05.1 Microbial Diversity and Natural Product Discovery

PROJECT LEADER | Prof. Dr. Rolf Müller | Research Group Microbial Drugs | [email protected]

PROJECT MEMBERS | Dr. Klaus Gerth | Dr. Herbert Irschik | Dr. Rolf Jansen | Wolfgang Kessler | Dr. Kathrin Mohr | Heinrich Steinmetz

Since the closure of the department of natural product research in 2006, the new group “Microbial Drugs” is focus- ing on the discovery and characterization of novel bioactive compounds from gliding bacteria. Since 2009, it concentrates on the reactivation, conservation, and screening of our strain collection. Elansolid A1 Elansolid A1 and A2 Elansolid A2

Strain collection: For the fi rst time we got a general idea on the status of the complete strain collection of gliding bacteria Fermentation: Two major tasks are covered by the fer- at HZI. We developed strategies how to improve the value of mentation facility, provision of raw material for structure the collection and at the same time reduce the number of elucidation and all other requests from the chemical pipeline strains. So far we saved about 700 strains and prepared fresh and external partners on one hand and the improvement of culture extracts which were analysed by modern techniques fermentation processes on the other hand. of HPLC-UV-HRMS. We isolated DNA for genome sequencing and the search for silent PKS gene clusters and then charac- Basic studies on the improvement of the fermentation pro- terized the isolates physiologically. cess were done with Chitinophaga Fx GBF13, the producer of elansolids. The infl uence of different carbon- and nitrogen Natural product biology: About 50 strains of myxobacteria sources, of different pH values of the culture broth and the have been isolated from fresh soil samples and screened. effect of oxygen limitations on elansolid A1 production were Based on morphology and 16S rDNA analysis, one of these studied systematically. strains could not be classifi ed to any known group of myxo- bacteria. First inhibition tests against different bacteria, yeasts and fungi revealed a strong unknown antimicrobial and fungicidal activity. New groups of myxobacteria hold a good potential for the discovery of new secondary metabolites.

Natural product chemistry: Gephyronic acid was charac- terized as a selective inhibitor of eukaryotic protein syn- thesis with a nanomolar cytostatic effect against a range of mammalian cell lines. Using new MS data and chemical derivatisation the structure was revised and the absolute confi guration of all asymmetric centres was determined.

Photo: HZI Parallel fermentations with Fx GBF 13 at different pO2.

1. Jansen, R., Irschik, H., Huch, V., Schummer, D., Steinmetz, H., Bock, M., Schmidt, T., Kirschning, A., & Müller, R. (2010) Carolacton -a macrolide ketocarbonic acid reducing biofi lm by the caries- and endocarditis-associated bacterium Streptococcus mutans. European Journal of Organic Chemistry 7, 1284.

2. Buntin, K., Irschik, H., Weissman, K. J., Luxenburger, E., Blöcker, H., & Müller, R. (2010) Biosynthesis of Thuggacins in Myxobacteria: Comparative cluster analysis reveals basis for natural product structural diversity. Chemistry & Biology 17, 342.

Development of an anhydrous isolation procedure for a novel 3. Irschik, H., Kopp, M., Weissman, K. J., Buntin, K., Piel, J., & Müller, R. (2010) Analysis elansolid A isomer surprisingly revealed a common precursor of the sorangicin gene cluster reinforces the utitlity of a combined hylogenetic/ retrobiosynthetic analysis for deciphering natural product assembly by transATPKS. of the atropisomers elansolids A1 and A2. Genetic studies ChemBioChem 11, 1840. now are expected to show which elansolid A isomer really 4. Nawrath, T., Gerth, K., Müller, R., & Schulz, S. (2010) The biosynthesis of the aroma volatile 2-methyltetrahydrothiophen-3-one in the bacterium Chitinophaga Fx 7914. represents the fi nal product of the biosynthesis. ChemBioChem 11, 1014.

5. Menche, D., & Irschik, H. (2010). Design, synthesis and biological evaluation of sim- plifi ed analogues of the RNA polymerase inhibitor etnangien. Bioorganic & Medicinal Chemistry Letters 20, 939.

6 . B ü l o w , L . , N i c k e l e i t , I . , G i r b i g , A . - K . , B r o d m a n n , T . , R e n t s c h , A . , E g g e r t , U . , S a s s e , F., Steinmetz, H., Frank, R., Carlomagno, T., Malek, N.P., & Kalesse, M. (2010).Synthesis and biological characterization of argyrin F. ChemMedChem 5, 832. SCIENTIFIC REPORTS | Infection and Immunity | Pharmaceutical Research 111

05.2 Medicinal Chemistry of Natural Products

PROJECT LEADER | Prof. Dr. Markus Kalesse | Department of Medicinal Chemistry | [email protected] | [email protected]

PROJECT MEMBERS | Christina Brünjes | Dr. Jutta Niggemann | Dr. Evgeny Prusov | Tang Wufeng

In the frame of this project, synthetic routes will be New projects which have begun are the syntheses of developed to provide new antibiotics. This will enable the pellasoren and angiolam. For both syntheses, the central construction of derivatives and analogues for improving the transformation should be an asymmetric protonation of an biological profi le of the lead structure as well as allowing aldehyde-enolate. conclusions about structure activity relationships to be drawn.

In this context, through synthesis the confi guration of the chonramides could be unambiguously assigned. In addition, through the synthesis of the four diastereomers in the polyketide segment of this natural product the contribution of these segments for its biological activity could be made. It was shown that the polyketide segment of this natural product is the structural element, adjusting the peptide por- tion of the molecule so that the optimal distance and torsion angle between the two ends of the peptide can be attained. Infl uence of chondramide C (30) on the actin cytoskeleton In the case of corallopyronin, a similar approach will be of A-498 kidney cancer after different incubation times. A,B: used. The fi rst task is to develop a stereoselective and control cells with a dividing cell in the centre, in metaphase (A), convergent synthesis to access the natural product. Initially, and in telophase (B). Cells that were incubated with chondra- the related natural product myxopyronin was synthesised. mide (100 ng/ml) showed abnormal metaphase cells (C, after 2 Through this synthesis we were able to successfully ad- hours), and a strengthened contractile ring in late telophase (E, dress the two main challenging aspects in the myxopyronin after 18 hours). Spot of F-actin became visible especially at focal structure and we are now in the process of transferring adhesion points (D, after 4 hours), stress fi bers became stronger these results to the synthesis of corallopyronin and its and fl akes of actin appeared (E and F, after 18 hours). (Kalesse derivatives. In comparison to myxopyronin, corallopyronin et al (2008) Angewandte Chemie International Edition 47, has an alternative side-chain which has also been synthe- 6478-6482) sised, and now the two segments need to be coupled.

In the synthesis of chloronitril derivatives it was necessary to make water soluble analogues. We envisioned that the decalin part of the molecule could be replaced by a simple double bond. Unexpected diffi culties were experienced in this synthesis and, therefore, to date, no derivatives have been obtained. This project could be taken further with an alternative synthesis concept.

The synthesis of chivasozole was successfully completed this year and confi rmed our structure proposal and the chirality at the 10 stereocentres. The convergent synthesis is characterised by an unusual Stille reaction which was used as the macrolactonisation step.

Brodmann, T., Janssen, D., & Kalesse, M. (2010) Total synthesis of chivosazole F. Journal of American Chemical Society 132, 13610-13611.

Schäckel, R., Hinkelmann, B., Sasse, F., & Kalesse, M. (2010) The synthesis of novel disorazoles. Angewandte Chemie 122, 1663-1666; Angewandte Chemie International Edition 49, 1619-1622.

Bülow, F.L., Nickeleit, I., Girbig, A.-K., Brodmann, T., Rentsch, A., Eggert, U., Sasse, f., Steinmetz, H., Frank, R., Carlomagno, T., Malek, N.P., & Kalesse, M. (2010) Synthesis and biological characterization of argyrin. ChemMedChem 5, 832-836. 112 SCIENTIFIC REPORTS | Infection and Immunity | Pharmaceutical Research

05.3 Chemical Biology of Infectious Disease

PROJECT LEADERS | Dr. Ronald Frank | Department of Chemical Biology | [email protected] Dr. Florenz Sasse | Department of Chemical Biology | [email protected]

PROJECT MEMBERS | Jihad Al-Qudsi | Ulrike Beutling | Randi Diestel | Dr. Michelle Fountain | Dr. Raimo Franke | Dr. Bernd Hofer | Denis Koska | Michael Mrosek | Dr. Irina Nickeleit | Dr. Mahtab Nourbakhsh | Dr. Marc Reboll | Saad Shaaban | Galina Sergeev | Dennis Schwab | Dr. Dr. Werner Tegge | Dr. Peter Washausen | Marina Wöhl

The aim of the project is to elucidate the molecular mecha- complex biochemical network of a target cell. This can lead nisms of infection processes by interfering small molecules. to striking changes in the morphology of the cell and the Special assay systems and screening techniques are devel- cell organelles or in the protein pattern. These phenotypical oped to select bioactive substances out of chemical libraries changes can be visualised via antibodies or specifi c dyes. whose mode of action will be further analysed. These Comparing phenotypes is one way to get hints about the analyses should lead to new antibiotics, chemotherapeutics, mode of action. Now we have strengthened this method and immune modulators. The knowledge about their mode by automated microscopy and statistical analysis of large of actions will open new ways of therapy. Within the frame- amounts of data. By automated microscopy we can take a lot work of the „Chemical Pipeline“, we built an infrastructure of pictures of treated cells, evaluate them by software-based that allows a rapid and effective search in miniaturised image analysis, and form a profi le of the biologically active assay systems. Our library of about 90,000 samples includes compound. By comparing the profi les of known and un- a unique collection of natural compounds isolated from known compounds we get hints about the mode of action of myxobacteria (see 05.1), substances from collaboration the new substance. This is done by software-based statisti- partners, purchased compound collections, and substances cal methods, e.g., hierarchical cluster analysis. Substances, that were synthesised by us. The infrastructure is also at which have the same mode of action, group together then. the disposal of external scientists and to other applications In the meantime we have also got useful hints concern- via the German ChemBioNet (www.chembionet.de). ing the mode of action of new substances. First follow-up experiments confi rmed the fi rst hints. Phenotypes characterise the mode of action There is no easy way to elucidate the mode of action of a biologi- Inhibitors of the adherence of Staphylococcus aureus to cally active compound. Each biologically active compound human epithelial cells In the context of the BMBF funded changes the metabolic and signalling pathways within the consortium “SkinStaph” a high-throughput amenable adhe- sion assay was established, which provides the possibility to search for new inhibitors of the interaction between the pathogenic bacterium Staphylococcus aureus and human epithelial cells (see Fig. page 109). Highly reliable data is obtained by a newly developed method that employs an automated microscope. Epithelial cells are incubated with the bacteria in the presence of potentially interfering com- pounds, followed by the removal of non-adherent bacteria and the staining of the cells. The nuclei of the epithelial cells are stained with a DNA specifi c dye (DAPI) and the bacteria are labelled with specifi c fl uorescent antibodies. Several pictures for every compound are taken with the auto mated microscope. With a software module the nuclei and the bacteria are automatically quantifi ed and set into relation. This allows the identifi cation of substances that reduce the adhesion of the bacteria. After screening more than 4,000 individual substances and mixtures, several promising compounds were identifi ed. These drug candi- Biologically active substances induce phenotypical changes in dates are currently investigated with primary human nasal treated cells which are characteristic for their mode of action. cells and their toxicity is determined with cell-based assays. This is shown for chivosazol A. The green actin fi lament struc- tures of the treated cells in B clearly differ from the control cells in A. In C we see a hierarchical cluster analysis of substances from myxobacteria. D gives a detail which shows that chivosazol A clusters together with a second sample that was given “blind”, Diestel, R., Irschik, H., Jansen, R., Khalil, M.W., Reichenbach, H., & Sasse, F. (2009) Chivosazole A and F, cytostatic macrolides from Myxobacteria, interfere with actin. a clear proof of principle. ChemBioChem 10, 2900-2903. SCIENTIFIC REPORTS | Infection and Immunity | Pharmaceutical Research 113

05.4 Identifi cation of Molecular Targets of Antiinfectives

PROJECT LEADER | Prof. Dr. Ursula Bilitewski | Research Group Biological Systems Analysis | [email protected]

PROJECT MEMBERS | Dr. Nina Klippel | Dr. Bianca Lüderitz | Anna Buschart | Shuna Cui | Mohammed El-Mowafy | Daniela Evers | Katja Gremmer | Rabeay Hassan | Hani Kaba | Carolin Lewark | Dörthe Sokolis

New therapies of infections require new antimicrobial compounds, which exploit new molecular targets to avoid already existing antibiotic resistances. We focused on com- mensal and persistent microorganisms, and chose the yeast Candida albicans and the bacteria Mycobacterium bovis (BCG) as substitute of the tuberculosis pathogen Mycobacte- rium tuberculosis. These microorganisms are adapted to the survival in the presence of host cells, and become infectious in patients with an impaired immune system.

Histidine kinases as molecular targets of anti-infectives Histidine kinases are mainly found in bacteria, but also in fungi. Mostly they are sensor proteins and induce the adaptation of the microorganisms to their environment. We had previously shown that the histidine kinase CaNik1 from Candida albicans is the molecular target of the myxobacte- rial secondary metabolites ambruticin VS3 and jerangolid The interaction between the yeast Candida albicans and cells A, as we transferred sensitivity for these fungicides to the of the epithelium (cell line A431) in the presence of the peptide otherwise resistant yeast S. cerevisiae by heterologous LL37. LL37 belongs to the anti-microbial peptides and does not functional expression of the CaNik1 gene. We identifi ed a inhibit the growth of C. albicans, but it supports the adhesion pairwise sequence of 9 so-called HAMP-domains, each of of the yeast to human cells. After an incubation time of 6 hours which comprises approximately 50 amino acids. Deletion of (conditions of cell cultures), C. albicans forms hyphen which selected pairs of these domains from CaNik1 reduced the can be found in the cells of the epithelium as well, where they sensitivity for fungicides, with the magnitude of the effect also continue to grow. Photo: HZI, Rohde being dependent on the compound. At present investigations are performed to elucidate further details of the interaction of the protein with fungicides. Gene expression analysis showed that in the S. cerevisiae-transformants fungicides, block the morphology change of C. albicans and the survival which target CaNik1, mainly activate the signal transduc- of dormant mycobacteria. Sources of those compounds are tion pathway of the osmotic stress defense network, which libraries of chemical compounds, extracts from myxobac- includes effects on cell cycle and thus on cell proliferation. teria and commercially available inhibitors. We could show that all steps of the infectious process can be infl uenced Exploitation of new targets for anti-infectives Candida by chemical compounds, some of which are endogenous albicans usually colonizes human skin and mucosa (mouth, products of the organisms. Human cells produce peptides, gastro-intestinal tract, vagina). Systemic infections which manipulate the adhesion of C. albicans to surfaces, in include penetration of the underlying cell layers, followed particular to components of the extracellular matrix. The by invasion of deeper tissues and spread to inner organs formation of hyphae is suppressed by compounds which are via the bloodstream. An essential pathogenicity factor is formed by C. albicans as quorum sensing compounds. Finally, the capability to switch morphology between yeast and we discovered that genistein, a well-known constituent of hyphal forms. Among primary immune defense reactions soybeans, stimulates phagocytosis of genistein-treated is phagocytosis and stimulation of the immune system by C. albicans. Further studies aim at the elucidation of under- activation of macrophages and neutrophils. Macrophages lying molecular mechanisms. are also involved in the immune defense against infections of the lung by mycobacteria. However, these bacteria can survive phagocytosis as persistent, dormant organisms and Wesolowski,J., Hassan,R.Y.A., Reinhardt,K., Hodde,S. & Bilitewski,U. (2010) Antifungal are hardly eliminated by present antibiotics. compounds redirect metabolic pathways in yeasts: Metabolites as indicators of modes of action, Journal of Applied Microbiology 108, 462 - 471

Klippel,N., Cui,S., Gröbe,L. & Bilitewski,U. (2010) Deletion of the Candida albicans hi- To empirically exploit new targets for anti-infectives, we stidine kinase gene CHK1 improves recognition by phagocytes through an increased simulate environmental conditions, which the microorgan- exposure of cell wall ß-glucans, Microbiology 156, 3432 – 3431 isms face in the human host. We test whether small mol- Buschart,A., Gremmer,K., v.d.Heuvel,J. Müller,P.P. & Bilitewski,U. (2011) Repetitive HAMP domains at the N-terminus of the CaNik1 histidine kinase of Candida albicans ecules can enhance the clearing effi ciency of phagocytes, mediate specifi c interactions with fungicides, Molecular Microbiology, submitted 114 SCIENTIFIC REPORTS | Infection and Immunity | New Project Groups

New Project Groups

The HZI has given young researchers with very good ideas the possibilities to do their research at the HZI facilities when their scope has been within the forthcoming R&D developments of the Centre. Since 2009 further new projects groups have started their work at the HZI. During the next 5 years they will try to establish new research ways and present excellent results. The fi r s t results and further ideas of this 2nd generation of new R&D groups are presented on the next pages.

The platform in the Gründerzentrum of the HZI, where new project groups have the possibilty of presenting their research results. The photo was taken during a visit of Nobel Laureate Prof. Dr. Kurt Wütherich (La Jolla, USA / Zürich, Switzerland) (left side at the fi rst poster) in 2010. Photo: HZI SCIENTIFIC REPORTS | Infection and Immunity | New Project Groups 115

01 Regulation and Herpesviral Immune Modulation of Toll-like Receptor Signalling

PROJECT LEADER | Dr. Melanie M. Brinkmann | Junior Research Group Viral Immune Modulation | [email protected]

PROJECT MEMBERS | Dr. Bettina Gruhne | Elisa Reimer

Herpesviruses Herpesviruses establish lifelong persist- ing infections. Whereas immunocompetent individuals are able to control herpesviral infections immunocompromised persons such as transplant recipients or AIDS patients can encounter severe medical problems. An infection with the betaherpesvirus cytomegalovirus (CMV) during pregnancy is the most prevalent cause for virus-induced abnormalities of the fetus. Further, the human gammaherpesviruses Epstein- Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvi- rus (KSHV) can cause tumours. In order to develop improved prophylactic and strategic therapies it is crucial to improve our understanding how herpesviruses are recognized by the host’s immune system and how herpesviruses modulate the immune response to avoid their detection and elimination. TLR9 localizes to the endolysosomal compartment after TLR agonist stimulation. Primary bone marrow derived macro- Innate Immune System The innate immune system allows phages were isolated from TLR9-GFP transgenic mice. Live an early and potent antiviral response and plays an impor- cells were stimulated with LPS and analysed with a spinning tant role for the induction of the adaptive immune response. disc confocal microscope in real-time. Endolysosomes were One group of pathogen sensors of the innate immune system visualised with lysotracker (in red). Copyright Melanie M. Brinkmann are members of the family of Toll-like receptors (TLRs). TLR9 senses herpesviral DNA and induces a potent antiviral inter- feron response. The cellular UNC93B protein is essential for is known about the regulation of intracellular traffi cking in proper functioning of TLR9: it delivers TLR9 from the endo- infected antigen presenting cells. plasmic reticulum, where it is synthesized and stored until needed, to a specialized intracellular compartment called The Project Our research focuses on the mechanisms the endosome. There TLR9 meets the DNA of the invading herpesviruses evolved to modulate their recognition by the pathogen and initiates a signalling cascade that results in host’s immune system, thereby allowing them to establish the activation of an antiviral response. Whereas the down- chronic infections. We have evidence that herpesviruses can stream signalling cascade of TLRs is well characterized little modulate the antiviral response initiated by TLRs. We aim to identify the responsible herpesviral genes and characterize their mechanism of action. We further study the regula- tion of intracellular traffi cking of TLR9 upon infection with herpesviruses. Using fl uorescently labelled virions and fl uorescently labelled TLR9 and UNC93B proteins, the group will analyse intracellular traffi cking in real-time in primary antigen presenting cells by confocal lasermicroscopy. In a biochemical approach, using herpesviral DNA as bait, we aim to identify further DNA sensors that are crucial for the detection of herpesviruses. Furthermore, we identifi ed novel interacting partners of the UNC93B protein and will charac- terize their role for traffi cking of the UNC93B-TLR9 complex The membrane protein UNC93B interacts with Toll-like recep- from the endoplasmic reticulum to the endolysosomal com- tor 9 (TLR9) in the endoplasmic reticulum and delivers it to partment where TLR9 signalling is initiated. the endolysosomal compartment where TLR9 meets DNA of invading pathogens. Upon arrival in the endolysosome, TLR9 Boyoun Park, Melanie M. Brinkmann, Eric Spooner, Clarissa C. Lee, You-Me Kim and Hidde L. Ploegh, (2008) Nature Immunology, 9 (12), 1407-1414 undergoes proteolytic cleavage which is the prerequisite for You-Me Kim, Melanie M. Brinkmann, Marie-Eve Paquet and Hidde L. Ploegh (2008) signalling and the induction of an antiviral response. Nature, 452 (7184), 234-238 Copyright Melanie M. Brinkmann Melanie M. Brinkmann, Eric Spooner, Kasper Hoebe, Bruce Beutler, Hidde L. Ploegh and You-Me Kim, (2007) Journal of Cell Biology, 177 (2), 265-275 116 SCIENTIFIC REPORTS | Infection and Immunity | New Project Groups

02 Systems Immunology

PROJECT LEADER | Prof. Dr. Michael Meyer-Hermann | Department of Systems Immunology | [email protected]

PROJECT MEMBERS | Dr. Marc Thilo Figge | Dr. Valerii Sukhorukov | Dr. Arndt Telschow | Sebastian Binder | Jaber Dehghany | Harald Kempf

Systems Immunology What is Systems Biology and why do of GC. We tested in silico with the aforementioned model we need it? It is not possible to understand the functionality how disruption of TLR4-signalling would change the GC of a system by just knowing its constituents, as derived from and predicted that the mutation rate has to be reduced. This „omics“-approaches. The major question is how all these prediction was confi rmed in experiment. [3] constituents interact with each other and how they give rise to a meaningful and functional concept like an organism. Directions The novel department Systems Immunology The key to this question is the dynamics of the system under at the HZI will work on models of dynamic systems in the investigation. Systems Immunology is the science of develop- immune system and in the fi eld of infection and infl amma- ing mathematical models of the dynamic behaviour and tion. In the coming year pre-established research fi elds, like interaction of complex systems related to health. The future modelling of adaptive immune responses and of pancreatic of research in Biology will rely on the iterative discussion of betacells in the context of diabetes, will be continued and it theoretical prediction and experimental result, inspired by is planned to develop new collaborations, in particular, with the success of Physics. emphasis on chronic infl ammatory diseases.

Methodology As it is impossible to understand the mecha- nisms of a biological system from the knowledge of its constituents alone, we are restricted to start from phenom- enological descriptions of the whole system. These should be as simple as possible and as complex as needed to allow comparison with available experimental data. In an iterative optimisation process the model assumptions are evolved up to the point that the in silico model behaves as the in vivo model. If theory and experiment disagree, research may take advantage of Systems Immunology: Either the model has to be extended or an assumption in the model has to be recon- sidered. In both cases the knowledge of how the biological system works is improved.

The advantage of such a phenomenological approach is that at any level of mathematical description reached so far, the in silico model remains functional. Ultimately this approach based on an increasingly complex picture of the biological system will enable to (i) design new experiments that are conclusive for specifi c questions, (ii) predict the outcome of planned experiments in order to verify the consistency of the In silico agent-based simulation of a germinal centre reaction. current knowledge, and (iii) predict optimal treatment strate- A 20 micron thick slice of the germinal centre at day 5 after gies in deregulated systems. onset of proliferation. Every object represents a cell: Dividing and mutating BC (blue), non-dividing Ig-expressing BC (dark Example generation of specifi c antibodies The immune green), BC with positive signals from FDC (light green), plas- system contains a specifi c environment, the germinal centre ma cells (white), T follicular helper cells (red), FDC (yellow) (GC), in which B lymphocytes (BC) are induced to mutate where dendrites are not shown. The cells interact according their antibody encoding genes. Subsequently, mutated BC rules derived from present knowledge and move in response undergo a selection process in order to either produce high- to chemokines (not shown) and in agreement with recent in- affi nity antibodies or to contribute to the immune memory. travital two-photon imaging data. Previously published in [4].

The general conviction was that selection is controlled by the availability of antigen. We have developed a mathemati- [1] Meyer-Hermann, M., Figge, M.T., & Toellner, K.M. (2009) Trends in Immunology 30, 157-164. cal model for the GC dynamics and predicted that it is not [2] Victora, G.D., Schwickert, T.A., Fooksman, D.R., Meyer-Hermann, M., Dustin, M.L., & Nussenzweig, M.C. (2010) Cell 143, 592-605. the antigen but affi nity dependent TC help that is limiting [3] Garin1, A., Meyer-Hermann1, M., Contie, M., Figge, M.T., Buatois, V., Gunzer, M., Toellner, [1]. This prediction was verifi ed in a series of experiments K.-M., Elson, G., & Kosco-Vilbois, M.H. (2010) Immunity 33, 84-95. 1: shared fi rst author. [2] and, thus, a new concept of the GC reaction was induced. [4] Figge, M.T., Garin, A., Gunzer, M., Kosco-Vilbois, M., Toellner, K.-M., & Meyer-Hermann, Also toll-like-receptor-4 (TLR4) impacts on the development M. (2008). Journal of Experimental Medicine 205, 3019-3029. SCIENTIFIC REPORTS | Infection and Immunity | New Project Groups 117

03 The National (“Helmholtz”) Cohort

PROJECT LEADER | Priv.-Doz. Dr. Frank Pessler | Department of Infection Genetics | [email protected]

PROJECT MEMBERS | Dr. Manas Akmatov | Matthias Preuße

Prospective cohort studies The rise of chronic diseases The HZI is and will be contributing to the Cohort as follows: such as cancer, atherosclerosis, diabetes, osteoarthritis and • By recruiting 10, 000 study participants during 2012- Alzheimer’s disease represents an ever-growing challenge 2017 as part of the Recruitment Cluster North-West to the health care systems of industrialized countries. The Germany. causes of these disorders are multi-factorial and consist of • By developing and optimizing protocols (SOPs) for collec- environmental, lifestyle and genetic risk factors. Epidemi- ting and storing biomaterials, in particular nasal swabs, ologists are presented with the challenge to develop strate- saliva, peripheral blood leukocytes, and stool. gies for the early detection of risk factors and diseases, • By initiating, developing and supporting aspects of the ideally at a time when interventions are most effective. Cohort that relate to infectious diseases and disorders of Because of their prospective character, which allows asses- immunity. Our approach will follow two tiers: fi rstly, to sing risk factors and biomarkers in the preclinical stage of evaluate microorganisms as risk factors for the develop- disease development, cohort studies are the most powerful ment of common chronic diseases such as cardiovascular tools of modern epidemiology for this purpose. and neurodegenerative diseases; secondly, to identify risk factors for the development of acute and chronic infec- The National (“Helmholtz”) Cohort The Helmholtz As so- tions and disorders of immunity. ciation has been spearheading a nationwide cohort study to • By implementing novel epidemiologic tools for the study address the health care challenges of the 21st century. Ulti- of infectious diseases and disorders of immunity. For mately, this undertaking will involve recruiting 200,000 instance, we are testing whether study participants can human volunteers throughout Germany, constructing a reliably collect their own biospecimens for diagnostic central biobank near Munich, and the follow-up of the testing (e.g., nasal swabs or stool samples) during an subjects for 10-20 years. A start-up budget of 20 million acute illness and then send it to the reference lab. In Euros from the Helmholtz Association is being used by the the same projects, we are studying the use of modern fi ve participating Helmholtz Centres to help plan and pilot communication tools such as SMS and email as reminder the study protocols for this largest health-research-related systems to maintain compliance and to obtain real-time endeavor in all of Germany’s history, to establish and equip information about symptomatic infections. study centres, and to begin recruiting and phenotyping • By evaluating and adapting novel biomarkers (e.g., micro- participants in the middle of 2012. RNAs) and biostatistical approaches.

Networking within the Cohort We represent the HZI in 2009 2012 2017 2022 2027 20+ the national Epidemiologic Planning Committee for the years? Cohort, in the writing committee for the international Planning Recruitment, Follow-up Follow-up evaluation which is anticipated in 2011, and in the Thematic & piloting; construction visit #1 visit #2 (?) feasibility of biobank Working Groups “Infection & Immunity”, “Musculoskeletal studies Follow-up Detection of incident cases of disease Disorders”, “Biobank” and “Questionnaires”. In the current feasibility phase of the Cohort we are coordinating a multi- Currently proposed time line for the planning, recruitment centre feasibility study on novel measurement instruments and follow-up phases of the Cohort. Graphic: HZI for infectious disease epidemiology.

Akmatov, M.K. & Pessler F. (2011) The potential of self-collected nasal swabs to detect infection and colonization in population-based epidemiological studies. International Journal of Infectious Diseases (accepted for publication).

Akmatov, M.K., Pessler, F. & Brzoska P. (2011) Attitudes among women in low- and middle-income countries towards people living with HIV/AIDS – the situation after three decades of AIDS. BMC Public Health (under review).

Ogdie, A., Li, J., Dai, L., Yu, X., Diaz-Torne, C., Akmatov, M., Schumacher, H.R., & Pessler, F. (2010) Identifi cation of broadly discriminatory synovial tissue biomarkers with binary and multicategory receiver operating characteristic analysis. Biomarkers 15(2), 183-90.

Slansky, E., Li, J., Häupl, T., Morawietz, L., Krenn, V., & Pessler, F. (2010) Quantitative determination of the diagnostic accuracy of the synovitis score and its components. Histopathology 57(3), 436-443. 118 SCIENTIFIC REPORTS | Infection and Immunity | New Project Groups

04 Immune Aging

PROJECT LEADER | Dr. Dr. Luka Cicin-Sain | Junior Research Group Aging and Chronic Infections | [email protected]

PROJECT MEMBERS | Dr. Linda Ebermann | Iryna Dekhtiarenko | Anja Drabig | Thomas Marandu | Ilona Paprotta | Franziska Schreiner

The loss of immunological function caused by the aging Our preliminary data show that mice carrying latent CMV process is termed immune senescence. There is a consensus exhibit weaker CD8 responses to superinfections with that the adaptive immune response to emerging infections is emerging pathogens, such as Infl uenza Virus, than control compromised in the elderly. Naïve lymphocytes can recog- (uninfected) animals. Therefore, chronic CMV infection may nize a broad and divergent array of potential pathogens contribute to the immune aging process. Our group aims to because they are clonally diverse. Aging results in a loss defi ne the effects of persistent virus infections on the host of naïve T-cells and of T-cell clonal diversity, increasing the immune system, by addressing two specifi c objectives: I. elu- susceptibility to novel infections and decreasing the ability cidate the cellular and molecular mechanisms of the induc- to generate protective immunity upon a vaccination. There- tion of the adaptive immune system by CMV infection, and II. fore, the loss of clonal diversity in the T-lymphocyte pool is a assess the broader biological relevance of this phenomenon. prominent change associated with immunosenescence. The causes of immune senescence remain incompletely under- I. Mechanism: It is not clear if the poor CD8 response in stood. While thymic involution plays a prominent role in the CMV infected mice is indeed caused by changes intrinsic aging process, the role of the oxidative metabolism and envi- to the CD8 subset, or by changes in their immunological ronmental stressors remain less defi ned. Understanding the environment. This question can be addressed by adoptive key environmental contributors to the aging process would transfer experiments. Moreover, it is not clear if this loss is allow us to defi ne targets for strategies delaying immune due to persistent antigenic stimulation or to infl ammation senescence. caused by the virus. This can be defi ned by recombinant viruses with conditional and/or gene-specifi c expression of Cytomegalovirus Cytomegalovirus (CMV) is a ubiquitous immunodominant T-cell Ag. pathogen, infecting the majority of the human population worldwide. CMV establishes a lifelong latent infection in II. Relevance: It is not clear if the poor immune response infected hosts, and can reactivate in immunosuppressed or relates exclusively to virus challenges of CMV infected hosts, immunodefi cient individuals. Interestingly, CMV elicits a or if this is a broader phenomenon. We will test the T-cell T-cell response that is unparalleled in its strength, which repertoire and immune function in other chronic infections, dominates the repertoire of the memory T-cell compart- and we will test if the chronic infections will result in poor ment, and where CMV-specifi c CD8 cells appear to accrue cellular immune responses to non-viral challenges, such as in older age groups. Hence, the immune system may devote challenges with (a) bacterial intracellular pathogens (e.g. a signifi cant fraction of its adaptive capacity to the control Listeria monocytogenes, Mycobacterium bovis), or (b) tumour of one single pathogen and the mechanisms leading to these antigens. uniquely strong immune responses may have consequences in our understanding of the homeostatic processes that govern the interaction between a host and its microbiological environment.

The Project Epidemiological longitudinal studies of elderly volunteers identifi ed immune-risk phenotypes that pre- dicted poor survival, and these phenotypes coincided with seropositivity to (CMV). If a ubiquitous agent, such as CMV, contributed to immune aging, this would have a huge impact on our understanding of the immune aging process in the general population. 1.Cicin-Sain, L., Bubic, I., Ruzsics, Z., Schnee, M., Mohr, C., Jonjic, S. & Koszinowski, U.H. (2007) Targeted deletion of regions rich in immune-evasive genes from the cytomegalovirus genome as a novel vaccine strategy. Journal of Virology 81(24),13825-34.

2.Cicin-Sain, L., Messaoudi, I., Park, B., Currier, N., Planer, S., Fischer, M, Tackitt, S., Nikolich-Zugich, D., Legasse, A., Axthelm, M.K., Picker, L.J., Mori, M. & Nikolich-Zugich J. (2007). Dramatic Increase in Naïve T-Cell Turnover is Linked to Loss of Naïve T-cells from Old Primates. PNAS 104(50),19960-5.

3.Cicin-Sain, L., Smyk-Pearson, S., Fisher, M.B., Currier, N., Koudelka, C., Nikolich-Zugich, D., Legasse, A., Tackit, S., Axthelm, M.K., Mori, M. Lewinsohn, D., & Nikolich-Zugich, J. (2010) Loss of naïve T-cells and repertoire constriction predict poor response to vaccination in old primates. Journal of Immunology 184(12), 6739-45. SCIENTIFIC REPORTS | Infection and Immunity | New Project Groups 119

05 Development of Novel Diagnostic and Therapeutic Tools for Tuberculosis Control and Prevention

PROJECT LEADER | Prof. Dr. Mahavir Singh | Department of Gene Regulation and Differentiation | [email protected]

PROJECT MEMBERS | Cyril Alozieuwa | Dr. Sabin Bhuju | Ayssar Elamin | Dr. Matthias Stehr | Hanaa Wanas

The aim of the project is to develop new diagnostics, thera- substances must be developed in order to prevent the further peutics and vaccines against tuberculosis and HIV. For the di- spread of drug resistant tuberculosis. agnosis and the development of a vaccine, we aim to identify new, highly specifi c antigens. The search for lead structures Identifi cation of new leads for the development of new is carried out by the screening of compound libraries against antibiotics Supported by the EU-funded project “NOPERSIST” virulence associated proteins and enzymes of the pathogen. we are identifying new lead compounds that act against two virulence associated target enzymes of the pathogen. The Tuberculosis Tuberculosis is a bacterial infectious disease. alanine dehydrogenase (AlaDH) synthesizes building blocks Every year two million people die from tuberculosis and for cell wall biosynthesis. We have developed a method, another nine million are newly infected. Africa and Asia which is capable to screen around 10,000 compounds from are the most affected regions in the world. In Germany the the HZI compound library per week. So far we have identi- number of new cases of tuberculosis is declining. However, fi ed 50 candidate inhibitors which are currently validated in about 15 years ago new, multi-resistant strains have emerged. follow-up assays for further analysis. The so-called MDR (multidrug resistant) and XDR (Extreme Drug Resistant) strains spread rapidly and can already be The second target is the thiol peroxidase (TPX), an enzyme of found worldwide. Most of the known antibiotics are not effec- the mycobacterial peroxide defense system. Currently, we are tive against MDR and XDR strains, thus new highly effective developing a high throughput assay for substance screening against the TPX enzyme.

New antigens for rapid diagnosis tools and vaccine candidates For tuberculosis control modern and rapid mo- lecular diagnostic tools are needed. Current methods take up to four weeks. The high-throughput cloning and expression allows the production of hundreds of pure mycobacterial pro- teins per week. The cellular and humoral immune response to these candidate proteins is analysed by the partners of the consortium of the EU funded project”NOPERSIST”. From the pool of candidate proteins the vaccine and diagnostic candidates will be identifi ed.

Transvac We are partners in an infrastructure project “TRANSVAC” funded by the EU. Transvac is the European platform for vaccine development for HIV, TB and malaria. We are currently performing transcriptome mapping of sam- ples from BCG vaccinated human individuals and healthy controls.

Rosenkrands, I., Aagaard, C., Weldingh, K., Brock, I., Dziegiel, M.H., Singh, M., Hoff, S., Ravn, P., & Andersen, P. (2008) Identifi cation of Rv0222 from RD4 as a novel serodiag- Schematic representation of the structure of the drug target nostic target for tuberculosis. Tuberculosis (Edinb) 88, 335-343. alanine dehydrogenase. The bound cofactor NAD+ and Ågren, D., Stehr, M., Berthold, C.L., Kapoor, S., Oehlmann, W., Singh, M., & Schneider, G. (2008) Three-dimensional structures of apo- and holo-L-alanine dehydrogenase from pyruvate are depicted as ball-and-stick. (Ågren, D., Stehr, M., Mycobacterium tuberculosis reveal conformational changes upon coenzyme binding. Berthold, C.L., Kapoor, S., Oehlmann, W., Singh, M. and Sch- Journal of Molecular Biology 377, 1161-1173. neider, G. (2008) Three-dimensional structures of apo- and Elamin, A.A., Stehr, M., Oehlmann, W., & Singh, M. (2009) The mycolyltransferase 85A, a putative drug target of Mycobacterium tuberculosis: Development of a novel assay and holo-L-alanine dehydrogenase from Mycobacterium tubercu- quantifi cation of glycolipid-status of the mycobacterial cell wall. Journal of Microbiologi- losis reveal conformational changes upon coenzyme binding. cal Methods 24, 358-363. Journal of Molecular Biology, 377, 1161-1173.) Von Groll A., Martin A., Stehr M., Singh M., Portaels F., da Silva P.E., & Palomino J. C. (2010) Fitness of Mycobacterium tuberculosis strains of the W-Beijing and Non-W-Beijing genotype. PLoS One 5, e10191. 120 SCIENTIFIC REPORTS | Infection and Immunity | New Project Groups

06 Exploring and Exploiting Microbial Proteomes

PROJECT LEADER | Prof. Dr. Katharina Riedel | Research Group Microbial Proteomics | [email protected] | [email protected]

PROJECT MEMBERS | Dr. Isabel Hartmann | Dr. Martin Kucklick | Thomas Langer | Christian Lassek

Proteomics – a powerful tool to investigate microbial pathogens Proteins are the effector molecules that medi- ate basic cellular functions and are often the direct targets of antimicrobial drugs. The analysis of the entirety of all expressed proteins of microbes (“microbial proteomics”) has been proven as an ideal tool to investigate various aspects in medical microbiology, e.g. the molecular basis of bacterial infections or the adaptation of microbes to antibiotic stresses. We employ two-dimensional polyacrylamide gel electrophore- sis, gel-free liquid chromatography, and mass spectrometry- Workfl ow of a typical proteomics experiment starting with based technologies for microbial proteome analyses. protein extraction, followed by separation via gel-based or gel- free approaches, identifi cation & quantifi cation by mass spec- Evaluation of novel anti-infectiva The emergence of drug- trometry, and data validation by complementary approaches resistant microbes has led to an urgent need of novel anti-in- (e.g. phenotypical assays). fective agents. Cell-to-cell communication systems (quorum sensing, QS), which operate in many pathogens as central regulators for the expression of virulence factors and biofi lm formation, represent attractive targets for the design of novel Metaproteome analyses of uropathogenic catheter bio- therapeutics. The major advantage of this strategy is that it fi lms Urinary tract infections are among the most common circumvents the problem of resistance, which is intimately nosocomial infections. Because of the ability of uropathogens connected to conventional antibiotics. We have developed to develop resistances during antibiotic treatment, catheter specifi c sensor strains to screen compound libraries for their biofi lms are diffi cult to eradicate and often become chronic. potential to interfere with QS of two important pathogens, P. aeruginosa as well as Escherichia coli have been reported Pseudomonas aeruginosa and Burkholderia cenocepacia. To to cause about 40% of complicated catheter-associated infec- elucidate the compounds effi cacy and specifi city compara- tions; however, recent investigations indicate that catheter tive proteome analyses are employed. Moreover, in close biofi lms are highly complex and consist of various different collaboration with Prof. J. Robinson (University of Zürich) we species. Our group is part of the UroGenOmics consortium, have recently unraveled the mode-of-action of peptidomimet- which aims on the elucidation of regulatory and metabolic ics that specifi cally inhibit P. aeruginosa. strategies during colonization and infection. We are cur- rently employing metaproteome analyses to investigate In situ proteomics to study the molecular basis of mi- the interplay between composition and function of mixed crobial infections Eighty percent of microbial infections in catheter biofi lms, to investigate temporal changes and spatial humans are mediated by biofi lms which are highly resistant distributions during the infections process, and to unravel against antimicrobial therapies. Deciphering the crucial strain-specifi c strategies to adapt to urinary tract conditions. command lines of biofi lm formation under infection-like con- The gained knowledge will help to develop new analytical ditions will therefore contribute to a better understanding of tools, to identify novel drug targets, and to improve catheter bacterial pathogens. We have established in situ proteome surface design. analyses of P. aeruginosa during colonization of non-mam- malian and mammalian pathogenicity models to identify virulence factors specifi cally expressed during the infec- tion process and to observe changes in the host proteome induced by the bacteria. A long term goal of the collaborative project with Prof. M. Givskov (University of Copenhagen) is Srinivas, N., Jetter, P., Ueberbacher, B.J., Werneburg, M., Zerbe, K., Steinmann, J., Van der to apply our in situ proteomics analyses for target validation Meijden, B., Bernardini, F., Lederer, A., Dias, R.L., Misson, P.E., Henze, H., Zumbrunn, J., Gombert, F.O., Obrecht, D., Hunziker, P., Schauer, S., Ziegler, U., Käch, A., Eberl, L., Riedel, of linked multi-drugs, which exhibit two or more antimicro- K., Demarco, S.J., Robinson, J.A. (2010). Peptidomimetic Antibiotics Target Outer-Membrane bial features. Biogenesis in Pseudomonas aeruginosa. Science 327: 1010-1013. Schneider, T., Riedel, K. (2010). Environmental proteomics: Analysis of structure and function of microbial communities. Proteomics 10: 785-798.

Riedel, K., Köthe, M., Kramer, B., Saeb, W., Gotschlich, A., Ammendola, A., Eberl, L. (2006). Computer-aided design of agents that inhibit the cep quorum sensing system of Burkholde- ria cenocepacia. Antimicrob. Agents Chemotherapy 50: 318-323. SCIENTIFIC REPORTS | PoFII Independent Research 121

POFII INDEPENDENT RESEARCH

Some projects at the HZI are indepedently performed of the PoF-programme. In this Research Report we present three projects. Ken Timmis, who is going to retire in 2011, presents fi nal results from the research of his group on “Functional genomics and niche specifi ty”.

Inari Kursula is coordinating a research group within the newly established “Centre for Structural Systems Biology” (CSSB) at the German Electron Synchrotron (DESY), Hamburg. This is a new external HZI research group after a joint venture agreement was signed in 2010. She is presenting results on “Structural biology of the cytoskeleton”.

Wolf-Dieter Schubert, until 2009 head of a research group at the HZI, presents research results which he obtained jointly with the group of Prof. Dirk Heinz fi nalizing his former activities at the HZI: “Structural analysis of the innate immune system”.

An aerial photograph of the German Electron Synchrotron, Hamburg, where Prof. Inari Kursula, HZI, is working. Photo: DESY 122 SCIENTIFIC REPORTS | PoFII Independent Research

01 Functional Genomics and Niche Specifi city

PROJECT LEADER | Prof. Dr. Kenneth N. Timmis | Research Group Environmental Microbiology | [email protected]

PROJECT MEMBERS | Dr. Sagrario Arias Rivas | Dr. Mónica Bassas Galia | Simrita Cheema | Dr. Thorben Dammeyer | Dr. Isabel Hartmann | Dr. Stefanie Libnow | Dr. Gabriella Molinari | Dr. Daniela Näther | Kateryna Selezska | Dr. Johannes Sikorski | Dr. Joao Vieira de Castro | Montri Yasawong.

The microbial biota of the human gastro-intestinal (GI) tract also analysed 8 fecal specimens from IBD patients who had has been designated an additional human organ and a major not undergone intestinal lavage. Halobacteriaceae-like se- compartment of the human “biome”, to refl ect the multitude quences were obtained from 2 of the 8 fecal samples (76 se- of diverse contributions and services it provides and the ex- quences) and from all of the lavage samples (125 sequences), tent to which it infl uences the human phenotype. Despite its the latter of which grouped into 17 phylotypes, none of which key roles in health and disease, it remains a diverse, variable, corresponded to phylotypes detected in patient samples. diffi cult to study and poorly understood entity. Current large scale metagenome projects aim to shed light on the commu- Another potential origin of halophiles in the human intestine nity composition and functional genomics of these microbial is the table salt assimilated in food. We repeated the proce- ecosystems, but tend to focus on the abundant members, dure used above with 3 table salt samples and obtained 34 which are more accessible and considered to play the more haloarchaeal sequences representing 11 phylotypes. None of important roles. these was the same as any phylotype from the lavage prepa- rations, and only one was the same as that from a patient. Infl ammatory bowel disease (IBD), encompassing the syn- dromes Crohn’s disease, ulcerative colitis and other undeter- The experimental approach used does not distinguish mined forms of colitis, are complex, multifactorial infl amma- between dead cells/free DNA and viable metabolically active tory diseases. They are known to be determined by multiple cells. Therefore we set up enrichment cultures for halophiles genetic loci and thought to triggered and/or exacerbated by from a patient biopsy. This yielded haloarchaeal sequence the microbial gut fl ora. As part of a collaborative study with types, and fl uorescence in situ hybridization analysis sub- the University Clinic Kiel (Schreiber, Ott) and the University sequently revealed viable archaeal cells in these cultures. of the Balearic Isles (Nogales, Lanfranconi) seeking microbial Thus: while Haloarchaea are continuously introduced into correlates of intestinal bowel disease, we obtained mucosal the GI tract via food, the lack of relatedness of table salt and biopsies from the infl amed parts of the sigmoid region of GI tract phylotypes found here failed to identify either table the colon of patients suffering from diverse forms of IBD. salt or lavage solutions as a source of GI tract phylotypes. We established 16S ribosomal RNA gene clone libraries, sequenced the clones, and determined their phylogenetic These results point to the possibility that haloarchaea are assignments. Though bacteria dominate the colonic micro- functional members of the human colonic biota, which would bial community, archaeal methanogens are minor members. seem to be counter-intuitive, given that this environment is Therefore, in addition to creating libraries of bacterial 16S not considered to be particularly salty. The colon environ- rRNA gene sequences, we also created libraries of archaeal ment is however rather heterogeneous: while the intestinal 16S rRNA genes obtained by low-stringency amplifi cation us- contents of healthy individuals have an average salinity simi- ing archaeal domain primers. Surprisingly, this yielded from lar to that of plasma (135–145 mM sodium), small pockets of a number of biopsies, in addition to expected sequences concentrated luminal ions (reported to be in the order of 15 of methanogens, sequences of members of the Halobacte- mM above that of the surrounding fl uids) may form within riaceae. This was unexpected not only because the GI tract the lateral intercellular spaces (LIS) and crypts as part of is not considered to be a salty environment and had not the normal processes of fl uid transport in the intestinal previously been reported to harbour halophilic archaea, but epithelium. Regions like the LIS may thus provide suitable also because of the high diversity and novelty of the cloned micro-niches favourable for colonization by haloarchaea. If sequences: 269 Halobacteriaceae-related sequences obtained the haloarchaea discovered here in colonic biopsies are from the biopsies grouped into 15 distinct phylotypes (which indeed active, then their metabolism and products may have covered almost the entire phylogenetic range of this family), signifi cant consequences for mucosal physiology in general, 9 of which were new. and the diseased bowel, in particular, and vice versa – as has been previously suggested for methanogens in conditions Colonoscopy requires prior intestinal lavage with a solution of colorectal cancer and diverticulosis. In this regard, it is containing polyethylene glycol and salts, so one possibility worth noting that altered bowel physiologies are a prominent was that the haloarchaeal sequences originated from the feature of IBD. Impaired absorption of sodium and organic lavage solution. We obtained two unopened lavage prepara- solutes, like short-chain fatty acids such as butyrate, result tions from the hospital in which the biopsies were collected in increased solute concentrations and osmolarity in the and, for comparison, one from the same manufacturer from a lumen. These results suggest that investigation of a potential pharmacy in Palma, Mallorca, and one from a different manu- physiological basis of the haloarchaeal-intestinal mucosal as- facturer from the same pharmacy, and repeated the DNA sociation may lead to new insights into GI health and disease. isolation, amplifi cation and sequencing of library clones. We SCIENTIFIC REPORTS | PoFII Independent Research 123

02 Structural Biology of the Cytoskeleton

PROJECT LEADER | Prof. Dr. Inari Kursula | Centre for Structural Systems Biology (CSSB) at DESY, Hamburg | [email protected]

PROJECT MEMBERS | Dr. Alexander Ignatev | Dr. Petri Kursula | Dr. Thorsten Mengesdorf | Saligram Prabhakar Bhargav | Moon Chatterjee | Gopinath Muruganandam | Nele Vervaet

We are interested in how cytoskeletal proteins recognize profi lin and actin and to determine the three-dimensional and bind to each other and how actin polymerization in structure of the complex. eukaryotic cells is regulated. In particular, we want to un- derstand how pathogenic parasites use their actin cytoskel- Once we have the crystal structure available, it is possible eton for motility and host cell invasion and to fi nd ways to to start looking for compounds, which specifi cally disrupt interfere with these processes. Another focal point in our the Plasmodium profi lin-actin complex. We are also working work is histone deacetylases, especially those with cytosolic on the two Plasmodium forming isoforms to fi nd out what substrates. We use X-ray crystallography and small-angle their roles in the regulation of actin polymerization are and X-ray scattering for protein structure determination and if they work together with profi lin in these parasites. complementary biophysical and biochemical methods for the functional characterization of proteins and complexes. Plasmodium has two actin depolymerization factors (ADFs), We are also interested in the development of new synchro- which differ from each other in structure and function. The tron-based applications for visualizing large cytoskeletal major isoform in apicomplexan parasites, ADF1, is essential complexes at high resolution. for survival, does not bind F-actin and seems to work rather as a nucleotide exchange factor than a fi lament destabilizing Actin-based motility of the malaria parasite Malaria is protein. ADF2 is present only in Plasmodium spp. and is not one of the most devastating worldwide health threats. More essential but is structurally similar to the canonical mem- than a million people die of malaria and up to 300 million bers of the ADF family. people are infected every year, most of them young children and pregnant women. Malaria is also a signifi cant economi- cal burden, trapping the poorest areas in a downward spiral of poverty. Resistance to existing anti-malarial drugs is a growing problem in nearly all malaria-endemic areas. Therefore, there is an urgent need for new drug and vaccine candidates.

Malaria is caused by Plasmodium spp., a group of unicel- lular, eukaryotic, intracellular parasites, belonging to the phylum Apicomplexa. These parasites use actin for both a) We have determined the crystal structure of Plasmodium motility and host cell invasion. Their cytoskeleton dif- falciparum profi lin in complex with an octa-proline peptide. fers signifi cantly from that of higher eukaryotes and is, The structure contains a large b-hairpin insertion, which is not therefore, an attractive target for anti-malarial research. present in any other profi lins, as seen in the superposition of The parasite actin fi laments are extremely short and their Plasmodium and mouse profi lins (small picture below, where rapid turnover is regulated by a limited set of actin-binding green is Plasmodium and gray mouse profi lin). proteins, which are poorly conserved with their mamma- b) Homology modeling suggests that the b-hairpin protrusion lian homologues. may be involved in actin binding. c) A close-up view of the putative interactions of the b-hairpin Plasmodium actin-binding proteins with divergent protrusion in Plasmodium profi lin (green) with actin (gray). structures and functions Profi lins are ubiquitous, small, The superimposed mouse profi lin is shown in pink. actin-binding proteins that work together with formins to Graphics: Inari and Petri Kursula facilitate actin polymerization by recruiting polymerizable actin monomers close to the growing end of the fi lament. We have determined the crystal structure of P. falciparum Kursula, P., Kursula, I., Massimi, M., Song, Y.H., Downer, J., Stanley, W.A., Witke, W. & profi lin (PfPfn) and shown that it possesses the key func- Wilmanns, M. (2008) High-resolution structural analysis of mammalian profi lin 2a complex formation with two physiological ligands: formin homology 1 domain of mDia1 tionalities of profi lins and is essential for the survival of and the proline rich domain of VASP. Journal of Molecular Biology 375, 270-290. the parasite. Structurally, PfPfn differs signifi cantly from Kursula, I., Kursula, P., Ganter, M., Panjikar, S., Matuschewski, K. & Schüler, H. (2008) all other profi lins. The large structural rearrangements Structural basis for parasite-specifi c functions of the divergent profi lin of Plasmodium close to the actin-binding face point towards a possibly falciparum. Structure 16, 1638-1648. novel binding mode to actin. Our work aims to fi nd out the Huttu, J., Singh, B., Bhargav, S.P., Sattler, J., Schüler, H. & Kursula, I. (2010) Crystalli- zation and preliminary structural characterization of the two actin depolymerization regions required for the interaction between Plasmodium factors of the malaria parasite. Acta Crystallographica Section F 66, 583-587. 124 SCIENTIFIC REPORTS | PoFII Independent Research

03 Structural Analysis of the Innate Immune System

PROJECT LEADER | Prof. Dr. Wolf-Dieter Schubert | Former Research Group Molecular Host-Pathogen Interactions | Department of Biotechnology, University of the Western Cape, Cape Town, South Africa | [email protected]

PROJECT MEMBERS | Nils Kuklik | Clive Mketsu | Mujaahida Mohammed | Edukondalu Mullapudi | Lilia Polle | Donné Simpson | Jason Stark

In the Research Group “Molecular Host-Pathogen Interac- Auto is an N-acetylglucosaminidase, meaning that it cleaves tions” (MHPI) we previously focussed on the molecular the sugar backbone of the cell wall exposing the reducing details of human defence mechanisms against invading end of N-acetylglucosamine. Solving the crystal structure pathogens (innate immunity) as well as the corresponding of Auto we observed that it is structurally related to lytic molecular strategies of pathogens in infecting humans. transglycosylases and some lysozymes despite catalysing an unrelated chemical reaction. By mutating residues potential- Novel Listerial Virulence Factors InlJ and Auto The bac- ly involved in catalysis, we identifi ed the glutamic acid resi- terium Listeria monocytogenes infects humans following the dues Glu122 and Glu156 as being essential for the enzymatic consumption of contaminated food. To breach the intestinal reaction. Fascinatingly, we found that Auto, as originally pro- barrier and for all steps of the infection process Listeria has duced, is autoinhibited by an N-terminal α-helix. Its specifi c evolved a dedicated set of highly specifi c protein factors that N-terminal cleavage by an as yet unidentifi ed protease fol- it produces and secretes in a highly coordinated manner. To lowing an extracellular signal may, therefore, allow its rapid understand how their physical structure relates to their phys- and coordinated activation. In addition, Auto has a highly iological function, we have undertaken to investigate their acidic pH optimum making it essentially inactive at a pH of 7. three-dimensional structure at the atomic level of resolution. Overall we interpret our data to indicate that Auto partici- pates in liberating the pathogen from the partially acidifi ed “Auto” The cell wall of Gram-positive bacteria is a complex phagolysosome. By increasing the porosity of the cell wall it envelope that helps cells to maintain their shape and to would help to coordinate the release of other virulence fac- counteract the turgor pressure of their cytosol. It consists of tors such as listeriolysin. Their activity in turn would result long, linear chains of alternating sugar residues (N-acetylglu- in the breaching of the phagolysosomal membrane, raising cosamine and N-acetylmuramic acid) crosslinked by short the pH to physiological levels and de-activating Auto. peptides. Cell growth, cell division and many other processes crucially depend on the constant remodelling of the cell wall. InlJ The internalin family of proteins constitutes a group Enzymes involved in this process are generally referred to as of virulence factors of Listeria monocytogenes. The founding autolysins. member, Internalin or InlA, is central to the forced uptake of the bacterium into epithelial cells of the human small in- Studies revealed that only one of the autolysins is involved testine. Only recently this family was found to possess an ad- in the pathogenesis of Listeria monocytogenes. This autolysin ditional, previously unrecognized member, InlJ. This protein named “Auto” is essential for listerial entry into numerous has been shown to be crucial to bacterial adhesion to host eukaryotic cell lines. cells. InlJ is distinct from other family members in that its 16 LRR units mostly comprise 21 rather than the standard 22 residues and by one LRR-defi ning hydrophobic residue being replaced with a conserved cysteine. Solving the crystal struc- ture of this protein we could show that the cysteines line up to form a unique cysteine ladder in consecutive repeats. InlJ thus presents a very rare case of an extracellular protein (oxidizing environment) bearing a large number of reduced cysteine residues. This implies that the cysteines must serve to signifi cantly stabilize the structure, offsetting the danger of its unfolding followed by cysteine oxidation.

Left: Crystal structure of autolysin Auto of L. monocytogenes is depicted in cartoon style and with transparent surface. Bröcker, M.J., Schomburg, S., Heinz, D.W., Jahn, D., Schubert, W.-D., & Moser, J (2010) The active site of the pro-enzyme is blocked by an N-terminal Crystal structure of the nitrogenase-like dark operative protochlorophyllide oxidoreduc- tase catalytic complex (ChlN/ChlB)2. Journal of Biological Chemistry 285, 27336-27345. α-helix (red). Right: Crystal structure of InlJ (right) with enlar- Heinemann, I.U., Schulz, C., Schubert, W.-D., Heinz, D.W., Wang, Y.G., Kobayashi, Y., ged view of the cysteine ladder (left): reduced cysteines align Awa, Y., Wachi, M., Jahn, D., & Jahn, M. (2010) Structure of the heme biosynthetic in the hydrophobic core (Sulphur - yellow). Graphics: HZI Pseudomonas aeruginosa porphobilinogen synthase in complex with the antibiotic alaremycin. Antimicrobial Agents and Chemotherapy 54, 267-272.

Bublitz, M., Polle, L., Holland, C., Heinz, D.W., Nimtz, M., & Schubert, W.-D. (2009) Structural basis for autoinhibition and activation of Auto, a virulence-associated pepti- doglycan hydrolase of L. monocytogenes. Molecular Microbiology 71, 1509–1522. SCIENTIFIC REPORTS | Technological Platforms 125

TECHNOLOGICAL PLATFORMS

A number of platform technologies essential for research and development carried out at the Helmholtz Centre for Infection Research are made available to the scientifi c projects as centralised facilities. In the context of national and international research programmes, these platforms provide services not only to internal projects, but also to scientifi c collaborators from other Helm- holtz research centres, universities, other public research institutes, and industry. On the following pages the most important platforms are described in detail.

Dr. Matthias Negri and Jennifer Hermann, HIPS, discussing a drug-target interaction. Photo: HIPS/HZI 126 SCIENTIFIC REPORTS | Technological Platforms

01 Central Animal Facility

HEAD | Dr. Hermann Riedesel | Central Animal Facility | [email protected]

The Central Animal Facility provides state-of-the-art In 2010 the Transgenic Unit has successfully generated 30 laboratory animal care and service for the scientifi c needs new knock out lines. 50 mouse lines from four new groups of the HZI in compliance with the animal welfare guide- have been successfully imported into our breeding units by lines. This facility consists of 24 animal and 13 adjacent means of IVF and/or Embryo transfer. The 2 step controlled procedure rooms with a total capacity of 15,000 cages which rate freezing method with propandiole as cryoprotectant equals 37,500 mice. Exclusively laboratory mice are housed was successfully implemented and is now also working suc- as single species. All animals are kept in individually cessfully in combination with IVF. ventilated cage systems in seven holding units of fi ve different hygienic and three biosafety levels. Five units are Animal house at TWINCORE At the TWINCORE Hanover constructed as a complete specifi c-pathogen-free (SPF)- the refurbished mouse facility was commissioned in summer barrier, two units are registered as animal biosafety level 2 and put into operation in September 2010. This satellite (ABSL2) and one as ABSL3 facility for infection experi- mouse facility consists of four animals rooms with fl oor ments. The average daily animal census is currently about area of 96,5 m2 and a cage capacity of 2,300 IVC cages and 15,000 mice consisting of 450 different strains and has ABSL 1, 2 and 3 capabilities. The core breeding of all genetically engineered lines. The health status, which is TWINCORE lines is done at the HZI, so that the TWIN- monitored on a quarterly basis, complies with the FELASA CORE facility will be mainly used for expansion breeding recommendations. and experimental holding. By this means mice kept at the TWINCORE will exhibit the same hygienic status as at the The following services are offered: HZI. Four animal technicians are working at the TWIN- • Basic animal care CORE facility offering basic animal care, breeding and • Breeding services and colony management colony management and experimental assistance. All the • Assistance with experimental procedures other above mentioned services are performed by the HZI • Health monitoring animal facility. • Animal procurement • Organization of national and international mouse shipments • Training programme for animal care technicians • Animal welfare services for about 60 animal experimen- tal protocols1 • Consultation and training in laboratory animal science • Quarantine and rederivation of imported 50 lines • In vitro fertilization (IVF) for rescue, speed expansion and rederivation of mouse lines • Embryo-cryopreservation of mouse lines and banking of germplasm • Generation of knock out lines by blastozyst injektion

In 2010 the new mouse facility T2 has been put into opera- tion starting with the core breeding unit in May. By the end The mouse house. Photo: HZI, Krämer of July all breeding colonies were completely housed in this new unit. In October the S2 unit in T2 has been started. The S3 unit will be operative by the beginning of 2011. After moving out most of the animals, the T1 building will be refurbished and reorganized for future use as experimental holding facility. One fl oor of this building can be used for preliminary S2 experiments with animals from outside sources. This unit harbours an imaging unit equipped with an IVIS system. SCIENTIFIC REPORTS | Technological Platforms 127

02 Recombinant Protein Expression

HEAD | Dr. Joop van den Heuvel | Research Group Recombinant Protein Expression | [email protected]

SCIENTIFIC COLLABORATOR | Dr. Konrad Büssow | Dr. Volker Jäger | Prof. Dr. Ursula Rinas

The recombinant protein expression platform (RPEX) is essential for the production of ultra pure protein for high resolution structural analysis using X-ray crystallography and NMR spectroscopy. Four major expression systems have been established in the RPEX facility: E. coli, P. pastoris, insect cell and mammalian cell culture. This allows the production of “simple proteins” as well as proteins with complicated modifi cations.

For production of the challenging mammalian protein targets an animal cell culture facility has been established. The size of the cell culture reactors ranges from 1.6 to 6 litres in order to meet the increasing requirements for routine production of proteins on a 10-50 mg scale. For the PSPF-Training course in basic techniques in insect cell purifi cation of poorly expressed recombinant multi-protein cultivation and baculoviral expression of recombinant protein. complexes a 30-litres-bioreactor has been set up that allows Photo: HZI, Bierstedt the pilot-scale production of protein from several hundred litres of medium.

The system includes all the necessary equipment for con- Support The Protein Sample Production Facility (PSPF) tinuous media exchange via tangential fl ow microfi ltration is a part of the HZI protein expression platform and was or internal membrane perfusion and subsequent protein established in 2007 in cooperation with the Max Delbrück purifi cation steps. As a supplement to recombinant protein Centre for Molecular Medicine (MDC). The main goal of this production, large amounts of HeLa cell biomass can be Helmholtz cooperation is the support of structural biologists produced as a source for purifi cation of naturally occurring in Germany via circumvention of the major bottleneck of human protein complexes. protein production. The PSPF service is offered either on a subsidised fee-for-service billing scheme or as a scientifi c Research A research group within the PSPF is focussing cooperation (www.pspf.de). Currently, 50% of the capacity of on the development of new and fast strategies for creating the unit is used for internal HZI projects. stable expression cell lines. This group has already estab- lished a new and unique methodology for rapidly generating During 2010, a total of 20 PSPF projects have been processed. highly overproducing stable mammalian cell lines (RMCE) Eight of these projects have been successfully fi nished, four (Wilke et al., 2010). The expression level varies between have been terminated and eight are still in progress. In 2010, 1-5 mg/L and can be easily, and reproducibly, scaled up to three scientists were trained and supported to establish the 10-100 L. In cooperation with the project group of K. Büs- expression of their target gene in the baculovirus expression sow the newly developed CHO-Lec-RMCE cell lines will be system in our lab. Several in-house HZI cooperation projects improved in robustness and validated for their effi ciency for the analysis of multi-protein complexes have been under- of generating a substantial set of new expression cell lines. taken and include: TLRs (Schubert (University of the Western The performance of these production cell lines in cell cul- Cape)/Heinz), HGF-cMet receptor interaction (Krausze/ ture bioreactors will be characterized. Development of new Heinz/Gherardi), HCV Protease NS3/4 complexed with novel cell lines for the co-expression of multi-protein complexes inhibitory peptides (Collins/Schmelz) and the ABCB6 mem- on the basis of the CHO-Lec-RMCE cell lines will be one of brane protein (Menzel/Heinz). The production of these pro- the major challenges in future. teins is based either on the baculovirus expression system or the Acembl/Multibac expression system, animal stable glyco- sylation defi cient CHO cell line cultivation or expression in Pichia pastoris. Many projects are on-going as the production of suffi cient amounts of soluble and stable protein is still limiting and requires extensive redesign and recloning of the expression systems. For several of the projects the process of crystallisation is still continuing and requires a continuous supply of fresh and pure material. 128 SCIENTIFIC REPORTS | Technological Platforms

03 Gene Expression Analysis

HEAD | Dr. Robert Geffers | Research Group Genome Analytics | [email protected]

This platform is a central service unit for our internal ray analysis determines target sequence enrichments based investigators as well as for HZI collaboration partners. We on chromatin immunoprecipitation (ChIP or MeDIP) of DNA are offering microarray technology for transcriptional and binding proteins resulting in the identifi cation of either ge- genomic analysis utilizing the following applications: nomic DNA transcription factor binding sites or epigenetic modifi cations within CpG islands. Some tiling arrays are Gene expression For gene expression analysis we provide designed to cover the complete genome (aCGH: array based microarray technology ordered from the market leaders comparative genome hybridization analysis). Comparative Affymetrix and Agilent Technologies, respectively. More- genome hybridization, i.e. tumour vs. normal tissue, can de- over, the core unit is furnished with a gene arrayer which termine genetic instability established during tumourigene- we use to manufacture microarrays in customer desired sis of individual tumour entities. The core unit offers besides format. Most of the microarrays used are so-called “whole catalog aCGH arrays also the opportunity to design focused genome” microarrays. More than 40,000 probes (probe versions with higher resolution. Thus, for any complexity of sets) arranged on each microarray are suffi cient to cover the project a proper design can be adapted. Moreover, the the transcriptomes of species like human and mouse. The core unit will support also the primary data mining process. straight forward Agilent microarray technology allows for processing of up to eight samples simultaneously on only In course of internal and external collaborations new analy- one slide. High fl exibility in probe design helps to generate ses were placed in several areas: tumourigenesis and tumour new microarray formats very cost effective in any complexi- typing, pathogen-host interaction of streptococcus, pseudo- ty for any organism. The core unit takes responsibility moniae and mycobacteria and on immunological issues. for the probe design and supervises the steps during the manufacturing processes. Gene expression analysis can be performed in different ways depending on the “Target” that should be measured. Usually 3´ expression arrays are used. Here the probe design depends on sequences nearby the 3´end of each transcript. Such microarray can be used for quantifi cation of the expressed RNA, but failed to detect splice variants per gene. Exon arrays (Affymetrix) are composed of probes tiled across the exon sequences of each gene in the genome. Consequently, these arrays can be used to quantify and qualify splice variants at a genome wide level. Also for this approach the core unit takes over Agilent arrayCGH: Human, Mouse and Rat microarrays can be responsibility, if desired, for experimental setup of the offered fort he Agilent SurePrint Systems. Investigators have projects and gives advice during the steps of primary data the choice between general catalogue arrays or customized acquisition. aCGH formats. Left: 244k aCGH; right: 105k aCGH. Graphic: HZI

microRNA expression MicroRNAs are known to play a pivotal role in gene regulation. Incorporated into the “RISC”-complex (RNA inducing Silencing Complex) miRNA molecules mediate the breakdown of mRNA or the reduc- tion of ribosomal RNA translation. Our core unit offers continuously updated murine and human microRNA expres- sion chips based on the newest releases from the Sanger miRBase. Complete service is provided including microRNA extraction and array analysis. Finally, a systematically approach linking changes of microRNA expression with predicted target gene modifi cation, aims to uncover their regulative effects applied to the transcriptome.

Tiling arrays Tiling arrays are composed of partially over- lapping probes across defi ned genomic sequences. These selected genomic regions either represent regulatory DNA regions (promoter, enhancer) or DNA regions underlying Genome-wide expression: Human, Mouse and Rat on either epigenetic modifi cations (CpG isles). Comparative microar- Affymetrix GeneChip or Agilent SuerPrint Systems.Graphic: HZI SCIENTIFIC REPORTS | Technological Platforms 129

04 Peptide Synthesis

HEAD | Dr. Dr. Werner Tegge | Department of Chemical Biology | [email protected]

SCIENTIFIC COLLABORATOR | Dr. Ronald Frank

Since its inauguration as a service unit in 1990, the plat- Soluble peptides To date, over 3,000 soluble peptides with form generates synthetic peptides, both in soluble form a length of two to over fi fty amino acids have been gener- and immobilised in the form of arrays, for many different ated in the platform. Soluble peptides are characterised HZI projects and external collaborations. State-of-the-art using HPLC and mass spectrometry. If necessary, further equipment is employed for the synthesis, characterisation characterisation is carried out by amino acid analysis, and purifi cation. By pursuing own research projects, the protein sequencing, special mass spectrometry techniques methodological repertoires are continuously updated and and NMR in the HZI Division of Structural Biology. extended. Depending on the intended usage and desired quality of the products, purifi cations are carried out, usually by pre- Developments in this context include: parative HPLC. For special applications, the platform also • New methodologies for the generation of peptide arrays offers peptide modifi cations like fl uorescence labelling, (e.g. the SPOT method); phosphorylation, biotinylation, lipid conjugation, PEGyla- • Methods for the preparation of phosphorylated and thio- tion, branched peptides and cyclisations. phosphorlylated peptides • The utilisation of new biocompatible solid supports SPOT-arrays In the platform, immobilised peptides in the • New selectively cleavable peptide linkers form of arrays are generated to facilitate the systematic • Methods for the synthesis of branched peptides and empirical search for ligands. For the successful design • Methods for the generation of libraries of linear and of such arrays a thorough understanding of the biological cyclic peptides problem is essential, which is attained via close co-oper- • Assays for the utilisation of soluble and immobilised ation and collaboration with the users. The SPOT-arrays peptides in biological systems are generated semi- and fully automatically on cellulose membranes or other polymeric supports. Each year, ap- proximately 15,000 peptides and peptide mixtures are generated in an array format and utilised for the investiga- tion of e.g. protein-protein interaction, including epitope mapping and enzyme-substrate recognition.

Method developments for parallel combinatorial chemical synthesis and screening are based on the SPOT synthesis performed on cellulose membranes. Photo: HZI, Bierstedt

Tegge, W., Bonafe, C.F.S., Teichmann, A., & Erck, C. (2010) Synthesis of peptides from α- and β-tubulin containing glutamic acid side chain linked oligo-Glu with defi ned length. International Journal of Peptides, Article ID 189396.

Nickl, C.K., Raidas, S.K., Zhao, Sausbier, M., Ruth, P., Tegge, W. Brayden, J.E. & Dostmann, W.R. (2010) (D)-Amino acid analogues of DT-2 as highly selective and superior inhibitors of cGMP-dependent protein kinase I-alpha. Biochimica et Biophysica Acta (BBA) – Proteins and Proteomics 1804, 524-532.

Weiß, S.M., Ladwein, M., Schmidt, D., Ehinger, J., Lommel, S., Städing, K., Beutling, U., Disanza, A., Frank, R., Jänsch, L., Scita, G., Gunzer, F., Rottner, K. & Stradal, T.E.B. (2009)

IRSp53 links Tir to EspFU /N-WASP-mediated actin assembly in EHEC pedestal formation. Cell Host Microbe 15, 244-258. 130 SCIENTIFIC REPORTS | Technological Platforms

05 Histo-Pathology Platform

HEAD | Prof. Dr. Klaus Schughart | Department of Infection Genetics | [email protected]

SCIENTIFIC COLLABORATOR | Dr. Verena Haist | University of Veterinary Medicine, Hannover

The histo-pathology platform represents a central service unit at the HZI. It supports several projects and research groups which require support for histological analyses and pathology expertise.

Many research projects at the HZI are performing infection challenge experiments in mice and studying mechanisms of the host defense in genetically diverse mouse strains and mutant lines. The histo-pathology unit offers a central customized service and provides the entire necessary infrastructure in a single unit. Scientists from the HZI can either use a complete service – from embedding, sectioning, staining, archiving of tissues, and review by a pathologist – or take advantage of its infrastructure and perform some of the tasks themselves. Immunohistochemical staining for infl uenza nuclear protein At present, paraffi n sections and histochemical staining are (brown) showing infection of cells in the lungs of mice. offered on a routine basis. Cryostats are available and a set Photo: HZI of immune-histochemical analyses is offered which allows the detection of immune cells in the mouse during infection and infl ammation. A new vibratom was recently acquired which allows to cut non-treated samples and therefore can be used for further experiments, for example in organ cultures.

The unit is operated by Anna Rinkel and it maintains close collaboration with experts from the Institute of Pathology at the University of Veterinary Medicine, Hannover, (Dr. Verena Haist) and the Institute of Veterinary Pathology at the Free University of Berlin (Prof. Dr. Achim Gruber), who provide pathological expertise and support for the planning of experiments and interpretation of results.

Ziehl-Neelsen staining of liver section showing the presence of acid-resistant bacteria (red). Photo: HZI SCIENTIFIC REPORTS | Technological Platforms 131

06 Analytical Instruments

HEAD | Dr. Victor Wray | Research Group Biophysical Analysis | [email protected]

SCIENTIFIC COLLABORATORS | Dr. Heinrich Lünsdorf | Dr. Manfred Nimtz | Priv.-Doz. Dr. Manfred Rohde

The platform provides a facility for determining the three X-ray crystallography The main emphasis in X-ray crystal- dimensional structure of all types of natural products and lography is the structural analysis of proteins. A pipette- has instrumentation for mass spectrometry (MS), nuclear robot and an X-ray unit with an area detector and rotating magnetic resonance (NMR) spectroscopy, X-ray crystallog- anode are available for crystallization and data collection. raphy (X-ray), protein sequencing (PS), electron microscopy The measurement of high resolution data and phase deter- (EM) and confocal laser microscopy. mination using anomalous dispersion is available through the use of external synchrotron facilities. MS and NMR spectroscopy For the majority of low- molecular weight natural products the total structure is Electron microscopy This technique includes a fi eld elucidated in a routine manner using a combination of MS emission scanning electron microscope (FESEM) with an and NMR spectroscopy. The direct analysis of large, intact installed EDX analysis system for elemental analysis and biomolecules such as proteins, oligonucleotides and com- an attached cryo-stage for cryo-FESEM work. The main plex carbohydrates, is routinely carried out using MALDI- mission is to provide a competitive modern spectrum of and ESI-MS. MS has the important advantage of providing methodology to develop and perform techniques for high information for very small amounts of compound. Automat- resolution morphological analysis and immune localization ed MS micro-techniques are used for the identifi cation and of proteins. High resolution FESEM techniques are especial- characterization of proteins from 2D gels and from “gel-less” ly used to visualize the adherence to and invasion of host techniques for “Proteomics”, through the determination of cells by a wide range of pathogens. Preparation protocols the molecular weight of their proteolytic fragments using have been customized to fulfi l the wide requests of differ- MALDI/TOF-MS/MS and HPLC-ESI-MS/MS. The second- ent researchers on the campus. New methodologies have ary and tertiary structure of peptides and proteins can be been developed to immune localize pathogenicity factors on elucidated when appropriately labeled material (15N and 13C) the bacterial cell surface or the interface between bacterial is available through the application of multidimensional and host cell membranes using FESEM. Furthermore, gold- NMR spectroscopy. particles coated with isolated proteinaceous pathogenicity factors have been proven to be a useful tool for studying the crosstalk between pathogen and host cell. In addition, Platform: Analytical Instruments conventional and an energy fi ltering transmission electron microscopes (TEM, EF-TEM) are used to support fi ndings by FESEM studies especially for immune localization of pathogenicity factors inside bacteria or host cells and for NMR Small Natural Products following the intracellular traffi cking of pathogens. TEM is used for studying the quaternary structure of proteins by Macromolecules: MS negative-staining techniques and is applied for high resolu- Proteins tion elemental localisation studies by electron spectroscopic imaging and electron energy loss spectroscopy. X-ray Molecular Complexes: PS Protein-ligand Protein-Protein

Super macromolecules: EM Organelles Bacteria

Platform techniques available for investigation of all types of Wang,Y., Edrada-Ebel,R., Tsevegsuren,N., Sendker,J., Braun,M., Wray,V., Lin,W., & Proksch,P. Photo: HZI natural products (2009) Dihydrostilbene derivatives from the Mongolian medicinal plant Scorzonera radiata. Journal of Natural Products 72, 671-675.

Xu,J., Kjer,J., Sendker,J., Wray,V., Guan,H., Edrada,R., Lin,W., Wu,J., & Proksch,P. (2009) Chromones from the endophytic fungus Pestalotiopsis sp. isolated from the chinese man- grove plant Rhizophora mucronata. Journal of Natural Products 72, 662-665.

Zelena,K., Zorn,H., Nimtz,M., & Berger,R.G. (2009) Heterologous expression of the msp2 gene from Marasmius scorodonius. Archives of Microbiology 191, 397-402. 132 SCIENTIFIC REPORTS | The Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS)

The Helmholtz-Institute for Pharmceutical Research Saarland (HIPS)

MANAGING DIRECTOR | Prof. Dr. Rolf Müller | Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS) within Helmholtz Centre for Infection Research (HZI) |

Campus Saarland University, Building C23, 66123 Saarbrücken | Germany | [email protected]

STAFF | Dr. Markus Ehses | David Hofmann | Birgitta Lelarge | Natja Mellendorf | Claudia Thiele

The Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS) was founded in August 2009 jointly by the Helmholtz Centre for Infection Research (HZI) and Saarland University (UdS). It is established as branch of HZI on the campus of UdS with the aim to accelerate the identifi cation of new drug candidates for antiinfective research, their optimization by methods of molecular biology and medicinal chemistry and the improvement of the transport to the site of action. It is especially the emerging of new and often multi-resistant pathogens that requires an effi cient approach to the development of antiinfectiva. The founding of HIPS is the result of an initiative by the Helmholtz Association to complement translational research within the research fi eld “health”. The combination of the internationally renowned high quality of infection research at HZI and of pharmaceutical sciences at UdS creates a signifi cant added value. Close cooperations with regional partner institutes allow covering the wide range of drug devel- opment starting from early drug candidate identifi cation to clinical studies all within one research centre. The implementation of the complete development pipeline will considerably speed up the use of natural products in clinical studies.

The range of scientifi c activities at HIPS comprises genetic and genome-analytic methods for optimizing natural pro- duct producers and lead compounds as well as methodologies to improve the transportation of pharmaceutical agents to their target. The combination of the expertise in infectious diseases and in pharmaceutical research at HZI and HIPS takes a unique position in Germany and Europe, especially regarding the development of antiinfectives. The three departments of HIPS, “Microbial Natural Products” (MINS, Prof. R. Müller), “Drug Design and Development” (DDOP, Prof. R.W. Hartmann), and “Drug Delivery” (DDEL, Prof. C.-M. Lehr), originate each from a pharmaceutical research group at UdS, with approximately 140 employees in total, of The founding heads of departments of HIPS (from left to right): which at the end of 2010 30 were fi nanced by HIPS. Finally, Claus-Michael Lehr (DDEL), Rolf Müller (MINS) and 100 collaborators will move to the new building, which Rolf W. Hartmann (DDOP) Photo: HZI should be constructed within three to four years on the campus of UdS.

Another mission of HIPS is the promotion of young scien- research approaches, methods and apparatus in this pool of tists. Currently, a structured doctoral programme is deve- pharmaceutical competence will work together synergisti- loped, which besides courses in soft and professional skills cally under the roof of the intended “Centre for Pharmaceu- will feature an optional one-year-stay abroad. Furthermore, tical Sciences Saarland”. three young researcher groups will be established. The Helmholtz Young Investigators Group “Metabolic Engineer- The close linking of HIPS to HZI is promoted with high ing of Actinomycetes” (AMEG), headed by Andriy Luzhet- priority and is being realized by initiation of research col- skyy, moved to HIPS in January 2011. By the follow-up laborations and implementation of compatible administra- appointments to the three vacant university chairs and tive workfl ows and infrastructure for controlling, admin- further new junior professor positions at UdS, the number istration of personnel, IT and public relations. For these of groups with pharmaceutical focus at Saarbrücken will measures an administrative manager, a scientifi c assistant raise from currently fi ve full professors and two young re- as well as an administrative assistant support the director searcher groups to a total of 15 groups in the mid-term. The of the institute. SCIENTIFIC REPORTS | The Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS) 133

The Department “Microbial Natural Products” (MINS)

Head of Department | Prof. Dr. Rolf Müller | Helmholtz- Institute for Pharmaceutical Research Saarland (HIPS) within Helmholtz Centre for Infection Research (HZI) | Department of Microbial Natural Products (MINS) [email protected] Scientifi c Collaborators | Dr. Daniel Krug | Dr. Silke Wenzel

The department “Microbial Natural Products” investigates the chemistry, biological activity, production and regulation of natural products mainly from myxobacteria. Myxobac- teria are soil-living organisms and represent a promising source of natural products, which are now as much valued as the long-established actinomycetes. Myxobacterial natural products exhibit diverse biological activities and modes-of-action and are thus useful for various therapeuti- cal applications – examples include their use as antibiotics, anti-cancer drugs, immunosuppressants or anti-parasitics. Myxobacteria are known to secrete a high number of natu- ral products, so-called secondary metabolites, at least in part to presumably inhibit growth of their competitors. In order to uncover the potential of myxobacteria as produ- cers of secondary metabolites, scientists at MINS employ (A) Swarming of the myxobacterium Byssovorax cruenta a broad spectrum of techniques including microbiological, starting from a piece of cellulose on agar plate and (B) micro- molecular-biological, genetic, biochemical, biotechnological scope image of a fruiting body from Chondromyces crocatus. and analytical methods. Photos: R. Garcia

Newly isolated myxobacterial strains from locations world- wide, together with the existing myxobacterial strain collec- tion at HZI (harbouring over 8000 isolates), form the basis organisms by actively degrading their prey. When starvation for our interdisciplinary efforts to discover novel natural conditions are encountered, many cells can aggregate to form products. To date, a number of entirely new myxobacterial a multicellular structure, termed a “fruiting body”, contain- species, genera and families as well as many potentially ing a high number of heat- and dryness resistant myxospores, novel natural products have been isolated successfully. which may survive unfavourable environmental conditions Isolation of a novel myxobacterium usually requires careful even for a prolonged period of time. optimization of microbiological methods and cultivation conditions, since these bacteria exhibit a rather complex Once a new myxobacterial strain has been successfully “lifestyle”: Myxobacteria are able to glide on surfaces in a adapted to growth under laboratory conditions, MINS uses swarm-like pattern and they collectively feed on other micro- state-of-the-art mass spectrometric techniques to screen the strain’s metabolite profi le with respect to the presence of known myxobacterial secondary metabolites. At the same time the department MINS performs a range of biological assays, using indicator organisms as well as cell-based screening approaches to discover novel compounds exhibit- ing a potentially interesting activity. Following optimization of the production of candidate compounds, cultivation of the producing strain is upscaled. The target compounds are purifi ed using liquid chromatography methods. Thereafter, structure elucidation using a panel of analytical techniques including multidimensional NMR spectroscopy is carried out. In addition, further cell-based experiments are performed in order to investigate in detail the mode-of-action of a novel natural product. This work is performed in close collabora- Sonja Burkhard checks a culture in the shaker. Photo: HZI, Bellhäuser tion with the work group MWIS at HZI. 134 SCIENTIFIC REPORTS | The Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS)

Another core expertise of MINS is the investigation of The treatment of infectious diseases is of growing signifi - the biosynthetic pathways underlying the formation of cance in health care as an increasing number of pathogens myxobacterial natural products, including the biochemical become multi-resistant. Thus, the development of new characterization of the involved enzymatic machinery. The antibiotic drugs has become one major topic for pharma- scientists at MINS aim to improve the fundamental under- ceutical research, in particular because effi cient medications standing of the mechanisms directing secondary meta- fi ghting against infectious diseases are still missing. Con- bolite biosynthesis in myxobacteria on the molecular level. ventional antibiotics target essential processes in bacteria Thereafter, we apply this knowledge in order to increase to kill these pathogens. An alternative strategy aims at product yields via the manipulation of regulatory processes blocking the cell-cell communication by inter ference of the and for the targeted manipulation of biosynthetic pathways, drug with the communication system. This communication resulting in the production of structurally modifi ed or novel system is based on the exchange of signal molecules and compounds. The researchers underpin their efforts with regulates the production of the virulence factor production sequencing of the exceptionally large genomes of a number and biofi lm formation, which could thus both be reduced or of myxobacterial model strains. Having reported the even prevented. sequence of the to date largest bacterial genome from Sorangium cellulosum a couple of years ago, every ad- Recent advances in microorganism research have resulted ditional genome highlights again the enormous genetic in the identifi cation of an increasing number of natural capacity of myxobacteria for the production of secondary compounds showing antibiotic activity. However, most of metabolites: the number of predicted biosynthetic pathways these structures are not suitable for becoming drugs due regularly outnumbers the natural products previously to unfavourable pharmacokinetic properties, such as poor known from a strain. Thus, the bioinformatic analysis of solubility or diffi cult large-scale synthesis. The department myxobacterial genomes forms the basis for the discovery of DDOP is focusing on the development of novel synthetic novel secondary metabolites by using a combined genome- antibiotics, which can either be derivatives of the natural mining and “secondary metabolome mining” approach, hit compounds with improved drug likeness, or synthetic implementing highly sensitive mass spectrometric methods. compounds based on a rational drug design. Different me- Moreover, the sequence data allow the researchers to opti- dicinal chemistry strategies are applied, ranging from sim- mize the production of myxobacterial metabolites and even plifi cation of the molecular complexity of the compounds to enable the expression of complete biosynthetic pathways computer-aided methods in drug design. in heterologous hosts, thereby increasing product yield. Furthermore, it is possible to introduce alterations to a At DDOP two main projects are currently developed, target- compound’s structure by genetic engineering. Last but not ing either the bacterial growth or their cell-cell communica- least, the availability of whole-genome information is an tion system. important prerequisite to deepen our understanding of the host microbiology of myxobacteria.

The Department “Drug Design and Optimisation” (DDOP)

Head of Department | Prof. Dr. Rolf W. Hartmann | Helm- holtz-Institute for Pharmaceutical Research Saarland (HIPS) within Helmholtz Centre for Infection Research (HZI) | Department of Drug Design and Development (DDOP) | [email protected]

Scientifi c Collaborators | Dr. Johannes De Jong | Dr. Jörg Haupenthal | Dr. Matthias Negri | Dr. Anke Steinbach | Dr. Christiane Zimmer Jeannine Jung and Cenbin Lu from the department DDOP discuss the synthesis of a new inhibitor. Photo: HZI, Bellhäuser SCIENTIFIC REPORTS | The Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS) 135

Development of potent bacterial RNA polymerase inhibitors The project focuses on the development of inhi bitors that block the transcription of bacterial genes. These molecules are intended to target the bacterial RNA polymerase (RNAP). The novel RNAP inhibitors should also be effective against bacteria that are resistant to other antiinfectives.

RNAP is the enzyme responsible for the formation of RNA in all living cells. The bacterial form is a complex multi- subunit enzyme with a molecular weight of about 400 kDa. It consists of four different subunits, which form the core enzyme (α2ββ‘ω). The latter is activated for the transcrip- tion by interaction with the so-called sigma factor. As RNAP is essential for bacterial growth and selective to bacteria, it is a highly attractive target for the development of a broad- spectrum antibiotic against severe diseases with humans. One important example is Mycobacterium tuberculosis (Mtb), the pathogen of tuberculosis, which causes two million deaths per year worldwide. However, RNAP inhibitors that are in clinical use have evoked resistant strains, which Structure of RNA polymerase exhibiting fi ve subunits. Ligand- increase the demand for new drugs enormously. The aim and structure- based approaches are followed in the design of of the project is to develop a new inhibitor of the bacterial new RNAP inhibitors. RNAP, to characterise its mode-of-action and to optimise the hits. By this approach, the inhibitor becomes a highly potent drug that invades pathogenic bacteria to fi nally unfold its antibiotic function in humans. is used by DDOP to identify a suitable regions for binding and hence to develop an appropriate inhibitor. First RNAP In the development of novel RNAP inhibitors DDOP follows inhibitors, in part short peptides, have already been identi- two strategies: fi ed. The aim of the project is to optimize the inhibition effect and various pharmacokinetic properties of these The fi rst strategy consists of direct inhibition of the core en- compounds. Finally, this should lead to a highly potent drug zyme. Different binding sites have been described for RNAP, for use in therapeutic applications in humans. which are prone to inhibitors that can act either by directly blocking the active site or by infl uencing the fl exibility of RNAP from both E. coli and Mycobacterium tuberculosis will RNAP via allosteric mechanisms. Since the 3D structure of be cloned and used for the biological testing of the syn- RNAP is known, computer-aided drug design strategies are thesized compounds. The characterization of the putative used to screen virtual compound databases and to rational- binding site(s) of novel potent inhibitors will be performed ly select compounds for subsequent experimental screening. by site-directed mutagenesis. Virtual hits showing effectiveness in vitro will then pass over to the hit-to-lead phase. First validated screening hits Development of compounds interfering with PQS quorum have already been identifi ed and novel synthetic projects sensing communication in Pseudomonas aeruginosa In have been launched to chemically optimize the hits. this project, the scientists at DDOP develop compounds that interfere with the PQS quorum sensing communication The second strategy aims at preventing the holoenzyme system in Pseudomonas aeruginosa. Lung infections by formation, i.e. the assembly of the core enzyme with the this opportunistic pathogen are considered a major cause sigma factor. Part of the interaction interface of the two of death for patients with cystic fi brosis. Resistance to subunits has been characterized by means of mutagenesis conventional antibiotics is often associated with the forma- studies, and it has been shown that this interface might be tion of biofi lms, which prevent the penetration of antibiotics a suitable target for enzyme inhibition. This information through this physical barrier and thus reduce the immune 136 SCIENTIFIC REPORTS | The Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS)

response. Both the formation of biofi lms and the production tion of fragments and their binding mode will be further ex- of virulence factors are controlled by a complex quorum plored by high resolution NMR-spectroscopy. Further, kinetic sensing communication system. By this communication studies, both computational and biochemical (biological as- system, bacteria detect their population density and control says, SPR, ITC), should promote the development of PqsD-in- a kind of population behaviour, amongst others. Besides the hibitors. Especially the evaluation of the kinetic mechanisms two quorum sensing systems, LasR and RHLR that exist in by molecular dynamic simulations will provide information many bacterial species, in P. aeruginosa, a third, unique about the fl exibility of the protein and may contribute to the communication system is regulated by PQS (Pseudomonas elucidation of possible induced-fi t mechanisms. Quinoline Signal, 2-heptyl-3-hydroxy-4-quinolone) as signal molecule. The aim of the project is the development of compounds that interfere with the PQS quorum sensing The Department “Drug Delivery” (DDEL) communication system in order to prevent the formation of biofi lms and the production of different virulence factors – Head of Department | Prof. Dr. Claus-Michael Lehr | this, however, without affecting the growth of bacteria. Two Helmholtz-Institute for Pharmaceutical Research Saarland approaches are followed in this project as well: On the one (HIPS) within Helmholtz Centre for Infection Research hand the effect of PQS on the receptor PqsR is antagonized (HZI) | Department of Drug Delivery (DDEL) | cle09@ and on the second hand, the biosynthesis of HHQ, the direct helmholtz-hzi.de precursor of PQS, should be blocked by inhibition of the enzyme PqsD. Scientifi c Collaborators | Dr. Eva-Maria Collnot | Dr. Nicole Daum | Dr. Steffi Hansen | Dr. Brigitta Loretz Development of antagonists of the transcription regula- tor PqsR Initial drug discovery efforts are focused on the Advances in molecular biotechnology and medicinal chem- synthesis of close analogues of PQS intended to bind to istry have led to the discovery of new drug candidates with and block PqsR. The transcription regulator PqsR drives potential affi nity to target receptors. However, developing the expression of virulence determinants after activation such molecules into drug products necessitates fi rst screen- by PQS. The PQS analogues will be evaluated in a recently ing of their per meability, transport and bioavailability established β-galactosidase reporter gene assay. After through biological barriers and second establishing new activation of the PqsR transcriptional regulator by PQS, the technologies to ensure their safe and effective delivery to transcriptional activation of the PqsA promoter should be the site of action. Therefore, the main focus of research in detected. Furthermore, the SPR methodology for the kinetic the department Drug Delivery is on the one hand exploring characterization of hits will be developed with the soluble the biological barriers, in particular the lungs, the gastro- truncated version of PqsR, (C87PqsR) and applied for medium intestinal tract, and the skin. On the other hand, DDEL throughput screening of fragment libraries. Hits from the develops the appropriate carriers capable of crossing these SPR screening are further thermodynamically character- epithelial barriers and delivering the active molecule to the ized using ITC (isothermal titration calorimetry). target.

Inhibition of the second enzyme of the PQS biosynthesis PqsD For the biochemical evaluation of potential PqsD inhibitors, a medium-throughput PqsD LC-MS/MS based inhibition assay has been established in our lab. Novel PqsD inhibitors, structural analogues of the native substrate anthraniloyl-CoA, have been synthesized. Currently, an SPR-based medium-throughput screening of the in-house library and of commercially available fragment-libraries is performed followed by the kinetic characterization of hits. The same libraries are also virtually screened on PqsD with the aim to identify the putative binding mode of the frag- ment hits. Initial active fragments are identifi ed and used as Nico Mell from the department DDEL attaches fl asks with novel scaffolds in ongoing synthesis projects. The identifi ca- samples at the lyophilisator for freeze drying. Photo: HZI, Bellhäuser SCIENTIFIC REPORTS | The Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS) 137

Development of a new in vitro model of the air-blood- barrier The so-called “air-blood-barrier” represented by the alveolar epithelium of the lung is crucial for the pulmonary delivery of drugs and plays an important role in the context of aerosol transmitted infectious diseases (e.g. swine fl u, tuberculosis). To mimic the in vivo situation several in vitro cell models are available (e.g., tumour cells, primary cells from animal origin), most of them lacking biological rele- vance due to impaired barrier properties. Primary human cells refl ect the in vivo situation best, but are not available in suffi cient amounts and the isolation procedure is time and cost intensive.

Hence, a new cell line is being developed to establish a non- tumour derived in vitro model with intact barrier properties. This approach will help to elucidate infection pathways across the respiratory tract and to facilitate the discovery of Caco-2 Zelle with propylstarch-nanopartikels adhered at the drug compounds capable of treating these diseases. cell membrane Photo: HIPS / HZI

Needle-free transdermal vaccination Traditional vaccina- tion requires intramuscular injection. However, this kind of vaccination does not ensure the optimal delivery of the diseases in the near future. Currently, such new formulations vaccine to the skin antigen presenting cells that elicit the are being developed for a number of anti-infl ammatory drugs immune response. Current strategies of needle-free vacci- at DDEL. To better understand the mechanism of particle ac- nation via the dermal route reduce the protective stratum cumulation in the infl amed tissue a novel cell culture model corneum barrier for a signifi cant time. This makes them of the infl amed intestinal mucosa has been established. suboptimal for certain applications, such as mass vaccina- tion campaigns in countries with critical hygienic conditions. Design of new gene delivery carriers In the fi eld of non- viral genetic therapies the current focus shifted from the Recently, an alternative pathway for targeting the skin cure of genetic disorders more towards vaccination or allergy immune system via the hair follicles by using nanoparticles treatment. Additional new interest arose with the discovery has been reported. As it is well known that pollen-antigens of RNA interference. It concerns a mechanism for sequence- rapidly cross the sebum fi lled follicle and cause an allergic specifi c inhibition of target genes, which can be used as reaction with susceptible persons, the scientists at DDEL therapy for the treatment of a variety of diseases. There hypothesize that mimicking the “delivery strategy” of pollen are physicochemical and biological barriers, which hinder with artifi cial particulate carriers is a promising strategy the delivery of free oligo- or poly-nucleotides. Their use is, for non-invasive vaccination across the intact skin barrier. therefore, reliant on suitable systems to achieve effi cient, safe and clinically appropriate delivery to overcome biologi- Drug loaded nano- and microparticles for the treatment cal barriers. The scientists at DDEL develop new formula- of infl ammatory bowel diseases Infl ammatory bowel tions from current approaches in polymer chemistry and diseases such as ulcerative colitis are autoimmune diseases nanotechnology. In these formulations, the drug is either of the intestine. Patients suffer from recurring acute pain, bound coordinatively (polyrotaxanes as cationic partner gastric and intestinal spasms, and heavy, often bloody diar- complexing anionic nucleic acid polymers) or encapsulated rhea. In addition, the risk of developing intestinal cancer is in nanoparticles (core-shell carrier particles on the basis substantially increased. of biodegradable polymers like chitosan-coated PLGA par- ticles). The aim is the development of carrier systems that It could be shown in animal studies that nano- to microsized can be adapted to various application routes by adjustable particles accumulate specifi cally in infl amed areas of the physical and pharmacokinetic properties. Such transport intestine. Therefore, nano- and microparticle based medi- systems are optimized for the intake via the lung or, in a cines may help to improve the therapy of infl ammatory bowel future project, via the intestinal tract. 138 SCIENTIFIC REPORTS | TWINCORE, Centre for Experimental and Clinical Infection Research GmbH

TWINCORE, Centre for Experimental and Clinical Infection Research GmbH

EXECUTIVE DIRECTOR | Prof. Dr. Ulrich Kalinke

HEAD OF EXPERIMENTAL INFECTION RESEARCH | Prof. Dr. Ulrich Kalinke HEAD OF EXPERIMENTAL VIROLOGY | Prof. Dr. Thomas Pietschmann HEAD OF INFECTION IMMUNOLOGY | Prof. Dr. Tim Sparwasser HEAD OF PATHOPHYSIOLOGY OF BACTERIAL BIOFILMS | Prof. Dr. Susanne Häußler HEAD OF CELL AND GENE THERAPY | Prof. Dr. Michael Ott

TWINCORE, the translation centre of HZI and MHH TWINCORE, Centre for Experimental and Clinical Infection Research GmbH, is a joint venture between the Helmholtz Centre for Infection Research (HZI), Braunschweig, and the Hannover Medical School (MHH). The goal pursued in the founding of TWINCORE is to promote and further develop the outstanding expertise of HZI and MHH in the fi eld of infection research in a joint centre with a specifi c focus on translational research. The purpose of translational research is a dual one, on one hand facilitating the path of the latest fi ndings from basic research to the patients and, on the other hand, enabling unanswered questions from clinical practice to make their way back to researchers. A key part of the work at TWINCORE is also the scientifi c investigation of regulatory concerns regarding approval and implementation of clinical trials. Complex issues frequently arise in advance of clinical trials, for example regarding the relevance of preclinical experiments for the safety and effi ciency of new medicines. TWINCORE helps to enable the development of new treatment options for the prophylaxis and therapy of infectious diseases ensuring that a solid scientifi c basis for the minimisation of risk exists prior to the testing of new approaches on humans. Research at TWINCORE focuses on the analysis of pathogen-host interactions. New fi ndings in this area lead to new approaches regarding mechanisms of pathogen inhibition and to the development of new vaccination strategies. TWINCORE holds laboratories which are adapted to conduct experiments up to bio safety level S3**. During the 2nd TWINCORE Symposium entitled “Antimicrobials and Vaccines”, which was held on August 12, 2010, the freshly renovated animal house was inaugurated (see Figure 1). The animal house can accommodate more than 2,000 mouse cages and is also adequate for perform- ing experiments up to bio safety level S3**. In addition to the annual TWINCORE Symposium a varied programme of lectures has been established, in which translational and basic researchers report on their recent research results.

1. Analysis of pathogen-host interactions During long have developed various strategies to evade host immunity. periods of co-evolution, pathogens and hosts developed Pathogen-encoded factors that modulate immune responses complex strategies to enable survival of both the host popu- are sought. Furthermore, the infl uence of regulatory cells lation and the pathogen population. At the cellular level on the course of infection is also analysed. intrinsic immune mechanisms play a role. At TWINCORE investigations are conducted to establish the infl uence such 2. New mechanisms of pathogen inhibition Following the factors have on host- and tissue-specifi city of pathogens. For triumphant march of antibiotics in the treatment of bacteri- several years it has been acknowledged that, in addition to al infections, over the past decades key breakthroughs have the recognition of “foreign”, the communication of “danger been made in the development of antiviral substances. New signals” via pattern recognition receptors (PRR) plays a approaches to the inhibition of pathogen reproduction are central role in the induction of protective immunity. The sought at TWINCORE. In collaboration with the HZI and the analysis of how innate immunity is triggered by stimulation University of Hannover, biological compound libraries are of PRR and the consequences on pathogen-specifi c immu- being examined for antiviral and antibacterial substan ces. nity are the subject of intensive investigations. In this pro- This involves the utilisation of new cell culture methods, for cess both acute and chronic courses of infections and their example permitting a targeted search for inhibitors of HCV associated infl ammatory reactions are examined. Pathogens replication. A further core focus is the search for inhibitors SCIENTIFIC REPORTS | TWINCORE, Centre for Experimental and Clinical Infection Research GmbH 139

Fig. 1. As part of the 2nd TWINCORE Symposium “Antimicrobials and Vaccines” the newly renovated animal house was inaugurated on August 12, 2010 at TWINCORE (left picture). The TWINCORE main entrance with the symposium transparent (right image). The “red tape” was jointly cut through at the opening of the new animal house by Prof. Bitter-Suermann (MHH), Prof. Wehland (HZI), Mr. Gevers (MWK) and Prof. Kalinke (TWINCORE) (from left to right). Photos: HZI/Twincore

of bacterial biofi lm formation which occur in chronic infec- human precursor cells contribute to the formation of liver tions. Similarly, new gene-therapy approaches are also being tissue. A further aspect is the genetic humanisation of mice, sought for the treatment of infectious diseases. Moreover, it for example by bacterial artifi cial chromosome (BAC)-medi- is also being investigated whether pathogen-encoded im- ated transgenesis. Using this method, human receptors and mune modulators constitute potential target structures for allelic forms of them found in the human population may be new therapeutic approaches. expressed in order to investigate their function in an animal model. Another subject is the investigation of effects that 3. New vaccination strategies To the general public, vacci- are mediated by constant portions of human antibodies. This nation is one of the most successful medical advances. Never - subject is particularly relevant in the context of the develop- theless, there are still numerous infectious diseases for ment of new therapeutic monoclonal antibodies. which no vaccine is available. Consequently, at TWINCORE new vaccination strategies are being developed. Virus-like particles are investigated for utilisation as vaccine vectors, as well as the specifi c in vivo charging of specialised cross- presenting dendritic cells with antigens. One interesting option is the reinforcement of immune responses by infl u- encing regulatory T cells. There are currently comparatively few approved adjuvants for the enhancement of immune responses following vaccination. Therefore, new adjuvants are being investigated in collaboration with partners at HZI. Similarly, investigations are also underway to determine whether cytokines are suitable natural adjuvants for certain vaccination protocols. A further key focus is the analysis of mechanisms that support the induction of long lasting IgG responses.

4. New preclinical models New therapeutic or prophylactic approaches developed in basic research need to be subjected to extensive preclinical testing prior to fi rst being tested in humans. At TWINCORE new models are being developed that should enable improved prediction with regard to reac- tions in humans. An important point in this respect is the A view of the TWINCORE building, Centre for Experimental and humanisation of mice. To this end, on the one hand, mice Clinical Infection Research GmbH. Alongside modern laborato- are treated with human cells to enable components of the ries and superb equipment, TWINCORE also offers optimal rooms human immune system to develop in the animals. On the in which to hold scientifi c meetings and seminars. Photo: HZI/Twincore other hand, mouse models are also being developed in which 140 SCIENTIFIC REPORTS | TWINCORE, Centre for Experimental and Clinical Infection Research GmbH

Research groups at TWINCORE Research group Prof. Dr. Ulrich Kalinke | TWINCORE was offi cially inaugurated in 2008 and all key [email protected] Following viral infection, leading positions were occupied in 2009. Currently, Prof. Dr. within hours type I interferon responses are usually Ulrich Kalinke is working as Executive Director of TWIN- induced which secure the initial survival of the host. It is CORE and Director of the Institute for Experimental Infec- only about a week later that adaptive immunity is activated tion Research, Prof. Dr. Thomas Pietschmann as Head of the to an extent that it is able to eradicate pathogens. In earlier Department for Experimental Virology, Prof. Dr. Tim Spar- projects we have shown that following an infection with the wasser as Director of the Institute for Infection Immunology, vesicular stomatitis virus (VSV) a small number of highly and Prof. Dr. Susanne Häußler as Head of the Department specialised cells, also addressed as plasmacytoid dendritic for Pathophysiology of Bacterial Biofi lms. Furthermore, the cells (pDC), are activated via PRR triggering to produce Translational Research Group for Cell and Gene Therapy large quantities of protective type I interferon. Interestingly, headed by Prof. Dr. Michael Ott of the Clinic for Gastroenter- practically all viruses examined more closely developed ology, Hepatology and Endocrinology of the MHH, directed countermeasures that inhibit the induction of such type I by Prof. Manns, has been delegated to TWINCORE. A total interferon responses. A key focus of our work is how differ- of 98 employees are working currently at TWINCORE. ent viruses induce type I interferon responses and which type I interferon-inhibiting factors they encode. Local condi- tions of type I interferon responses decisively infl uence the course of disease. In that line we are investigating how type I interferon inhibits the spread of pathogens within the central nervous system. In more recent investigations, we discovered that type I interferon can also have direct effects on immune cell functions. We are currently study- ing how type I interferon induced upon vaccination with virus-like particles infl uences the formation of long lasting IgG antibody responses. Antibodies are comparatively large molecules, the variable parts of which bind antigens specifi cally, whereas the constant parts may be bound via so-called Fc receptors expressed by certain immune cells and other body cells and confer antibody function. We The TWINCORE research group leaders (from left to right): investigate how Fc receptors interact with different sub- Profs. Susanne Häußler, Michael Ott, Thomas Pietschmann, classes of IgG antibodies and which immune functions are Executive Director Ulrich Kalinke, and Tim Sparwasser. infl uenced this way. This issue is particularly relevant for Photo: HZI, Gramann therapeutically-employed monoclonal antibodies because such reagents play an increasingly important role as in- novative therapeutic agents in the treatment of tumours,

Theresa Frenz working at the clean bench Photo: Twincore/HZI Claudia Soldner analysing the results of an experiment Photo: Twincore/HZI SCIENTIFIC REPORTS | TWINCORE, Centre for Experimental and Clinical Infection Research GmbH 141

are involved in the development of a new immuno therapy approach for the treatment of chronic HCV infection and HCV-associated hepatocellular carcinoma. A new focus is forming our investigations of the HCV species and tis- sue tropism. HCV replicates in the liver and only infects humans and chimpanzees. The mechanisms which are responsible for this restricted species and tissue tropism have been scarcely studied up to now and thus are only barely understood. With our research on this topic we aim to understand how extra hepatic virus reservoirs can affect virus pathogenesis and the response to therapy, how HCV makes use of essential cellular cofactors, and which host factors control the HCV replication. The workgroup of Prof. Dr. Ulrich Kalinke: (from left to right) Ulrich Kalinke, Robert Bittorf (last row), Linda Semmler, Theresa Frenz, Marius Döring (3rd row), Claudia Soldner, Anette Kessler, Christian Mers, Jennifer Paijo, Claudia Detje, Lukas Graalmann, Patrick Bartholomäus (2nd row), Sabrina Heindorf, Stephanie Vogel, Julia Heinrich (1st row). Photo: Twincore/HZI

autoimmune diseases and infections. In order to make even better use of the results of the above mentioned approaches for the application and implementation of clinical trials, regulatory research is performed in collaboration with the The workgroup of Prof. Dr. Pietschmann: (from left to right) Paul-Ehrlich-Institut, Germany. Kathrin Hüging, Nikoleta Bodosoglou, Juliane Dörrbecker, Dorothea Bankwitz, Christina Grethe, Juliane Gentzsch, Sibylle Research group Prof. Dr. Thomas Pietschmann | Haid, Nicolas Menzel, Patrick Chhatwal, Eike Steinmann, Tho- [email protected] Infection with HCV mas Pietschmann, Gabrielle Vieyres, Stephanie Pfänder, Anne which belongs to the family of fl aviviruses is one of the Frentzen (missing: Joachim Hain, Martina Friesland, Sandra major causes of chronic liver disease. According to WHO Ciesek) Photo: Twincore/HZI estimates, up to 170 million people have had HCV contact worldwide. Of these, around 100 to 130 million people are considered to be chronically infected. We develop new cell culture techniques for the investigation of HCV Research group Prof. Dr. Tim Sparwasser | replication. The goal of this research is to investigate the [email protected] Our focus lies upon the in- molecular mechanisms of HCV replication in liver cells. In vestigation of the signifi cance of PRR, for example from the particular, early stages of HCV infection are studied which family of Toll-like receptors (TLRs) and the C-type lectins, are critically involved in virus entry into liver cells. We for the activation of the most important positive regulators are also analysing processes that lead to the packing of the of the immune system and initiators of adaptive immunity, viral genetic material in progeny viruses and their release i.e. the dendritic cell system (DCs). A further focus is upon from the host cell. In this manner we aim to draw up the regulatory T cells (Tregs) which may be regarded as the fundamental basis of the infection strategy of this human- principal counterparts to DCs: Tregs use mechanisms as pathogenic virus in order to subsequently generate new ap- yet not completely understood to inhibit an overshooting proaches and perspectives for the development of therapies. immune response and limit the proliferation of T effector In TWINNING projects with partners at the MHH and the cells. Optimal vaccination strategies against pathogens HZI we employ the HCV cell culture system to identify new may comprise the activation of specifi c DC subpopulations agents that inhibit HCV replication. Furthermore, within whilst avoiding Treg expansion or induction. Vaccination the Helmholtz Alliance on Immunotherapy of Cancer we studies aimed towards Tregs and DCs in the murine model 142 SCIENTIFIC REPORTS | TWINCORE, Centre for Experimental and Clinical Infection Research GmbH

Matthias Lochner is currently establishing a junior research team on the analysis of the role of DCs in the induction and control of infl ammatory (Th17) and regulatory T-cell popula- tions in infection and infl ammation reaction of the intestine.

Research group Prof. Dr. Susanne Häußler | [email protected] The successes of modern medicine are increasingly affected by opportunistic bacterial infections. In chronic bacterial infections the pathogens may often come together within so-called biofi lms and are then better protected against attack by cells of the immune system or antibiotics. The life within the population of the bacteria also provides additional adaptation mechanisms to stress Jointly analysing scatter plots (from left to right): Nicole Fisch, situations that go beyond the usual reactions of individual Venkateswaran Ganesh, Wiebke Ginter, Catharina Schrauf, cells. Pseudomonas aeruginosa is the most dominant bacte- Christian Mayer und Ina Lu Photo: Twincore/HZI rial pathogen causing chronic lung infection in cystic fi brosis (CF) patients. Although most patients are colonised with only one P. aeruginosa clone, various morphotypes can be isolated. This diversity seems to play an important role in the persist- system have several limitations, one of which is that in vivo ence of the germ and thus in the formation of a chronic infec- analysis of Tregs and DC subpopulations is extremely dif- tion. Our research focuses on the elucidation of the molecular fi cult. For example, subpopulations of DCs exist in extremely mechanism underlying this diversity. In CF patients with a low numbers in various lymphatic organs that have highly chronic P. aeruginosa infection of the lungs so-called small specialised tasks in several cases including the induction colony variants (SCVs) are frequently found which form of tolerance. As these “regulatory immune cells” usually biofi lms particularly effi ciently. To identify the mutations react highly sensitively to ex vivo isolation and have thus far that lead to the formation of such SCVs, we sequenced the proved only inadequately manipulable in vivo, the knowledge genomes of P. aeruginosa clinical isolates using the so-called of function and signifi cance of Tregs and DC subpopulations next generation sequencing technology. The comparative for adaptive immunity remains incomplete. A further compli- analysis of chromosomal DNA sequence of P. aeruginosa cating factor is the expression of different pattern recognition isolates with similar phenotypic characteristics; we look for molecules or different expression profi les of PRRs to DC sub - typical base substitutions and check whether they are caus- populations between humans and mice. A key objective is, ally involved in the progression of the phenotype. therefore, the development of molecular tools that allow the genetic manipulation of DCs and Tregs. We wish to use these In the future we aim to study clinically relevant mutations models to investigate the function of Tregs and subpopulations that occur in P. aeruginosa under in vitro biofi lm growth of DCs in infection, allergy and tolerance. In “humanised” conditions and in vivo in the course of a chronic infection. models the role of PRRs such as human TLR9 and DC-SIGN The knowledge of the genotypes that are selected at different is analysed and vaccination strategies aimed at these mole- stages of infection can be helpful for the development of cules tested in vivo. Within the Sparwasser Institute Dr. new, promising therapy strategies. In addition, to two

The workgroup of Prof. Dr. Sparwasser: (from left to right) Christina Hesse, Christian Klemann, Amrita Nandan, Matthias Lochner, Stefanie Pohl, Christian Mayer, Zuobai Wang, Franz Puttur, Catharina Schrauf, Luciana Berod, Christopher van Helt, Julia Huntenburg, Wiebke Ginter, Nicole Fisch, Martina Thiele, Stephanie Dippel, Christine Jänke, Janika Quindt, Esther Ermeling, Abdul Mannan Baru, Venkateswaran Ganesh, Siona Hauer, Tim Sparwasser. Photo: Twincore/HZI SCIENTIFIC REPORTS | TWINCORE, Centre for Experimental and Clinical Infection Research GmbH 143

well-characterized homoserine lactone signal molecules P. aeruginosa produces a third interbacterial signal molecule, the Pseudomonas quinolone signal (PQS). PQS is involved in cell density dependent virulence factor regulation – as well as the homoserine lactone – and is essentially involved in the establishment of P. aeruginosa biofi lms. However, the molecular mechanism of the implementation of the PQS sig- nal in a bacterial behaviour at the signal cell level is largely unknown. Here, a pqsE encoded enzyme, the last gene in the PQS biosynthetic operon, seems to play a central role. The elucidation of the function of PqsE is a key research focus of our group. The workgroup of Prof. Dr. Ott: (from left to right) Johan Waern, Michael Rothe, Martin Pacher, Michael Ott, Urda Rüdrich, Gesa Riedel, Quinggong Yuan, Ina Rittelmeyer (missing: Michael Bock). Photo: Twincore/HZI

of human blood stem cells and foetal liver tissue to a newly- developed mouse strain are set to be performed shortly. Due to the lack of availability of primary human cell material for the “humanisation” of mice, as well as for cellular therapies in humans, the research group is conducting intensive re- search into alternative cell sources. In association with MHH groups and international partners, research is underway into hepatic differentiation protocols for embryonic stem cells Prof. Häußler and her team: (from left to right) Vera Nöding, and iPS cells. In another project the risk of insertional mu- Kathi Klimmek, Susanne Häußler, Mathias Müsken, Andrea tagenesis in lentiviral gene transfer is being analysed. Serial Blanka Photo: Twincore/HZI transplantation of ex vivo genetically transduced hepatocytes enables the incidence of liver tumours in dependence on the number of lentiviral insertions to be investigated. Further- more, the research group is supervising a clinical study on Research group Prof. Dr. Michael Ott | cell transplantation in patients with urea cycle disorder. [email protected] We develop cell and gene This project marks the world’s fi rst controlled study on cell therapy procedures for the treatment of hereditary liver therapy of hereditary metabolic diseases of the liver. disease. Another focus lies on the development of mouse models with chimeric human/murine liver tissue and hu- TWINCORE key publications In 2010 employees working at man immune system for researching vaccination strategies TWINCORE published a total of 54 articles. In 2011 already against HIV and HCV. The repopulation of the liver with 24 articles have either been published or are “in press” (see human liver cells and the transplantation of human blood “Complete list of articles published in 2010 as well as arti- stem cells into immune defi cient mice continue to represent cles published an “in press” in 2011 by employees working a major scientifi c challenge. In order to investigate vaccina- at TWINCORE”). Particularly important research results tion strategies in the alb-uPA transgene immune defi cient have been compiled in the following areas: (RAGyc) mouse it is necessary that human cells of a donor are utilised in the repopulation of the mice. Where primary 1. Regulatory T cells in the immune modulation therapy tissue is used as starting material the isolation of both cell So far, proven therapeutic vaccination against tumours populations using foetal liver tissue exclusively is possible. have shown only minimal or no success. Now we have suc- In our transplantation experiments it emerged that the trans- ceeded in improving the therapeutic vaccination success plantation of foetal hepatoblasts is signifi cantly less effi cient in the treatment of malignant melanoma in a mouse model than that of adult primary hepatocytes. Alternatively, the by selective depletion of regulatory T cells (Klages et al., research group enabled the transplantation of foetal human 2010). Another project made an important contribution to a liver tissue beneath the capsule of the recipient liver to be better understanding of the regulatory mechanisms of the tested for the fi rst time. The fi rst combined transplantations immune system in the intestine (Sawa et al., 2011). 144 SCIENTIFIC REPORTS | TWINCORE, Centre for Experimental and Clinical Infection Research GmbH

2. Mechanisms of hepatitis C virus replication and • Bitzegeio J, Bankwitz D, Hueging K, Haid S, Brohm C, Zeisel MB, Herrmann E, Iken M, Ott M & Baumert TF, Pietschmann T (2010) immune control In a hepatitis C virus (HCV) infection Adaptation of hepatitis C virus to mouse CD81 permits infection of species-specifi c interactions with surface receptors such mouse cells in the absence of human entry factors. PLoS Pathogens 6: e1000978. as CD81 play a central role. According to this, only chim- panzees and humans are naturally infected with HCV. • Frenz T, Waibler Z, Hofmann J, Hamdorf M, Lantermann M, Reizis B, Tovey MG, Aichele P, Sutter G & Kalinke U (2010) Concomitant After adaptation of HCV to a mouse CD81, it was possible IFNAR-triggering of T cells and of DC is required to promote maxi- to identify three mutations in the envelope glycoprotein of mal MVA-induced T-Cell expansion. European Journal of Immuno- logy 40(10), 2769-2777. HCV which enhance the infection of cells with murine CD81 100-fold and thereby allow the HC cell entry into mouse • Klages K, Mayer CT, Lahl K, Loddenkemper C, Teng MW, Ngiow SF, Smyth MJ, Hamann A, Huehn J & Sparwasser T (2010) Selective cells without using human cofactors (Bitzegeio et al., 2010). depletion of Foxp3+ regulatory T cells improves effective therapeutic These studies raise the hope that it will one day be possible vaccination against established melanoma. Cancer Research 70(20), 7788-7799. to establish a permissive immuno-competent small animal model for HCV infection. • Pommerenke C, Müsken M, Becker T, Dotsch A, Klawonn F & Häus- sler S (2010) Global genotype-phenotype correlations in Pseu- domonas aeruginosa. PLoS Pathogens 6(8). pii: e1001074. 3. Pathogen recognition and immune stimulation by den- • Sancho-Bru P, Roelandt P, Narain N, Pauwelyn K, Notelaers T, dritic cells Virus-induced type I interferon (IFN) responses Shimizu T, Ott M & Verfaillie C (2010) Directed differentiation of may enhance cytotoxic T cell responses dramatically. This murine-induced pluripotent stem cells to functional hepatocyte-like cells. Journal of Hepatology 54(1), 98-107. fi nding is surprising because IFN-responses reach peak serum levels within hours of infection, while cytotoxic T • Sawa S, Cherrier M, Lochner M, Satoh-Takayama N, Fehling HJ, Langa F, Di Santo JP & Eberl G (2010) Lineage relationship analysis cell responses are maximally induced after approximately of ROR t+ innate lymphoid cells. Science 330(6004), 665-669. one week. The work by Frenz et al. has shown that IFN- responses enhance T cell proliferation by the stimulation of antigen-presenting cells as well as through the direct stimulation of T cells (Frenz et al., 2010).

4. Immune subversion strategies In the context of chronic Contact bacterial infections biofi lms may develop which cannot be TWINCORE GmbH treated with normal antibiotics. We have succeeded in de- Zentrum für Experimentelle und Klinische veloping a new in vitro test system that allows the targeted Infektionsforschung search of novel compounds that inhibit biofi lm formation Feodor-Lynen-Str. 7 | 30625 Hannover (Pommerenke et al., 2010). Telefon 0511 2200 27 102 | Fax 0511 2200 27 186 [email protected] | www.twincore.de 5. Humanised mouse models Treatment options for vari- ous liver diseases are limited by the fact that syngeneic hepatocytes are not usually available. In the mouse system we have succeeded in developing a new process that allows the differentiation of induced pluripotent stem (iPS) in hepatocyte-like cells (Sancho-Bru et al., 2010). SCIENTIFIC REPORTS | TWINCORE, Centre for Experimental and Clinical Infection Research GmbH 145

Complete list of articles published in 2010 as well as • 13. Kern M, Popov A, Scholz K, Schumak B, Djandji D, Limmer A, Eggle D, Sacher T, Zawatzky R, Holtappels R, Reddehase MJ, articles published and “in press” in 2011 by employees Hartmann G, Debey-Pascher S, Diehl L, Kalinke U, Koszinowski U, working at TWINCORE Schultze J & Knolle PA (2010) Virally infected mouse liver endothelial cells trigger CD8+ T-cell immunity. Gastroenterology 138(1), 336-346. • 14. Malinarich FH, Grabski E, Worbs T, Chennupati V, Haas JD, Research group Prof. Kalinke Schmitz S, Candia E, Quera R, Malissen B, Förster R, Hermoso M & Prinz I (2010) Constant TCR triggering suggests that the TCR • 1. Williams SK, Fairless R, Weise J, Kalinke U, Schulz-Schaeffer W & expressed on intestinal intraepithelial γδ T cells is functional in vivo. Diem R (2011) Neuroprotective effects of the cellular prion protein in European Journal of Immunology 40(12), 3378-88. autoimmune optic neuritis. American Journal of Pathology (in press). • 15. Schneider CK, Salmikangas P, Jilma B, Flamion B, Todorova LR, • 2. Gratz MS, Suezer Y, Kremer M, Volz A, Majzoub M, Hanschmann Paphitou A, Haunerova I, Maimets T, Trouvin JH, Flory E, Tsiftsoglou KM, Kalinke U, Schwantes A & Sutter G (2011) N1L is a virulence A, Sarkadi B, Gudmundsson K, O’Donovan M, Migliaccio G, Ancans factor of ectromelia virus and essential for in vivo spread upon J, Maciulaitis R, Robert JL, Samuel A, Ovelgonne JH, Hystad M, Fal respiratory infection. Journal of Virology (in press). AM, Lima BS, Moraru AS, Turcani P, Zorec R, Ruiz S, Akerblom L, Narayanan G, Kent A, Bignami F, Dickson JG, Niederwieser D, • 3. Grabski E, Waibler Z, Schule S, Kloke BP, Sender LY, Panitz S, Figuerola-Santos MA, Reischl IG, Beuneu C, Georgiev R, Vassiliou Cichutek K, Schweizer M & Kalinke U (2011) Comparative analysis M, Pychova A, Clausen M, Methuen T, Lucas S, Schussler-Lenz M, of transduced primary human dendritic cells generated by the use Kokkas V, Buzas Z, MacAleenan N, Galli MC, Line A, Gulbinovic J, of three different lentiviral vector systems. Molecular Biotechnology Berchem G, Fraczek M, Menezes-Ferreira M, Vilceanu N, Hrubisko 47(3), 262-269. M, Marinko P, Timon M, Cheng W, Crosbie GA, Meade N, di Paola ML, VandenDriessche T, Ljungman P, D’Apote L, Oliver-Diaz O, Buttel • 4. Kochs G, Bauer S, Vogt C, Frenz T, Tschopp J, Kalinke U & I & Celis P (2010) Challenges with advanced therapy medicinal Waibler Z (2010) Thogoto virus infection induces sustained type I products and how to meet them. Nature Reviews Drug Discovery 9(3), interferon responses that depend on RIG-I-like helicase signaling of 195-201. Review. conventional dendritic cells. Journal of Virology 84(23), 12344-12350. • 16. Buttel IC, Voller K & Schneider CK (2010) Immunogenicity and • 5. Frenz T, Waibler Z, Hofmann J, Hamdorf M, Lantermann M, Reizis its impact on benefi t/risk considerations in the authorisation of B, Tovey MG, Aichele P, Sutter G & Kalinke U (2010) Concomitant biopharmaceuticals. Current Drug Safety 5(4), 287-292. Review. IFNAR-triggering of T cells and of DC is required to promote maximal MVA-induced T-Cell expansion. European Journal of • 17. Goetz KB, Pfl eiderer M & Schneider CK (2010) First-in- Immunology 40(10), 2769-2777. human clinical trials with vaccines-what regulators want. Nature Biotechnology 28(9), 910-916. Review. • 6. Goossens P, Gijbels MJ, Zernecke A, Eijgelaar W, Vergouwe MN, van der Made I, Vanderlocht J, Beckers L, Buurman WA, Daemen • 18. Schussler-Lenz M & Schneider CK (2010) [Clinical trials with MJ, Kalinke U, Weber C, Lutgens E & de Winther MP (2010) Myeloid advanced therapy medicinal products]. Bundesgesundheitsblatt type I interferon signaling promotes atherosclerosis by stimulating Gesundheitsforschung Gesundheitsschutz 53(1), 68-74. Review. macrophage recruitment to lesions. Cell Metabolism 12(2), 142-153. • 7. Lang PA, Recher M, Honke N, Scheu S, Borkens S, Gailus N, Krings C, Meryk A, Kulawik A, Cervantes-Barragan L, Van Rooijen Research group Prof. Pietschmann N, Kalinke U, Ludewig B, Hengartner H, Harris N, Häussinger D, Ohashi PS, Zinkernagel RM & Lang KS (2010) Tissue macrophages • 1. Frentzen A, Hüging K, Bitzegeio J, Friesland M, Haid S, Gentzsch suppress viral replication and prevent severe immunopathology in J, Hoffmann M, Lindemann D, Zimmer G, Zielecki F, Weber F, an interferon-I-dependent manner in mice. Hepatology 52(1), 25-32. Steinmann E & Pietschmann T (2011) Completion of hepatitis C virus replication cycle in heterokaryons excludes dominant restrictions in • 8. Prinz M & Kalinke U (2010) New lessons about old molecules: how human non-liver and mouse liver cell lines. PLoS Pathog (in press). type I interferons shape Th1/Th17-mediated autoimmunity in the CNS. Trends in Molecular Medicine 16(8), 379-386. Review. • 2. Gentzsch J, Hinkelmann B, Kaderali L, Irschik H, Jansen R, Sasse F, Frank R & Pietschmann T (2011) Hepatitis C virus complete • 9. Murikinati S, Juttler E, Keinert T, Ridder DA, Muhammad S, life cycle screen for identifi cation of small molecules with pro- or Waibler Z, Ledent C, Zimmer A, Kalinke U & Schwaninger M (2010) antiviral activity. Antiviral Research 89(2),136-48. Epub 2010 Dec 15. Activation of cannabinoid 2 receptors protects against cerebral ischemia by inhibiting neutrophil recruitment. FASEB Journal 24(3), • 3. Montserret R, Saint N, Vanbelle C, Salvay AG, Simorre JP, Ebel 788-798. C, Sapay N, Renisio JG, Bockmann A, Steinmann E, Pietschmann T, Dubuisson J, Chipot C & Penin F (2010) NMR structure and ion • 10. Sender LY, Gibbert K, Suezer Y, Radeke HH, Kalinke U & Waibler channel activity of the p7 protein from hepatitis C virus. Journal of Z (2010) CD40 ligand-triggered human dendritic cells mount Biological Chemistry 285(41), 31446-31461. interleukin-23 responses that are further enhanced by danger signals. Molecular Immunology 47(6), 1255-1261. • 4. Steinmann E & Pietschmann T (2010) Hepatitis C virus P7 - a viroporin crucial for virus assembly and an emerging target for • 11. Al Moussawi K, Ghigo E, Kalinke U, Alexopoulou L, Mege JL antiviral therapy. Viruses 2010 2(9), 2078-2095 (Review). & Desnues B (2010) Type I interferon induction is detrimental during infection with the Whipple’s disease bacterium, Tropheryma • 5. Bitzegeio J, Bankwitz D, Hueging K, Haid S, Brohm C, Zeisel MB, whipplei. PLoS Pathogens 6(1): e1000722. Herrmann E, Iken M, Ott M & Baumert TF, Pietschmann T (2010) Adaptation of hepatitis C virus to mouse CD81 permits infection of • 12. Ilchmann A, Burgdorf S, Scheurer S, Waibler Z, Nagai R, Wellner mouse cells in the absence of human entry factors. PLoS Pathogens A, Yamamoto Y, Yamamoto H, Henle T, Kurts C, Kalinke U, Vieths S 6: e1000978. & Toda M (2010) Glycation of a food allergen by the Maillard reaction enhances its T-cell immunogenicity: role of macrophage scavenger • 6. Bürgel B, Friesland M, Koch A, Manns MP, Wedemeyer H, receptor class A type I and II. Journal of Allergy and Clinical Weissenborn K, Schulz-Schaeffer WJ, Pietschmann T, Steinmann Immunology 125(1), 175-183 e171-111. E & Ciesek S (2010) Hepatitis C virus enters human peripheral neuroblastoma cells - evidence for extra-hepatic cells sustaining hepatitis C virus penetration. Journal of Viral Hepatology Jun 23 [Epub ahead of print]. 146 SCIENTIFIC REPORTS | TWINCORE, Centre for Experimental and Clinical Infection Research GmbH

• 7. Ciesek S, Friesland M, Steinmann J, Becker B, Wedemeyer H, • 6. Stagg J, Divisekera U, Duret H, Sparwasser T, Teng MW, Darcy PK Manns MP, Pietschmann T & Steinmann E (2010) How stable is the & Smyth MJ (2011) CD73-defi cient mice have increased anti-tumor hepatitis C virus (HCV)? Environmental stability of HCV and its immunity and are resistant to experimental metastasis. Cancer susceptibility to chemical biocides. Journal of Infectious Diseases Research Feb 3 [Epub ahead of print]. 201(12), 1859-1866. • 7. Ohnmacht C*, Marques R*, Presley L*, Sawa S*, Lochner M* & • 8. Lemon SM, McKeating JA, Pietschmann T, Frick DN, Glenn JS, Eberl G (2011) Intestinal microbiota, evolution of the immune system Tellinghuisen TL, Symons J & Furman PA (2010) Development of and the bad reputation of pro-infl ammatory immunity. Cellular novel therapies for hepatitis C. Antiviral Research 86(1), 79-92 Microbiology Jan 28 [Epub ahead of print] Review. (Review). • 8. Mayer CT, Floess S, Baru AM, Lahl K, Huehn J & Sparwasser T • 9. Bankwitz D, Steinmann E, Bitzegeio J, Ciesek S, Friesland M, (2011) CD8+Foxp3+ T cells share developmental and phenotypic Herrmann E, Zeisel MB, Baumert TF, Keck ZY, Foung SK, Pecheur features with classical CD4+Foxp3+ regulatory T cells but lack EI & Pietschmann T (2010) Hepatitis C virus hypervariable region 1 potent suppressive activity. European Journal of Immunology 41(3), modulates receptor interactions, conceals the CD81 binding site, and 716-725. protects conserved neutralizing epitopes. Journal of Virology 84(11), 5751-5763. • 9. Lahl K & Sparwasser T (2011) In vivo depletion of Foxp3+ Tregs using the DEREG mouse model. Methods in Molecular Biology 707, • 10. Stegmann KA, Bjorkstrom NK, Veber H, Ciesek S, Riese P, 157-172 Review. Wiegand J, Hadem J, Suneetha PV, Jaroszewicz J, Wang C, Schlaphoff V, Fytili P, Cornberg M, Manns MP, Geffers R, Pietschmann T, • 10. Berod L, Heinemann C, Heink S, Escher A, Stadelmann C, Drube Guzman CA, Ljunggren HG & Wedemeyer H (2010) Interferon-alpha- S, Wetzker R, Norgauer J & Kamradt T (2011) PI3K defi ciency delays induced TRAIL on natural killer cells is associated with control of the onset of experimental autoimmune encephalomyelitis and hepatitis C virus infection. Gastroenterology 138(5), 1885-1897. ameliorates its clinical outcome. European Journal of Immunology 41(3), 833-844. • 11. Ciesek S, Steinmann E, Iken M, Ott M, Helfritz FA, Wappler I, Manns MP, Wedemeyer H & Pietschmann T (2010) • 11. Pellegrini M, Calzascia T, Toe JG, Preston SP, Lin AE, Elford Glucocorticosteroids increase cell entry by hepatitis C virus. AR, ShahinianA, Lang PA, Lang KS, Morre M, Assouline B, Lahl K, Gastroenterology 138(5), 1875-1884. Sparwasser T, Tedder TF, Paik J, DePinho RA, Basta S, Ohashi PS & Mak TW (2011) IL-7 engages multiple mechanisms to overcome • 12. von Hahn T, Steinmann E, Ciesek S & Pietschmann T (2010) chronic viral infection and limit organ pathology. Cell 144(4), 601- Know your enemy: translating insights about the molecular biology 613. of hepatitis C virus into novel therapeutic approaches. Expert Review on Gastroenterology & Hepatology 4(1), 63-79. • 12. Dietze KK, Zelinskyy G, Gibbert K, Schimmer S, Francois S, Myers L, Sparwasser T, Hasenkrug KJ & Dittmer U (2011) Transient • 13. Steinmann J, Becker B, Bischoff B, Paulmann D, Friesland M, depletion of regulatory T cells in transgenic mice reactivates virus- Pietschmann T, Steinmann E (2010) Virucidal activity of 2 alcohol- specifi c CD8+ T cells and reduces chronic retroviral set points. based formulations proposed as hand rubs by the World Health Proceedings of the National Academy of Sciences U S A 108(6), 2420- Organization. American Journal of Infection Control 38(1), 66-68. 2425. • 14. Haid S, Windisch MP, Bartenschlager R & Pietschmann T (2010) • 13. Vaeth M, Gogishvili T, Bopp T, Klein M, Berberich-Siebelt F, Mouse-specifi c residues of claudin-1 limit hepatitis C virus genotype Gattenloehner S, Avots A, Sparwasser T, Grebe N, Schmitt E, Hunig 2a infection in a human hepatocyte cell line. Journal of Virology T, Serfl ing E & Bodor J (2011) Regulatory T cells facilitate the 84(2), 964-975. nuclear accumulation of inducible cAMP early repressor (ICER) and suppress nuclear factor of activated T cell c1 (NFATc1). Proceedings of the National Academy of Sciences U S A 108(6), 2480-2485. Research group Prof. Sparwasser • 14. Lochner M, Bérard M, Sawa S, Hauer S, Gaboriau-Routhiau V, Fernandez F, Bousso P, Cerf-Bensussan N & Eberl G (2011) Restricted • 1. Hackl D, Loschko J, Sparwasser T, Reindl W & Krug A (2011) microbiota and absence of cognate TCR antigen leads to an Activation of dendritic cells via TLR7 reduces Foxp3 expression unbalanced generation of Th17 cells. Journal of Immunology 186(3), and suppressive function in induced Tregs. European Journal of 1531-1537 (Epub 2010 Dec 22) Immunology (in press). • 15. Lochner M, Ohnmacht C, Presley L, Bruhns P, Si-Tahar M, Sawa • 2. Paust HJ, Ostmann A, Erhardt A, Turner JE, Mittrücker HW, S & Eberl G (2011) Microbiota-induced tertiary lymphoid tissues Sparwasser T, Panzer U & Tiegs G (2011) Regulatory T cells control aggravate infl ammatory disease in the absence of RORgt and LTi the Th1 immune response in murine crescentic glomerulonephritis. cells. Journal of Experimental Medicine 208(1), 125-134 (Epub 20 Dec Kidney International (in press). 2010) • 3. Sawa S, Lochner M, Satoh-Takayama N, Gaboriau-Routhiau V, • 16. Haque A, Best SE, Amante FH, Mustafah S, Desbarrieres L, de Dulauroy S, Bérard M, Kleinschek M, Cerf-Bensussan N, Cua D, Di Labastida F, Sparwasser T, Hill GR & Engwerda CR (2010) CD4+ Santo JP & Eberl G (2011) RORgt+ innate lymphoid cells regulate natural regulatory T cells prevent experimental cerebral malaria via intestinal homeostasis by integrating negative signals from the CTLA-4 when expanded in vivo. PLoS Pathogens 6(12), e1001221. symbiotic microbiota. Nature Immunology Feb 20 [Epub ahead of print]. • 17. Engel, D, Koscielny A, Wehner S, Maurer J, Schiwon M, Franken L, Schumak B, Limmer A, Sparwasser T, Hirner A, Knolle P, Kalff J • 4. Blankenhaus B, Klemm U, Eschbach ML, Sparwasser T, & Kurts C (2010) Th1 memory cells disseminate postoperative ileus Huehnn J, Kühl AA, Loddenkemper C, Jacobs T & Breloer M (2011) over the entire intestinal tract. Nature Medicine 16(12), 1407-1413. Strongyloides ratti infection induces expansion of Foxp3+ regulatory T cells that interfere with immune response and parasite clearance • 18. Hubert S, Rissiek B, Klages K, Hühn J, Sparwasser T, Haag F, in BALB/c mice. Journal of Immunology Feb 18 [Epub ahead of print]. Koch-Nolte F, Boyer O, Seman M & Adriouch S (2010) Extracellular NAD+ shapes the Foxp3+ regulatory T cell compartment through • 5. Hadis U, Wahl B, Schulz O, Schippers A, Wagner N, Müller W, the ART2/P2X7 pathway. Journal of Experimental Medicine 207(12), Sparwasser T, Förster R & Pabst O (2011) Cooperation of lymph nodes 2561-2568. and intestinal lamina propria in FoxP3+ regulatory T cell mediated intestinal tolerance. Immunity 34(2), 237-246. SCIENTIFIC REPORTS | TWINCORE, Centre for Experimental and Clinical Infection Research GmbH 147

• 19. Teng MW, Ngiow SF, von Scheidt B, McLaughlin N, Sparwasser T • 4. Müsken M, Di Fiore S, Römling U & Häussler S (2010) A 96-well- & Smyth MJ (2010) Conditional regulatory T-cell depletion releases plate-based optical method for the quantitative and qualitative adaptive immunity preventing carcinogenesis and suppressing evaluation of Pseudomonas aeruginosa biofi lm formation and its established tumor growth. Cancer Research 70(20), 7800-7809. application to susceptibility testing. Nature Protocols 5(8), 1460-1469. • 20. Klages K, Mayer CT, Lahl K, Loddenkemper C, Teng MW, Ngiow • 5. Häussler S & Parsek MR (2010) Biofi lms 2009: new perspectives SF, Smyth MJ, Hamann A, Huehn J & Sparwasser T (2010) Selective at the heart of surface-associated microbial communities. Journal of depletion of Foxp3+ regulatory T cells improves effective therapeutic Bacteriology 192(12), 2941-2949. vaccination against established melanoma. Cancer Research 70(20), 7788-7799. • 6. Dötsch A, Klawonn F, Jarek M, Scharfe M, Blocker H & Häussler S (2010) Evolutionary conservation of essential and highly expressed • 21. Sawa S, Cherrier M, Lochner M, Satoh-Takayama N, Fehling HJ, genes in Pseudomonas aeruginosa. BMC Genomics 11, 234. Langa F, Di Santo JP & Eberl G (2010) Lineage relationship analysis of ROR t+ innate lymphoid cells. Science 330(6004), 665-669. • 7. Müsken M, Di Fiore S, Dotsch A, Fischer R & Häussler S (2010) Genetic determinants of Pseudomonas aeruginosa biofi lm • 22. Navarro S, Cossalter G, Chiavaroli C, Kanda A, Fleury S, Lazzari establishment. Microbiology 156(Pt 2), 431-441. A, Cazareth J, Sparwasser T, Dombrowicz D, Glaichenhaus N & Julia V (2010) The oral administration of bacterial extracts prevents asthma via the recruitment of regulatory T cells to the airways. Mucosal Immunology 4(1), 53-65 (Epub 01 Sept. 2010). Research group Prof. Ott • 23. Arnold I, Lee J, Amieva M, Roers A, Flavell R, Sparwasser T & Müller A (2010) Tolerance rather than immunity protects from • 1. Sharma AD, Narain N, Händel EM, Iken M, Singhal N, Cathomen Helicobacter pylori-induced gastric preneoplasia. Gastroenterology T, Manns M, Schöler HR, Ott M & Cantz T (2011) MicroRNA-221 140(1), 199-209 (Epub 22 June 2010). regulates FAS-induced fulminant liver failure. Hepatology [Epub ahead of print]. • 24. Baru AM. Hartl A, Lahl K, Krishnaswamy JK, Fehrenbach H, Yildirim A, Garn H, Renz H, Behrens G & Sparwasser T (2010) • 2. Sharma AD, Razvan I, Bock M, Cantz T, Manns MP & Ott M (2011) Selective depletion of Foxp3+ regulatory T cells during sensitization Liver (chapter 33); G. Steinhoff (ed.) Regenerative Medicine: From phase aggravates experimental allergic airway infl ammation. protocol to patient, pp. 773-803. European Journal of Immunology 40(8), 2259-2266. • 3. Razvan I, Rüdrich U, Rothe M, Kirsch S, Maasoumy B, Narain • 25. Pechloff K, Holch J, Ferch U, Schweneker M, Brunner K, Kremer N, Verfaillied CM, Sancho-Bru P, Iken M, Popescu I, Schambach M, Sparwasser T, Quintanilla-Martinez L, Zimber-Strobl U, Streubel A, Manns MP & Bock M (2011) Induction of a mature hepatocyte B, Gewies A, Peschel C & Ruland J (2010) The fusion kinase ITK- phenotype in adult liver derived progenitor cells by ectopic SYK mimics a T-cell receptor signal and drives oncogenesis in expression of transcription factors. Stem Cell Research Feb 24 [Epub conditional mouse models of peripheral T cell lymphoma. Journal of ahead of print]. Experimental Medicine 207(5), 1031-1044. • 4. Becker PD, Legrand N, van Geelen CM, Noerder M, Huntington • 26. Suttner K, Depner M, Wetzke M, Klopp N, von Mutius E, Illig ND, Lim A, Yasuda E, Diehl SA, Scheeren FA, Ott M, Weijer K, T, Sparwasser T & Kabesch M (2010) Genetic variants harbored in Wedemeyer H, Di Santo JP, Beaumont T, Guzman CA & Spits H (2010) the forkhead box protein 3 locus increase hay fever risk. Journal of Generation of human antigen-specifi c monoclonal IgM antibodies Allergy and Clinical Immunology 125(6), 1395-1399. using vaccinated “human immune system” mice. PLoS One 5(10). • 27. Boissonnas A, Scholer-Dahirel A, Simon-Blancal V, Pace L, Valet • 5. Sancho-Bru P, Roelandt P, Narain N, Pauwelyn K, Notelaers T, F, Kissenpfennig A, Sparwasser T, Malissen B, Fetler L & Amigorenga Shimizu T, Ott M & Verfaillie C (2010) Directed differentiation of S (2010) FoxP3+ T cells induce perforin-dependent dendritic cell murine-induced pluripotent stem cells to functional hepatocyte-like death in tumor-draining lymph nodes. Immunity 32(2), 266-278. cells. Journal of Hepatology 54(1), 98-107. • 28. Schildknecht A, Brauer S, Brenner C, Lahl K, Schild H, • 6. Asgari S, Pournasr B, Salekdeh GH, Ghodsizadeh A, Ott M & Sparwasser T*, Probst HC* & van den Broek M* (2010) FoxP3+ Baharvand H (2010) Induced pluripotent stem cells: a new era for regulatory T cells essentially contribute to peripheral CD8+ T cell hepatology. Journal of Hepatology 53(4), 738-751. tolerance induced by steady state dendritic cells. Proceedings of the National Academy of Sciences U S A 107(1), 199-203 *(equally • 7. Bitzegeio J, Bankwitz D, Hueging K, Haid S, Brohm C, Zeisel MB, contributed) (Epub 2009). Herrmann E, Iken M, Ott M, Baumert TF & Pietschmann T (2010) Adaptation of hepatitis C virus to mouse CD81 permits infection of • 29. Anz D, Koelzer VH, Moder S, Thaler R, Schwerd T, Lahl mouse cells in the absence of human entry factors. PLoS Pathogens K, Sparwasser T, Besch R, Poeck H, Hornung V, Hartmann G, 6, e1000978. Rothenfusser S, Bourquin C & Endres S (2010) Immunostimulatory RNA blocks suppression by regulatory T cells. Journal of Immunology • 8. Ciesek S, Steinmann E, Iken M, Ott M, Helfritz FA, 184(2), 939-946 (Epub 2009). Wappler I, Manns MP, Wedemeyer H & Pietschmann T (2010) Glucocorticosteroids increase cell entry by hepatitis C virus. Gastroenterology 138(5), 1875-1884.

Research group Prof. Häußler • 9. Hettwer M, Reis-Fernandes MA, Iken M, Ott M, Steinberg P & Nau H (2010) Metabolic activation capacity by primary hepatocytes • 1. Schmidt, J, Müsken M, Becker T, Magnowska Z, Bertinetti D, expands the applicability of the embryonic stem cell test as Möller S, Zimmermann B, Herberg FW, Jänsch L & Häussler S (2011). alternative to experimental animal testing. Reproductive Toxicology The Pseudomonas aeruginosa chemotaxis methyltransferase CheR1 30(1), 113-120. impacts on bacterial surface sampling. PLoS One (in press). • 10. Stange MA, Tutarel O, Pischke S, Schneider A, Strassburg CP, • 2. Häussler S (2010) Multicellular signalling and growth of Becker T, Barg-Hock H, Basturk M, Wursthorn K, Cornberg M, Ott Pseudomonas aeruginosa. International Journal of Medical M, Greten TF, Manns MP & Wedemeyer H (2010) Fulminant hepatic Microbiology 300(8), 544-548. failure due to chemotherapy-induced hepatitis B reactivation: role of rituximab. Zeitschrift für Gastroenterologie 48(2), 258-263. • 3. Pommerenke C, Müsken M, Becker T, Dotsch A, Klawonn F & Häussler S (2010) Global genotype-phenotype correlations in Pseudomonas aeruginosa. PLoS Pathogens 6(8). pii: e1001074. 148 SCIENTIFIC REPORTS | Publications

Publications 2010-2011

Dept. Molecular Infection Biology | Prof. Dr. Petra Dersch • Müsken,M., Di,F.S., Römling,U., & Häußler,S. (2010) A 96-well-plate- based optical method for the quantitative and qualitative evaluation • Driouch,H., Roth,A., Dersch,P., & Wittmann,C. (2010) Filamentous of Pseudomonas aeruginosa biofi lm formation and its application to fungi in good shape: Microparticles for tailor-made fungal morpho- susceptibility testing. Nature Protocols 5, 1460-1469. logy and enhanced enzyme production. Bioengineered Bugs 2. • Pommerenke,C., Müsken,M., Becker,T., Dötsch,A., Klawonn,F., & • Driouch,H., Roth,A., Dersch,P., & Wittmann,C. (2010) Optimized bio - Häußler,S. (2010) Global genotype-phenotype correlations in Pseu- process for production of fructofuranosidase by recombinant Asper- domonas aeruginosa. PLoS Pathogens 6, 89-90. gillus niger. Applied Microbiology and Biotechnology 87, 2011-2024. • Haddad,A., Jensen,V., Becker,T., & Häußler,S. (2011) The Pho regu- • Fleissner,A. & Dersch,P. (2010) Expression and export: recombinant lon infl uences biofi lm formation and type three secretion in Pseu- protein production systems for Aspergillus. Applied Microbiology and domonas aeruginosa. Environmental Microbiology Reports 1, 488-494. Biotechnology 87, 1255-1270. • Schmidt,J., Müsken,M., Becker,T., Magnowska,Z., Bertinetti,D., • Göpel,Y., Luttmann,D., Heroven,A.K., Reichenbach,B., Dersch,P., & Moller,S., Zimmermann,B., Herberg,F.W., Jansch,L., & Häußler,S. Görke,B. (2010) Common and divergent features in transcriptional (2011) The Pseudomonas aeruginosa chemotaxis methyltransferase control of the homologous small RNAs GlmY and GlmZ in Enterobac- CheR1 impacts on bacterial surface sampling. PLoS ONE 6, e18184. teriaceae. Nucleic Acids Research 39, 1294-1309.

• Heroven,A.K. & Dersch,P. (2010) Global virulence gene regulation networks in enteropathogenic Yersiniae. In: Research Advances in RG Microbial Communication | Prof. Dr. Irene Wagner-Döbler Molecular Microbiology (Mohan,R.M., ed.), Global Research Network, Kerala, Indien, pp. 1-17. • Bodor,A.M., Jänsch,L., Wissing,J., & Wagner-Döbler,I. (2011) The luxS mutation causes loosely-bound biofi lms in Shewanella oneidensis. • Homann,A., Qamar,R.-U., Serim,S., Dersch,P., & Seibel,J. (2010) BMC Research Notes 4, 180. Bioorthogonal metabolic glycoengineering of human larynx carci- noma (HEp-2) cells targeting sialic acid. Beilstein Journal of Organic • Jaramillo-Colorado,B., Olivero-Verbel,J., Stashenko,E.E., Wagner- Chemistry 6. Döbler,I., & Kunze,B. (2011) Anti-quorum sensing activity of essential oils from Colombian plants. Natural Product Research, DOI:10.1080/1 • Quade,N., Dieckmann,M., Haffke,M., Heroven,A.K., Dersch,P., & 4786419.2011.557376 Heinz,D.W.*. (2011) Structure of the effector-binding domain of the LysR-type transcription factor RovM from Yersinia pseudotuberculo- • Kunze,B., Reck,M., Dotsch,A., Lemme,A., Schummer,D., Irschik,H., sis. Acta Crystallographica Section D: Biological Crystallography 67, Steinmetz,H., & Wagner-Döbler,I. (2010) Damage of Streptococcus 81-90. mutans biofi lms by carolacton, a secondary metabolite from the myxobacterium Sorangium cellulosum. BMC Microbiology 10, 199. • Dersch,P. & Jahn,D. (2011) Mikrobielle Genetik. In Mikrobiologie (Munk,K., ed.), Thieme., in press • Lemme,A., Grobe,L., Reck,M., Tomasch,J., & Wagner-Döbler,I. (2011) Subpopulation-specifi c transcriptome analysis of competence-stimu- lating-peptide induced Streptococcus mutans. Journal of Bacteriology 193, 1863-1877. RG Chronic Pseudomonas Infections | Prof. Dr. Susanne Häußler • Lemme,A., Sztajer,H., & Wagner-Döbler,I. (2010) Characterization of • Dötsch,A., Klawonn,F., Jarek,M., Scharfe,M., Blöcker,H., & Häußler,S. mleR, a positive regulator of malolactic fermentation and part of the (2010) Evolutionary conservation of essential and highly expressed acid tolerance response in Streptococcus mutans. BMC Microbiology genes in Pseudomonas aeruginosa. BMC GENOMICS 11, 234. 10, 58.

• Häussler,S. (2010) Multicellular signalling and growth of Pseudo- • Nawrath,T., Dickschat,J.S., Kunze,B., & Schulz,S. (2010) The biosyn- monas aeruginosa. International Journal of Medical Microbiology thesis of branched dialkylpyrazines in myxobacteria. Chemistry and 300, 544-548. Biodiversity 7, 2129-2144.

• Häußler,S. & Parsek,M.R. (2010) Biofi lms 2009: new perspectives at • Reck,M., Rutz,K., Kunze,B., Tomasch,J., Surapaneni,S.K., Schulz,S., the heart of surface-associated microbial communities. Journal of & Wagner-Döbler,I. (2011) The biofi lm inhibitor carolacton disturbs Bacteriology 192, 2941-2949. membrane integrity and cell division of Streptococcus mutans through the serine/threonine protein kinase PknB. Journal of • Häußler,S. (2010) Multicellular signalling and growth of Pseu- Bac teriology 193, 5692-5706. domonas aeruginosa. International Journal of Medical Microbiology 300, 544-548. • Riedel,T., Tomasch,J., Buchholz,I., Jacobs,J., Kollenberg,M., Gerdts,G., Wichels,A., Brinkhoff,T., Cypionka,H., & Wagner-Döbler,I. (2010) Con- • Moreno,A.M., Matz,C., Kjelleberg,S., & Manefi eld,M. (2010) Identifi - stitutive expression of the proteorhodopsin gene by a fl avobacterium cation of ciliate grazers of autotrophic bacteria in ammonia-oxidizing strain representative of the proteorhodopsin-producing microbial activated sludge by RNA stable isotope probing. Applied and Environ- community in the North Sea. Applied in Environmental Microbiology mental Microbiology 76, 2203-2211. 76, 3187-3197.

• Müsken,M., Di,F.S., Dötsch,A., Fischer,R., & Häußler,S. (2010) Genetic • Schulz,S., Dickschat,J.S., Kunze,B., Wagner-Döbler,I., Diestel,R., determinants of Pseudomonas aeruginosa biofi lm establishment. & Sasse,F. (2010) Biological activity of volatiles from marine and Microbiology 156, 431-441. terrestrial bacteria. Marine Drugs 8, 2976-2987. SCIENTIFIC REPORTS | Publications 149

• Thiel,V., Brinkhoff,T., Dickschat,J.S., Wickel,S., Grunenberg,J., • Coroadinha,A.S., Gama-Norton,L., Amaral,A.I., Hauser,H., Alves,P.M., Wagner-Döbler,I., Simon,M., & Schulz,S. (2010) Identifi cation and & Cruz,P.E. (2010) Production of retroviral vectors: review. Current biosynthesis of tropone derivatives and sulfur volatiles produced by Gene Therapy 10, 456-473. bacteria of the marine Roseobacter clade. Organic and Biomolecular Chemistry 8, 234-246. • Courties,G., Seiffart,V., Presumey,J., Escriou,V., Scherman,D., Zwerina,J., Ruiz,G., Zietara,N., Jablonska,J., Weiss,S., Hoffmann,A., • Tomasch,J., Gohl,R., Bunk,B., Suarez-Diez,M., & Wagner-Döbler,I. Jorgensen,C., Apparailly,F., & Gross,G. (2010) In vivo RNAi-mediated (2011) Transcriptional response of the photoheterotrophic marine silencing of TAK1 decreases infl ammatory Th1 and Th17 cells bacterium Dinoroseobacter shibae to changing light regimes. ISME through targeting of myeloid cells. Blood 116, 3505-3516. Journal, DOI:10.1038/ismej.2011.68 • Dietrich,N., Rohde,M., Geffers,R., Kroger,A., Hauser,H., Weiss,S., & • Vilchez,R., Lemme,A., Ballhausen,B., Thiel,V., Schulz,S., Jansen,R., Gekara,N.O. (2010) Mast cells elicit proinfl ammatory but not type I Sztajer,H., & Wagner-Döbler,I. (2010) Streptococcus mutans inhibits interferon responses upon activation of TLRs by bacteria. Proceeding Candida albicans hyphal formation by the fatty acid signaling mole- of the National Academy of Sciences of the United States of America cule trans-2-decenoic acid (SDSF). ChemBioChem 11, 1552-1562. 107, 8748-8753.

• Wagner-Döbler,I., Ballhausen,B., Berger,M., Brinkhoff,T., Buchholz,I., • Dittmar,K.E.*., Simann,M., Zghoul,N., Schön,O., Meyring,W., Bunk,B., Cypionka,H., Daniel,R., Drepper,T., Gerdts,G., Hahnke,S., Hannig,H., Macke,L., Dirks,W.G., Miller,K., Garritsen,H.S.P., & Han,C., Jahn,D., Kalhoefer,D., Kiss,H., Klenk,H.P., Kyrpides,N., Lindenmaier,W. (2010) Quality of cell products: Authenticity, iden- Liebl,W., Liesegang,H., Meincke,L., Pati,A., Petersen,J., Piekarski,T., tity, genomic stability and status of differentiation. Transfusion Pommerenke,C., Pradella,S., Pukall,R., Rabus,R., Stackebrandt,E., Medicine and Hemotherapy 37, 57-64. Thole,S., Thompson,L., Tielen,P., Tomasch,J., von,J.M., Wanphrut,N., Wichels,A., Zech,H., & Simon,M. (2010) The complete genome se- • Ehlert,N., Hoffmann,A., Luessenhop,T., Gross,G., Mueller,P.P.*., quence of the algal symbiont Dinoroseobacter shibae: a hitchhiker’s Stieve,M., Lenarz,T., & Behrens,P. (2010) Amino-modifi ed silica sur- guide to life in the sea. ISME Journal 4, 61-77. faces effi ciently immobilize bone morphogenetic protein 2 (BMP2) for medical purposes. Acta Biomaterialia 7, 1772-1779. • Xue,X., Sztajer,H., Buddruhs,N., Petersen,J., Rohde,M., Talay,S.R., & Wagner-Döbler,I. (2011) Lack of the delta subunit of RNA poly- • Ehlert,N., Müller,P.P.*., Stieve,M., & Behrens,P. (2010) Immobilization merase increases virulence related traits of Streptococcus mutans. of alkaline phosphatase on modifi ed silica coatings. Microporous and PLoS ONE 6, e20075. Mesoporous Materials 131, 51-57.

• Xue,X., Tomasch,J., Sztajer,H., & Wagner-Döbler,I. (2010) The delta • Gama-Norton,L., Herrmann,S., Schucht,R., Coroadinha,A.S., Low,R., subunit of RNA polymerase, RpoE, is a global modulator of Strepto- Alves,P.M., Bartholomae,C.C., Schmidt,M., Baum,C., Schambach,A., coccus mutans environmental adaptation. Journal of Bacteriology Hauser,H., & Wirth,D. (2010) Retroviral vector performance in de- 192, 5081-5092. fi ned chromosomal Loci of modular packaging cell lines. Human Gene Therapy 21, 979-991. • Brinkhoff,T., Fischer,D., Vollmers,J., Vogel,S., Beardsley,C., Thole,S., Mussmann,M., Kunze,B., Wagner-Döbler,I., Daniel,R., & Simon,M. • Garritsen,H.S., Macke,L., Meyring,W., Hannig,H., Pagelow,U., (2011) Biogeography and phylogenetic diversity of a cluster of exclu- Wormann,B., Geffers,R., Dittmar,K.E.*., & Lindenmaier,W. (2010) Effi - sively marine myxobacteria. ISME Journal, in press cient generation of clinical-grade genetically modifi ed dendritic cells for presentation of multiple tumor-associated proteins. Trans fusion • Xue,X., Li,J., Wang,W., Sztajer,H., & Wagner-Döbler,I. (2011) The 50, 831-842. global impact of the delta subunit RpoE of the RNA polymerase on the proteome of Streptococcus mutans. Microbiology, in press • Kirschning,C.J., Dreher,S., Maass,B., Fichte,S., Schade,J., Koster,M., Noack,A., Lindenmaier,W., Wagner,H., & Boldicke,T. (2010) Genera- tion of anti-TLR2 intrabody mediating inhibition of macrophage surface TLR2 expression and TLR2-driven cell activation. BMC Bio- Dept. of Gene Regulation and Differentiation | technology 10, 31. Prof. Dr. Hansjörg Hauser • Loew,R., Meyer,Y., Kuehlcke,K., Gama-Norton,L., Wirth,D., Hauser,H., • Badar,M., Hemmen,K., Nimtz,M., Stieve,M., Stiesch,M., Lenarz,T., Stein,S., Grez,M., Thornhill,S., Thrasher,A., Baum,C., & Schambach,A. Hauser,H., Möllmann,U., Vogt,S., Schnabelrauch,M., & Müller,P.P.*. (2010) A new PG13-based packaging cell line for stable production of (2010) Evaluation of madurahydroxylactone as a slow release anti- clinical-grade self-inactivating gamma-retroviral vectors using tar- bacterial implant coating. Open Biomedical Engineering Journal 4, geted integration. Gene Therapy 17, 272-280. 263-270. • Macke,L., Garritsen,H.S., Meyring,W., Hannig,H., Pagelow,U., • Becker,J., Pavlakovic,H., Ludewig,F., Wilting,F., Weich,H.A.*., Wormann,B., Piechaczek,C., Geffers,R., Rohde,M., Lindenmaier,W., Albuquerque,R., Ambati,J., & Wilting,J. (2010) Neuroblastoma pro- & Dittmar,K.E.*. (2010) Evaluating maturation and genetic modifi - gression correlates with downregulation of the lymphangiogenesis cation of human dendritic cells in a new polyolefi n cell culture bag inhibitor sVEGFR-2. Clinical Cancer Research 16, 1431-1441. system. Transfusion 50, 843-855.

• Bergmann,A., Ahmad,S., Cudmore,M., Gruber,A.D., Wittschen,P., • May,T., Butueva,M., Bantner,S., Markusic,D., Seppen,J., Lindenmaier,W., Christofori,G., Gross,V., Gonzalves,A.C., Grone,H.J., MacLeod,R.A., Weich,H., Hauser,H., & Wirth,D. (2010) Synthetic gene Ahmed,A., & Weich,H.A.*. (2010) Reduction of circulating soluble regulation circuits for control of cell expansion. Tissue Engineering – Flt-1 alleviates preeclampsia-like symptoms in a mouse model. Part A 16, 441-452. Journal of Cellular and Molecular Medicine 14, 1857-1867. • Nehlsen,K., Herrmann,S., Zauers,J., Hauser,H., & Wirth,D. (2010) • Charbord,P., Livne,E., Gross,G., Haupl,T., Neves,N.M., Marie,P., Toxin-antitoxin based transgene expression in mammalian cells. Bianco,P., & Jorgensen,C. (2010) Human bone marrow mesenchymal Nucleic Acids Research 38, e32. stem cells: a systematic reappraisal via the genostem experience. Stem Cell Reviews and Reports 7, 32-42. • Pulverer,J.E., Rand,U., Lienenklaus,S., Kugel,D., Zietara,N., Kochs,G., Naumann,R., Weiss,S., Staeheli,P., Hauser,H., & Koster,M. (2010) • Cohen-Haguenauer,O., Creff,N., Cruz,P., Tunc,C., Aiuti,A., Baum,C., Temporal and spatial resolution of type I and III interferon responses Bosch,F., Blomberg,P., Cichutek,K., Collins,M., Danos,O., Dehaut,F., in vivo. Journal of Virology 84, 8626-8638. Federspiel,M., Galun,E., Garritsen,H., Hauser,H., Hildebrandt,M., Klatzmann,D., Merten,O.W., Montini,E., O’Brien,T., Panet,A., • Roming,M., Lünsdorf,H., Dittmar,K.E.*., & Feldmann,C. (2010) Rasooly,L., Scherman,D., Schmidt,M., Schweitzer,M., Tiberghien,P., ZrO(HPO(4))(1-x)(FMN)(x): quick and easy synthesis of a nanoscale Vandendriessche,T., Ziehr,H., Yla-Herttuala,S., von,K.C., Gahrton,G., luminescent biomarker. Angewandte Chemie International Edition & Carrondo,M. (2010) Relevance of an academic GMP Pan-European 49, 632-637. vector infra-structure (PEVI). Current Gene Therapy 10, 414-422. 150 SCIENTIFIC REPORTS | Publications

• Sandhu,U., Cebula,M., Behme,S., Riemer,P., Wodarczyk,C., • Thorey,F., Menzel,H., Lorenz,C., Gross,G., Hoffmann,A., & Metzger,D., Reimann,J., Schirmbeck,R., Hauser,H., & Wirth,D. (2010) Windhagen,H. (2011) Osseointegration by bone morphogenetic Strict control of transgene expression in a mouse model for sensitive protein-2 and transforming growth factor beta2 coated titanium biological applications based on RMCE compatible ES cells. Nucleic implants in femora of New Zealand white rabbits. Indian Journal of Acids Research 39, e1. Orthopaedics 45, 57-62.

• Schniedermann,J., Rennecke,M., Buttler,K., Richter,G., Stadtler,A.M., Norgall,S., Badar,M., Barleon,B., May,T., Wilting,J., & Weich,H.A.*. (2010) Mouse lung contains endothelial progenitors with high capac- RG Molecular Immunology | Dr. Siegfried Weiss ity to form blood and lymphatic vessels. BMC Cell Biology 11, 50. • Courties,G., Seiffart,V., Presumey,J., Escriou,V., Scherman,D., • Schucht,R., Wirth,D., & May,T. (2010) Precise regulation of transgene Zwerina,J., Ruiz,G., Zietara,N., Jablonska,J., Weiss,S., Hoffmann,A., expression level and control of cell physiology. Cell Biology and Jorgensen,C., Apparailly,F., & Gross,G. (2010) In vivo RNAi-mediated Toxicology 26, 29-42. silencing of TAK1 decreases infl ammatory Th1 and Th17 cells through targeting of myeloid cells. Blood 116, 3505-3516. • Shahab-Osterloh,S., Witte,F., Hoffmann,A., Winkel,A., Laggies,S., Neumann,B., Seiffart,V., Lindenmaier,W., Gruber,A.D., Ringe,J., • Dietrich,N., Rohde,M., Geffers,R., Kröger,A., Hauser,H., Weiss,S., & Haupl,T., Thorey,F., Willbold,E., Corbeau,P., & Gross,G. (2010) Mes- Gekara,N.O. (2010) Mast cells elicit proinfl ammatory but not type I enchymal stem cell-dependent formation of heterotopic tendon-bone interferon responses upon activation of TLRs by bacteria. Proceeding insertions (osteotendinous junctions). Stem Cells 28, 1590-1601. of the National Academy of Sciences of the United States of America 107, 8748-8753. • Stammen,S., Schuller,F., Dietrich,S., Gamer,M., Biedendieck,R., & Jahn,D. (2010) Application of Escherichia coli phage K1E DNA- • Dietrich,N., Lienenklaus,S., Weiss,S., & Gekara,N. (2010) Murine toll- dependent RNA polymerase for in vitro RNA synthesis and in vivo like receptor 2 activation induces type I interferon responses from protein production in Bacillus megaterium. Applied Microbiology and endolysosomal compartments. PLoS ONE 5, e10250. Biotechnology 88, 529-539. • Dolowschiak,T., Chassin,C., Ben,M.S., Fuchs,T.M., Weiss,S., • Stirnweiss,A., Ksienzyk,A., Klages,K., Rand,U., Grashoff,M., Vandewalle,A., & Hornef,M.W. (2010) Potentiation of epithelial innate Hauser,H., & Kröger,A. (2010) IFN regulatory factor-1 bypasses IFN- host responses by intercellular communication. PLoS Pathogens 6, mediated antiviral effects through viperin gene induction. Journal of e1001194. Immunology 184, 5179-5185. • Düber,S. & Weiss,S. (2010) Inducible B cell development. G. I. T. • Sun,L., Hemgard,G.V., Susanto,S.A., & Wirth,M. (2010) Caveolin-1 Laboratory Journal Europe 7-8, 28-29. infl uences human infl uenza A virus (H1N1) multiplication in cell culture. Virology Journal 7, 108. • Gekara,N.O., Zietara,N., Geffers,R., & Weiss,S. (2010) Listeria mono- cytogenes induces T cell receptor unresponsiveness through pore- • Thorey,F., Menzel,H., Lorenz,C., Gross,G., Hoffmann,A., & forming toxin listeriolysin O. Journal of Infectious Diseases 202, Windhagen,H. (2010) Enhancement of endoprosthesis anchoring 1698-1707. using BMP-2. Technology and Health Care 18, 217-229. • Jablonska,J., Leschner,S., Westphal,K., Lienenklaus,S., & Weiss,S. • Wirth,D., Gama-Norton,L., Schucht,R., Nehlsen,K., & Hauser,H. (2010) (2010) Neutrophils responsive to endogenous IFN-beta regulate Site-directed engineering of defi ned chromosomal sites for recombi- tumor angiogenesis and growth in a mouse tumor model. Journal of nant protein and virus expression. BioPharm International 22, 32-39. Clinical Investigation 120, 1151-1164.

• Buttler,K., Schniedermmann,J., Weich,H.A.*., & Wilting,J. (2011) • Jellusova,J., Duber,S., Guckel,E., Binder,C.J., Weiss,S., Voll,R., & Isolierung bipotenter endothelialer Vorläuferzellen aus der Lunge Nitschke,L. (2010) Siglec-G regulates B1 cell survival and selection. adulter Mäuse [Isolation of bipotent endothelial precursor cells from Journal of Immunology 185, 3277-3284. lungs of adult mice]. Lymphologie in Forschung und Praxis 14, 58-64. • Leschner,S. & Weiss,S. (2010) Salmonella-allies in the fi ght against • Carrondo,M., Panet,M., Wirth,D., Coroadinha,A.S., Cruz,P., Falk,H., cancer. Journal of Molecular Medicine 88, 763-773. Schucht,R., Dupont,F., Geny-Fiamma,C., Merten,O.W., & Hauser,H. (2011) Integrated strategy for the production of therapeutic retroviral • Leschner,S., Wolf,K., & Weiss,S. (2010) Bakterielle Infektion auf vectors. Human Gene Therapy 22, 370-379. Rezept. labor&more 5, 14-17.

• Ehlert,N., Badar,M., Christel,A., Lohmeier,S.J., Luessenhop,T., • Leschner,S. & Weiss,S. (2010) Bacteria - fi ghters against cancer. Stieve,M., Lenarz,T., Müller,P.P.*., & Behrens,P. (2011) Mesoporous G. I. T. Laboratory Journal Europe 9, 20-21. silica coatings for controlled release of the antibiotic ciprofl oxacin from implants. Journal of Materials Chemistry 21, 752-760. • Prajeeth,C.K., Jirmo,A.C., Krishnaswamy,J.K., Ebensen,T., Guzmán,C.A.*., Weiss,S., Constabel,H., Schmidt,R.E., & Behrens,G.M. • Kugel,D., Pulverer,J.E., Koster,M., Hauser,H., & Staeheli,P. (2011) (2010) The synthetic TLR2 agonist BPPcysMPEG leads to effi cient Novel nonviral bioassays for mouse type I and type III interferon. cross-priming against co-administered and linked antigens. European Journal of Interferone and Cytokine Research 31, 345-349. Journal of Immunology 40, 1272-1283.

• Lindenmaier,W., Lachmann,K., Meyring,W., Garritsen,H.S.P., • Pulverer,J.E., Rand,U., Lienenklaus,S., Kugel,D., Zietara,N., Kochs,G., Thomas,M., & Dittmar,K.J.*. (2011) Innenbeschichtung von Beuteln Naumann,R., Weiss,S., Staeheli,P., Hauser,H., & Koster,M. (2010) für die Kultur adhärenter humaner Zellen. Biospektrum 17, 32-34. Temporal and spatial resolution of type I and III interferon responses in vivo. Journal of Virology 84, 8626-8638. • Lohmeyer,J.A., Liu,F., Kruger,S., Lindenmaier,W., Siemers,F., & Machens,H.G. (2011) Use of gene-modifi ed keratinocytes and fi brob- • Steidle,S., Martinez-Sobrido,L., Mordstein,M., Lienenklaus,S., Garcia- lasts to enhance regeneration in a full skin defect. Langenbeck’s Sastre,A., Staheli,P., & Kochs,G. (2010) Glycine 184 in nonstructural Archives of Surgery. protein NS1 determines the virulence of infl uenza A virus strain PR8 without affecting the host interferon response. Journal of Virology 84, • Lorenz,C., Hoffmann,A., Gross,G., Windhagen,H., Dellinger,P., 12761-12770. Mohwald,K., Dempwolf,W., & Menzel,H. (2011) Coating of titanium implant materials with thin polymeric fi lms for binding the signaling • Lyszkiewicz,M., Zietara,N., Rohde,M., Gekara,N.O., Jablonska,J., protein BMP2. Macromolecular Bioscience 11, 234-244. Dittmar,K.E.*., & Weiss,S. (2011) SIGN-R1+MHC II+ cells of the splenic marginal zone--a novel type of resident dendritic cells. Jour- • Lyszkiewicz,M., Zietara,N., Rohde,M., Gekara,N.O., Jablonska,J., nal of Leukocyte Biology 89, 607-615. Dittmar,K.E.*., & Weiss,S. (2011) SIGN-R1+MHC II+ cells of the splenic marginal zone--a novel type of resident dendritic cells. Journal of Leukocyte Biology 89, 607-615. SCIENTIFIC REPORTS | Publications 151

• Crull,K., Bumann,D., & Weiss,S. (2011) Infl uence of infection route • Xu,M.Z., Chan,S.W., Liu,A.M., Wong,K.F., Fan,S.T., Chen,J., Poon,R.T., and virulence factors on colonization of solid tumors by Salmonella Zender,L., Lowe,S.W., Hong,W., & Luk,J.M. (2011) AXL receptor ki- enterica serovar Typhimurium. FEMS Immunology and Medical Micro- nase is a mediator of YAP-dependent oncogenic functions in hepato- biology, in press cellular carcinoma. Oncogene 30(10), 1229-1240

• Solodova,E., Jablonska,J., Weiss,S., & Lienenklaus,S. (2011) Produc- • Sawey,E., Chanrion,M., Cai,C., Wu,G., Zhang,J., Zender,L., Zhao,A., tion of IFN-beta Listeria monocytogenes infection is restricted to the Busuttil,R., Yee,H., Stein,L., French,D., Finn,R., Lowe,S. & Powers,S. monocyte/macrophage lineage. PLOS ONE, in press (2011) Identifi cation of a Therapeutic Strategy Targeting Amplifi ed FGF19 in Liver Cancer by Oncogenomic Screening. Cancer Cell 19(3), 347-358

RG Model Systems for Infection | Dr. Dagmar Wirth

• Gama-Norton,L., Herrmann,S., Schucht,R., Coroadinha,A.S., Low,R., Dept. of Molecular Structural Biology | Prof. Dr. Dirk Heinz Alves,P.M., Bartholomae,C.C., Schmidt,M., Baum,C., Schambach,A., Hauser,H., & Wirth,D. (2010) Retroviral vector performance in de- • Brocker,M.J., Schomburg,S., Heinz,D.W.*, Jahn,D., Schubert,W.D., & fi ned chromosomal Loci of modular packaging cell lines. Human Moser,J. (2010) Crystal structure of the nitrogenase-like dark opera- gene therapy 21, 979-991. tive protochlorophyllide oxidoreductase catalytic complex (ChlN/ ChlB)2. Journal of Biological Chemistry 285, 27336-27345. • Loew,R., Meyer,Y., Kuehlcke,K., Gama-Norton,L., Wirth,D., Hauser,H., Stein,S., Grez,M., Thornhill,S., Thrasher,A., Baum,C., & Schambach,A. • Carius,Y., Christian,H., Faust,A., Zander,U., Klink,B.U.*, (2010) A new PG13-based packaging cell line for stable production of Kornberger,P., Kohring,G.W., Giffhorn,F., & Scheidig,A.J. (2010) Struc- clinical-grade self-inactivating gamma-retroviral vectors using tar- tural insight into substrate differentiation of the sugar-metabolizing geted integration. Gene Therapy 17, 272-280. enzyme galactitol dehydrogenase from Rhodobacter sphaeroides D. Journal of Biological Chemistry 285, 20006-20014. • May,T., Butueva,M., Bantner,S., Markusic,D., Seppen,J., MacLeod,R.A., Weich,H., Hauser,H., & Wirth,D. (2010) Synthetic gene • Ferraris,D.M., Gherardi,E., Di,Y., Heinz,D.W.*, & Niemann,H. (2010) regulation circuits for control of cell expansion. Tissue Engineering - Ligand-mediated dimerization of the Met receptor tyrosine kinase by Part A 16, 441-452. the bacterial invasion protein InIB. Journal of Molecular Biology 395, 522-532. • Nehlsen,K., Herrmann,S., Zauers,J., Hauser,H., & Wirth,D. (2010) Toxin-antitoxin based transgene expression in mammalian cells. • Haffke,M., Menzel,A., Carius,Y., Jahn,D., & Heinz,D.W.* (2010) Struc- Nucleic Acids Research 38, e32. tures of the nucleotide-binding domain of the human ABCB6 trans- porter and its complexes with nucleotides. Acta Crystallographica • Sandhu,U., Cebula,M., Behme,S., Riemer,P., Wodarczyk,C., Section D: Biological Crystallography 66, 979-987. Metzger,D., Reimann,J., Schirmbeck,R., Hauser,H., & Wirth,D. (2010) Strict control of transgene expression in a mouse model for sensitive • Heinemann,I.U., Schulz,C., Schubert,W.D., Heinz,D.W.*, Wang,Y.G., biological applications based on RMCE compatible ES cells. Nucleic Kobayashi,Y., Awa,Y., Wachi,M., Jahn,D., & Jahn,M. (2010) Structure Acids Research 39, e1. of the heme biosynthetic Pseudomonas aeruginosa porphobilinogen synthase in complex with the antibiotic alaremycin. Antimicrobial • Schucht,R., Wirth,D., & May,T. (2010) Precise regulation of transgene Agents and Chemotherapy 54, 267-272. expression level and control of cell physiology. Cell Biology and Toxicology 26, 29-42. • Heinz,D.W.*., Betzel,C., & Wilmanns,M. (2010) Highlight: of systems and structures. Biological Chemistry 391, 717-718. • Wirth,D., Gama-Norton,L., Schucht,R., Nehlsen,K., & Hauser,H. (2010) Site-directed engineering of defi ned chromosomal sites for recombi- • Hertiani,T., Edrada-Ebel,R., Ortlepp,S., van Soest,R.W., de Voogd,N.J., nant protein and virus expression. BioPharm International 22, 32-39. Wray,V., Hentschel,U., Kozytska,S., Muller,W.E., & Proksch,P. (2010) From anti-fouling to biofi lm inhibition: New cytotoxic secondary • Carrondo,M., Panet,M., Wirth,D., Coroadinha,A.S., Cruz,P., Falk,H., metabolites from two Indonesian Agelas sponges. Bioorganic and Schucht,R., Dupont,F., Geny-Fiamma,C., Merten,O.W., & Hauser,H. Medicinal Chemistry 18, 1297-1311. (2011) Integrated strategy for the production of therapeutic retroviral vectors. Human Gene Therapy 22, 370-379. • Ibrahim,S.R., Min,C.C., Teuscher,F., Ebel,R., Kakoschke,C., Lin,W., Wray,V., Edrada-Ebel,R., & Proksch,P. (2010) Callyaerins A-F and H, new cytotoxic cyclic peptides from the Indonesian marine sponge Callyspongia aerizusa. Bioorganic and Medicinal Chemistry 18, 4947- JRG Chronic Infections and Cancer | Prof. Dr. Lars Zender 4956.

• Gurlevik,E., Woller,N., Struver,N., Schache,P., Kloos,A., Manns,M.P., • Kampfer,P., Busse,H.J., Tindall,B.J., Nimtz,M., & Grun-Wollny,I. (2010) Zender,L., Kuhnel,F., & Kubicka,S. (2010) Selectivity of oncolytic viral Nonomuraea rosea sp. nov. International Journal of Systematic and replication prevents antiviral immune response and toxicity, but Evolutionary Microbiology 60, 1118-1124. does not improve antitumoral immunity. Molecular Therapy 18, 1972- 1982. • Klink,B.U., Barden,S., Heidler,T.V., Borchers,C., Ladwein,M., Stradal,T.B.*, Rottner,K., & Heinz,D.W.* (2010) Structure of Shigella • Klawonn,F., Wüstefeld,T., & Zender,L. (2010) Statistical modelling for IpgB2 in complex with human RhoA: implications for the mechanism data from experiments with short haripin RNAs. LNCS (Lecture Notes of bacterial guanine nucleotide exchange factor mimicry. Journal of in Computer Science (including subseries Lecture Notes in Artifi cial Biological Chemistry 285, 17197-17208. Intelligence and Lecture Notes in Bioinformatics)) 6065, 79-90. • Klink,B.U.* & Scheidig,A.J. (2010) New insight into the dynamic • Wuestefeld,T. & Zender,L. (2010) DLC1 and liver cancer: the Akt properties and the active site architecture of H-Ras p21 revealed by connection. Gastroenterology 139, 1093-1096. X-ray crystallography at very high resolution. BMC Structural Biology 10, 38. • Zender,L., Villanueva,A., Tovar,V., Sia,D., Chiang,D.Y., & Llovet,J.M. (2010) Cancer gene discovery in hepatocellular carcinoma. Journal of • Klodmann,J., Sunderhaus,S., Nimtz,M., Jänsch,L., & Braun,H.P. (2010) Hepatology 52, 921-929. Internal architecture of mitochondrial complex I from Arabidopsis thaliana. Plant Cell 22, 797-810. • Kühnel,F., Gürvelik,E., Wirth,T.C., Strüver,N., Malek,N.P., Müller- Schilling,M., Manns,M.P., Carnero,A., Zender,L. & Kubicka,S. (2010) • Layer,G., Reichelt,J., Jahn,D., & Heinz,D.W.* (2010) Structure and Targeting of p53-transcriptional dysfunction by conditionally rep- function of enzymes in heme biosynthesis. Protein Science 19, 1137- licating adenovirus is not limited by p53-homologues. Molecular 1161. Therapie 18(5), 936-946 152 SCIENTIFIC REPORTS | Publications

• Marin,M., Perez-Pantoja,D., Donoso,R., Wray,V., Gonzalez,B., & • Shiloach,J. & Rinas,U. (2011) Bacterial cultivation for producing Pieper,D.H.* (2010) Modifi ed 3-oxoadipate pathway for the biodegra- of proteins and other biological products. In Manual of industrial dation of methylaromatics in Pseudomonas reinekei MT1. Journal of microbiology and biotechnology (Demain,A.L., ed), American Society Bacteriology 192, 1543-1552. for Microbiology (ASM), Washington DC., in press

• Moghaddam,F.M., Farimani,M.M., Seirafi ,M., Taheri,S., Khavasi,H.R., • Fraatz,M.A., Riemer,S.J.L., Stober,R., Kaspera,R., Nimtz,M., Sendker,J., Proksch,P., Wray,V., & Edrada,R. (2010) Sesterterpenoids Berger,R.G., & Zorn,H. (2011) A novel oxygenase from Pleurotus and other constituents of Salvia sahendica. Journal of Natural Prod- sapidus transforms valencene to nootkatone. Journal of Molecular ucts 73, 1601-1605. Catalysis B: Enzymatic, in press

• Palme,O., Comanescu,G., Stoineva,I., Radel,S., Benes,E., Develter,D., Wray,V., & Lang,S. (2010) Sophorolipids from Candida bombicola: Cell separation by ultrasonic particle manipulation. European Journal of RG Biophysical Analysis | Dr. Victor Wray Lipid Science and Technology 112, 663-673. • Hertiani,T., Edrada-Ebel,R., Ortlepp,S., van Soest,R.W., de Voogd,N.J., • Rand,K., Noll,C., Schiebel,H.M., Kemken,D., Dulcks,T., Kalesse,M., Wray,V., Hentschel,U., Kozytska,S., Muller,W.E., & Proksch,P. (2010) Heinz,D.W.*, & Layer,G. (2010) The oxygen-independent copropor- From anti-fouling to biofi lm inhibition: New cytotoxic secondary phyrinogen III oxidase HemN utilizes harderoporphyrinogen as a metabolites from two Indonesian Agelas sponges. Bioorganic and reaction intermediate during conversion of coproporphyrinogen III to Medicinal Chemistry 18, 1297-1311. protoporphyrinogen IX. Biological Chemistry 391, 55-63. • Ibrahim,S.R., Min,C.C., Teuscher,F., Ebel,R., Kakoschke,C., Lin,W., • Solbak,S.M., Reksten,T.R., Wray,V., Bruns,K., Horvli,O., Raae,A.J., Wray,V., Edrada-Ebel,R., & Proksch,P. (2010) Callyaerins A-F and H, Henklein,P., Henklein,P., Roder,R., Mitzner,D., Schubert,U., & new cytotoxic cyclic peptides from the Indonesian marine sponge Fossen,T. (2010) The intriguing cyclophilin A-HIV-1 Vpr interaction: Callyspongia aerizusa. Bioorganic and Medicinal Chemistry 18, 4947- prolyl cis/trans isomerisation catalysis and specifi c binding. BMC 4956. Structural Biology 10, 31. • Marin,M., Perez-Pantoja,D., Donoso,R., Wray,V., Gonzalez,B., & • Tomala,M., Lavrentieva,A., Moretti,P., Rinas,U., Kasper,C., Stahl,F., Pieper,D.H.*. (2010) Modifi ed 3-oxoadipate pathway for the biodegra- Schambach,A., Warlich,E., Martin,U., Cantz,T., & Scheper,T. (2010) dation of methylaromatics in Pseudomonas reinekei MT1. Journal of Preparation of bioactive soluble human leukemia inhibitory factor Bacteriology 192, 1543-1552. from recombinant Escherichia coli using thioredoxin as fusion part- ner. Protein Expression and Purifi cation 73, 51-57. • Moghaddam,F.M., Farimani,M.M., Seirafi ,M., Taheri,S., Khavasi,H.R., Sendker,J., Proksch,P., Wray,V., & Edrada,R. (2010) Sesterterpenoids • Wei,M.X., Hu,P., Wang,P., Naruse,S., Nokihara,K., Wray,V., & Ozaki,T. and other constituents of Salvia sahendica. Journal of Natural (2010) Possible key residues that determine left gastric artery blood Products 73, 1601-1605. fl ow response to PACAP in dogs. World Journal of Gastroenterology 16, 4865-4870. • Palme,O., Comanescu,G., Stoineva,I., Radel,S., Benes,E., Develter,D., Wray,V., & Lang,S. (2010) Sophorolipids from Candida bombicola: • Wilke,S., Krausze,J., Gossen,M., Gröbe,L., Jager,V., Gherardi,E., van Cell separation by ultrasonic particle manipulation. European Journal den Heuvel, J., & Bussow,K. (2010) Glycoprotein production for of Lipid Science and Technology 112, 663-673. structure analysis with stable, glycosylation mutant CHO cell lines established by fl uorescence-activated cell sorting. Protein Science 19, • Rand,K., Noll,C., Schiebel,H.M., Kemken,D., Dulcks,T., Kalesse,M., 1264-1271. Heinz,D.W.*, & Layer,G. (2010) The oxygen-independent copropor- phyrinogen III oxidase HemN utilizes harderoporphyrinogen as a • Wilkens,A., Paulsen,J., Wray,V., & Winterhalter,P. (2010) Structures reaction intermediate during conversion of coproporphyrinogen III to of two novel trimeric stilbenes obtained by horseradish peroxidase protoporphyrinogen IX. Biological Chemistry 391, 55-63. catalyzed biotransformation of trans-resveratrol and (-)-epsilon- viniferin. Journal of Agricultural and Food Chemistry 58, 6754-6761. • Solbak,S.M., Reksten,T.R., Wray,V., Bruns,K., Horvli,O., Raae,A.J., Henklein,P., Henklein,P., Roder,R., Mitzner,D., Schubert,U., & • Wolfram,K., Schmidt,J., Wray,V., Milkowski,C., Schliemann,W., & Fossen,T. (2010) The intriguing cyclophilin A-HIV-1 Vpr interaction: Strack,D. (2010) Profi ling of phenylpropanoids in transgenic low- prolyl cis/trans isomerisation catalysis and specifi c binding. BMC sinapine oilseed rape (Brassica napus). Phytochemistry 71, 1076-1084. Structural Biology 10, 31.

• Xu,J., Aly,A.H., Wray,V., & Proksch,P. (2010) Polyketide derivatives of • Tomala,M., Lavrentieva,A., Moretti,P., Rinas,U., Kasper,C., Stahl,F., endophytic fungus Pestalotiopsis sp. isolated from the Chinese man- Schambach,A., Warlich,E., Martin,U., Cantz,T., & Scheper,T. (2010) grove plant Rhizophora mucronata. Tetrahedron Letters 52, 21-25. Preparation of bioactive soluble human leukemia inhibitory factor from recombinant Escherichia coli using thioredoxin as fusion part- • Zakikhany,K., Harrington,C.R., Nimtz,M., Hinton,J.C., & Römling,U. ner. Protein Expression and Purifi cation 73, 51-57. (2010) Unphosphorylated CsgD controls biofi lm formation in Sal- monella enterica serovar Typhimurium. Molecular Microbiology 77, • Wei,M.X., Hu,P., Wang,P., Naruse,S., Nokihara,K., Wray,V., & Ozaki,T. 771-786. (2010) Possible key residues that determine left gastric artery blood fl ow response to PACAP in dogs. World Journal of Gastroenterology • Aly,A.H., Debbab,A., Clements,C., Edrada-Ebel,R., Orlikova,B., 16, 4865-4870. Diederich,M., Wray,V., Lin,W., & Proksch,P. (2011) NF kappa B in- hibitors and antitrypanosomal metabolites from endophytic fungus • Wilke,S., Krausze,J., Gossen,M., Gröbe,L., Jager,V., Gherardi,E., van Penicillium sp. isolated from Limonium tubifl orum. Bioorganic and den Heuvel, J., & Büssow,K. (2010) Glycoprotein production for Medicinal Chemistry 19, 414-421. structure analysis with stable, glycosylation mutant CHO cell lines established by fl uorescence-activated cell sorting. Protein Science 19, • Esatbeyoglu,T., Jaschok-Kentner,B., Wray,V., & Winterhalter,P. (2011) 1264-1271. Structure elucidation of procyanidin oligomers by low-temperature 1H NMR spectroscopy. Journal of Agricultural and Food Chemistry • Wilkens,A., Paulsen,J., Wray,V., & Winterhalter,P. (2010) Structures 59, 62-69. of two novel trimeric stilbenes obtained by horseradish peroxidase catalyzed biotransformation of trans-resveratrol and (-)-epsilon- • Quade,N., Dieckmann,M., Haffke,M., Heroven,A.K., Dersch,P., & viniferin. Journal of Agricultural and Food Chemistry 58, 6754-6761. Heinz,D.W.* (2011) Structure of the effector-binding domain of the LysR-type transcription factor RovM from Yersinia pseudotuberculo- • Wolfram,K., Schmidt,J., Wray,V., Milkowski,C., Schliemann,W., & sis. Acta Crystallographica Section D: Biological Crystallography 67, Strack,D. (2010) Profi ling of phenylpropanoids in transgenic low-si- 81-90. napine oilseed rape (Brassica napus). Phytochemistry 71, 1076-1084. SCIENTIFIC REPORTS | Publications 153

• Xu,J., Aly,A.H., Wray,V., & Proksch,P. (2010) Polyketide derivatives of • Klodmann,J., Sunderhaus,S., Nimtz,M., Jänsch,L., & Braun,H.P. (2010) endophytic fungus Pestalotiopsis sp. isolated from the Chinese man- Internal architecture of mitochondrial complex I from Arabidopsis grove plant Rhizophora mucronata. Tetrahedron Letters 52, 21-25. thaliana. Plant Cell 22, 797-810.

• Zakikhany,K., Harrington,C.R., Nimtz,M., Hinton,J.C., & Romling,U. • Lu,X., Sun,J., Nimtz,M., Wissing,J., Zeng,A.-P., & Rinas,U. (2010) The (2010) Unphosphorylated CsgD controls biofi lm formation in Sal- intra- and extracellular proteome of Aspergillus niger growing on de- monella enterica serovar Typhimurium. Molecular Microbiology 77, fi ned medium with xylose or maltose as carbon substrate. Microbial 771-786. Cell Factories 9, 1-13.

• Aly,A.H., Debbab,A., Clements,C., Edrada-Ebel,R., Orlikova,B., • Maahs,D.M., Siwy,J., Argiles,A., Cerna,M., Delles,C., Dominiczak,A.F., Diederich,M., Wray,V., Lin,W., & Proksch,P. (2011) NF kappa B in- Gayrard,N., Iphöfer,A., Jänsch,L., Jerums,G., Medek,K., Mischak,H., hibitors and antitrypanosomal metabolites from endophytic fungus Navis,G.J., Roob,J.M., Rossing,K., Rossing,P., Rychlik,I., Schiffer,E., Penicillium sp. isolated from Limonium tubifl orum. Bioorganic and Schmieder,R.E., Wascher,T.C., Winklhofer-Roob,B.M., Zimmerli,L.U., Medicinal Chemistry 19, 414-421. Zurbig,P., & Snell-Bergeon,J.K. (2010) Urinary collagen fragments are signifi cantly altered in diabetes: a link to pathophysiology. • Esatbeyoglu,T., Jaschok-Kentner,B., Wray,V., & Winterhalter,P. (2011) PLoS ONE 5. Structure elucidation of procyanidin oligomers by low-temperature 1H NMR spectroscopy. Journal of Agricultural and Food Chemistry • Mischak,H., Kolch,W., Aivaliotis,M., Bouyssie,D., Court,M., Dihazi,H., 59, 62-69. Dihazi,G.H., Franke,J., Garin,J., de Peredo,A.G., Iphöfer,A., Jänsch,L., Lacroix,C., Makridakis,M., Masselon,C., Metzger,J., Monsarrat,B., • Quade,N., Dieckmann,M., Haffke,M., Heroven,A.K., Dersch,P., & Mrug,M., Norling,M., Novak,J., Pich,A., Pitt,A., Bongcam-Rudloff,E., Heinz,D.W.* (2011) Structure of the effector-binding domain of the Siwy,J., Suzuki,H., Thongboonkerd,V., Wang,L.-S., Zoidakis,J., LysR-type transcription factor RovM from Yersinia pseudotuberculo- Zurbig,P., Schanstra,J.P., & Vlahou,A. (2010) Comprehensive human sis. Acta Crystallographica Section D: Biological Crystallography 67, urine standards for comparability and standardization in clinical 81-90. proteome analysis. Proteomics - Clinical Applications 4, 464-478.

• Shiloach,J. & Rinas,U. (2011) Bacterial cultivation for producing • Pommerenke,C., Müsken,M., Becker,T., Dötsch,A., Klawonn,F., & of proteins and other biological products. In Manual of industrial Häußler,S. (2010) Global genotype-phenotype correlations in Pseu- microbiology and biotechnology (Demain,A.L., ed), American Society domonas aeruginosa. PLoS Pathogens 6, 89-90. for Microbiology (ASM), Washington DC., in press • Steinke,N., Kaller,M., Nimtz,M., Baro,A., & Laschat,S. (2010) Colum- • Fraatz,M.A., Riemer,S.J.L., Stober,R., Kaspera,R., Nimtz,M., nar liquid crystals derived from crown ethers with two lateral ester- Berger,R.G., & Zorn,H. (2011) A novel oxygenase from Pleurotus substituted ortho-terphenyl units: Unexpected destabilisation of the sapidus transforms valencene to nootkatone. Journal of Molecular mesophase by potassium iodide. Liquid Crystals 37, 1139-1149. Catalysis B: Enzymatic, in press • Stepanova,T., Smal,I., van,H.J., Akinci,U., Liu,Z., Miedema,M., Limpens,R., van,H.M., van der,R.M., Poot,R., Grosveld,F., Mommaas,M., Meijering,E., & Galjart,N. (2010) History-dependent RG Recombinant Protein Expression | Dr. Joop van den Heuvel catastrophes regulate axonal microtubule behavior. Current biology 20, 1023-1028. • Lu,X., Sun,J., Nimtz,M., Wissing,J., Zeng,A.-P., & Rinas,U. (2010) The intra- and extracellular proteome of Aspergillus niger growing on de- • Trunk,K., Benkert,B., Quack,N., Munch,R., Scheer,M., Garbe,J., fi ned medium with xylose or maltose as carbon substrate. Microbial Jänsch,L., Trost,M., Wehland,J., Buer,J., Jahn,M., Schobert,M., & Cell Factories 9, 1-13. Jahn,D. (2010) Anaerobic adaptation in Pseudomonas aeruginosa: defi nition of the Anr and Dnr regulons. Environmental Microbiology • Wilke,S., Krausze,J., Gossen,M., Gröbe,L., Jager,V., Gherardi,E., van 12, 1719-1733. den Heuvel, J., & Bussow,K. (2010) Glycoprotein production for structure analysis with stable, glycosylation mutant CHO cell lines • Tschumitschew,K. & Klawonn,F. (2010) Incremental quantile estima- established by fl uorescence-activated cell sorting. Protein Science 19, tion. Evolving Systems 1, 253-264. 1264-1271. • Tschumitschew,K. & Klawonn,F. (2010) The need for benchmarks • Bals,C., Schambach,A., Meyer,J., Scheper,T., & Rinas,U. (2011) Ex- with data from stochastic processes and meta-models in evolving pression and purifi cation of bioactive soluble murine stem cell factor systems. In Proceedings of the International Symposium on Evolving from recombinant Escherichia coli using thioredoxin as fusion part- Intelligent Systems (Angelov,P., Filev,D., & Kasabov,N., eds), pp. 30- ner. Journal of Biotechnology 152, 1-8. 33. SSAISB, Leicester.

• Shiloach,J. & Rinas,U. (2011) Bacterial cultivation for producing • Tschumitschew,K. & Klawonn,F. (2010) AVEDA: Statistical tests for of proteins and other biological products. In: Manual of industrial fi nding interesting visulatisations. 5711 LNAI (Part 1) Lecture Notes microbiology and biotechnology (Demain,A.L., ed.), American in Computer Science (including subseries Lecture Notes in Artifi cial Society for Microbiology (ASM), Washington DC, in press. Intelligence and Lecture Notes in Bioinformatics), 235-242.

• Krone,M. & Klawonn,F. (2011) Rank correlation coeffi cient correction by removing worst cases. In: Information Processing and Manage- RG Cellular Proteome Research | Dr. Lothar Jänsch ment of Uncertainty in Knowledge-Based Systems: Theory and Meth- ods (Hüllermeier,E., Kruse,R., & Hoffmann,F., eds.), Springer, Berlin, • Badar,M., Hemmen,K., Nimtz,M., Stieve,M., Stiesch,M., Lenarz,T., pp. 356-364. Hauser,H., Möllmann,U., Vogt,S., Schnabelrauch,M., & Müller,P.P.* (2010) Evaluation of madurahydroxylactone as a slow release anti- • Winkler,R., Klawonn,F., & Kruse,R. (2011) Fuzzy clustering with poly- bacterial implant coating. Open Biomedical Engineering Journal 4, nomial fuzzifi er function in connection with m-estimators. Applied 263-270. and Computational Mathematics 10, 146-163.

• Eisele,N., Linke,D., Bitzer,K., Na’amnieh,S., Nimtz,M., & Berger,R.G. • Eberl,L. & Riedel,K. (2011) Mining quorum sensing regulated pro- (2010) The fi rst characterized asparaginase from a basidiomycete, teins - role of bacterial cell-to-cell communication in global gene Flammulina velutipes. Bioresource Technology 102, 3316-3321. regulation as assessed by proteomics. Proteomics, in press

• El-Banna,A., Hajirezaei,M.R., Wissing,J., Ali,Z., Vaas,L., Heine-Dobber • Hebecker,S., Arendt,W., Heinemann,I.U., Tiefenau,J.H., Nimtz,M., -nack,E., Jacobsen,H.J., Schumacher,H.M., & Kiesecker,H. (2010) Rohde,M., Soll,D., & Moser,J. (2011) Alanyl-phosphatidylglycerol syn- Over-expression of PR-10a leads to increased salt and osmotic toler- thase: mechanism of substrate recognition during tRNA-dependent ance in potato cell cultures. Journal of Biotechnology 150, 277-287. lipid modifi cation in Pseudomonas aeruginosa. Molecular Micro- biology, in press. 154 SCIENTIFIC REPORTS | Publications

• Hemmen,K., Reinl,T., Buttler,K., Behler,F., Dieken,H., Jänsch,L., JRG Centre for Structural Systems Biology at DESY | Wilting,J., & Weich,H.A.*. (2011) High-resolution mass spectrometric Prof. Dr. Inari Kursula analysis of the secretome from mouse lung endothelial progenitor cells. Angiogenesis, in press. • Chen,W.Q., Salmazo,A., Myllykoski,M., Sjoblom,B., Bidlingmaier,M., Pollak,A., Baumgartel,P., Djinovic-Carugo,K., Kursula,P.*., & Lubec,G. (2010) Purifi cation of recombinant growth hormone by clear native gels for conformational analyses: preservation of conformation and RG Microbial Proteomics | Prof. Dr. Katharina Riedel receptor binding. Amino Acids 39, 859-869.

• Bosshard,F., Riedel,K., Schneider,T., Geiser,C., Bucheli,M., & Egli,T. • Huttu,J., Singh,B.K., Bhargav,S.P., Sattler,J.M., Schuler,H., & Kursula,I. (2010) Protein oxidation and aggregation in UVA-irradiated (2010) Crystallization and preliminary structural characterization of Escherichia coli cells as signs of accelerated cellular senescence. the two actin-depolymerization factors of the malaria parasite. Acta Environmental Microbiology 12, 2931-2945. Crystallographica Section F: Structural Biology and Crystallisation Community 66, 583-587. • Carranza,P., Grunau,A., Schneider,T., Hartmann,I., Lehner,A., Stephan,R., Gehrig,P., Grossmann,J., Groebel,K., Hoelzle,L.E., • Majava,V., Polverini,E., Mazzini,A., Nanekar,R., Knoll,W., Peters,J., Eberl,L., & Riedel,K. (2010) A gel-free quantitative proteomics ap- Natali,F., Baumgartel,P., Kursula,I., & Kursula,P. (2010) Structural proach to investigate temperature adaptation of the food-borne patho- and functional characterization of human peripheral nervous system gen Cronobacter turicensis 3032. Proteomics 10, 3248-3261. myelin protein P2. PLoS ONE 5, e10300.

• Grunau,A. & Riedel,K. (2011) Exoenzymes. In Encyclopaedia of Geo- • Myllykoski,M. & Kursula,P.*. (2010) Expression, purifi cation, and biology (Reiter,J. & Thiel,V., eds), pp. 355-359. Springer. initial characterization of different domains of recombinant mouse 2’,3’-cyclic nucleotide 3’-phosphodiesterase, an enigmatic enzyme • Hartmann,I., Carranza,P., Lehner,A., Stephan,R., Eberl,L., & Riedel,K. from the myelin sheath. BMC Research Notes 3, 12. (2010) Genes involved in Cronobacter sakazakii biofi lm formation. Applied and Environmental Microbiology 76, 2251-2261. • Suresh,S., Wang,C., Nanekar,R., Kursula,P.*., & Edwardson,J.M. (2010) Myelin basic protein and myelin protein 2 act synergistically • Lumjiaktase,P., Aguilar,C., Battin,T., Riedel,K., & Eberl,L. (2010) to cause stacking of lipid bilayers. Biochemistry 49, 3456-3463. Construction of self-transmissible green fl uorescent protein-based bi- osensor plasmids and their use for identifi cation of N-acyl homoser- • Kursula,P.*. (2011) Structural biology insights into the autoantingens ine-producing bacteria in lake sediments. Applied and Environmental of the myelin sheath. In Autoimmune Diseases: Symptoms, Diagno- Microbiology 76, 6119-6127. sis and Treatment Nova, in press.

• Moretti,M., Grunau,A., Minerdi,D., Gehrig,P., Roschitzki,B., Eberl,L., Garibaldi,A., Gullino,M.L., & Riedel,K. (2010) A proteomics approach to study synergistic and antagonistic interactions of the fungal-bac- Dept. of Medical Microbiology | Prof. Dr. G. Singh Chhatwal terial consortium Fusarium oxysporum wild-type MSA 35. Proteomics 10, 3292-3320. • Abt,B., Foster,B., Lapidus,A., Clum,A., Sun,H., Pukall,R., Lucas,S., Glavina Del,R.T., Nolan,M., Tice,H., Cheng,J.F., Pitluck,S., Liolios,K., • Schneider,T. & Riedel,K. (2010) Environmental proteomics: analysis Ivanova,N., Mavromatis,K., Ovchinnikova,G., Pati,A., Goodwin,L., of structure and function of microbial communities. Proteomics 10, Chen,A., Palaniappan,K., Land,M., Hauser,L., Chang,Y.J., Jeffries,C.D., 785-798. Rohde,M., Göker,M., Woyke,T., Bristow,J., Eisen,J.A., Markowitz,V., Hugenholtz,P., Kyrpides,N.C., & Klenk,H.P. (2010) Complete genome • Schneider,T. & Riedel,K. (2010) Environmental proteomics: Study- sequence of Cellulomonas fl avigena type strain (134). Standards in ing structure and function of microbial communities. In Geomicro- Genomic Sciences 3, 15-25. biology: Molecular and Environmental Perspective (Barton,L.L., Mandl,M., & Loy,A., eds), pp. 91-108. Springer. • Anderson,I., Djao,O.D., Misra,M., Chertkov,O., Nolan,M., Lucas,S., Lapidus,A., Del Rio,T.G., Tice,H., Cheng,J.F., Tapia,R., Han,C., Good - • Schneider,T., Gerrits,B., Gassmann,R., Schmid,E., Gessner,M.O., win,L., Pitluck,S., Liolios,K., Ivanova,N., Mavromatis,K., Mikhailova,N., Richter,A., Battin,T., Eberl,L., & Riedel,K. (2010) Proteome analysis of Pati,A., Brambilla,E., Chen,A., Palaniappan,K., Land,M., Hauser,L., fungal and bacterial involvement in leaf litter decomposition. Pro- Chang,Y.J., Jeffries,C.D., Sikorski,J., Spring,S., Rohde,M., Eichinger,K., teomics 10, 1819-1830. Huber,H., Wirth,R., Göker,M., Detter,J.C., Woyke,T., Bristow,J., Eisen,J.A., Markowitz,V., Hugenholtz,P., Klenk,H.P., & Kyrpides,N.C. • Srinivas,N., Jetter,P., Ueberbacher,B.J., Werneburg,M., Zerbe,K., (2010) Complete genome sequence of Methanothermus fervidus type Steinmann,J., Van der,M.B., Bernardini,F., Lederer,A., Dias,R.L., strain (V24S). Standards in Genomic Sciences 3, 315-324. Misson,P.E., Henze,H., Zumbrunn,J., Gombert,F.O., Obrecht,D., Hunziker,P., Schauer,S., Ziegler,U., Kach,A., Eberl,L., Riedel,K., • Aziz,R.K., Kansal,R., Aronow,B.J., Taylor,W.L., Rowe,S.L., Kubal,M., DeMarco,S.J., & Robinson,J.A. (2010) Peptidomimetic antibiotics tar- Chhatwal,G.S*., Walker,M.J., & Kotb,M. (2010) Microevolution of get outer-membrane biogenesis in Pseudomonas aeruginosa. Science group A streptococci in vivo: capturing regulatory networks engaged 327, 1010-1013. in sociomicrobiology, niche adaptation, and hypervirulence. PLoS ONE 5, e9798.

• Bunk,B., Schulz,A., Stammen,S., Münch,R., Warren,M.J., Rohde,M., JRG Macromolecular Interactions | Prof. Dr. Christiane Ritter Jahn,D., & Biedendieck,R. (2010) A short story about a big magic bug. Bioengineered Bugs 1, 85-91. • Greenwald,J., Buhtz,C., Ritter,C., Kwiatkowski,W., Choe,S., Maddelein,M.L., Ness,F., Cescau,S., Soragni,A., Leitz,D., Saupe,S.J., • Candela,M., Centanni,M., Fiori,J., Biagi,E., Turroni,S., Orrico,C., & Riek,R. (2010) The mechanism of prion inhibition by HET-S. Mol- Bergmann,S., Hammerschmidt,S., & Brigidi,P. (2010) DnaK from ecules and Cells 38, 889-899. Bifi dobacterium animalis subsp. lactis is a surface-exposed human plasminogen receptor upregulated in response to bile salts. Micro- • Wasmer,C., Zimmer,A., Sabate,R., Soragni,A., Saupe,S.J., Ritter,C., & biology 156, 1609-1618. Meier,B.H. (2010) Structural similarity between the prion domain of HET-s and a homologue can explain amyloid cross-seeding in spite of • Chang,Y.J., Pukall,R., Saunders,E., Lapidus,A., Copeland,A., Nolan,M., limited sequence identity. Journal of Molecular Biology 402, 311-325. Glavina Del,R.T., Lucas,S., Chen,F., Tice,H., Cheng,J.F., Han,C., Detter,J.C., Bruce,D., Goodwin,L., Pitluck,S., Mikhailova,N., Liolios,K., Pati,A., Ivanova,N., Mavromatis,K., Chen,A., Palaniappan,K., Land,M., Hauser,L., Jeffries,C.D., Brettin,T., Rohde,M., Göker,M., Bristow,J., Eisen,J.A., Markowitz,V., Hugenholtz,P., Kyrpides,N.C., & Klenk,H.P. (2010) Complete genome sequence of Acidaminococcus fermentans type strain (VR4). Standards in Genomic Sciences 3, 1-14. SCIENTIFIC REPORTS | Publications 155

• Chertkov,O., Sikorski,J., Brambilla,E., Lapidus,A., Copeland,A., Glavi- • Göker,M., Held,B., Lapidus,A., Nolan,M., Spring,S., Yasawong,M., na Del,R.T., Nolan,M., Lucas,S., Tice,H., Cheng,J.F., Han,C., Detter,J.C., Lucas,S., Glavina Del,R.T., Tice,H., Cheng,J.F., Goodwin,L., Tapia,R., Bruce,D., Tapia,R., Goodwin,L., Pitluck,S., Liolios,K., Ivanova,N., Pitluck,S., Liolios,K., Ivanova,N., Mavromatis,K., Mikhailova,N., Mavromatis,K., Ovchinnikova,G., Pati,A., Chen,A., Palaniappan,K., Pati,A., Chen,A., Palaniappan,K., Brambilla,E., Land,M., Hauser,L., Land,M., Hauser,L., Chang,Y.J., Jeffries,C.D., Spring,S., Rohde,M., Chang,Y.J., Jeffries,C.D., Brettin,T., Detter,J.C., Han,C., Rohde,M., Göker,M., Bristow,J., Eisen,J.A., Markowitz,V., Hugenholtz,P., Sikorski,J., Woyke,T., Bristow,J., Eisen,J.A., Markowitz,V., Kyrpides,N.C., & Klenk,H.P. (2010) Complete genome sequence of Hugenholtz,P., Kyrpides,N.C., & Klenk,H.P. (2010) Complete genome Aminobacterium colombiense type strain (ALA-1). Standards in Ge- sequence of Ignisphaera aggregans type strain (AQ1.S1). Standards in nomic Sciences 2, 280-289. Genomic Sciences 3, 66-75.

• Clum,A., Tindall,B.J., Sikorski,J., Ivanova,N., Mavromatis,K., • Göker,M., Held,B., Lucas,S., Nolan,M., Yasawong,M., Glavina Del,R.T., Lucas,S., Glavina Del,R.T., Nolan,M., Chen,F., Tice,H., Pitluck,S., Tice,H., Cheng,J.F., Bruce,D., Detter,J.C., Tapia,R., Han,C., Goodwin,L., Cheng,J.F., Chertkov,O., Brettin,T., Han,C., Detter,J.C., Kuske,C., Pitluck,S., Liolios,K., Ivanova,N., Mavromatis,K., Mikhailova,N., Bruce,D., Goodwin,L., Ovchinikova,G., Pati,A., Mikhailova,N., Pati,A., Chen,A., Palaniappan,K., Land,M., Hauser,L., Chang,Y.J., Chen,A., Palaniappan,K., Land,M., Hauser,L., Chang,Y.J., Jeffries,C.D., Jeffries,C.D., Rohde,M., Sikorski,J., Pukall,R., Woyke,T., Bristow,J., Chain,P., Rohde,M., Göker,M., Bristow,J., Eisen,J.A., Markowitz,V., Eisen,J.A., Markowitz,V., Hugenholtz,P., Kyrpides,N.C., Klenk,H.P., & Hugenholtz,P., Kyrpides,N.C., Klenk,H.P., & Lapidus,A. (2010) Er- Lapidus,A. (2010) Complete genome sequence of Olsenella uli type ratum to: Complete genome sequence of Pirellula staleyi type strain strain (VPI D76D-27C). Standards in Genomic Sciences 3, 76-84. (ATCC 27377). Standards in Genomic Sciences 2, 228-288. • Gronow,S., Welnitz,S., Lapidus,A., Nolan,M., Ivanova,N., Glavina • Copeland,A., Sikorski,J., Lapidus,A., Nolan,M., Glavina,T., Del,R., Del,R.T., Copeland,A., Chen,F., Tice,H., Pitluck,S., Cheng,J.F., Lucas,S., Chen,F., Tice,H., Pitluck,S., Cheng,J.F., Pukall,R., Saunders,E., Brettin,T., Han,C., Detter,J.C., Bruce,D., Goodwin,L., Chertkov,O., Brettin,T., Han,C., Kuske,C., Bruce,D., Goodwin,L., Land,M., Hauser,L., Chang,Y.J., Jeffries,C.D., Pati,A., Mavromatis,K., Ivanova,N., Mavromatis,K., Mikhailova,N., Chen,A., Palaniappan,K., Mikhailova,N., Chen,A., Palaniappan,K., Chain,P., Rohde,M., Chain,P., Rohde,M., Göker,M., Bristow,J., Eisen,J.A., Markowitz,V., Göker,M., Bristow,J., Eisen,J.A., Markowitz,V., Hugenholtz,P., Hugenholtz,P., Kyrpides,N.C., Klenk,H.P., & Detter,J.C. (2010) Erratum Kyrpides,N.C., Klenk,H.P., & Lucas,S. (2010) Complete genome se- to: Complete genome sequence of Atopobium parvulum type strain quence of Veillonella parvula type strain (Te3). Standards in Genomic (IPP 1246). Standards in Genomic Sciences 2, 361-362. Sciences 2, 57-65.

• Del Rio,T.G., Chertkov,O., Yasawong,M., Lucas,S., Deshpande,S., • Han,C., Gu,W., Zhang,X., Lapidus,A., Nolan,M., Copeland,A., Lucas,S., Cheng,J.F., Detter,C., Tapia,R., Han,C., Goodwin,L., Pitluck,S., Del Rio,T.G., Tice,H., Cheng,J.F., Tapia,R., Goodwin,L., Pitluck,S., Liolios,K., Ivanova,N., Mavromatis,K., Pati,A., Chen,A., Pagani,I., Ivanova,N., Mavromatis,K., Mikhailova,N., Pati,A., Chen,A., Palaniappan,K., Land,M., Hauser,L., Chang,Y.J., Jeffries,C.D., Palaniappan,K., Land,M., Hauser,L., Chang,Y.J., Jeffries,C.D., Rohde,M., Pukall,R., Sikorski,J., Göker,M., Woyke,T., Bristow,J., Schneider,S., Rohde,M., Göker,M., Pukall,R., Woyke,T., Bristow,J., Eisen,J.A., Markowitz,V., Hugenholtz,P., Kyrpides,N.C., Klenk,H.P., Eisen,J.A., Markowitz,V., Hugenholtz,P., Kyrpides,N.C., Klenk,H.P., & Lapidus,A. (2010) Complete genome sequence of Intrasporangium & Detter,J.C. (2010) Complete genome sequence of Thermaerobacter calvum type strain (7 KIP). Standards in Genomic Sciences 3, 294-303. marianensis type strain (7p75a). Standards in Genomic Sciences 3, 337-345. • Dietrich,N., Rohde,M., Geffers,R., Kroger,A., Hauser,H., Weiss,S., & Gekara,N.O. (2010) Mast cells elicit proinfl ammatory but not type I • Hänisch,J., Ehinger,J., Ladwein,M., Rohde,M., Derivery,E., Bosse,T., interferon responses upon activation of TLRs by bacteria. Proceeding Steffen,A., Bumann,D., Misselwitz,B., Hardt,W.D., Gautreau,A., of the National Academy of Sciences of the United States of America Stradal,T.B.*., & Rottner,K. (2010) Molecular dissection of Salmonel- 107, 8748-8753. la-induced membrane ruffl ing versus invasion. Cellular Microbiology 12, 84-98. • Djao,O.D., Zhang,X., Lucas,S., Lapidus,A., Del Rio,T.G., Nolan,M., Tice,H., Cheng,J.F., Han,C., Tapia,R., Goodwin,L., Pitluck,S., Liolios,K., • Hoffmann,C., Berking,A., Agerer,F., Buntru,A., Neske,F., Ivanova,N., Mavromatis,K., Mikhailova,N., Ovchinnikova,G., Chhatwal,G.S*., Ohlsen,K., & Hauck,C.R. (2010) Caveolin limits Pati,A., Brambilla,E., Chen,A., Palaniappan,K., Land,M., Hauser,L., membrane microdomain mobility and integrin-mediated uptake of Chang,Y.J., Jeffries,C.D., Rohde,M., Sikorski,J., Spring,S., Göker,M., fi bronectin-binding pathogens. Journal of Cell Science 123, 4280-4291. Detter,J.C., Woyke,T., Bristow,J., Eisen,J.A., Markowitz,V., Hugenholtz,P., Kyrpides,N.C., & Klenk,H.P. (2010) Complete genome • Hu,Y., van der,G.R., Besra,G.S., Gurcha,S.S., Liu,A., Rohde,M., sequence of Syntrophothermus lipocalidus type strain (TGB-C1). Singh,M., & Coates,A. (2010) 3-Ketosteroid 9alpha-hydroxylase is an Standards in Genomic Sciences 3, 268-275. essential factor in the pathogenesis of Mycobacterium tuberculosis. Molecular Microbiology 75, 107-121. • Foster,B., Pukall,R., Abt,B., Nolan,M., Glavina Del,R.T., Chen,F., Lucas,S., Tice,H., Pitluck,S., Cheng,J.F., Chertkov,O., Brettin,T., • Itzek,A., Gillen,C.M., Fulde,M., Friedrichs,C., Rodloff,A.C., Han,C., Detter,J.C., Bruce,D., Goodwin,L., Ivanova,N., Mavromatis,K., Chhatwal,G.S*., & Nitsche-Schmitz,D.P. (2010) Contribution of plas- Pati,A., Mikhailova,N., Chen,A., Palaniappan,K., Land,M., Hauser,L., minogen activation towards the pathogenic potential of oral strepto- Chang,Y.J., Jeffries,C.D., Chain,P., Rohde,M., Goker,M., Bristow,J., cocci. PLoS ONE 5, e13826. Eisen,J.A., Markowitz,V., Hugenholtz,P., Kyrpides,N.C., Klenk,H.P., & Lapidus,A. (2010) Complete genome sequence of Xylanimonas cellu- • Ivanova,N., Sikorski,J., Jando,M., Munk,C., Lapidus,A., Glavina losilytica type strain (XIL07). Standards in Genomic Sciences 2, 1-8. Del,R.T., Copeland,A., Tice,H., Cheng,J.F., Lucas,S., Chen,F., Nolan,M., Bruce,D., Goodwin,L., Pitluck,S., Mavromatis,K., Mikhailova,N., • Garbe,J., Wesche,A., Bunk,B., Kazmierczak,M., Selezska,K., Rohde,C., Pati,A., Chen,A., Palaniappan,K., Land,M., Hauser,L., Chang,Y.J., Sikorski,J., Rohde,M., Jahn,D., & Schobert,M. (2010) Characterization Jeffries,C.D., Meincke,L., Brettin,T., Detter,J.C., Rohde,M., Göker,M., of JG024, a Pseudomonas aeruginosa PB1-like broad host range Bristow,J., Eisen,J.A., Markowitz,V., Hugenholtz,P., Kyrpides,N.C., & phage under simulated infection conditions. BMC Microbiology 10 ,301. Klenk,H.P. (2010) Complete genome sequence of Geodermatophilus obscurus type strain (G-20). Standards in Genomic Sciences 2, 158-167. • Glavina Del,R.T., Abt,B., Spring,S., Lapidus,A., Nolan,M., Tice,H., Copeland,A., Cheng,J.F., Chen,F., Bruce,D., Goodwin,L., Pitluck,S., • Ivanova,N., Sikorski,J., Jando,M., Lapidus,A., Nolan,M., Lucas,S., Ivanova,N., Mavromatis,K., Mikhailova,N., Pati,A., Chen,A., Del Rio,T.G., Tice,H., Copeland,A., Cheng,J.F., Chen,F., Bruce,D., Palaniappan,K., Land,M., Hauser,L., Chang,Y.J., Jeffries,C.D., Chain,P., Goodwin,L., Pitluck,S., Mavromatis,K., Ovchinnikova,G., Pati,A., Saunders,E., Detter,J.C., Brettin,T., Rohde,M., Göker,M., Bristow,J., Chen,A., Palaniappan,K., Land,M., Hauser,L., Chang,Y.J., Jeffries,C.D., Eisen,J.A., Markowitz,V., Hugenholtz,P., Kyrpides,N.C., Klenk,H.P., & Chain,P., Saunders,E., Han,C., Detter,J.C., Brettin,T., Rohde,M., Lucas,S. (2010) Complete genome sequence of Chitinophaga pinensis Göker,M., Bristow,J., Eisen,J.A., Markowitz,V., Hugenholtz,P., type strain (UQM 2034). Standards in Genomic Sciences 2, 87-95. Klenk,H.P., & Kyrpides,N.C. (2010) Complete genome sequence of Gordonia bronchialis type strain (3410). Standards in Genomic Sciences 2, 19-28. 156 SCIENTIFIC REPORTS | Publications

• Ivanova,N., Daum,C., Lang,E., Abt,B., Kopitz,M., Saunders,E., • Macke,L., Garritsen,H.S., Meyring,W., Hannig,H., Pagelow,U., Lapidus,A., Lucas,S., Glavina Del,R.T., Nolan,M., Tice,H., Copeland,A., Wormann,B., Piechaczek,C., Geffers,R., Rohde,M., Lindenmaier,W., Cheng,J.F., Chen,F., Bruce,D., Goodwin,L., Pitluck,S., Mavromatis,K., & Dittmar,K.E.*. (2010) Evaluating maturation and genetic modifi ca- Pati,A., Mikhailova,N., Chen,A., Palaniappan,K., Land,M., Hauser,L., tion of human dendritic cells in a new polyolefi n cell culture bag Chang,Y.J., Jeffries,C.D., Detter,J.C., Brettin,T., Rohde,M., Göker,M., system. Transfusion 50, 843-855. Bristow,J., Markowitz,V., Eisen,J.A., Hugenholtz,P., Kyrpides,N.C., & Klenk,H.P. (2010) Complete genome sequence of Haliangium ochra- • Marjenberg,Z.R., Ellis,I.R., Hagan,R.M., Prabhakaran,S., Hook,M., ceum type strain (SMP-2). Standards in Genomic Sciences 2, 96-106. Talay,S.R.*., Potts,J.R., Staunton,D., & Schwarz-Linek,U. (2010) Coop- erative binding and activation of fi bronectin by a bacterial surface • Jensch,I., Gamez,G., Rothe,M., Ebert,S., Fulde,M., Somplatzki,D., protein. Journal of Biological Chemistry 286, 1884-1894. Bergmann,S., Petruschka,L., Rohde,M., Nau,R., & Hammerschmidt,S. (2010) PavB is a surface-exposed adhesin of Streptococcus pneumo- • Mavromatis,K., Sikorski,J., Pabst,E., Teshima,H., Lapidus,A., Lucas,S., niae contributing to nasopharyngeal colonization and airways infec- Nolan,M., Glavina Del,R.T., Cheng,J.F., Bruce,D., Goodwin,L., tions. Molecular Microbiology 77, 22-43. Pitluck,S., Liolios,K., Ivanova,N., Mikhailova,N., Pati,A., Chen,A., Palaniappan,K., Land,M., Hauser,L., Chang,Y.J., Jeffries,C.D., • Kaur,S.J., Nerlich,A., Bergmann,S., Rohde,M., Fulde,M., Zahner,D., Rohde,M., Spring,S., Göker,M., Wirth,R., Woyke,T., Bristow,J., Hanski,E., Zinkernagel,A., Nizet,V., Chhatwal,G.S.*., & Talay,S.R. Eisen,J.A., Markowitz,V., Hugenholtz,P., Klenk,H.P., & Kyrpides,N.C. (2010) The CXC chemokine-degrading protease SpyCep of Streptococ- (2010) Complete genome sequence of Vulcanisaeta distributa type cus pyogenes promotes its uptake into endothelial cells. Journal of strain (IC-017). Standards in Genomic Sciences 3, 117-125. Biological Chemistry 285, 27798-27805. • Mavromatis,K., Abt,B., Brambilla,E., Lapidus,A., Copeland,A., • Kiss,H., Lang,E., Lapidus,A., Copeland,A., Nolan,M., Glavina Deshpande,S., Nolan,M., Lucas,S., Tice,H., Cheng,J.F., Han,C., Del,R.T., Chen,F., Lucas,S., Tice,H., Cheng,J.F., Han,C., Goodwin,L., Detter,J.C., Woyke,T., Goodwin,L., Pitluck,S., Held,B., Brettin,T., Pitluck,S., Liolios,K., Pati,A., Ivanova,N., Mavromatis,K., Chen,A., Tapia,R., Ivanova,N., Mikhailova,N., Pati,A., Liolios,K., Chen,A., Palaniappan,K., Land,M., Hauser,L., Chang,Y.J., Jeffries,C.D., Palaniappan,K., Land,M., Hauser,L., Chang,Y.J., Jeffries,C.D., Detter,J.C., Brettin,T., Spring,S., Rohde,M., Göker,M., Woyke,T., Rohde,M., Göker,M., Bristow,J., Eisen,J.A., Markowitz,V., Bristow,J., Eisen,J.A., Markowitz,V., Hugenholtz,P., Kyrpides,N.C., Hugenholtz,P., Klenk,H.P., & Kyrpides,N.C. (2010) Complete genome & Klenk,H.P. (2010) Complete genome sequence of Denitrovibrio sequence of Coraliomargarita akajimensis type strain (04OKA010- acetiphilus type strain (N2460). Standards in Genomic Sciences 2, 24). Standards in Genomic Sciences 2, 290-299. 270-279. • Mavromatis,K., Sikorski,J., Lapidus,A., Glavina Del,R.T., Copeland,A., • Kotz,A., Wagener,J., Engel,J., Routier,F., Echtenacher,B., Pich,A., Tice,H., Cheng,J.F., Lucas,S., Chen,F., Nolan,M., Bruce,D., Rohde,M., Hoffmann,P., Heesemann,J., & Ebel,F. (2010) The mitA Goodwin,L., Pitluck,S., Ivanova,N., Ovchinnikova,G., Pati,A., Chen,A., gene of Aspergillus fumigatus is required for mannosylation of inosi- Palaniappan,K., Land,M., Hauser,L., Chang,Y.J., Jeffries,C.D., Chain,P., tol-phosphorylceramide, but is dispensable for pathogenicity. Fungal Meincke,L., Sims,D., Chertkov,O., Han,C., Brettin,T., Detter,J.C., Genetics and Biology 47, 169-178. Wahrenburg,C., Rohde,M., Pukall,R., Göker,M., Bristow,J., Eisen,J.A., Markowitz,V., Hugenholtz,P., Klenk,H.P., & Kyrpides,N.C. (2010) Com- • Labutti,K., Sikorski,J., Schneider,S., Nolan,M., Lucas,S., Glavina plete genome sequence of Alicyclobacillus acidocaldarius type strain Del,R.T., Tice,H., Cheng,J.F., Goodwin,L., Pitluck,S., Liolios,K., (104-IA). Standards in Genomic Sciences 2, 9-18. Ivanova,N., Mavromatis,K., Mikhailova,N., Pati,A., Chen,A., Palaniappan,K., Land,M., Hauser,L., Chang,Y.J., Jeffries,C.D., • Mavromatis,K., Yasawong,M., Chertkov,O., Lapidus,A., Lucas,S., Tindall,B.J., Rohde,M., Göker,M., Woyke,T., Bristow,J., Eisen,J.A., Nolan,M., Del Rio,T.G., Tice,H., Cheng,J.F., Pitluck,S., Liolios,K., Markowitz,V., Hugenholtz,P., Kyrpides,N.C., Klenk,H.P., & Lapidus,A. Ivanova,N., Tapia,R., Han,C., Bruce,D., Goodwin,L., Pati,A., Chen,A., (2010) Complete genome sequence of Planctomyces limnophilus type Palaniappan,K., Land,M., Hauser,L., Chang,Y.J., Jeffries,C.D., strain (Mu 290). Standards in Genomic Sciences 3, 47-56. Detter,J.C., Rohde,M., Brambilla,E., Spring,S., Göker,M., Sikorski,J., Woyke,T., Bristow,J., Eisen,J.A., Markowitz,V., Hugenholtz,P., • Labutti,K., Mayilraj,S., Clum,A., Lucas,S., Glavina Del,R.T., Nolan,M., Klenk,H.P., & Kyrpides,N.C. (2010) Complete genome sequence of Spi- Tice,H., Cheng,J.F., Pitluck,S., Liolios,K., Ivanova,N., Mavromatis,K., rochaeta smaragdinae type strain (SEBR 4228). Standards in Genomic Mikhailova,N., Pati,A., Goodwin,L., Chen,A., Palaniappan,K., Land,M., Sciences 3, 136-144. Hauser,L., Chang,Y.J., Jeffries,C.D., Rohde,M., Spring,S., Göker,M., Woyke,T., Bristow,J., Eisen,J.A., Markowitz,V., Hugenholtz,P., • Moeller,R., Rohde,M., & Reitz,G. (2010) Effects of ionizing radioation Kyrpides,N.C., Klenk,H.P., & Lapidus,A. (2010) Permanent draft ge- on the survival of bacterial spores in artifi cial martian regolith. nome sequence of Dethiosulfovibrio peptidovorans type strain (SEBR Icarus - International Journal of Solar System Studies 206, 783-786. 4207). Standards in Genomic Sciences 3, 85-92. • Nolan,M., Sikorski,J., Davenport,K., Lucas,S., Del Rio,T.G., • Lail,K., Sikorski,J., Saunders,E., Lapidus,A., Glavina Del,R.T., Tice,H., Cheng,J.F., Goodwin,L., Pitluck,S., Liolios,K., Ivanova,N., Copeland,A., Tice,H., Cheng,J.F., Lucas,S., Nolan,M., Bruce,D., Mavromatis,K., Ovchinnikova,G., Pati,A., Chen,A., Palaniappan,K., Goodwin,L., Pitluck,S., Ivanova,N., Mavromatis,K., Ovchinnikova,G., Land,M., Hauser,L., Chang,Y.J., Jeffries,C.D., Tapia,R., Brettin,T., Pati,A., Chen,A., Palaniappan,K., Land,M., Hauser,L., Chang,Y.J., Detter,J.C., Han,C., Yasawong,M., Rohde,M., Tindall,B.J., Göker,M., Jeffries,C.D., Chain,P., Brettin,T., Detter,J.C., Schutze,A., Rohde,M., Woyke,T., Bristow,J., Eisen,J.A., Markowitz,V., Hugenholtz,P., Tindall,B.J., Göker,M., Bristow,J., Eisen,J.A., Markowitz,V., Kyrpides,N.C., Klenk,H.P., & Lapidus,A. (2010) Complete genome Hugenholtz,P., Kyrpides,N.C., Klenk,H.P., & Chen,F. (2010) Complete sequence of Ferrimonas balearica type strain (PAT). Standards in genome sequence of Spirosoma linguale type strain (1). Standards in Genomic Sciences 3, 174-182. Genomic Sciences 2, 176-185. • Nolan,M., Sikorski,J., Jando,M., Lucas,S., Lapidus,A., Glavina Del,R.T., • Liolios,K., Sikorski,J., Jando,M., Lapidus,A., Copeland,A., Glavina,T., Chen,F., Tice,H., Pitluck,S., Cheng,J.F., Chertkov,O., Sims,D., Meincke,L., Del,R., Nolan,M., Lucas,S., Tice,H., Cheng,J.F., Han,C., Woyke,T., Brettin,T., Han,C., Detter,J.C., Bruce,D., Goodwin,L., Land,M., Hauser,L., Goodwin,L., Pitluck,S., Ivanova,N., Mavromatis,K., Mikhailova,N., Chang,Y.J., Jeffries,C.D., Ivanova,N., Mavromatis,K., Mikhailova,N., Chertkov,O., Kuske,C., Chen,A., Palaniappan,K., Land,M., Hauser,L., Chen,A., Palaniappan,K., Chain,P., Rohde,M., Göker,M., Bristow,J., Chang,Y.J., Jeffries,C.D., Detter,J.C., Brettin,T., Rohde,M., Göker,M., Eisen,J.A., Markowitz,V., Hugenholtz,P., Kyrpides,N.C., & Klenk,H.P. Bristow,J., Eisen,J.A., Markowitz,V., Hugenholtz,P., Klenk,H.P., & (2010) Complete genome sequence of Streptosporangium roseum type Kyrpides,N.C. (2010) Complete genome sequence of Thermobispora strain (NI 9100). Standard in Genomic Sciences 2, 29-37. bispora type strain (R51). Standards in Genomic Sciences 2, 318-326. • Pati,A., Gronow,S., Lapidus,A., Copeland,A., Glavina Del,R.T., • Maamary,P.G., Sanderson-Smith,M.L., Aziz,R.K., Hollands,A., Nolan,M., Lucas,S., Tice,H., Cheng,J.F., Han,C., Chertkov,O., Cole,J.N., McKay,F.C., Mcarthur,J.D., Kirk,J.K., Cork,A.J., Keefe,R.J., Bruce,D., Tapia,R., Goodwin,L., Pitluck,S., Liolios,K., Ivanova,N., Kansal,R.G., Sun,H., Taylor,W.L., Chhatwal,G.S*., Ginsburg,D., Mavromatis,K., Chen,A., Palaniappan,K., Land,M., Hauser,L., Nizet,V., Kotb,M., & Walker,M.J. (2010) Parameters governing inva- Chang,Y.J., Jeffries,C.D., Detter,J.C., Rohde,M., Göker,M., Bristow,J., sive disease propensity of non-M1 serotype group A streptococci. Eisen,J.A., Markowitz,V., Hugenholtz,P., Klenk,H.P., & Kyrpides,N.C. Journal of Innate Immunology 2, 596-606. (2010) Complete genome sequence of Arcobacter nitrofi gilis type strain (CI). Standards in Genomic Sciences 2, 300-308. SCIENTIFIC REPORTS | Publications 157

• Pati,A., Sikorski,J., Gronow,S., Munk,C., Lapidus,A., Copeland,A., • Sikorski,J., Chertkov,O., Lapidus,A., Nolan,M., Lucas,S., Del Rio,T.G., Glavina Del,T.T., Nolan,M., Lucas,S., Chen,F., Tice,H., Cheng,J.F., Tice,H., Cheng,J.F., Tapia,R., Han,C., Goodwin,L., Pitluck,S., Han,C., Detter,J.C., Bruce,D., Tapia,R., Goodwin,L., Pitluck,S., Liolios,K., Ivanova,N., Mavromatis,K., Mikhailova,N., Pati,A., Liolios,K., Ivanova,N., Mavromatis,K., Mikhailova,N., Chen,A., Chen,A., Palaniappan,K., Land,M., Hauser,L., Chang,Y.J., Jeffries,C.D., Palaniappan,K., Land,M., Hauser,L., Chang,Y.J., Jeffries,C.D., Brambilla,E., Yasawong,M., Rohde,M., Pukall,R., Spring,S., Göker,M., Spring,S., Rohde,M., Göker,M., Bristow,J., Eisen,J.A., Markowitz,V., Woyke,T., Bristow,J., Eisen,J.A., Markowitz,V., Hugenholtz,P., Hugenholtz,P., Kyrpides,N.C., & Klenk,H.P. (2010) Complete genome Kyrpides,N.C., & Klenk,H.P. (2010) Complete genome sequence of sequence of Brachyspira murdochii type strain (56-150). Standards in Ilyobacter polytropus type strain (CuHbu1). Standards in Genomic Genomic Sciences 2, 260-269. Sciences 3, 304-314.

• Pati,A., Labutti,K., Pukall,R., Nolan,M., Glavina Del,R.T., Tice,H., • Sikorski,J., Lapidus,A., Chertkov,O., Lucas,S., Copeland,A., Glavina Cheng,J.F., Lucas,S., Chen,F., Copeland,A., Ivanova,N., Mavromatis,K., Del,R.T., Nolan,M., Tice,H., Cheng,J.F., Han,C., Brambilla,E., Mikhailova,N., Pitluck,S., Bruce,D., Goodwin,L., Land,M., Hauser,L., Pitluck,S., Liolios,K., Ivanova,N., Mavromatis,K., Mikhailova,N., Chang,Y.J., Jeffries,C.D., Chen,A., Palaniappan,K., Chain,P., Brettin,T., Pati,A., Bruce,D., Detter,C., Tapia,R., Goodwin,L., Chen,A., Sikorski,J., Rohde,M., Göker,M., Bristow,J., Eisen,J.A., Markowitz,V., Palaniappan,K., Land,M., Hauser,L., Chang,Y.J., Jeffries,C.D., Hugenholtz,P., Kyrpides,N.C., Klenk,H.P., & Lapidus,A. (2010) Com- Rohde,M., Göker,M., Spring,S., Woyke,T., Bristow,J., Eisen,J.A., plete genome sequence of Sphaerobacter thermophilus type strain (S Markowitz,V., Hugenholtz,P., Kyrpides,N.C., & Klenk,H.P. (2010) 6022). Standards in Genomic Sciences 2, 49-56. Complete genome sequence of Acetohalobium arabaticum type strain (Z-7288). Standards in Genomic Sciences 3, 57-65. • Pitluck,S., Yasawong,M., Munk,C., Nolan,M., Lapidus,A., Lucas,S., Glavina Del,R.T., Tice,H., Cheng,J.F., Bruce,D., Detter,C., Tapia,R., • Sikorski,J., Lapidus,A., Copeland,A., Glavina Del,R.T., Nolan,M., Han,C., Goodwin,L., Liolios,K., Ivanova,N., Mavromatis,K., Lucas,S., Chen,F., Tice,H., Cheng,J.F., Saunders,E., Bruce,D., Mikhailova,N., Pati,A., Chen,A., Palaniappan,K., Land,M., Hauser,L., Goodwin,L., Pitluck,S., Ovchinnikova,G., Pati,A., Ivanova,N., Chang,Y.J., Jeffries,C.D., Rohde,M., Spring,S., Sikorski,J., Göker,M., Mavromatis,K., Chen,A., Palaniappan,K., Chain,P., Land,M., Woyke,T., Bristow,J., Eisen,J.A., Markowitz,V., Hugenholtz,P., Hauser,L., Chang,Y.J., Jeffries,C.D., Brettin,T., Detter,J.C., Han,C., Kyrpides,N.C., & Klenk,H.P. (2010) Complete genome sequence of Rohde,M., Lang,E., Spring,S., Göker,M., Bristow,J., Eisen,J.A., Thermosediminibacter oceani type strain (JW/IW-1228P). Standards Markowitz,V., Hugenholtz,P., Kyrpides,N.C., & Klenk,H.P. (2010) Com- in Genomic Sciences 3, 108-116. plete genome sequence of Sulfurospirillum deleyianum type strain (5175). Standards in Genomic Sciences 2, 149-157. • Pitluck,S., Yasawong,M., Held,B., Lapidus,A., Nolan,M., Copeland,A., Lucas,S., Del Rio,T.G., Tice,H., Cheng,J.F., Chertkov,O., • Sikorski,J., Munk,C., Lapidus,A., Ngatchou Djao,O.D., Lucas,S., Goodwin,L., Tapia,R., Han,C., Liolios,K., Ivanova,N., Mavromatis,K., Glavina Del,R.T., Nolan,M., Tice,H., Han,C., Cheng,J.F., Tapia,R., Ovchinnikova,G., Pati,A., Chen,A., Palaniappan,K., Land,M., Goodwin,L., Pitluck,S., Liolios,K., Ivanova,N., Mavromatis,K., Hauser,L., Chang,Y.J., Jeffries,C.D., Pukall,R., Spring,S., Rohde,M., Mikhailova,N., Pati,A., Sims,D., Meincke,L., Brettin,T., Detter,J.C., Sikorski,J., Göker,M., Woyke,T., Bristow,J., Eisen,J.A., Markowitz,V., Chen,A., Palaniappan,K., Land,M., Hauser,L., Chang,Y.J., Jeffries,C.D., Hugenholtz,P., Kyrpides,N.C., & Klenk,H.P. (2010) Non-contiguous Rohde,M., Lang,E., Spring,S., Göker,M., Woyke,T., Bristow,J., fi nished genome sequence of Aminomonas paucivorans type strain Eisen,J.A., Markowitz,V., Hugenholtz,P., Kyrpides,N.C., & Klenk,H.P. (GLU-3). Standards in Genomic Sciences 3, 285-293. (2010) Complete genome sequence of Sulfurimonas autotrophica type strain (OK10). Standards in Genomic Sciences 3, 194-202. • Pukall,R., Lapidus,A., Glavina Del,R.T., Copeland,A., Tice,H., Cheng,J.F., Lucas,S., Chen,F., Nolan,M., Labutti,K., Pati,A., Ivanova,N., • Sikorski,J., Tindall,B.J., Lowry,S., Lucas,S., Nolan,M., Copeland,A., Mavromatis,K., Mikhailova,N., Pitluck,S., Bruce,D., Goodwin,L., Glavina Del,R.T., Tice,H., Cheng,J.F., Han,C., Pitluck,S., Liolios,K., Land,M., Hauser,L., Chang,Y.J., Jeffries,C.D., Chen,A., Palaniappan,K., Ivanova,N., Mavromatis,K., Mikhailova,N., Pati,A., Goodwin,L., Chain,P., Rohde,M., Göker,M., Bristow,J., Eisen,J.A., Markowitz,V., Chen,A., Palaniappan,K., Land,M., Hauser,L., Chang,Y.J., Jeffries,C.D., Hugenholtz,P., Kyrpides,N.C., Klenk,H.P., & Brettin,T. (2010) Com- Rohde,M., Göker,M., Woyke,T., Bristow,J., Eisen,J.A., Markowitz,V., plete genome sequence of Kribbella fl avida type strain (IFO 14399). Hugenholtz,P., Kyrpides,N.C., Klenk,H.P., & Lapidus,A. (2010) Com- Standards in Genomic Sciences 2, 186-193. plete genome sequence of Meiothermus silvanus type strain (VI-R2). Standards in Genomic Sciences 3, 37-46. • Pukall,R., Lapidus,A., Glavina Del,R.T., Copeland,A., Tice,H., Cheng,J.F., Lucas,S., Chen,F., Nolan,M., Bruce,D., Goodwin,L., • Sikorski,J., Lapidus,A., Copeland,A., Misra,M., Glavina Del,R.T., Pitluck,S., Mavromatis,K., Ivanova,N., Ovchinnikova,G., Pati,A., Nolan,M., Lucas,S., Chen,F., Tice,H., Cheng,J.F., Jando,M., Chen,A., Palaniappan,K., Land,M., Hauser,L., Chang,Y.J., Jeffries,C.D., Schneider,S., Bruce,D., Goodwin,L., Pitluck,S., Liolios,K., Chain,P., Meincke,L., Sims,D., Brettin,T., Detter,J.C., Rohde,M., Mikhailova,N., Pati,A., Ivanova,N., Mavromatis,K., Chen,A., Göker,M., Bristow,J., Eisen,J.A., Markowitz,V., Kyrpides,N.C., Palaniappan,K., Chertkov,O., Land,M., Hauser,L., Chang,Y.J., Klenk,H.P., & Hugenholtz,P. (2010) Complete genome sequence of Jeffries,C.D., Brettin,T., Detter,J.C., Han,C., Rohde,M., Göker,M., Conexibacter woesei type strain (ID131577). Standards in Genomic Bristow,J., Eisen,J.A., Markowitz,V., Hugenholtz,P., Kyrpides,N.C., & Sciences 2, 212-219. Klenk,H.P. (2010) Complete genome sequence of Segniliparus rotun- dus type strain (CDC 1076). Standards in Genomic Sciences 2, 203-211. • Reißmann,S., Friedrichs,C., Rajkumari,R., Itzek,A., Fulde,M., Rodloff,A.C., Brahmadathan,K.N., Chhatwal,G.S.*., & Nitsche- • Spring,S., Nolan,M., Lapidus,A., Glavina Del,R.T., Copeland,A., Schmitz,D.P. (2010) Contribution of Streptococcus anginosus to Tice,H., Cheng,J.F., Lucas,S., Land,M., Chen,F., Bruce,D., Goodwin,L., infections caused by groups C and G Streptococci, Southern India. Pitluck,S., Ivanova,N., Mavromatis,K., Mikhailova,N., Pati,A., Emerging Infectious Diseases 16, 656-663. Chen,A., Palaniappan,K., Hauser,L., Chang,Y.J., Jeffries,C.D., Munk,C., Kiss,H., Chain,P., Han,C., Brettin,T., Detter,J.C., • Rohde,M., Graham,R.M., Branitzki-Heinemann,K., Borchers,P., Schuler,E., Göker,M., Rohde,M., Bristow,J., Eisen,J.A., Markowitz,V., Preuss,C., Schleicher,I., Zahner,D., Talay,S.R., Fulde,M., Dinkla,K., & Hugenholtz,P., Kyrpides,N.C., & Klenk,H.P. (2010) Complete genome Chhatwal,G.S.*. (2010) Differences in the aromatic domain of ho- sequence of Desulfohalobium retbaense type strain (HR(100)). Stand- mologous streptococcal fi bronectin-binding proteins trigger different ards in Genomic Siences 2, 38-48. cell invasion mechanisms and survival rates. Cellular Microbiology 13, 450-468. • Spring,S., Scheuner,C., Lapidus,A., Lucas,S., Glavina Del,R.T., Tice,H., Copeland,A., Cheng,J.F., Chen,F., Nolan,M., Saunders,E., • Saunders,E., Tindall,B.J., Fahnrich,R., Lapidus,A., Copeland,A., Del Pitluck,S., Liolios,K., Ivanova,N., Mavromatis,K., Lykidis,A., Pati,A., Rio,T.G., Lucas,S., Chen,F., Tice,H., Cheng,J.F., Han,C., Detter,J.C., Chen,A., Palaniappan,K., Land,M., Hauser,L., Chang,Y.J., Jeffries,C.D., Bruce,D., Goodwin,L., Chain,P., Pitluck,S., Pati,A., Ivanova,N., Goodwin,L., Detter,J.C., Brettin,T., Rohde,M., Göker,M., Woyke,T., Mavromatis,K., Chen,A., Palaniappan,K., Land,M., Hauser,L., Bristow,J., Eisen,J.A., Markowitz,V., Hugenholtz,P., Kyrpides,N.C., Chang,Y.J., Jeffries,C.D., Brettin,T., Rohde,M., Göker,M., Bristow,J., & Klenk,H.P. (2010) The Genome Sequence of Methanohalophilus Eisen,J.A., Markowitz,V., Hugenholtz,P., Klenk,H.P., & Kyrpides,N.C. mahii SLP Reveals Differences in the Energy Metabolism among (2010) Complete genome sequence of Haloterrigena turkmenica type Members of the Methanosarcinaceae Inhabiting Freshwater and strain (4k). Standards in Genomic Sciences 2, 107-116. Saline Environments. Archaea 2010, 690737. 158 SCIENTIFIC REPORTS | Publications

• Sun,H., Spring,S., Lapidus,A., Davenport,K., Del Rio,T.G., Tice,H., • Bergmann,R., Dinkla,K., Nitsche-Schmitz,D.P., Graham,R.M., Nolan,M., Copeland,A., Cheng,J.F., Lucas,S., Tapia,R., Goodwin,L., Luttge,M., Sanderson-Smith,M.L., Nerlich,A., Rohde,M., & Pitluck,S., Ivanova,N., Pagani,I., Mavromatis,K., Ovchinnikova,G., Chhatwal,G.S.*. (2011) Biological functions of GCS3, a novel plas- Pati,A., Chen,A., Palaniappan,K., Hauser,L., Chang,Y.J., Jeffries,C.D., minogen-binding protein of Streptococcus dysgalactiae ssp. equisi- Detter,J.C., Han,C., Rohde,M., Brambilla,E., Göker,M., Woyke,T., milis. International Journal of Medical Microbiology 301, 157-164. Bristow,J., Eisen,J.A., Markowitz,V., Hugenholtz,P., Kyrpides,N.C., Klenk,H.P., & Land,M. (2010) Complete genome sequence of Desul- • Fulde,M., Rohde,M., Hitzmann,A., Preissner,K.T., Nitsche- farculus baarsii type strain (2st14). Standards in Genomic Sciences 3, Schmitz,D.P., Nerlich,A., Chhatwal,G.S.*., & Bergmann,S. (2011) 276-284. SCM, a novel M-like protein from Streptococcus canis, binds (mini)- plasminogen with high affi nity and facilitates bacterial transmigra- • Sun,H., Lapidus,A., Nolan,M., Lucas,S., Del Rio,T.G., Tice,H., tion. Biochemical Journal 434, 523-535. Cheng,J.F., Tapia,R., Han,C., Goodwin,L., Pitluck,S., Pagani,I., Ivanova,N., Mavromatis,K., Mikhailova,N., Pati,A., Chen,A., • Härtel,T., Klein,M., Koedel,U., Rohde,M., Petruschka,L., & Palaniappan,K., Land,M., Hauser,L., Chang,Y.J., Jeffries,C.D., Hammerschmidt,S. (2011) Impact of glutamine transporters on pneu- Djao,O.D., Rohde,M., Sikorski,J., Göker,M., Woyke,T., Bristow,J., mococcal fi tness under infection-related conditions. Infection and Eisen,J.A., Markowitz,V., Hugenholtz,P., Kyrpides,N.C., & Klenk,H.P. Immunity 79, 44-58. (2010) Complete genome sequence of Nocardiopsis dassonvillei type strain (IMRU 509). Standards in Genomic Sciences 3, 325-336. • Lyszkiewicz,M., Zietara,N., Rohde,M., Gekara,N.O., Jablonska,J., Dittmar,K.E.*., & Weiss,S. (2011) SIGN-R1+MHC II+ cells of the • Tegtmeyer,N., Hartig,R., Delahay,R.M., Rohde,M., Brandt,S., splenic marginal zone--a novel type of resident dendritic cells. Conradi,J., Takahashi,S., Smolka,A.J., Sewald,N., & Backert,S. (2010) Journal of Leukocyte Biology 89, 607-615. A small fi bronectin-mimicking protein from bacteria induces cell spreading and focal adhesion formation. Journal of Biological • Gupta,A.K., Dharne,M.S., Rangrez,A.Y., Verma,P., Ghate,H.V., Chemistry 285, 23515-23526. Rohde,M., Patole,M.S., & Shouche,Y.S. (2010) Ignatzschineria indica sp. nov. and Ignatzschineria ureaclastica sp. nov., isolated from adult • Tice,H., Mayilraj,S., Sims,D., Lapidus,A., Nolan,M., Lucas,S., Glavina fl esh fl y (Diptera: Sarcophagidae). International Journal of Systematic Del,R.T., Copeland,A., Cheng,J.F., Meincke,L., Bruce,D., Goodwin,L., and Evolutionary Microbiology, in press. Pitluck,S., Ivanova,N., Mavromatis,K., Ovchinnikova,G., Pati,A., Chen,A., Palaniappan,K., Land,M., Hauser,L., Chang,Y.J., Jeffries,C.D., • von Jan,M., Riegger,N., Potter,G., Schumann,P., Verbarg,S., Sproer,C., Detter,J.C., Brettin,T., Rohde,M., Goker,M., Bristow,J., Eisen,J.A., Rohde,M., Lauer,B., Labeda,D.P., & Klenk,H.P. (2010) Kroppenstedtia Markowitz,V., Hugenholtz,P., Kyrpides,N.C., Klenk,H.P., & Chen,F. eburnea gen. nov., sp. nov., a novel thermoactinomycete isolated by (2010) Complete genome sequence of Nakamurella multipartita type environmental screening, and emended description of the family strain (Y-104). Standards in Genomic Sciences 2, 168-175. Thermoactinomycetaceae Matsuo et al. 2006 emend. Yassin et al. 2009. International Journal of Systematic and Evolutionary Micro- • Tindall,B.J., Sikorski,J., Lucas,S., Goltsman,E., Copeland,A., Glavina biology, in press. Del,R.T., Nolan,M., Tice,H., Cheng,J.F., Han,C., Pitluck,S., Liolios,K., Ivanova,N., Mavromatis,K., Ovchinnikova,G., Pati,A., Fahnrich,R., • Abel,J., Goldmann,O., Ziegler,C., Höltje,C., Smeltzer,M.S., Goodwin,L., Chen,A., Palaniappan,K., Land,M., Hauser,L., Chang,Y.J., Cheung,A.L., Bruhn,D., Rohde,M., & Medina,E. (2011) Staphylococcus Jeffries,C.D., Rohde,M., Göker,M., Woyke,T., Bristow,J., Eisen,J.A., aureus evades the extracellular antimicrobial activity of mast cells Markowitz,V., Hugenholtz,P., Kyrpides,N.C., Klenk,H.P., & Lapidus,A. by promoting its own uptake. Journal of Innate Immunity, in press. (2010) Complete genome sequence of Meiothermus ruber type strain (21). Standards in Genomic Sciences 3, 26-36. • Hebecker,S., Arendt,W., Heinemann,I.U., Tiefenau,J.H., Nimtz,M., Rohde,M., Soll,D., & Moser,J. (2011) Alanyl-phosphatidylglycerol syn- • Traverso,F.R., Bohr,U.R.M., Oyarzabal,O.A., Rohde,M., Clarici,A., thase: mechanism of substrate recognition during tRNA-dependent Wex,T., Kuester,D., Malfertheiner,P., Fox,J.G., & Backert,S. (2010) Mor- lipid modifi cation in Pseudomonas aeruginosa. Molecular Micro- phologic, genetic, and biochemical characterization of Helicobacter biology, in press. magdeburgensis, a novel species isolated from the intestine of labora- tory mice. Helicobacter 15, 403-415. • Willenborg,J., Fulde,M., de,G.A., Rohde,M., Smith,H.E., Valentin- Weigand,P., & Goethe,R. (2011) Role of glucose and CcpA in capsule • von Jan,M., Lapidus,A., Del Rio,T.G., Copeland,A., Tice,H., Cheng,J.F., expression and virulence of Streptococcus suis. Microbiology, in press. Lucas,S., Chen,F., Nolan,M., Goodwin,L., Han,C., Pitluck,S., Liolios,K., Ivanova,N., Mavromatis,K., Ovchinnikova,G., Chertkov,O., Pati,A., Chen,A., Palaniappan,K., Land,M., Hauser,L., Chang,Y.J., Jeffries,C.D., Saunders,E., Brettin,T., Detter,J.C., Chain,P., Eichinger,K., Huber,H., RG Infection Immunology | Priv.-Doz. Dr. Eva Medina Spring,S., Rohde,M., Göker,M., Wirth,R., Woyke,T., Bristow,J., Eisen,J.A., Markowitz,V., Hugenholtz,P., Kyrpides,N.C., & Klenk,H.P. • Goldmann,O., Lehne,S., & Medina,E. (2010) Age-related susceptibil- (2010) Complete genome sequence of Archaeoglobus profundus type ity to Streptococcus pyogenes infection in mice: underlying immune strain (AV18). Standards in Genomic Sciences 2, 327-346. dysfunction and strategy to enhance immunity. Journal of Pathology 220, 521-529. • Wirth,R., Sikorski,J., Brambilla,E., Misra,M., Lapidus,A., Copeland,A., Nolan,M., Lucas,S., Chen,F., Tice,H., Cheng,J.F., Han,C., Detter,J.C., • Goldmann,O., Hertzen,E., Hecht,A., Schmidt,H., Lehne,S., Norrby- Tapia,R., Bruce,D., Goodwin,L., Pitluck,S., Pati,A., Anderson,I., Teglund,A., & Medina,E. (2010) Inducible cyclooxygenase released Ivanova,N., Mavromatis,K., Mikhailova,N., Chen,A., Palaniappan,K., prostaglandin E2 modulates the severity of infection caused by Bilek,Y., Hader,T., Land,M., Hauser,L., Chang,Y.J., Jeffries,C.D., Streptococcus pyogenes. Journal of Immunology 185, 2372-2381. Tindall,B.J., Rohde,M., Göker,M., Bristow,J., Eisen,J.A., Markowitz,V., Hugenholtz,P., Kyrpides,N.C., & Klenk,H.P. (2010) Complete genome • Loof,T.G., Goldmann,O., Gessner,A., Herwald,H., & Medina,E. (2010) sequence of Thermocrinis albus type strain (HI 11/12). Standards in Aberrant infl ammatory response to Streptococcus pyogenes in mice Genomic Sciences 2, 194-202. lacking myeloid differentiation factor 88. American Journal of Pathol- ogy 176, 754-763. • Yasawong,M., Teshima,H., Lapidus,A., Nolan,M., Lucas,S., Glavina Del,R.T., Tice,H., Cheng,J.F., Bruce,D., Detter,C., Tapia,R., Han,C., • Medina,E. & Goldmann,O. (2011) In vivo and ex vivo protocols for Goodwin,L., Pitluck,S., Liolios,K., Ivanova,N., Mavromatis,K., measuring the killing of extracellular pathogens by macrophages. Mikhailova,N., Pati,A., Chen,A., Palaniappan,K., Land,M., Hauser,L., Current Protocols in Immunology Chapter 14, Unit14.19. Chang,Y.J., Jeffries,C.D., Rohde,M., Sikorski,J., Pukall,R., Göker,M., Woyke,T., Bristow,J., Eisen,J.A., Markowitz,V., Hugenholtz,P., • Medina,E. (2010) Murine model of cutaneous infection with Strepto- Kyrpides,N.C., & Klenk,H.P. (2010) Complete genome sequence of coccus pyogenes. Methods in Molecular Biology 602, 395-403. Arcanobacterium haemolyticum type strain (11018). Standards in Genomic Sciences 3, 126-135. • Medina,E. (2010) Murine model of pneumococcal pneumonia. Meth- ods in Molecular Biology 602, 405-410. SCIENTIFIC REPORTS | Publications 159

• Medina,E. (2010) Murine model of polymicrobial septic peritonitis • Vilchez,R., Gomez-Silvan,C., Purswani,J., Gonzalez-Lopez,J., & using cecal ligation and puncture (CLP). Methods in Molecular Biol- Rodelas,B. (2011) Characterization of bacterial communities exposed ogy 602, 411-415. to Cr(III) and Pb(II) in submerged fi xed-bed biofi lms for groundwater treatment. Ecotoxicology, in press. • Nippe,N., Varga,G., Holzinger,D., Loffl er,B., Medina,E., Becker,K., Roth,J., Ehrchen,J.M., & Sunderkotter,C. (2010) Subcutaneous infec- tion with S. aureus in mice reveals association of resistance with infl ux of neutrophils and Th2 response. Journal of Investigative Der- Dept. of Vaccinology and Applied Microbiology | matology 131, 125-132. Prof. Dr. Dr. Carlos Guzmán

• Tuchscherr,L., Medina,E., Hussain,M., Volker,W., Heitmann,V., • Becker,P.D., Royo,J.L., & Guzmán,C.A.* (2010) Exploitation of Niemann,S., Holzinger,D., Roth,J., Proctor,R.A., Becker,K., Peters,G., prokaryotic expression systems based on the salicylate-dependent & Loffl er,B. (2011) Staphylococcus aureus phenotype switching: an control circuit encompassing nahRPsal::xylS2 for biotechnological effective bacterial strategy to escape host immune response and applications. Bioengineered Bugs 1, 246-253. establish a chronic infection. EMBO Molecular Medicine 3, 129-141. • Becker,P.D., Legrand,N., van Geelen,C.M., Noerder,M., • Medina,E. (2011) Novel experimental models for dissecting genetic Huntington,N.D., Lim,A., Yasuda,E., Diehl,S.A., Scheeren,F.A., Ott,M., susceptibility of superantigen-mediated diseases. In: Superanti- Weijer,K., Wedemeyer,H., Di Santo,J.P., Beaumont,T., Guzmán,C.A.*, gens: Molecular Basis for Their Role in Human Disease (Kotb,M. & & Spits,H. (2010) Generation of human antigen-specifi c monoclonal Frase,J.D., eds.), ASM Press, Washington, D.C., in press. IgM antibodies using vaccinated “human immune system” mice. PLoS ONE 5. • Abel,J., Goldmann,O., Ziegler,C., Höltje,C., Smeltzer,M.S., Cheung,A.L., Bruhn,D., Rohde,M., & Medina,E. (2011) Staphylococcus • Bjorkstrom,N.K., Riese,P., Heuts,F., Andersson,S., Fauriat,C., aureus evades the extracellular antimicrobial activity of mast cells Ivarsson,M.A., Bjorklund,A.T., Flodstrom-Tullberg,M., Michaelsson,J., by promoting its own uptake. Journal of Innate Immunity, in press. Rottenberg,M.E., Guzmán,C.A.*, Ljunggren,H.G., & Malmberg,K.J. (2010) Expression patterns of NKG2A, KIR, and CD57 defi ne a proc- ess of CD56dim NK-cell differentiation uncoupled from NK-cell education. Blood 116, 3853-3864. RG Microbial Interactions and Processes | Prof. Dr. Dietmar Pieper • Caro-Quintero,A., Deng,J., Auchtung,J., Brettar,I., Höfl e,M.G.*, Klappenbach,J., & Konstantinidis,K.T. (2010) Unprecedented levels • Gomes,N.C., Flocco,C.G., Costa,R., Junca,H., Vilchez,R., Pieper,D.H.*., of horizontal gene transfer among spatially co-occurring Shewanella Krogerrecklenfort,E., Paranhos,R., Mendonca-Hagler,L.C., & Smalla,K. bacteria from the Baltic Sea. ISME Journal 5, 140. (2010) Mangrove microniches determine the structural and function- al diversity of enriched petroleum hydrocarbon-degrading consortia. • Cazorla,S.I., Frank,F.M., Becker,P.D., Arnaiz,M., Mirkin,G.A., FEMS Microbiology Ecology 74, 276-290. Corral,R.S., Guzmán,C.A.*, & Malchiodi,E.L. (2010) Redirection of the immune response to the functional catalytic domain of the cystein • Marin,M., Perez-Pantoja,D., Donoso,R., Wray,V., Gonzalez,B., & proteinase cruzipain improves protective immunity against Trypano- Pieper,D.H.*. (2010) Modifi ed 3-oxoadipate pathway for the biodegra- soma cruzi infection. Journal of Infectious Diseases 202, 136-144. dation of methylaromatics in Pseudomonas reinekei MT1. Journal of Bacteriology 192, 1543-1552. • Chen,H.L., Lünsdorf,H., Hecht,H.J., & Tsai,H. (2010) Porcine pulmo- nary angiotensin I-converting enzyme-biochemical characterization • Vilchez-Vargas,R., Junca,H., & Pieper,D.H.*. (2010) Metabolic net- and spatial arrangement of the N- and C-domains by three-dimen- works, microbial ecology and ‘omics’ technologies: towards under- sional electron microscopic reconstruction. Micron 41, 674-685. standing in situ biodegradation processes. Environental Microbiology 12, 3089-3104. • Ebensen,T., Libanova,R., & Guzman,C.A.* (2010) Bis-(3’,5’)-cyclic di-GMP: promising adjuvant for vaccine design. In The Second Mes- • Wos-Oxley,M.L., Plumeier,I., von,E.C., Taudien,S., Platzer,M., Vilchez- senger Cyclic di-GMP (Wolf,A.J. & Visick,K.L.I., eds), pp. 311-319. Vargas,R., Becker,K., & Pieper,D.H.*. (2010) A poke into the diversity ASM Press, Washington, D.C., USA. and associations within human anterior nare microbial communi- ties. ISME Journal 4, 839-851. • Fritzer,A., Senn,B.M., Minh,D.B., Hanner,M., Gelbmann,D., Noiges,B., Henics,T., Schulze,K., Guzmán,C.A.*, Goodacre,J., von,G.A., Nagy,E., • Junca,H. & Pieper,D.H.* (2011) Functional marker gene assays for hy- & Meinke,A.L. (2010) Novel conserved group A streptococcal proteins drocarbon degrading microbial communities - aerobic. In: Handbook identifi ed by the antigenome technology as vaccine candidates for a of Hydrocarbon Microbiology (Timmis,K.N., ed.), Springer, in press. non-M protein-based vaccine. Infection and Immunity 78, 4051-4067.

• Palleroni,N., Pieper,D.H.*, & Moore,E.R.B. (2011) Microbiology of • Guzmán,C.A.*. (2010) Research needs in vaccinology. In: Handbook hydrocarbon-degrading Pseudomonas. In: Handbook of Hydrocarbon of Hydrocarbon and Lipid Microbiology (Timmis,K.N., ed.), Springer- Microbiology (Timmis,K.N., ed.), Springer, in press. Verlag, Berlin Heidelberg, pp. 3383-3385.

• Perez-Pantoja,D., Gonzalez,B., & Pieper,D.H.* (2011) Aerobic degrada- • Guzmán,C.A.* (2010) Optimizing rational vaccine design. Microbial tion of aromatic hydrocarbons. In: Handbook of Hydrocarbon Micro- Biotechnology 2, 136-138. biology (Timmis,K.N., ed.), Springer, in press. • Hahn,M.W., Lang,E., Brandt,U., Lünsdorf,H., Wu,Q.L., & • Pieper,D.H.*., Gonzalez,B., Camara,B., Perez-Pantoja,D., & Reineke,W. Stackebrandt,E. (2010) Polynucleobacter cosmopolitanus sp. nov., free- (2011) Aerobic degradation of chloroaromatics. In: Handbook of living planktonic bacteria inhabiting freshwater lakes and rivers. Hydrocarbon Microbiology (Timmis,K.N., ed.), Springer, in press. International Journal of Systematic and Evolutionary Microbiology 60, 166-173. • Seeger,M. & Pieper,D.H.* (2011) Genetics of biphenyl biodegradation and co-metabolism of PCBs. In: Handbook of Hydrocarbon Microbiol- • Kahlisch,L., Henne,K., Draheim,J., Brettar,I., & Höfl e,M.G.* (2010) ogy (Timmis,K.N., ed.), Springer, in press. High-resolution in situ genotyping of Legionella pneumophila popula- tions in drinking water by multiple-locus variable-number tandem- • Becker,K., Mutter,W., Daniel,R., Rudack,C., Peschel,A., & Pieper,D. repeat analysis using environmental DNA. Applied and Environmen- (2011) Bakterielle Untermieter in der menschlichen Nase. GenomX- tal Microbiology 76, 6186-6195. Press, in press. • Kahlisch,L., Henne,K., Gröbe,L., Draheim,J., Höfl e,M.G.*, & Brettar,I. • Pfi ngsten-Würzburg,S., Pieper,D., Bautsch,W., & Probst-Kepper,M. (2010) Molecular analysis of the bacterial drinking water community (2011) Prevelence and molecular epidemiology of meticillin-resistant with respect to live/dead status. Water Science and Technology 61, Staphylococcus aureus in nursing home residents in northern Ger- 9-14. many. Journal of Hospital Infection, in press. 160 SCIENTIFIC REPORTS | Publications

• Knothe,S., Mutschler,V., Rochlitzer,S., Winkler,C., Ebensen,T., RG Chemical Microbiology | Dr. Wolf-Rainer Abraham Guzmán,C.A.*, Hohlfeld,J., Braun,A., & Muller,M. (2010) Local treat- ment with BPPcysMPEG reduces allergic airway infl ammation in • Abraham,W.-R., Estrela,A.B., Nikitin,D.I., Smit,J., & Vancanneyt,M. sensitized mice. Immunobiology 216, 110-117. (2010) Brevundimonas halotolerans sp. nov., Brevundimonas poindex- terae sp. nov. and Brevundimonas staleyi sp. nov., prosthecate bac- • Libanova,R., Ebensen,T., Schulze,K., Bruhn,D., Nörder,M., Yevsa,T., teria from aquatic habitats. International Journal of Systematic and Morr,M., & Guzmán,C.A.* (2010) Corrigendum to “The member of Evolutionary Microbiology 60, 1837-1843. the cyclic di-nucleotide family bis- (3’,5’)-cyclic dimeric inosine monophosphate exerts potent activity as mucosal adjuvant [Vaccine • Abraham,W.-R., Macedo,A.J., Lünsdorf,H., & Nikitin,D. (2011) Genus 28 (2010) 2249-2258]. Vaccine 28, 3625. V. Arcicella Nikitin, Strömpl, Oranskaya and Abraham. 2004, 683VP. (Arcocella Nitikin, Oranskaya, Pitryuk, Chernykh and Lysenko. 1994, • Libanova,R., Ebensen,T., Schulze,K., Bruhn,D., Norder,M., Yevsa,T., 152). In: Bergey’s Manual of Systematic Bacteriology (Garrity,G.M., & Morr,M., & Guzmán,C.A.* (2010) The member of the cyclic di-nucle- Ed.of Vol.4: Hedlund,B., Krieg,N.R., Ludwig,W., Paster,B.J., Staley,J.T., otide family bis-(3’, 5’)-cyclic dimeric inosine monophosphate exerts Ward,N., & Whitman,W.B., eds.), pp. 377-380. potent activity as mucosal adjuvant. Vaccine 28, 2249-2258. • Estrela,A.B. & Abraham,W.-R. (2010) Brevundimonas vancanneytii sp. • Nörder,M., Becker,P.D., Drexler,I., Link,C., Erfl e,V., & Guzmán,C.A.* nov., isolated from blood of a patient with endocarditis. International (2010) Modifi ed vaccinia virus Ankara exerts potent immune modu- Journal of Systematic and Evolutionary Microbiology 60, 2129-2134. latory activities in a murine model. PLoS ONE 5, e11400. • Estrela,A.B. & Abraham,W.-R. (2010) Combining biofi lm-controlling • Prajeeth,C.K., Jirmo,A.C., Krishnaswamy,J.K., Ebensen,T., compounds and antibiotics as a promising new way to control bio- Guzmán,C.A.*, Weiss,S., Constabel,H., Schmidt,R.E., & Behrens,G.M. fi lm infections. Pharmaceuticals 3, 1374. (2010) The synthetic TLR2 agonist BPPcysMPEG leads to effi cient cross-priming against co-administered and linked antigens. European • Estrela,A.B.*., Seixas,A., Teixeira,V.O., Pinto,A.F., & Termignoni,C. Journal of Immunology 40, 1272-1283. (2010) Vitellin- and hemoglobin-digesting enzymes in Rhipicephalus (Boophilus) microplus larvae and females. Comparative Biochemistry • Roming,M., Lünsdorf,H., Dittmar,K.E.*, & Feldmann,C. (2010) and Physiology - B Biochemistry and Molecular Biology 157, 326-335. ZrO(HPO(4))(1-x)(FMN)(x): quick and easy synthesis of a nanoscale luminescent biomarker. Angewandte Chemie International Edition • Jakobs-Schonwandt,D., Mathies,H., Abraham,W.-R., Pritzkow,W., 49, 632-637. Stephan,I., & Noll,M. (2010) Biodegradation of a biocide (Cu-N-cy- clohexyldiazenium dioxide) component of a wood preservative by a • Stegmann,K.A., Björkström,N.K., Liermann,H., Ciesek,S., Riese,P., defi ned soil bacterial community. Applied and Environment Microbiol- Wiegand,J., Hadem,J., Suneetha,P.V., Jaroszewicz,J., Wang,C., ogy 76, 8076-8083. Schlaphoff,V., Fytili,P., Cornberg,M., Manns,M.P., Geffers,R., Pietschmann,T., Guzmán,C.A.*, Ljunggren,H.-G., & Wedemeyer,H. • Menyailo,O.V., Abraham,W.-R., & Conrad,R. (2010) Tree species affect (2010) IFN alpha-induced TRAIL on human NK cells is associated atmospheric CH4 oxidation without community composition of soil with control of hepatitis C virus infection. Gastroenterology 138, methanotrophs. Soil Biology and Biochemistry 42, 101-107. 1885-1897. • Stiesch,M., Heuer,W., Kohorst,P., Winkel,A., Stumpp,S.N., Menzel,H., • Switalla,S., Lauenstein,L., Prenzler,F., Knothe,S., Forster,C., Pfaffenroth,C., Behrens,P., Chichkov,B., & Abraham,W.-R. (2010) Bio- Fieguth,H.G., Pfennig,O., Schaumann,F., Martin,C., Guzmán,C.A.*, fi lmbildung auf dentalen Implantaten - Biofi lm formation on dental Ebensen,T., Muller,M., Hohlfeld,J.M., Krug,N., Braun,A., & Sewald,K. implants. Biomedizinische Technik - Biomedical Engineering 55. (2010) Natural innate cytokine response to immunomodulators and adjuvants in human precision-cut lung slices. Toxicology and Applied • Heuer,W., Stiesch,M., & Abraham,W.-R. (2011) Microbial diversity Pharmacology 246, 107-115. of supra- and subgingival biofi lms on freshly colonized titanium implant abutments in the human mouth. European Journal of Clinical • Ebensen,T., Libanova,R., & Guzmán,C.A.* (2011) Infection preven- Microbiology and Infectious Diseases, in press. tion: oil- and lipid-containing products in vaccinology. In Handbook of Hydrocarbon and Lipid Microbiology (Timmis,K.N., ed), pp. 3311- • Abraham,W.-R. (2011) Megacities as sources for pathogenic bacteria 3331. Springer-Verlag, Berlin, Heidelberg. in rivers and their fate downstream. International Journal of Micro- biology, in press. • Knothe,S., Mutschler,V., Rochlitzer,S., Winkler,C., Ebensen,T., Guzmán,C.A.*, Hohlfeld,J., Braun,A., & Muller,M. (2011) Local treat- • Peres de Carvalo,M. & Abraham,W.-R. (2011) Antimicrobial second- ment with BPPcysMPEG reduces allergic airway infl ammation in ary metabolites from fungi for food safety. In: Natural Antimicrobials sensitized mice. Immunobiology 216, 110-117. for Biosafety and Quality (Rai,M., ed.), CABI, UK, in press.

• Guzmán,C.A.* (2009) Research needs in vaccinology. In: Handbook of hydrocarbon and lipid microbiology (Timmis,K.N., ed.), Springer, in press. JRG Phagosome Biology | Dr. Maximiliano Gutierrez

• Guzmán,C.A.* & Timmis,K.N.*. (2011) Towards intelligent vaccines: • Bleck,C.K., Merz,A., Gutierrez,M.G.*., Walther,P., Dubochet,J., the VAC-CHIP. In the “Crystal ball - 2011” section. Microbial Biotech- Zuber,B., & Griffi ths,G. (2010) Comparison of different methods for nology, in press. thin section EM analysis of Mycobacterium smegmatis. Journal of Microscopy 237, 23-38. • Huntington,N.D., Alves,N.L., Legrand,N., Lim,A., Strick-Marchand,H., Mention,J.J., Plet,A., Weijer,K., Jacques,Y., Becker,P.D., Guzmán,C. • Gutierrez,M.G.*. (2010) Salmonella vacuole maturation: PIKfyve A.*, Soussan,P., Kremsdorf,D., Spits,H., & Di Santo,J.P. (2011) IL-15 leads the way. Embo Journal 29, 1316-1317. transpresentation promotes both human T-cell reconstitution and T- cell-dependent antibody responses in vivo. Proceeding of the National • Wahe,A., Kasmapour,B., Schmaderer,C., Liebl,D., Sandhoff,K., Academy of Sciences USA, in press. Nykjaer,A., Griffi ths,G., & Gutierrez,M.G.*. (2010) Golgi-to-phago- some transport of acid sphingomyelinase and prosaposin is mediated • TrehanPati,N., Sukriti,S., Geffers,R., Hissar,S., Riese,P., Toepfer,T., by sortilin. Journal of Cell Science 123, 2502-2511. Buer,J., Adams,D.H., Guzmán,C.A.*, & Sarin,S.K. (2011) Acute and resolving phase of HEV infected patients and its cellular immune and global gene expression patterns. Journal of Clinical Immunology, in press.

• Zygmunt,B.M., Gröbe,L., & Guzmán,C.A.* (2011) Peritoneal cavity is dominated by IFNgamma-secreting CXCR3+ Th1 cells. PLOS ONE, in press. SCIENTIFIC REPORTS | Publications 161

JRG Immune Aging and Chronic Infections | • Holz,A., Kollmus,H., Ryge,J., Niederkofl er,V., Dias,J., Ericson,J., Dr. Dr. Luka Cicin-Sain Stoeckli,E.T., Kiehn,O. & Arnold,H.H. (2010) The transcription factors Nkx2.2 and Nkx2.9 play a novel role in fl oor plate development and • Cicin-Sain,L., Smyk-Pearson,S., Currier,N., Byrd,L., Koudelka,C., commissural axon guidance. Development 137, 4249-4260. Robinson,T., Swarbrick,G., Tackitt,S., Legasse,A., Fischer,M., Nikolich-Zugich,D., Park,B., Hobbs,T., Doane,C.J., Mori,M., • Kim,J.K., Kayali,G., Walker,D., Forrest,H.L., Ellebedy,A.H., Griffi n,Y.S., Axthelm,M.K., Lewinsohn,D.A., & Nikolich-Zugich,J. (2010) Loss of Rubrum,A., Bahgat,M.M.*., Kutkat,M.A., Ali,M.A., Aldridge,J.R., naive T cells and repertoire constriction predict poor response to vac- Negovetich,N.J., Krauss,S., Webby,R.J. & Webster,R.G. (2010) Puz- cination in old primates. Journal of Immunology 184, 6739-6745. zling ineffi ciency of H5N1 infl uenza vaccines in Egyptian poultry. Proceeding of the National Academy of Sciences of the United States of • Voss,A., Gescher,K., Hensel,A., Nacken,W.& Kerkhoff,C. (2011) Double- America 107, 11044-11049. Stranded RNA InducesMMP-9 Gene Expression in HaCaT Keratinocytes by Tumor Necrosis Factor-α. Infl amm Allergy Drug Targets, in press. • Meier,S., Mikolajczyk,R.T., Helmer,S., Akmatov,M., Steinke,B. & Krämer,A. (2010) Health status of students - Results from a multi- centre cross-sectional study in North Rhine-Westphalia, Germany [Prävalenz von Erkrankungen und Beschwerden bei Studierenden Dept. Infection Genetics | Prof. Dr. Klaus Schughart in NRW - Ergebnisse des Gesundheitssurveys NRW]. Prävention und Gesundheitsforderung 5, 257-264. • Alberts,R. & Schughart,K. (2010) QTLminer: identifying genes regu- lating quantitative traits. BMC Bioinformatics 11, 516. • Michaelson,J.J., Alberts,R., Schughart,K. & Beyer,A. (2010) Data-driv- en assessment of eQTL mapping methods. BMC GENOMICS 11, 502. • Alberts,R., Srivastava,B., Wu,H., Viegas,N., Geffers,R., Klawonn,F., Novoselova,N., Zaverucha,d., V, Panthier,J.J. & Schughart,K. (2010) • Moller,J., Girschick,H.J., Hahn,G. & Pessler,F. (2010) [Steroid-induced Gene expression changes in the host response between resistant spinal epidural lipomatosis in pediatric patients]. Zeitschrift Rheuma- and susceptible inbred mouse strains after infl uenza A infection. tologie 69, 447-449. Microbes and Infection 12, 309-318. • Ogdie,A., Schumacher,H.R., Jr., Dai,L., Chen,L.X., Einhorn,E. & • Bertram,S., Glowacka,I., Blazejewska,P., Soilleux,E., Allen,P., Pessler,F. (2010) Synovial biopsy fi ndings in arthritis associated with Danisch,S., Steffen,I., Choi,S.Y., Park,Y., Schneider,H., Schughart,K. & hepatitis C virus infection. Journal of Rheumatology 37, 1361-1363. Pohlmann,S. (2010) TMPRSS2 and TMPRSS4 facilitate trypsin-inde- pendent spread of infl uenza virus in Caco-2 cells. Journal of Virology • Ogdie,A., Li,J., Dai,L., Paessler,M.E., Yu,X., az-Torne,C., Akmatov,M., 84, 10016-10025. Schumacher,H.R. & Pessler,F. (2010) Identifi cation of broadly dis- criminatory tissue biomarkers of synovitis with binary and multi- • Bruck,N., Gahr,M. & Pessler,F. (2010) Transient oligoarthritis of the category receiver operating characteristic analysis. Biomarkers 15, lower extremity following infl uenza B virus infection: Case report. 183-190. Pediatric Rheumatology 8, 4. • Schofi eld,P.N., Eppig,J., Huala,E., De Angelis,M.H., Harvey,M., • Bucher,K., Dietz,K., Lackner,P., Pasche,B., Fendel,R., Mordmuller,B. Davidson,D., Weaver,T., Brown,S., Smedley,D., Rosenthal,N., Ben-Smith,A., & Hoffmann,W.H. (2010) Schistosoma co-infection Schughart,K., Aidinis,V., Tocchini-Valentini,G. & Hancock,J.M. (2010) protects against brain pathology but does not prevent severe disease Research funding. Sustaining the data and bioresource commons. and death in a murine model of cerebral malaria. International Jour- Science 330, 592-593. nal for Parasitology 41, 21-31. • Schughart,K. (2010) SYSGENET: a meeting report from a new Euro- • Dai,L., Zhu,L.J., Zheng,D.H., Mo,Y.Q., Wei,X.N., Su,J.H., Pessler,F. & pean network for systems genetics. Mammalian Genome 21, 331-336. Zhang,B.Y. (2010) Elevated serum glucose-6-phosphate isomerase correlates with histological disease activity and clinical improve- • Slansky,E., Li,J., Haupl,T., Morawietz,L., Krenn,V. & Pessler,F. (2010) ment after initiation of therapy in patients with rheumatoid arthritis. Quantitative determination of the diagnostic accuracy of the syno- Journal of Rheumatology 37, 2452-2461. vitis score and its components. Histopathology 57, 436-443.

• do Valle,T.Z., Billecocq,A., Guillemot,L., Alberts,R., Gommet,C., • Smedley,D., Schofi eld,P., Chen,C.K., Aidinis,V., Ainali,C., Bard,J., Geffers,R., Calabrese,K., Schughart,K., Bouloy,M., Montagutelli,X., & Balling,R., Birney,E., Blake,A., Bongcam-Rudloff,E., Brookes,A.J., Panthier,J.J. (2010) A new mouse model reveals a critical role for host Cesareni,G., Chandras,C., Eppig,J., Flicek,P., Gkoutos,G., innate immunity in resistance to Rift Valley fever. Journal of Immu- Greenaway,S., Gruenberger,M., Heriche,J.K., Lyall,A., Mallon,A.M., nology 185, 6146-6156. Muddyman,D., Reisinger,F., Ringwald,M., Rosenthal,N., Schughart,K., Swertz,M., Thorisson,G.A., Zouberakis,M. & Hancock,J.M. (2010) • Durzynska,J., Blazejewska,P., Szydlowski,J. & Gozdzicka-Jozefi ak,A. Finding and sharing: new approaches to registries of databases and (2010) Detection of anti-HPV11-L1 antibodies in immune sera from services for the biomedical sciences. Database (Oxford) 2010, baq014. patients suffering from recurrent respiratory papillomatosis using ELISA. Viral Immunology 23, 415-423. • Swertz,M.A., Velde,K.J., Tesson,B.M., Scheltema,R.A., Arends,D., Vera,G., Alberts,R., Dijkstra,M., Schofi eld,P., Schughart,K., • Faller,G., Mikolajczyk,R.T., Akmatov,M.K.*., Meier,S. & Kramer,A. Hancock,J.M., Smedley,D., Wolstencroft,K., Goble,C., de Brock,E.O., (2010) Accidents in the context of study among university students- Jones,A.R., Parkinson,H.E. & Jansen,R.C. (2010) XGAP: a uniform and -a multicentre cross-sectional study in North Rhine-Westphalia, extensible data model and software platform for genotype and phe- Germany. Accidents Analysis Prevention 42, 487-491. notype experiments. Genome Biology 11, R27.

• Gruenberger,M., Alberts,R., Smedley,D., Swertz,M., Schofi eld,P. & • Wu,H., Haist,V., Baumgartner,W., & Schughart,K. (2010) Sustained Schughart,K. (2010) Towards the integration of mouse databases - viral load and late death in Rag2-/- mice after infl uenza A virus defi nition and implementation of solutions to two use-cases in mouse infection. Virology Journal 7, 172. functional genomics. BMC Research Notes 3, 16. • Zouberakis,M., Chandras,C., Swertz,M., Smedley,D., Gruenberger,M., • Hahn,P., Wegener,I., Burrells,A., Bose,J., Wolf,A., Erck,C., Butler,D., Bard,J., Schughart,K., Rosenthal,N., Hancock,J.M., Schofi eld,P.N., Schofi eld,C.J., Bottger,A. & Lengeling,A. (2010) Analysis of Jmjd6 cel- Kollias,G. & Aidinis,V. (2010) Mouse Resource Browser – a database lular localization and testing for its involvement in histone demethy- of mouse databases. Database (Oxford) 2010, baq010. lation. PLoS ONE 5, e13769. • Akmatov,M.K.*. (2011) Child abuse in 28 developing and transitional • Hauck,F., Lee-Kirsch,M.A., Aust,D., Roesler,J. & Pessler,F. (2010) Com- countries – results from the Multiple Indicator Cluster Surveys. plement C2 defi ciency disarranging innate and adaptive humoral International Journal of Epidemiology 40, 219-227. immune responses in a pediatric patient: Treatment with rituximab. Arthritis Care Res (Hoboken.) 63, 454-459. • Bahgat,M.M., Blazejewska,P. & Schughart,K. (2011) Inhibition of lung serine proteases in mice: a potentially new approach to control infl u- enza infection. Virology Journal 8, 27. 162 SCIENTIFIC REPORTS | Publications

• Blazejewska,P., Koscinski,L., Viegas,N., Anhlan,D., Ludwig,S. & • Klages,K., Mayer,C.T., Lahl,K., Loddenkemper,C., Teng,M.W., Schughart,K. (2011) Pathogenicity of different PR8 infl uenza A virus Ngiow,S.F., Smyth,M.J., Hamann,A., Hühn,J., & Sparwasser,T. (2010) variants in mice is determined by both viral and host factors. Virol- Selective depletion of Foxp3+ regulatory T cells improves effective ogy 412, 36-45. therapeutic vaccination against established melanoma. Cancer Research 70, 7788-7799. • Bruck,N., Suttorp,M., Kabus,M., Heubner,G., Gahr,M. & Pessler,F. (2011) Rapid and sustained remission of systemic juvenile idiopathic • Mayer,C.T., Floess,S., Baru,A.M., Lahl,K., Hühn,J., & Sparwasser,T. arthritis-associated macrophage activation syndrome through treat- (2011) CD8(+) Foxp3(+) T cells share developmental and phenotypic ment with anakinra and corticosteroids. Journal of Clinical Rheuma- features with classical CD4(+) Foxp3(+) regulatory T cells but lack tology 17, 23-27. potent suppressive activity. European Journal of Immunology 41, 716- 725. • Gemulla,G. & Pessler,F. (2011) Can norovirus infection lead to a postinfectious arthritis? Report of 2 possible cases. Klinische Pädi- • Menning,A., Loddenkemper,C., Westendorf,A.M., Szilagyi,B., Buer,J., atrie 223, 43-44. Siewert,C., Hamann,A., & Hühn,J. (2010) Retinoic acid-induced gut tropism improves the protective capacity of Treg in acute but not • Schneider,J., Fricke,C., Overwin,H. & Hofer,B. (2011) High level ex- in chronic gut infl ammation. European Journal of Immunology 40, pression of a recombinant amylosucrase gene and selected proper- 2539-2548. ties of the enzyme. Applied Microbiology and Biotechnology, in press. • Polansky,J.K., Schreiber,L., Thelemann,C., Ludwig,L., Kruger,M., • Akmatov,M., Mikolajczyk,R.T., Meier,S. & Krämer,A. (2011) Alcohol Baumgrass,R., Cording,S., Floess,S., Hamann,A., & Hühn,J. (2010) consumption among university students in North Rhine-Westphalia, Methylation matters: binding of Ets-1 to the demethylated Foxp3 Germany - results from a multicentre cross-sectional study. Journal gene contributes to the stabilization of Foxp3 expression in regula- of American College Health, in press. tory T cells. Journal of Molecular Medicine 88, 1029-1040.

• Alberts,R. & Schughart,K. (2011) High throughput gene expression • Toker,A. & Hühn,J. (2011) To be or not to be a Treg cell: lineage deci- analysis and the identifi cation of expression QTLs. In Gene Discov- sions controlled by epigenetic mechanisms. Science Signaling 4, e4. ery for Disease Models John Wiley & Sons, Inc., Hoboken, New Jersey, USA, in press.

• Dai,L., Wei,X.N., Zheng,D.H., Mo,Y.Q., Pessler,F. & Zhang,B.Y. (2011) RG Immunoregulation | Priv.-Doz. Dr. Dunja Bruder Effective treatment of Kimura’s disease with lefl unomide in combi- nation with glucocorticoids. Clinical Rheumatology, in press. • Bar-On,L., Birnberg,T., Lewis,K.L., Edelson,B.T., Bruder,D., Hildner,K., Buer,J., Murphy,K.M., Reizis,B., & Jung,S. (2010) CX3CR1+ CD8al- • Hauck,F., Lee-Kirsch,M.A., Aust,D., Roesler,J. & Pessler,F. (2011) pha+ dendritic cells are a steady-state population related to plas- Disarranged innate and adaptive humoral immune responses in macytoid dendritic cells. Proceedings of the National Acadamy of complement 2 defi ciency: treatment with rituximab. Arthritis Care Sciences of the United States of America 107, 14745-14750. and Research (Hoboken), in press. • Bruns,S., Kniemeyer,O., Hasenberg,M., Aimanianda,V., Nietzsche,S., • Pessler,F. & Sherry,D.D. (2011) Rheumatic Fever. In The Merck Thywissen,A., Jeron,A., Latge,J.P., Brakhage,A.A., & Gunzer,M. (2010) Manual, in press. Production of extracellular traps against Aspergillus fumigatus in vit- ro and in infected lung tissue is dependent on invading neutrophils • von Jagwitz,M., Pessler,F. & Akmatov,M. (2011) Exhaled breath con- and infl uenced by hydrophobin RodA. PLoS Pathogens 6, e1000873. densate pH as risk factor for asthma in asymptomatic children with prior wheezing. Journal of Allergy and Clinical Immunology, in press. • Liesz,A., Zhou,W., Mracskó,E., Karcher,S., Doerr,H., Schwarting,S., Sun,L., Bruder,D., Stegemann,S., Cerwenka,A., Sommer,C., Dalpke,A., & Veltkamp,R. (2011) Inhibition of lymphocyte traffi cking shields the brain against deleterious neuroinfl ammation after stroke. Brain 134, AG Systemorientierte Immunologie und Entzündungsforschung | 704-720. Prof. Dr. Ingo Schmitz • Hasenberg,M., Köhler,A., Jeron,A., Bonifatius,S., & Gunzer,M. (2011) • Brandt,S., Ellwanger,K., Beuter-Gunia,C., Schuster,M., Hausser,A., Direct observation of phagocytosis and NET formation by neutrophils Schmitz,I., & Beer-Hammer,S. (2010) SLy2 targets the nuclear in infected lungs using 2-photon microscopy. Journal of Visualized SAP30/HDAC1 complex. International Journal of Biochemistry and Experiments, in press. Cell Biology 42, 1472-1481. • Tosiek,M.J., Gruber,A.D., Bader,S., Hoymann,H.G., Mauel,S., • Emadi,B.M., Soheili,Z.S., Schmitz,I., Sameie,S., & Schulz,W.A. (2010) Tschwernig,T., Buer,J., Gereke,M., & Bruder,D. (2011) CD4+CD25+Foxp3+ Snail regulates cell survival and inhibits cellular senescence in hu- regulatory T cells are dispensable for controlling CD8+ T cell- man metastatic prostate cancer cell lines. Cell Biology and Toxicology mediated lung infl ammation. Journal of Immunology, in press. 26, 553-567.

• Smith,A.J., Dai,H., Correia,C., Takahashi,R., Lee,S.H., Schmitz,I., & Kaufmann,S.H. (2011) Noxa/Bcl-2 interactions contribute to bort- Dept. of Systems Immunology | Prof. Dr. Michael Meyer-Hermann ezomib resistance in human lymphoid cells. Journal of Biological Chemistry, in press. • Garin,A., Meyer-Hermann,M., Contie,M., Figge,M.T., Buatois,V., Gunzer,M., Toellner,K.M., Elson,G., & Kosco-Vilbois,M.H. (2010) Toll-like receptor 4 signaling by follicular dendritic cells is pivotal for germinal center onset and affi nity maturation. Immunity 33, 84-95. Abt. für Experimentelle Immunologie | Prof. Dr. Jochen Hühn • Jain,H.V. & Meyer-Hermann,M. (2010) The molecular asis of syner- • Hubert,S., Rissiek,B., Klages,K., Hühn,J., Sparwasser,T., Haag,F., Koch- gism between carboplatin and ABT-737 therapy targeting ovarian Nolte,F., Boyer,O., Seman,M., & Adriouch,S. (2010) Extracellular NAD+ carcinomas. Cancer Research 71, 705-715. shapes the Foxp3+ regulatory T cell compartment through the ART2- P2X7 pathway. Journal of Experimental Medicine 207, 2561-2568. • Kempf,H., Bleicher,M., & Meyer-Hermann,M. (2010) Spatio-temporal cell dynamics in tumour spheroid irradiation. European Physical • Humrich,J.Y., Morbach,H., Undeutsch,R., Enghard,P., Rosenberger,S., Journal D 60, 177-193. Weigert,O., Kloke,L., Heimann,J., Gaber,T., Brandenburg,S., Scheffold,A., Hühn,J., Radbruch,A., Burmester,G.R., & Riemekasten,G. • Kobayashi,Y. & Telschow,A. (2010) Cytoplasmic feminizing elements (2010) Homeostatic imbalance of regulatory and effector T cells due in a two-population model: infection dynamics, gene fl ow modifi ca- to IL-2 deprivation amplifi es murine lupus. Proceedings of the Nation- tion, and the spread of autosomal suppressors. Journal of Evolution- al Academy of Sciences of the United States of America 107, 204-209. ary Biology 23, 2558-2568. SCIENTIFIC REPORTS | Publications 163

• Meyer-Hermann,M. & Benninger,R.K. (2010) A mathematical model • Heinzelmann,K., Scholz,B.A., Nowak,A., Fossum,E., Kremmer,E., of beta-cells in an islet of Langerhans sensing a glucose gradient. Haas,J., Frank,R., & Kempkes,B. (2010) Kaposi’s sarcoma-associated HFSP Journal 4, 61-71. herpesvirus viral interferon regulatory factor 4 (vIRF4/K10) is a novel interaction partner of CSL/CBF1, the major downstream effec- • Victora,G.D., Schwickert,T.A., Fooksman,D.R., Kamphorst,A.O., Mey- tor of Notch signaling. Journal of Virology 84, 12255-12264. er-Hermann,M., Dustin,M.L., & Nussenzweig,M.C. (2010) Germinal center dynamics revealed by multiphoton microscopy with a photo- • Kubicek,K., Grimm,S.K., Orts,J., Sasse,F., & Carlomagno,T. (2010) activatable fl uorescent reporter. Cell 143, 592-605. The tubulin-bound structure of the antimitotic drug tubulysin. Ange- wandte Chemie International Edition 49, 4809-4812. • Grise,G. & Meyer-Hermann,M. (2011) Surface reconstruction using Delaunay triangulation for applications in life sciences. Computer • Meens,J., Bolotin,V., Frank,R., Bohmer,J., & Gerlach,G.F. (2010) Char- Physics Communications 182, 967-977. acterization of a highly immunogenic Mycoplasma hyopneumoniae lipoprotein Mhp366 identifi ed by peptide-spot array. Veterinary • Kobayashi,Y. & Telschow,A. (2011) The concept of effective recom- Microbiology 142, 293-302. bination rate and its application in speciation theory. Evolution 65, 617-628. • Meyer,T., Stratmann-Selke,J., Meens,J., Schirrmann,T., Gerlach,G.F., Frank,R., Dubel,S., Strutzberg-Minder,K., & Hust,M. (2010) Isolation of scFv fragments specifi c to OmpD of Salmonella typhimurium. Veterinary Microbiology 147, 162-169. Abt. für Chemische Biologie | Dr. Ronald Frank, Dr. Florenz Sasse • Micklinghoff,J.C., Schmidt,M., Geffers,R., Tegge,W., & Bange,F.C. • Andreetto,E., Yan,L.M., Tatarek-Nossol,M., Velkova,A., Frank,R., & (2010) Analysis of expression and regulatory functions of the Kapurniotu,A. (2010) Identifi cation of hot regions of the abeta-IAPP ribosome-binding protein TypA in Mycobacterium tuberculosis under interaction interface as high-affi nity binding sites in both cross- and stress conditions. Archives of Microbiology 192, 499-504. self-association. Angewandte Chemie International Edition 49, 3081- • Nickl,C.K., Raidas,S.K., Zhao,H., Sausbier,M., Ruth,P., Tegge,W., • Barnickel,B., Bayliffe,F., Diestel,R., Kempf,K., Laschat,S., Pachali,S., Brayden,J.E., & Dostmann,W.R. (2010) (d)-Amino acid analogues of Sasse,F., Schlenk,A., & Schobert,R. (2010) Structure-activity relation- DT-2 as highly selective and superior inhibitors of cGMP-dependent ships of precursors and analogs of natural 3-enoyl-tetramic acids. protein kinase Ialpha. Biochimica et Biophysica Acta 1804, 524-532. Chemistry & Biodiversity 7, 2830-2845. • Nicolas,L., Anderl,T., Sasse,F., Steinmetz,H., Jansen,R., Hofl e,G., • Beutling,U. & Frank,R. (2010) Epitope analysis using synthetic pep- Laschat,S., & Taylor,R.E. (2010) Gephyronic acid, a missing link tide repertoires prepared by SPOT synthesis technology. In: Antibody between polyketide inhibitors of eukaryotic protein synthesis (Part I): Engineering (Kontermann,R. & Dübel,S., eds.), Springer-Verlag, Structural revision and stereochemical assignment of gephyronic Berlin, Heidelberg. acid. Angewandte Chemie International Edition 50, 938-941.

• Bourbeillon,J., Orchard,S., Benhar,I., Borrebaeck,C., de,D.A., • Rink,C., Sasse,F., Zubriene,A., Matulis,D., & Maier,M.E. (2010) Prob- Dubel,S., Frank,R., Gibson,F., Gloriam,D., Haslam,N., Hiltker,T., ing the infl uence of an allylic methyl group in zearalenone analogues Humphrey-Smith,I., Hust,M., Juncker,D., Koegl,M., Konthur,Z., on binding to Hsp90. Chemistry 16, 14469-14478. Korn,B., Krobitsch,S., Muyldermans,S., Nygren,P.A., Palcy,S., Polic,B., Rodriguez,H., Sawyer,A., Schlapshy,M., Snyder,M., Stoevesandt,O., • Schackel,R., Hinkelmann,B., Sasse,F., & Kalesse,M. (2010) The syn- Taussig,M.J., Templin,M., Uhlen,M., van der,M.S., Wingren,C., thesis of novel disorazoles. Angewandte Chemie International Edition Hermjakob,H., & Sherman,D. (2010) Minimum information about a 49, 1619-1622. protein affi nity reagent (MIAPAR). Nature Biotechnology 28, 650-653. • Schlenk,A., Diestel,R., Sasse,F., & Schobert,R. (2010) A selective • Brodmann,T., Janssen,D., Sasse,F., Irschik,H., Jansen,R., Müller,R., & 3-acylation of tetramic acids and the fi rst synthesis of ravenic acid. Kalesse,M. (2010) Isolation and synthesis of chivotriene, a chivosa- Chemistry 16, 2599-2604. zole shunt product from Sorangium cellulosum. European Journal of Organic Chemistry 27, 5155-5159. • Schobert,R., Sasse,F., Biersack,B., Effenberger,K., Breyer,S., & Diestel,R. (2010) Tumor-selective amphiphilic para-quinones and • Buchner,A., Pohla,H., Willimsky,G., Frankenberger,B., Frank,R., Baur- tetramic acids. International Journal of Clinical Pharmacology and Melnyk,A., Siebels,M., Stief,C.G., Hofstetter,A., Kopp,J., Pezzutto,A., Therapeutics 48, 459-461. Blankenstein,T., Oberneder,R., & Schendel,D.J. (2010) Phase 1 trial of allogeneic gene-modifi ed tumor cell vaccine RCC-26/CD80/IL-2 in • Schulz,S., Dickschat,J.S., Kunze,B., Wagner-Döbler,I., Diestel,R., & patients with metastatic renal cell carcinoma. Human Gene Therapy Sasse,F. (2010) Biological activity of volatiles from marine and terres- 21, 285-297. trial bacteria. Marine Drugs 8, 2976-2987.

• Bulow,L., Nickeleit,I., Girbig,A.K., Brodmann,T., Rentsch,A., • Sharma,R.K., Sundriyal,S., Wangoo,N., Tegge,W., & Jain,R. (2010) Eggert,U., Sasse,F., Steinmetz,H., Frank,R., Carlomagno,T., New antimicrobial hexapeptides: synthesis, antimicrobial activities, Malek,N.P., & Kalesse,M. (2010) Synthesis and biological characteri- cytotoxicity, and mechanistic studies. ChemMedChem 5, 86-95. zation of argyrin F. ChemMedChem 5, 832-836. • Voigt,J., Kiess,M., Getzlaff,R., Wostemeyer,J., & Frank,R. (2010) Gen- • Frank,R., Theis,F.J., & Klamt,S. (2010) From binary to multivalued to eration of the heterodimeric precursor GP3 of the Chlamydomonas continuous models: the lac operon as a case study. Journal of Integra- cell wall. Molecular Microbiology 77, 1512-1526. tive Bioinformatics 7, 151-169. • Capell,A., Liebscher,S., Fellerer,K., Brouwers,N., Willem,M., • Gloriam,D.E., Orchard,S., Bertinetti,D., Bjorling,E., Bongcam- Lammich,S., Gijselinck,I., Bittner,T., Carlson,A.M., Sasse,F., Kunze,B., Rudloff,E., Borrebaeck,C.A., Bourbeillon,J., Bradbury,A.R., de,D.A., Steinmetz,H., Jansen,R., Dormann,D., Sleegers,K., Cruts,M., Herms,J., Dubel,S., Frank,R., Gibson,T.J., Gold,L., Haslam,N., Herberg,F.W., Van,B.C., & Haass,C. (2011) Rescue of progranulin defi ciency associ- Hiltke,T., Hoheisel,J.D., Kerrien,S., Koegl,M., Konthur,Z., Korn,B., ated with frontotemporal lobar degeneration by alkalizing reagents Landegren,U., Montecchi-Palazzi,L., Palcy,S., Rodriguez,H., Schweins- and inhibition of vacuolar ATPase. Journal of Neuroscience 31, 1885- berg,S., Sievert,V., Stoevesandt,O., Taussig,M.J., Ueffi ng,M., Uhlen,M., 1894. van der,M.S., Wingren,C., Woollard,P., Sherman,D.J., & Hermjakob,H. (2010) A community standard format for the representation of pro- • Elsebai,M.F., Kehraus,S., Lindequist,U., Sasse,F., Shaaban,S., tein affi nity reagents. Molecular and Cellular Proteomics 9, 1-10. Gutschow,M., Josten,M., Sahl,H.G., & Konig,G.M. (2011) Antimicrobial phenalenone derivatives from the marine-derived fungus Coniothy- • Harmrolfs,K., Brunjes,M., Drager,G., Floss,H.G., Sasse,F., Taft,F., & rium cereale. Organic and Biomolecular Chemistry 9, 802-808. Kirschning,A. (2010) Cyclization of synthetic seco-proansamitocins to ansamitocin macrolactams by Actinosynnema pretiosum as bio- catalyst. ChemBioChem 11, 2517-2520. 164 SCIENTIFIC REPORTS | Publications

• Knobloch,T., Harmrolfs,K., Taft,F., Thomaszewski,B., Sasse,F., & • Bulow,L., Nickeleit,I., Girbig,A.K., Brodmann,T., Rentsch,A., Kirschning,A. (2011) Mutational biosynthesis of ansamitocin antibi- Eggert,U., Sasse,F., Steinmetz,H., Frank,R., Carlomagno,T., otics: A diversity-oriented approach to exploit biosynthetic fl exibility. Malek,N.P., & Kalesse,M. (2010) Synthesis and biological characteri- ChemBioChem 13, 540-547. zation of argyrin F. ChemMedChem 5, 832-836.

• Omnus,D.J., Mehrtens,S., Ritter,B., Resch,K., Yamada,M., Frank,R., • Kena,D.A., Noll,C., Richter,M., Gieseler,M.T., & Kalesse,M. (2010) Nourbakhsh,M., & Reboll,M.R. (2011) JKTBP1 is involved in stabiliza- Intramolecular stereoselective protonation of aldehyde-derived eno- tion and IRES-dependent translation of NRF mRNAs by binding to 5’ lates. Angewandte Chemie International Edition 49, 8367-8369. and 3’ untranslated regions. Journal of Molecular Biology 407, 492- 504. • Li,P., Li,J., Arikan,F.*., Ahlbrecht,W., Dieckmann,M., & Menche,D. (2010) Stereoselective total synthesis of etnangien and etnangien • Wunderlich,K., Juozapaitis,M., Ranadheera,C., Kessler,U., Martin,A., methyl ester. Journal of Organic Chemistry 75, 2429-2444. Eisel,J., Beutling,U., Frank,R., & Schwemmle,M. (2011) Identifi cation of high-affi nity PB1-derived peptides with enhanced affi nity to the • Menche,D., Li,P., & Irschik,H. (2010) Design, synthesis and biological PA protein of Infl uenza A virus polymerase. Antimicrobial Agents and evaluation of simplifi ed analogues of the RNA polymerase inhibitor Chemotherapy 55, 696-702. etnangien. Bioorganic and Medicinal Chemistry Letters 20, 939-941.

• Anderl,T., Nicolas,L., Munkemer,J., Baro,A., Sasse,F., Steinmetz,H., • Mohamed,I.E., Kehraus,S., Krick,A., Konig,G.M., Kelter,G., Maier,A., Jansen,R., Hofl e,G., Taylor,R.E., & Laschat,S. (2011) Gephyronic acid, Fiebig,H.H., Kalesse,M., Malek,N.P., & Gross,H. (2010) Mode of action a missing link between polyketide inhibitors of eukaryotic protein of epoxyphomalins A and B and characterization of related metabo- synthesis (Part II): Total synthesis of gephyronic acid. Angewandte lites from the marine-derived fungus Paraconiothyrium sp. Journal of Chemie International Edition, in press. Naturald Products 73, 2053-2056.

• Bremer,C.M., Sominskaya,I., Skrastina,D., Pumpens,P., Wahed,A.A., • Rand,K., Noll,C., Schiebel,H.M., Kemken,D., Dulcks,T., Kalesse,M., Beutling,U., Frank,R., Fritz,H.J., Hunsmann,G., Gerlich,W.H., & Heinz,D.W.*., & Layer,G. (2010) The oxygen-independent copropor- Glebe,D. (2011) N-terminal myristoylation-dependent masking of phyrinogen III oxidase HemN utilizes harderoporphyrinogen as a neutralizing epitopes in the preS1 attachment site of hepatitis B reaction intermediate during conversion of coproporphyrinogen III to virus. Journal of Hepatology, in press. protoporphyrinogen IX. Biological Chemistry 391, 55-63.

• Kraemer,S., Lue,H., Zernecke,A., Kapurniotu,A., Andreetto,E., • Schackel,R., Hinkelmann,B., Sasse,F., & Kalesse,M. (2010) The syn- Frank,R., Lennartz,B., Weber,C., & Bernhagen,J. (2011) MIF-chem- thesis of novel disorazoles. Angewandte Chemie International Edition okine receptor interactions in atherogenesis are dependent on an 49, 1619-1622. N-loop-based 2-site binding mechanism. FASEB Journal, in press. • Stauch,B., Simon,B., Basile,T., Schneider,G., Malek,N.P., Kalesse,M., & • Maenz,B., Goetz,V., Wunderlich,K., Eisel,J., Kirchmair,J., Stech,J., Carlomagno,T. (2010) Elucidation of the structure and intermolecular Stech,O., Chase,G., Frank,R., & Schwemmle,M. (2011) Disruption of interactions of a reversible cyclic-peptide inhibitor of the proteasome the viral polymerase complex assembly as a novel approach to at- by NMR spectroscopy and molecular modeling. Angewandte Chemie tenuate infl uenza a virus. Journal of Biological Chemistry, in press. International Edition 49, 3934-3938.

• Ritter,B., Kilian,P., Reboll,M.R., Resch,K., Distefano,J.K., Frank,R., Beil,W., & Nourbakhsh,M. (2011) Differential effects of multiplicity of infection on Helicobacter pylori-induced signaling pathways and RG Environmental Microbiology | Prof. Dr. Ken N. Timmis interleukin-8 gene transcription. Journal of Clinical Immunology, in press. • Beloqui,A., Nechitaylo,T.Y., Lopez-Cortes,N., Ghazi,A., Guazzaroni,M.E., Polaina,J., Strittmatter,A.W., Reva,O., Waliczek,A., • Schneider,J., Fricke,C., Overwin,H., & Hofer,B. (2011) High level Yakimov,M.M., Golyshina,O.V., Ferrer,M., & Golyshin,P.N. (2010) Di- expression of a recombinant amylosucrase gene and selected proper- versity of glycosyl hydrolases from cellulose-depleting communities ties of the enzyme. Applied Microbiology and Biotechnology, in press. enriched from casts of two earthworm species. Applied and Environ- mental Microbiology 76, 5934-5946. • Westermann,J., Florcken,A., Willimsky,G., van,L.A., Kopp,J., Takvorian,A., Johrens,K., Lukowsky,A., Schonemann,C., Sawitzki,B., • Beloqui,A., Polaina,J., Vieites,J.M., Reyes-Duarte,D., Torres,R., Pohla,H., Frank,R., Dorken,B., Schendel,D.J., Blankenstein,T., & Golyshina,O.V., Chernikova,T.N., Waliczek,A., Aharoni,A., Pezzutto,A. (2011) Allogeneic gene-modifi ed tumor cells (RCC-26/ Yakimov,M.M., Timmis,K.N.*., Golyshin,P.N., & Ferrer,M. (2010) IL-7/CD80) as a vaccine in patients with metastatic renal cell cancer: Novel hybrid esterase-haloacid dehalogenase enzyme. ChemBioChem a clinical phase-I study. Gene Therapy, in press. 11, 1975-1978.

• Fazzini,R.A., Preto,M.J., Quintas,A.C., Bielecka,A., Timmis,K.N.*., & dos Santos,V.A.P.M.*. (2010) Consortia modulation of the stress RG Biological Systems Analysis | Prof. Dr. Ursula Bilitewski response: proteomic analysis of single strain versus mixed culture. Environmental Microbiology 12, 2436-2449. • Klippel,N., Cui,S., Groebe,L., & Bilitewski,U. (2010) Deletion of the Candida albicans histidine kinase gene CHK1 improves recognition • Gertler,C., Näther,D.J., Gerdts,G., Malpass,M.C., & Golyshin,P.N.*. by phagocytes through an increased exposure of cell wall beta-1,3- (2010) A mesocosm study of the changes in marine fl agellate and glucans. Microbiology 156, 3432-3444. ciliate communities in a crude oil bioremediation trial. Microbial Ecology 60, 180-191.

• Martin-Arjol,I., Bassas-Galia,M.*., Bermudo,E., Garcia,F., & Dept. of Medicinal Chemistry | Prof. Dr. Markus Kalesse Manresa,A. (2010) Identifi cation of oxylipins with antifungal activity by LC-MS/MS from the supernatant of Pseudomonas 42A2. Chemis- • Brodmann,T., Janssen,D., Sasse,F., Irschik,H., Jansen,R., Müller,R., & try and Physics of Lipids 163, 341-346. Kalesse,M. (2010) Isolation and synthesis of chivotriene, a chivosa- zole shunt product from Sorangium cellulosum. European Journal of • Nechitaylo,T.Y., Yakimov,M.M., Godinho,M., Timmis,K.N.*., Organic Chemistry 27, 5155-5159. Belogolova,E., Byzov,B.A., Kurakov,A.V., Jones,D.L., & Golyshin,P.N. (2010) Effect of the earthworms Lumbricus terrestris and Aporrecto- • Brodmann,T., Janssen,D., & Kalesse,M. (2010) Total synthesis of dea caliginosa on bacterial diversity in soil. Microbial Ecology 59, chivosazole F. Journal of the American Chemical Society 132, 13610- 574-587. 13611. SCIENTIFIC REPORTS | Publications 165

• Nechitaylo,T.Y., Timmis,K.N.*., Byzov,B.A., Kurakov,A.V., Jones,D.L., • Varbiro,S., Biro,A., Cervenak,J., Cervenak,L., Singh,M., Banhidy,F., Ferrer,M., Belogolova,E., & Golyshin,P.N. (2010) Fate of prions in soil: Sebestyen,A., Fust,G., & Prohaszka,Z. (2010) Human anti-60 kD heat Degradation of recombinant prion in aqueous extracts from soil and shock protein autoantibodies are characterized by basic features of casts of two earthworm species. Soil Biology and Biochemistry 42, natural autoantibodies. Acta Physiologica Hungarica 97, 1-10. 116 8 -1171. • von Groll,A., Martin,A., Stehr,M., Singh,M., Portaels,F., da Silva,P.E., • Oxley,A.P., Lanfranconi,M.P., Wurdemann,D., Ott,S., Schreiber,S., & Palomino,J.C. (2010) Fitness of Mycobacterium tuberculosis strains McGenity,T.J., Timmis,K.N.*., & Nogales,B. (2010) Halophilic archaea of the W-Beijing and Non-W-Beijing genotype. PLoS ONE 5, e10191. in the human intestinal mucosa. Environmental Microbiology 12, 2398-2410. • Adhikary,T., Kaddatz,K., Finkernagel,F., Schonbauer,A., Meissner,W., Scharfe,M., Jarek,M., Blöcker,H., Muller-Brusselbach,S., & Müller,R. • Timmis,K.N.*. (2010) Human biome biotechnology and the personali- (2011) Genomewide analyses defi ne different modes of transcrip- zation of odour profi les. Microbial Biotechnology 2, 150-152. tional regulation by peroxisome proliferator-activated receptor-beta/ delta (PPARbeta/delta). PLoS ONE 6, e16344. • Wiethaus,J., Busch,A.W., Dammeyer,T., & Frankenberg-Dinkel,N. (2010) Phycobiliproteins in Prochlorococcus marinus: biosynthesis of pigments and their assembly into proteins. European Journal of Cell Biology 89, 1005-1010. Former Dept. of Cell Biology | Prof. Dr. Jürgen Wehland

• Ferrer,M., Beloqui,A., Zumárraga,M., Alcalde,M., & Golyshin,P.N.* • Alberts,R., Srivastava,B., Wu,H., Viegas,N., Geffers,R., Klawonn,F., (2011) Microbes and enzymes: recent trends and new directions to Novoselova,N., Zaverucha,d., V, Panthier,J.J., & Schughart,K. (2010) explore protein diversity space. In: Protein Engineering Handbook Gene expression changes in the host response between resistant (Bornscheuer,U., ed.), Wiley, in press and susceptible inbred mouse strains after infl uenza A infection. Microbes and Infection 12, 309-318. • Gertler,C., Gerdts,G., Yakimov,M.M., Timmis,K.N.*., & Golyshin,P.N.*. (2011) Populations of heavy fuel oil-degrading marine microbial com- • Bruns,S., Kniemeyer,O., Hasenberg,M., Aimanianda,V., Nietzsche,S., munity on oil-degrading marine microbial community on oil sorbent Thywissen,A., Jeron,A., Latge,J.P., Brakhage,A.A., & Gunzer,M. (2010) material surface. Journal of Applied Microbiology, in press. Production of extracellular traps against Aspergillus fumigatus in vit- ro and in infected lung tissue is dependent on invading neutrophils • Guzmán,C.A.*. & Timmis,K.N.* (2011) Towards intelligent vaccines: and infl uenced by hydrophobin RodA. PLoS Pathogens 6, e1000873. the VAC-CHIP. In the “Crystal ball - 2011” section. Microbial Biotech- nology, in press • Christgen,M., Geffers,R., Ballmaier,M., Christgen,H., Poczkaj,J., Krech,T., Kreipe,H., & Lehmann,U. (2010) Down-regulation of the • Sabirova,J.S., Haddouche,R., Van Bogaert,I.N., Mulaa,F., Verstraete,W., fetal stem cell factor SOX17 by H33342: a mechanism responsible for Timmis,K.N.*, Schmid-Dannert,C., Nicaud,J.M., & Soetaert,W. (2011) differential gene expression in breast cancer side population cells. The Lipo Yeast project: using the oleaginous yeast Yarrowia lipolytica Journal of Biological Chemistry 285, 6412-6418. in combination with specifi c bacterial genes for the bioconversion of lipids, fats and oils into high-value products. Microbial Biotechnology, • Dietrich,N., Rohde,M., Geffers,R., Kroger,A., Hauser,H., Weiss,S., & in press. Gekara,N.O. (2010) Mast cells elicit proinfl ammatory but not type I interferon responses upon activation of TLRs by bacteria. Proceeding of the National Academy of Sciences of the United States of America 107, 8748-8753. Dept. Genome Analysis | Dr. Helmut Blöcker • do Valle,T.Z., Billecocq,A., Guillemot,L., Alberts,R., Gommet,C., • Buntin,K., Irschik,H., Weissman,K.J., Luxenburger,E., Blöcker,H., & Geffers,R., Calabrese,K., Schughart,K., Bouloy,M., Montagutelli,X., & Müller,R. (2010) Biosynthesis of thuggacins in myxobacteria: com- Panthier,J.J. (2010) A new mouse model reveals a critical role for host parative cluster analysis reveals basis for natural product structural innate immunity in resistance to Rift Valley fever. Journal of Immu- diversity. Chemistry and Biology 17, 342-356. nology 185, 6146-6156.

• Danilowicz,E., Martinez-Arias,R., Dolf,G., Singh,M., Probst,I., • Fleissner,D., Hansen,W., Geffers,R., Buer,J., & Westendorf,A.M. (2010) Tummler,B., Holtig,D., Waldmann,K.H., Gerlach,G.F., Stanke,F., & Local induction of immunosuppressive CD8+ T cells in the gut-asso- Leeb,T. (2010) Characterization of the porcine transferrin gene (TF) ciated lymphoid tissues. PLoS ONE 5, e15373. and its association with disease severity following an experimental Actinobacillus pleuropneumoniae infection. Animal Genetics 41, 424- • Garin,A., Meyer-Hermann,M., Contie,M., Figge,M.T., Buatois,V., 427. Gunzer,M., Toellner,K.M., Elson,G., & Kosco-Vilbois,M.H. (2010) Toll-like receptor 4 signaling by follicular dendritic cells is pivotal for • Deyneko,I.V., Kalybaeva,Y.M., Kel,A.E., & Blöcker,H. (2010) Human- germinal center onset and affi nity maturation. Immunity 33, 84-95. chimpanzee promoter comparisons: Property-conserved evolution? Genomics 96, 129-133. • Garritsen,H.S., Macke,L., Meyring,W., Hannig,H., Pagelow,U., Wormann,B., Geffers,R., Dittmar,K.E.*., & Lindenmaier,W. (2010) • Dötsch,A., Klawonn,F., Jarek,M., Scharfe,M., Blöcker,H., & Häußler,S. Effi cient generation of clinical-grade genetically modifi ed dendritic (2010) Evolutionary conservation of essential and highly expressed cells for presentation of multiple tumor-associated proteins. Transfu- genes in Pseudomonas aeruginosa. BMC GENOMICS 11, 234. sion 50, 831-842.

• Hu,Y., van der,G.R., Besra,G.S., Gurcha,S.S., Liu,A., Rohde,M., • Gekara,N.O., Zietara,N., Geffers,R., & Weiss,S. (2010) Listeria mono- Singh,M., & Coates,A. (2010) 3-Ketosteroid 9alpha-hydroxylase is an cytogenes induces T cell receptor unresponsiveness through pore- essential factor in the pathogenesis of Mycobacterium tuberculosis. forming toxin listeriolysin O. Journal of Infectious Diseases 202, Molecular Microbiology 75, 107-121. 1698-1707.

• Lin,L., Flisikowski,K., Schwarzenbacher,H., Scharfe,M., Severitt,S., • Hansen,W., Westendorf,A.M., Toepfer,T., Mauel,S., Geffers,R., Blöcker,H., & Fries,R. (2010) Characterization of the procine AMPK Gruber,A.D., & Buer,J. (2010) Infl ammation in vivo is modulated by alpha 2 catalytic subunitgene (PRKAA2): genome structure, polymor- GPR83 isoform-4 but not GPR83 isoform-1 expression in regulatory phism detection and association study. Animal Genetics 41(2), 203- T cells. Genes and Immunity 11, 357-361. 207 • Kahlisch,L., Henne,K., Gröbe,L., Draheim,J., Höfl e,M.G.*., & Brettar,I. • Scholler,J., Singh,M., Bergmeier,L., Brunstedt,K., Wang,Y., Whittall,T., (2010) Molecular analysis of the bacterial drinking water community Rahman,D., Pido-Lopez,J., & Lehner,T. (2010) A recombinant human with respect to live/dead status. Water Science and Technology 61, HLA-class I antigen linked to dextran elicits innate and adaptive im- 9-14. mune responses. Journal of Immunological Methods 360, 1-9. 166 SCIENTIFIC REPORTS | Publications

• Lefever,T., Pedersen,E., Basse,A., Paus,R., Quondamatteo,F., • Kusch,K., Hanke,K., Holtfreter,S., Schmudde,M., Kohler,C., Erck,C., Stanley,A.C., Langbein,L., Wu,X., Wehland,J., Lommel,S., & Wehland,J., Hecker,M., Ohlsen,K., Bröker,B. & Engelmann,S. (2011) Brakebusch,C. (2010) N-WASP is a novel regulator of hair-follicle The infl uence of SaeRS and σB on the expression of superantigens cycling that controls antiproliferative TGF{beta} pathways. Journal of in different Staphylococcus aureus isolates. International Journal of Cell Science 123, 128-140. Microbiology, in press

• Leonhardt,J., Kuebler,J.F., Turowski,C., Tschernig,T., Geffers,R., & • TrehanPati,N., Sukriti,S., Geffers,R., Hissar,S., Riese,P., Toepfer,T., Petersen,C. (2010) Susceptibility to experimental biliary atresia Buer,J., Adams,D.H., Guzmán,C.A.*, & Sarin,S.K. (2011) Acute and linked to different hepatic gene expression profi les in two mouse resolving phase of HEV infected patients and its cellular immune strains. Hepatology Research 40, 196-203. and global gene expression patterns. Journal of Clinical Immunology, in press • Macke,L., Garritsen,H.S., Meyring,W., Hannig,H., Pagelow,U., Wormann,B., Piechaczek,C., Geffers,R., Rohde,M., Lindenmaier,W., & Dittmar,K.E.*. (2010) Evaluating maturation and genetic modifi ca- tion of human dendritic cells in a new polyolefi n cell culture bag Former RG Cytoskeleton Dynamics | Prof. Dr. Klemens Rottner system. Transfusion 50, 843-855. • Hanisch,J., Ehinger,J., Ladwein,M., Rohde,M., Derivery,E., Bosse,T., • Micklinghoff,J.C., Schmidt,M., Geffers,R., Tegge,W., & Bange,F.C. Steffen,A., Bumann,D., Misselwitz,B., Hardt,W.D., Gautreau,A., (2010) Analysis of expression and regulatory functions of the Stradal,T.B.*., & Rottner,K. (2010) Molecular dissection of Salmonel- ribosome-binding protein TypA in Mycobacterium tuberculosis under la-induced membrane ruffl ing versus invasion. Cellular Microbiology stress conditions. Archives of Microbiology 192, 499-504. 12, 84-98

• Pils,M.C., Pisano,F., Fasnacht,N., Heinrich,J.M., Gröbe,L., • Hertzog,M., Milanesi,F., Hazelwood,L., Disanza,A., Liu,H., Perlade,E., Schippers,A., Rozell,B., Jack,R.S., & Muller,W. (2010) Monocytes/ Malabarba,M.G., Pasqualato,S., Maiolica,A., Confalonieri,S., Le,C.C., macrophages and/or neutrophils are the target of IL-10 in the LPS Offenhauser,N., Block,J., Rottner,K., Di Fiore,P.P., Carlier,M.F., endotoxemia model. European Journal of Immunology 40, 443-448. Volkmann,N., Hanein,D., & Scita,G. (2010) Molecular basis for the dual function of Eps8 on actin dynamics: bundling and capping. • Quiel,A., Jurgen,B., Piechotta,G., Le Foll,A.P., Ziebandt,A.K., Kohler,C., PLoS Biology 8, e1000387. Koster,D., Engelmann,S., Erck,C., Hintsche,R., Wehland,J., Hecker,M., & Schweder,T. (2010) Electrical protein array chips for the detection • Klink,B.U., Barden,S., Heidler,T.V., Borchers,C., Ladwein,M., of staphylococcal virulence factors. Applied Microbiology and Biotech- Stradal,T.B.*., Rottner,K., & Heinz,D.W.*. (2010) Structure of Shigella nology 85, 1619-1627. IpgB2 in complex with human RhoA: implications for the mechanism of bacterial guanine nucleotide exchange factor mimicry. Journal of • Stegmann,K.A., Björkström,N.K., Liermann,H., Ciesek,S., Riese,P., Biological Chemistry 285, 17197-17208. Wiegand,J., Hadem,J., Suneetha,P.V., Jaroszewicz,J., Wang,C., Schlaphoff,V., Fytili,P., Cornberg,M., Manns,M.P., Geffers,R., • Marg,S., Winkler,U., Sestu,M., Himmel,M., Schonherr,M., Bar,J., Pietschmann,T., Guzmán,C.A.*., Ljunggren,H.-G., & Wedemeyer,H. Mann,A., Moser,M., Mierke,C.T., Rottner,K., Blessing,M., Hirrlinger,J., (2010) IFN alpha-induced TRAIL on human NK cells is associated & Ziegler,W.H. (2010) The vinculin-DeltaIn20/21 mouse: characteris- with control of hepatitis C virus infection. Gastroenterology 138, tics of a constitutive, actin-binding defi cient splice variant of vincu- 1885-1897. lin. PLoS ONE 5, e11530.

• Steinweg,C., Kuenne,C.T., Billion,A., Mraheil,M.A., Domann,E., • Rottner,K., Hanisch,J., & Campellone,K.G. (2010) WASH, WHAMM Ghai,R., Barbuddhe,S.B., Karst,U., Goesmann,A., Puhler,A., and JMY: regulation of Arp2/3 complex and beyond. Trends in Cell Weisshaar,B., Wehland,J., Lampidis,R., Kreft,J., Goebel,W., Biology 20, 650-661. Chakraborty,T., & Hain,T. (2010) Complete genome sequence of Liste- ria seeligeri, a nonpathogenic member of the genus Listeria. Journal • Jackson,B., Peyrollier,K., Pedersen,E., Basse,A., Karlsson,R., Wang,Z., of Bacteriology 192, 1473-1474. Lefever,T., Ochsenbein,A.M., Schmidt,G., Aktories,K., Stanley,A., Quondamatteo,F., Ladwein,M., Rottner,K., van,H.J., & Brakebusch,C. • Trunk,K., Benkert,B., Quack,N., Munch,R., Scheer,M., Garbe,J., (2011) RhoA is dispensable for skin development, but crucial for con- Jänsch,L., Trost,M., Wehland,J., Buer,J., Jahn,M., Schobert,M., & traction and directed migration of keratinocytes. Molecular Biology of Jahn,D. (2010) Anaerobic adaptation in Pseudomonas aeruginosa: the Cell 22, 593-605. defi nition of the Anr and Dnr regulons. Environmental Microbiology 12, 1719-1733.

• Wilke,S., Krausze,J., Gossen,M., Gröbe,L., Jager,V., Gherardi,E., van Former RG Signalling and Motility | Prof. Dr. Theresia Stradal den,H.J., & Bussow,K. (2010) Glycoprotein production for structure analysis with stable, glycosylation mutant CHO cell lines established • Hanisch,J., Ehinger,J., Ladwein,M., Rohde,M., Derivery,E., Bosse,T., by fl uorescence-activated cell sorting. Protein Science 19, 1264-1271. Steffen,A., Bumann,D., Misselwitz,B., Hardt,W.D., Gautreau,A., Stradal,T.B.*., & Rottner,K. (2010) Molecular dissection of Salmonel- • Froese,N., Kattih,B., Breitbart,A., Grund,A., Geffers,R., Molkentin,J.D., la-induced membrane ruffl ing versus invasion. Cellular Microbiology Kispert,A., Wollert,K.C., Drexler,H., & Heineke,J. (2011) GATA6 12, 84-98. promotes angiogenic function and survival in endothelial cells by suppression of autocrine transforming growth factor beta/activin • Klink,B.U., Barden,S., Heidler,T.V., Borchers,C., Ladwein,M., receptor-like kinase 5 signaling. Journal of Biological Chemistry Stradal,T.B.*., Rottner,K., & Heinz,D.W.*. (2010) Structure of Shigella 286(7), 5680-5690 IpgB2 in complex with human RhoA: implications for the mechanism of bacterial guanine nucleotide exchange factor mimicry. Journal of • Lorenz,U., Lorenz,B., Schmitter,T., Streker,K., Erck,C., Wehland,J., Biological Chemistry 285, 17197-17208. Nickel,J., Zimmermann,B., & Ohlsen,K. (2011) Functional antibod- ies targeting IsaA of Staphylococcus aureus augment host immune • Stradal,T.B.*. & Backert,S. (2010) Host-pathogen interaction: How response and open new perspectives for antibacterial therapy. Anti- EHEC infl uence the actin cytoskeleton of the host cell [Wirt-Patho- microbial Agents and Chemotherapy 55, 165-173. gen-Interaktion wie EHEC das Aktinzytoskelett der Wirtszelle beein- fl ussen]. Biospektrum 16, 624-627. • Schenk,U., Frascoli,M., Proietti,M., Geffers,R., Traggiai,E., Buer,J., Ricordi,C., Westendorf,A.M., & Grassi,F. (2011) ATP inhibits the • Tahirovic,S., Hellal,F., Neukirchen,D., Hindges,R., Garvalov,B.K., generation and function of regulatory T cells through the activation Flynn,K.C., Stradal,T.E.*., Chrostek-Grashoff,A., Brakebusch,C., & of purinergic P2X receptors. Science Signaling 4, ra12. Bradke,F. (2010) Rac1 regulates neuronal polarization through the WAVE complex. Journal of Neuroscience 30, 6930-6943. SCIENTIFIC REPORTS | Publications 167

• Breitsprecher,D., Kiesewetter,A.K., Linkner,J., Vinzenz,M., • Erol,O., Schaberle,T.F., Schmitz,A., Rachid,S., Gurgui,C., El,O.M., Stradal,T.E.*., Small,J.V., Curth,U., Dickinson,R.B., & Faix,J. (2011) Lohr,F., Kehraus,S., Piel,J., Müller,R., & Konig,G.M. (2010) Biosynthe- Molecular mechanism of Ena/VASP-mediated actin-fi lament elonga- sis of the myxobacterial antibiotic corallopyronin A. ChemBioChem tion. Embo Journal 30, 456-467. 11, 1253-1265.

• Garcia,R., Gerth,K., Stadler,M., Dogma,I.J., Jr., & Müller,R. (2010) Expanded phylogeny of myxobacteria and evidence for cultivation of Former RG Systems and Synthetic Biology | Prof. Dr. Vitor the ‘unculturables’. Molecular Phylogenetics and Evolution 57, 878- Martins dos Santos 887.

• Fazzini,R.A., Preto,M.J., Quintas,A.C., Bielecka,A., Timmis,K.N.*., • Irschik,H., Kopp,M., Weissman,K.J., Buntin,K., Piel,J., & Müller,R. & dos Santos,V.A.P.M.*. (2010) Consortia modulation of the stress (2010) Analysis of the sorangicin gene cluster reinforces the utility of response: proteomic analysis of single strain versus mixed culture. a combined phylogenetic/retrobiosynthetic analysis for deciphering Environmental Microbiology 12, 2436-2449. natural product assembly by trans-AT PKS. ChemBioChem 11, 1840- 1849. • Koutinas,M., Kiparissides,A., Lam,M.-C., Silva-Rocha,R., de Lorenzo,V., dos Santos,V.A.P.M.*., Pistikopoulos,E.N., & Mantalaris,A. • Khatri,Y., Hannemann,F., Ewen,K.M., Pistorius,D., Perlova,O., (2010) Combining genetic circuit and microbial growth kinetic mod- Kagawa,N., Brachmann,A.O., Müller,R., & Bernhardt,R. (2010) The els: A challenge for biological modelling. Computer Aided Chemical CYPome of Sorangium cellulosum So ce56 and identifi cation of Engineering 28, 301-306. CYP109D1 as a new fatty acid hydroxylase. Chemistry and Biology 17, 1295-1305. • Koutinas,M., Lam,M.C., Kiparissides,A., Silva-Rocha,R., Godinho,M., Livingston,A.G., Pistikopoulos,E.N., de,L., V, Dos,S.V., & Mantalaris,A. • Kunze,B., Reck,M., Dotsch,A., Lemme,A., Schummer,D., Irschik,H., (2010) The regulatory logic of m-xylene biodegradation by Pseu- Steinmetz,H., & Wagner-Döbler,I. (2010) Damage of Streptococcus domonas putida mt-2 exposed by dynamic modelling of the principal mutans biofi lms by carolacton, a secondary metabolite from the node Ps/Pr of the TOL plasmid. Environmental Microbiology 12, 1705- myxobacterium Sorangium cellulosum. BMC Microbiology 10, 199. 1718. • Li,Y., Weissman,K.J., & Müller,R. (2010) Insights into multienzyme • Lam,C.M.C., Puchalka,J., Diez,M.S., & dos Santos,V.A.P.M.*. (2010) docking in hybrid PKS-NRPS megasynthetases revealed by heterolo- Exploring networks at the genome scale. European Biotechnology gous expression and genetic engineering. ChemBioChem 11, 1069- Science and Industry News 9, 40-42. 1075.

• Menche,D., Li,P., & Irschik,H. (2010) Design, synthesis and biological evaluation of simplifi ed analogues of the RNA polymerase inhibitor Dept. of Microbial Natural Products – HIPS & RG Microbial etnangien. Bioorganic and Medicinal Chemistry Letters 20, 939-941. Drugs – HZI | Prof. Dr. Rolf Müller • Nawrath,T., Gerth,K., Müller,R., & Schulz,S. (2010) The biosynthesis • Arp,G., Bissett,A., Brinkmann,N., Cousin,S., de Beer,D., Friedl,T., of the aroma volatile 2-methyltetrahydrothiophen-3-one in the bacte- Mohr,K.I.*., Neu,T.R., Reimer,A., Shiraishi,F., Stackebrandt,E., rium Chitinophaga Fx7914. ChemBioChem 11, 1914-1919. & Zippel,B. (2010) Tufa-forming biofi lms of German karstwater streams: Microorganisms, exopolymers, hydrochemistry and calcifi - • Nawrath,T., Gerth,K., Müller,R., & Schulz,S. (2010) Volatile methyl cation. Geological Society Stecial Publication 336, 83-118. esters of medium chain length from the bacterium Chitinophaga Fx7914. Chemistry and Biodiversity 7, 2228-2253. • Binz,T.M., Maffi oli,S.I., Sosio,M., Donadio,S., & Müller,R. (2010) In- sights into an unusual nonribosomal peptide synthetase biosynthe- • Nicolas,L., Anderl,T., Sasse,F., Steinmetz,H., Jansen,R., Höfl e,G., sis: identifi cation and characterization of the GE81112 biosynthetic Laschat,S., & Taylor,R.E. (2010) Gephyronic Acid, a Missing Link be- gene cluster. Journal of Biological Chemistry 285, 32710-32719. tween Polyketide Inhibitors of Eukaryotic Protein Synthesis (Part I): Structural Revision and Stereochemical Assignment of Gephyronic • Brodmann,T., Janssen,D., Sasse,F., Irschik,H., Jansen,R., Müller,R., & Acid. Angewandte Chemie International Edition 50, 938-941. Kalesse,M. (2010) Isolation and synthesis of chivotriene, a chivosa- zole shunt product from Sorangium cellulosum. European Journal of • Rachid,S., Revermann,O., Dauth,C., Kazmaier,U., & Müller,R. (2010) Organic Chemistry 27, 5155-5159. Characterization of a novel type of oxidative decarboxylase involved in the biosynthesis of the styryl moiety of chondrochloren from an • Bulow,L., Nickeleit,I., Girbig,A.K., Brodmann,T., Rentsch,A., acylated tyrosine. Journal of Biological Chemistry 285, 12482-12489. Eggert,U., Sasse,F., Steinmetz,H., Frank,R., Carlomagno,T., Malek,N.P., & Kalesse,M. (2010) Synthesis and biological characteri- • Vilchez,R., Lemme,A., Ballhausen,B., Thiel,V., Schulz,S., Jansen,R., zation of argyrin F. ChemMedChem 5, 832-836. Sztajer,H., & Wagner-Döbler,I. (2010) Streptococcus mutans inhibits Candida albicans hyphal formation by the fatty acid signaling mol- • Buntin,K., Irschik,H., Weissman,K.J., Luxenburger,E., Blöcker,H., & ecule trans-2-decenoic acid (SDSF). ChemBioChem 11, 1552-1562. Müller,R. (2010) Biosynthesis of thuggacins in myxobacteria: com- parative cluster analysis reveals basis for natural product structural • Weissmann,K.J. & Müller,R. (2010) Myxobacterial secondary metabo- diversity. Chemistry and Biology 17, 342-356. lites: Bioactivities and modes-of-action. Natural Product Reports 27, 1276-1295. • Buntin,K., Weissman,K.J., & Müller,R. (2010) An unusual thioesterase promotes isochromanone ring formation in ajudazol biosynthesis. • Adhikary,T., Kaddatz,K., Finkernagel,F., Schonbauer,A., Meissner,W., ChemBioChem 11, 1137-1146. Scharfe,M., Jarek,M., Blöcker,H., Muller-Brusselbach,S., & Müller,R. (2011) Genomewide analyses defi ne different modes of transcrip- • Chai,Y., Pistorius,D., Ullrich,A., Weissman,K.J., Kazmaier,U., & tional regulation by peroxisome proliferator-activated receptor-beta/ Müller,R. (2010) Discovery of 23 natural tubulysins from Angiococcus delta (PPARbeta/delta). PLoS ONE 6, e16344. disciformis An d48 and Cystobacter SBCb004. Chemistry and Biology 17, 296-309. • Capell,A., Liebscher,S., Fellerer,K., Brouwers,N., Willem,M., Lammich,S., Gijselinck,I., Bittner,T., Carlson,A.M., Sasse,F., Kunze,B., • Daum,M., Schnell,H.J., Herrmann,S., Gunther,A., Murillo,R., Steinmetz,H., Jansen,R., Dormann,D., Sleegers,K., Cruts,M., Herms,J., Müller,R., Bisel,P., Muller,M., & Bechthold,A. (2010) Functions of Van,B.C., & Haass,C. (2011) Rescue of progranulin defi ciency associ- genes and enzymes involved in phenalinolactone biosynthesis. ated with frontotemporal lobar degeneration by alkalizing reagents ChemBioChem 11, 1383-1391. and inhibition of vacuolar ATPase. Journal of Neuroscience 31, 1885- 1894. 168 SCIENTIFIC REPORTS | Publications

• Dehn,R., Katsuyama,Y., Weber,A., Gerth,K., Jansen,R., Steinmetz,H., • Gaschen,A., Lang,D., Kalberer,M., Savi,M., Geiser,T., Gazdhar,A., Höfl e,G., Müller,R., & Kirschning,A. (2011) Molecular basis of elan- Lehr,C.-M., Bur,M., Dommen,J., Baltensperger,U., & Geiser,M. (2010) solid biosynthesis: evidence for an unprecedented quinone methide Cellular responses after exposure of lung cell cultures to secondary inititiated intramolecular Diels-Alder cycloaddition / macrolacto- organic aerosol particles. Environmental Science and Technology 44, nization. Angewandte Chemie 50, 532-536. 1424-1430.

• Huntley,S., Hamann,N., Wegener-Feldbrugge,S., Treuner-Lange,A., • Henning,A., Schneider,M., Nafee,N., Muijs,L., Rytting,E., Wang,X., Kube,M., Reinhardt,R., Klages,S., Müller,R., Ronning,C.M., Kissel,T., Grafahrend,D., Klee,D., & Lehr,C.-M. (2010) Infl uence of Nierman,W.C., & Sogaard-Andersen,L. (2011) Comparative genomic particle size and material properties on mucociliary clearance from analysis of fruiting body formation in Myxococcales. Molecular the airways. Journal of Aerosol Medicine and Pulmonary Drug Delivery Biology and Evolution 28, 1083-1097. 23, 233-241.

• Steinmetz,H., Gerth,K., Jansen,R., Schlager,N., Dehn,R., Reinecke,S., • Henning,A., Hein,S., Schneider,M., Bur,M., & Lehr,C.-M. (2010) Pul- Kirschning,A., & Müller,R. (2011) Elansolid A, a unique macrolide an- monary drug delivery: medicines for inhalation. Handbook of Experi- tibiotic from Chitinophaga sancti isolated as two stable atropisomers. mental Pharmacology 171-192. Angewandte Chemie International Edtion 50, 532-536. • Lehmann,A.D., Daum,N., Bur,M., Lehr,C.-M., Gehr,P., & Rothen- • Wenzel,S.C. & Müller,R. (2011) Myxobacteria - Unique microbial Rutishauser,B.M. (2010) An in vitro triple cell co-culture model with secondary metabolic factories. In: Comprehensive Natural Products primary cells mimicking the human alveolar epithelial barrier. Euro- II: Chemistry and Biology, Elsevier Science & Technology, Oxford, pp. pean Journal of Pharmaceutics and Biopharmaceutics 77, 398-406. 189-222. • Leonard,F., Collnot,E.M., & Lehr,C.-M. (2010) A three-dimensional • Anderl,T., Nicolas,L., Munkemer,J., Baro,A., Sasse,F., Steinmetz,H., coculture of enterocytes, monocytes and dendritic cells to model Jansen,R., Hofl e,G., Taylor,R.E., & Laschat,S. (2011) Gephyronic Acid, infl amed intestinal mucosa in vitro. Molecular Pharmaceutics 7, a Missing Link between Polyketide Inhibitors of Eukaryotic Protein 2103-2119. Synthesis (Part II): Total Synthesis of Gephyronic Acid. Angewandte Chemie International Edition, in press. • Melero,A., Lehr,C.-M., Schafer,U.F., & Garrigues,T.M. (2010) Wistar rat skin as surrogate for human skin in nortriptyline hydrochloride • Garcia,R., Pistorius,D., Stadler,M., & Müller,R. (2011) Fatty acid Re- patch studies. International Journal of Pharmaceutics 384, 137-139. lated Phylogeny of Myxobacteria as an Approach to Discover Polyun- saturated Omega-3/6 Fatty Acids. Journal of Bacteriology, in press. • Muendoerfer,M., Schaefer,U.F., Koenig,P., Walk,J.S., Loos,P., Balbach,S., Eichinger,T., & Lehr,C.-M. (2010) Online monitoring of • Okanya,P.W., Mohr,K.I., Gerth,K., Jansen,R., & Müller,R. (2011) Mari- transepithelial electrical resistance (TEER) in an apparatus for com- noquinolines A-F, Pyrroloquinolines from Ohtaekwangia kribbensis bined dissolution and permeation testing. International Journal of (Bacteroidetes). Journal of Natural Products, in press. Pharmaceutics 392, 134-140.

• Pistorius,D., Ullrich,A., Lucas,S., Hartmann,R.W., Kazmaier,U., & • Neumeyer,A., Bukowski,M., Veith,M., Lehr,C.-M., & Daum,N. (2010) Müller,R. (2011) Biosynthesis of 2-Alkyl-4(1H)-Quinolones in Pseu- Interaction of nanoparticles and cells - cellular uptake and cytotoxic domonas aeruginosa: Potential for Therapeutic Interference with effects in vitro. In Modern Polymeric Materials for Environmental Pathogenicity. ChemBioChem, in press. Applications (Pielichowski,K., ed), pp. 239-246. Cracow.

• Rachid,S., Huo,L., Herrmann,J., Stadler,M., Kopcke,B., Bitzer,J., & • Philippi,C., Loretz,B., Schaefer,U.F., & Lehr,C.-M. (2010) Telomerase Müller,R. (2011) Mining the cinnabaramide biosynthetic pathway to as an emerging target to fi ght cancer - Opportunities and challenges generate novel proteasome inhibitors. ChemBioChem, in press. for nanomedicine. Journal of Controlled Release 146, 228-240.

• Zander,W., Gerth,K., Mohr,K.I., Kessler,W., Jansen,R., & Müller,R. • Reum,N., Fink-Straube,C., Klein,T., Hartmann,R.W.*., Lehr,C.-M., & (2011) Roimatacene, an antibiotic against gram-negative bacteria Schneider,M. (2010) Multilayer coating of gold nanoparticles with isolated from cystobacter Cb G35 (myxobacteria). Chemistry - A Euro- drug-polymer coadsorbates. Langmuir 26, 16901-16908. pean Journal, in press. • Rytting,E., Bur,M., Cartier,R., Bouyssou,T., Wang,X., Kruger,M., Lehr,C.-M., & Kissel,T. (2010) In vitro and in vivo performance of biocompatible negatively-charged salbutamol-loaded nanoparticles. Dept. of Drug Delivery – HIPS | Prof. Dr. Claus-Michael Lehr Journal of Controlled Release 141, 101-107.

• Beisner,J., Dong,M., Taetz,S., Nafee,N., Griese,E.U., Schaefer,U., • Santander-Ortega,M.J., Stauner,T., Loretz,B., Ortega-Vinuesa,J.L., Lehr,C.-M., Klotz,U., & Murdter,T.E. (2010) Nanoparticle mediated Bastos-Gonzalez,D., Wenz,G., Schaefer,U.F., & Lehr,C.-M. (2010) Na- delivery of 2’-O-methyl-RNA leads to effi cient telomerase inhibition noparticles made from novel starch derivatives for transdermal drug and telomere shortening in human lung cancer cells. Lung Cancer delivery. Journal of Controlled Release 141, 85-92. 68, 346-354. • Hahn,T., Hansen,S., Neumann,D., Kostka,K.-H., Lehr,C.-M., Muys,L., • Bur,M., Huwer,H., Muys,L., & Lehr,C.-M. (2010) Drug transport across & Schaefer,U.F. (2011) Infrared densitometry: A fast and non-destruc- pulmonary epithelial cell monolayers: effects of particle size, apical tive method for exact stratum corneum depth calculation for in vitro liquid volume, and deposition technique. Journal of Aerosol Medicine tape-stripping. Skin Pharmacology and Physiology 23, 183-192. and Pulmonary Drug Delivery 23, 119-127. • Hahn,T., Winkler,K., Lehr,C.-M., & Schäfer,U.F. (2011) Ointment bases • Ciana,A., Meier,K., Daum,N., Gerbes,S., Veith,M., Lehr,C.-M., & and the rate of water loss of the skin [Salbengrundlagen und die Minetti,G. (2010) A dynamic ratio of the alpha+ and alpha- isoforms Wasserabgaberate der Haut]. Deutsche Apotheker Zeitung 150, 59-62. of the tight junction protein ZO-1 is characteristic of Caco-2 cells and correlates with their degree of differentiation. Cell Biology Interna- • Hansen,S., Selzer,D., Schaefer,U.F., & Kasting,G.B. (2011) An extend- tional 34, 669-678. ed database of keratin binding. Journal of Pharmaceutical Sciences 100, 1712-1726. • Collnot,E.M., Baldes,C., Schaefer,U.F., Edgar,K.J., Wempe,M.F., & Lehr,C.-M. (2010) Vitamin E TPGS P-glycoprotein inhibition mecha- • Hein,S., Bur,M., Schaefer,U.F., & Lehr,C.-M. (2011) A new Pharma- nism: infl uence on conformational fl exibility, intracellular ATP lev- ceutical Aerosol Deposition Device on Cell Cultures (PADDOCC) to els, and role of time and site of access. Molecular Pharmaceutics 7, evaluate pulmonary drug absorption for metered dose dry powder 642-651. formulations. European Journal of Pharmacology and Biopharma- cology 77, 132-138. SCIENTIFIC REPORTS | Publications 169

• Khvedelidze,M., Mdzinarashvili,T., Partskhaladze,T., Nafee,N., • Heinzerling,L., Hartmann,R.W.*., Frotscher,M., & Neumann,D. (2011) Schaefer,U.F., Lehr,C.-M., & Schneider,M. (2011) Calorimetric and Predicting putative inhibitors of 17beta-HSD1. Molecular Informatics spectrophotometric investigation of PLGA nanoparticles and their 29, 695-705. complex with DNA. Journal of Thermal Analysis and Calorimetry 99, 337-348. • Hille,U.E., Zimmer,C., Vock,C.A., & Hartmann,R.W.*. (2011) First selective CYP11B1 inhibitors for the treatment of cortisol-dependent • Maurer,F., Daum,N., Schaefer,U.F., Lehr,C.-M., & Bauer,P. (2011) Plant diseases. ACS Medicinal Chemistry Letters 2, 2-6. genetic factors for iron homeostasis affect iron bioavailability in Caco-2 cells. Food Research International 43, 1661-1665. • Marchais-Oberwinkler,S., Wetzel,M., Ziegler,E., Kruchten,P., Werth,R., Henn,C., Hartmann,R.W.*., & Frotscher,M. (2011) New • Patzelt,A., Richter,H., Knorr,F., Schafer,U., Lehr,C.-M., Dahne,L., drug-like hydroxyphenylnaphthol steroidomimetics as potent and Sterry,W., & Lademann,J. (2011) Selective follicular targeting by selective 17beta-hydroxysteroid dehydrogenase type 1 inhibitors for modifi cation of the particle sizes. Journal of Control Release 150, the treatment of estrogen-dependent diseases. Journal of Medicinal 45-48. Chemistry 54, 534-547.

• Schulze,C., Schaefer,U.F., Ruge,C.A., Wohlleben,W., & Lehr,C.-M. • Oster,A., Klein,T., Henn,C., Werth,R., Marchais-Oberwinkler,S., (2011) Interaction of metal oxide nanoparticles with lung surfactant Frotscher,M., & Hartmann,R.W.*. (2011) Bicyclic substituted hydroxy- protein A. European Journal of Pharmaceutics and Biopharmaceutics phenylmethanone type inhibitors of 17 beta-hydroxysteroid dehy- 77, 376-383. drogenase Type 1 (17 beta-HSD1): the role of the bicyclic moiety. ChemMedChem 6, 476-487.

• Stefanachi,A., Favia,A.D., Nicolotti,O., Leonetti,F., Pisani,L., Catto,M., Dept. of Drug Design and Optimisation – HIPS | Zimmer,C., Hartmann,R.W.*., & Carotti,A. (2011) Design, Synthesis, Prof. Dr. Rolf Hartmann and Biological Evaluation of Imidazolyl Derivatives of 4,7-Disubsti- tuted Coumarins as Aromatase Inhibitors Selective over 17-alpha- • Gobbi,S., Zimmer,C.*., Belluti,F., Rampa,A., Hartmann,R.W.*., Hydroxylase/C17-20 Lyase. Journal of Medicinal Chemistry 54, 1613- Recanatini,M., & Bisi,A. (2010) Novel highly potent and selective 1625. nonsteroidal aromatase inhibitors: synthesis, biological evaluation and structure-activity relationships investigation. Journal of • Wetzel,M., Marchais-Oberwinkler,S., & Hartmann,R.W.*. (2011) Medicinal Chemistry 53, 5347-5351. 17beta-HSD2 inhibitors for the treatment of osteoporosis: Identifi ca- tion of a promising scaffold. Bioorganic and Medicinal Chemistry 19, • Haller,F., Moman,E., Hartmann,R.W.*., Adamski,J., & Mindnich,R. 807-815. (2010) Molecular framework of steroid/retinoid discrimination in 17beta-hydroxysteroid dehydrogenase type 1 and photoreceptor- • Yadav,M.R., Sabale,P.M., Giridhar,R., Zimmer,C., Haupenthal,J., & associated retinol dehydrogenase. Journal of Molecular Biology 399, Hartmann,R.W.*. (2011) Synthesis of some novel androstanes as 255-267. potential aromatase inhibitors. Steroids 76, 464-470.

• Hu,Q., Yin,L., Jagusch,C., Hille,U.E., & Hartmann,R.W.*. (2010) Iso- propylidene substitution increases activity and selectivity of biphe- nylmethylene 4-pyridine type CYP17 inhibitors. Journal of Medicinal Chemistry 53, 5049-5053.

• Hu,Q., Jagusch,C., Hille,U.E., Haupenthal,J., & Hartmann,R.W.*. (2010) Replacement of imidazolyl by pyridyl in biphenylmethylenes results in selective CYP17 and dual CYP17/CYP11B1 inhibitors for the treatment of prostate cancer. Journal of Medicinal Chemistry 53, 5749-5758.

• Hu,Q., Negri,M., Olgen,S., & Hartmann,R.W. (2010) The role of fl uorine substitution in biphenyl methylene imidazole-type CYP17 inhibitors for the treatment of prostate carcinoma. ChemMedChem 5, 899-910.

• Negri,M., Recanatini,M., & Hartmann,R.W.*. (2010) Insights in 17beta-HSD1 enzyme kinetics and ligand binding by dynamic motion investigation. PLoS ONE 5, e12026.

• Oster,A., Hinsberger,S., Werth,R., Marchais-Oberwinkler,S., Frotscher,M., & Hartmann,R.W.*. (2010) Bicyclic substituted hy- droxyphenylmethanones as novel inhibitors of 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) for the treatment of estrogen- dependent diseases. Journal of Medicinal Chemistry 53, 8176-8186.

• Oster,A., Klein,T., Werth,R., Kruchten,P., Bey,E., Negri,M., Marchais- Oberwinkler,S., Frotscher,M., & Hartmann,R.W.*. (2010) Novel estrone mimetics with high 17beta-HSD1 inhibitory activity. Bio- organic and Medicinal Chemistry 18, 3494-3505.

• Reum,N., Fink-Straube,C., Klein,T., Hartmann,R.W.*., Lehr,C.-M., & Schneider,M. (2010) Multilayer coating of gold nanoparticles with drug-polymer coadsorbates. Langmuir 26, 16901-16908.

• Zimmer,C., Hafner,M., Zender,M., Ammann,D., Hartmann,R.W.*., & Vock,C.A. (2010) N-(Pyridin-3-yl)benzamides as selective inhibitors of human aldosterone synthase (CYP11B2). Bioorganic and Medicinal Chemistry Letters 21, 186-190. FOCUS RESEARCH REVIEWS SPECIAL FEATURES

Photos from left to right: Kurt Dittmar and Werner Lindenmaier investigate cells for immunotherapy using confocal laser scanning microscopy. | The BioTechnikum bus of the BMBF during the Biotech-Fair organized by OMNILAB at the HZI Campus. | During the opening ceremony of the symposium “Networking and Technology Transfer”, Bangkok, Thailand. In the 1st row (from left) Dr. Peter Winter (InWEnt/GIZ), Prof. Dr. Visith Sitprija (Director of QSMI, Bangkok), Mr. Stefan Duppel (Deputy Ambassador, German Embassy, Bangkok), in the 2nd row on the left: Dr. Suparp Artjariyasripong (Deputy Governor of TISTR). (right) Photos: HZI, Scheibe (le) | OMNILAB (ce) | QSMI, Thai Red Cross (ri) SCIENTIFIC REPORTS FACTS AND FIGURES 172 FACTS AND FIGURES

Facts and Figures

Prof. Dr. Rainer Jonas | Department of Scientifi c Information and Internationalization | [email protected]

In 1965 the HZI was founded as “Centre for Molecular Biological Research” (GMBF) with fi nancial support by the Volkswagen Foundation. In 1976 the Federal Government through the Ministry for Research and Technology (BMFT) together with the State of Niedersachsen took over the Centre, now called “German Research Centre for Biotechnology” (GBF). Since then the BMFT/BMBF as well as the State of Niedersachsen have jointly fi nanced the GBF/HZI. In 2006 it was the fi rst research centre of the Helmholtz Association to change its name into a Helmholtz Centre institution: the Helmholtz Centre for Infection Research – HZI.

Research Financing In 2010 the total costs of the HZI External Funding for Research More than 75% of the amounted to 62.9 Mio. € external funding came from national research programmes. About 14% and 5% were from EU programmes and industry, respectively. Costs per programme (in T€)

Research Area Programme Full Cost External fi nancing of research (in T€) Health Infection and Immunity 56 460 Source Full Cost Technology Transfer 580 BMBF 7 041.96 Special Tasks 3 901 DFG 2 761.78 Others 1 955 EU 2 465.56 Total Sum 62 896 Industry 898.9 HGF 3 133.37 DAAD 3.33 State of Niedersachsen 128.33 Full Costs 2010 Others 1 105.28 Infection and Immunity 89.8 % Total Sum 17 538.60

External funding 2010 – by source

BMBF 40.2 % HGF 17.9 % Special Tasks 6.2 % DFG 15.8 % Others 3.1 % EU 14.1 % Technology Transfer 0.9 % Others 6.3 % Industry 5.1 % State of Niedersachsen 0.7 % DAAD <0.1 %

Further External Funding of 2,133 T€ was acquired for infrastructural developments, including the construction of buildings. The money came among other things from the “Konjunkturprogramm II” and the City of Braunschweig. FACTS AND FIGURES 173

Property Rights / Licences In 2010, six patents were ap- BMBF plied for, fi ve of them outside of Germany. AiF-Dechema Cellular Screening Systems Basic Innovation Genome Research Candida Therapy Patents and property rights, licences, year 2010 Basic Innovation Genome Research Tuberculosis Therapy Bioprofi le TransMedLab Total number Germany ERA-Net SPATELIS Priority based applications 6 1 5 ERA-Net METAGUT (2010) ERA-Net LISTRESS Priority based applications, 102 35 67 FORSYS Metabilic Balance in E. coli total number FORSYS T cell Talk Granted patents (2010) 16 1 15 FORSYS SysLogics Total number of held 553 46 507 GenoMik ProTumor property rights GenoMik – Transfer MiPro Licence agreements, 41 32 9 GenoMik – Transfer ExpressSys total number GerontoSys GerontoMitoSys € Licence proceeds* (in T ) 631 161 470 GerontoSys GerontoSHIELD * Including revenues from other “know-how” transfer agreements InnoNet InnoSurf Innovative Therapies Armed Bacteria KMU innovative De novo – Gencluster-Synthese Publications, Professorships, DFG-Programmes, and Guest Scientists The HZI has further increased the impact KMU innovative Dis-Z-Konjugate of its scientifi c output in recent years. Several articles have Medicinal Infection Genomic Nasal Metagenomic been published in highly renowned journals of the Nature Medicinal Infection Genomic Metagenome and Interactome or Cell Press groups. Medicinal Infection Genomic LegioProtect MedSys Jekyll & Hyde Many HZI scientists are participating in important national MedSys BioInSys and international research programmes. NGFN Plus German Mouse Clinic NGFN Plus Adipositas Network Participation of HZI scientists in national and inter- Excellence Research New States “Taschentuchlabor” national research programmes Susceptbility & Resistance against SkinStaph Infections Quantitative Category 2008 2009 2010 Susceptbility & Resistance against PROGRESS Zoonosis / Systemsbiology PBA-ZooZooMAPInfl uenza Infections Parameters Susceptbility & Resistance against Resistance Susceptibility Infections Publications ISI-listed Papers pub- 205 229 281 Susceptbility & Resistance against SkinStaph2 lished by the Centre Infections SysMO PSYSMO Peer-reviewed non-ISI 5 11 66 publications in journals Zoonosis Zoo MAP Zoonosis PBA-Zoo Total number 210 240 347 Zoonosis Infl uenza Habilitations 2 1 4 Zoonosis ZooMAP2 Dissertations 40 34 40 Zoonosis Infl uenza 2 PhD Students 219 223 291 Zoonosis FBI-Zoo II Full Professor- Calls for professorship 4 7 2 ship Offerings (W2/W3) Special DFG- DFG-SFBs, Transregios, Graduate Schools STREP Fastest TB Programmes Excellence clusters 8 8 10 Helmholtz Graduate School for STREP PANFLUVAC DFG-Research Focus 2 3 3 Infection Research HIGS Graduiertenkollegs 3 4 4 Helmholtz-Kolleg for Infection Biology H-IRISIB Total number 13 15 17 DFG-Graduate School GRK 653 “Pseudomonas” Guest Scientists 98 87 123 DFG-Graduate School GRK1273 “Chronical Infections” 174 FACTS AND FIGURES

EU Frame Programmes DFG (German Research Foundation) CA CASIMIR SFB 566 Cytokin-Receptors COST Sysgenet SFB 578 From Gene to Product CP FAST-XDR-DETECT SFB 587 Lung Immunity CP FLUINHIBIT SFB 599 Permanent Implantates CP BACSIN SFB 621 Pathobiology of the Intestinal Mucosa CP HEPTROMIC SFB 738 Optimization of Conventional and Innovative CP OPTISTEM Transplants CP MagicPAH SFB 854 Molecular Organisation Cellular CP CAREPNEUMO Communication in the Immune System CSA TARPOL SFB 900 Chronic Infections: Microbial Persistence ERC StG RESISTOME and its Control ERC StG CMVAgSTIMULUS SFB/TR 51 Ecology, Physiology and Molecular Biology IMI EMTRAIN of the Roseobacter-Group Infrastructures EATRIS SPP 1258 Sensory and regulatory RNAs in Prokaryotes Infrastructures Infrafrontier SPP 1316 Host-adapted Metabolism of Bacterial Infrastructures ProteomeBinders Pathogens Infrastructures Instruct – Associated SPP 1394 Mast-cells – Promoters of Hhealth and Infrastructures TRANSVAC Modulators of Disease Infrastructures EU-Openscreen EXC 62 ReBirth IP EUMODIC FOR 629 Antibodies and Proteinanalysis Marie Curie IOF APPI FOR 1220 PROTRAIN NoE EuroPathoGenomics EPG FOR 1406 Exploiting the Potential of Natural NoE Clinigene Compounds: Myxobacteria SME driven NOPERSIST KFO 250 Genetic and Cellular Mechanisms of STREP Healthy-Water Autoimmune Diseases STREP ASSIST STREP Fastest TB STREP PANFLUVAC Intellectual Property Since 2002 the Ascenion Ltd. Co. offers services principally for the four Helmholtz Research Centres in the area of health care: GSF, HZI, MDC, and DKFZ. The headquarters are in Munich, but an offi ce with Technology Transfer The HZI has a great potential for the two employees works on the HZI-Campus. development of innovative products, processes and services, especially in cooperation with industrial partners. There- Ascenion Ltd. Co. manages the following areas for the HZI: fore, an important goal is to foster the transfer of research results into industrial applications through technology • Acquisition and management of intellectual property transfer. Thus, the establishment of spin-off and start-up • Evaluation of the commercial potential of an invention biotech companies, licence agreements as well as service before patent fi ling contracts with industrial partners are important elements • Development and employment of strategies for the for the transfer of R&D results. In order to further support exploitation of the HZI patent folio technology transfer activities, the HZI is a member of BioRegioN and the “Transferkolleg Biotechnologie e.V.”.

The HZI-Biotech Campus A new container for the admi- Biotech Fair on the HZI Campus On September 16, 2010 nistration was ready in 2009, and also a new S3-laboratory OMNILAB organized, with the support of HZI and DSMZ, is ready for use. The laboratory building D will get a new its 6th biotech-fair and symposium in the FORUM. It was the facility for offi ce work, which is under construction and celebration of its 10th anniversary and the 75th anniversary should be ready at the end of 2011. of OMNILAB. About 50 enterprises and several research institutions of the region presented themselves to about 600 visitors, a new record. The visitors came from the Braunschweig-Hannover-Magdeburg-Göttingen region. A special highlight was the BIOTechnikum of the BMBF. The next fair is foreseen to take place at the HZI facilities on September 20, 2012. FACTS AND FIGURES 175

Open Day at HZI On Saturday, May 08, 2010, HZI opened its doors for everybody to have a look at the activities of the infection research centre. More than 1,800 visitors from the region came to get to know more details about infections, vaccines, biofi lms, etc.

A scientifi c presentation during the Biotech Fair organized by OMNILAB at the HZI FORUM in September 2010 Photo: OMNILAB

Biotech Network ASEAN-MERCOSUL – Germany The internet platform of the ASEAN-South American-German Dr. Torsten Lührs explains visitors during the Open Day 2010 Biotechnetwork (www.asag-biotech.net) was further impro- how a NMR spectrometer is working. Photo: HZI ved. Now one can search for most of German institutes that are working in the biotech area, sensu latu. Furthermore, several institutes from Latin American countries as well as from ASEAN countries are listed. Also biotech-industries Crèche and Kindergarten for Children of HZI Employees and events can be searched for. The platform allows direct The HZI in cooperation with the Sterntaler Kindergarten in acces to the most important institutions in Germany and Braunschweig-Stöckheim offers childcare for those aged one other countries, to organisations that offer fellowships, year and older. In 2010, 19 children were admitted to the calls, and possibilities of networking. The publication kindergarten, 5 of them in the crèche and the others in the databasis of the Helmholtz Institutions, several patent data kindergarten. basis, some university libraries can be accessed easily. In June 2010, HZI together with InWEnt and other partners HGF-Mentoring Programme 2010/11 In 2010 three organized a big symposium on “Networking and Technology female employees participated in the programme, and it is Transfer”, which took place in Bangkok, Thailand. More expected that another fi ve will participate in 2011. than 150 scientists from Germany, Southeast Asian and Mercosur countries participated. Audit “berufundfamilie” On November 27, 2010 the HZI was certifi cated again for further 3 years for that pro- gramme.

Future Day for girls and boys In 2011 nearly 100 pupils between 12 and 16 from various types of schools visited the HZI at the Future Day. Fifteen different work groups participated and about 60 staff members showed the pupils interesting experiments and/or gave them ideas of the work at the HZI.

Prof. Dr. Yongyuth Yuthavong, former Minister of Science and Technology of Thailand and Professor at NSTDA, Thailand, during his keynote speech on “Aspects of the future develop- ment of biotechnology” at the symposium “Networking and Technology Transfer” held in Bangkok in June 2010. (Photo: QSMI, Thai Red Cross).

Girls and boys are very curious to learn more about what HZI is doing in its research. Photo: HZI 176 FACTS AND FIGURES

Personnel At the end of 2010 the HZI staff comprised 731 persons with full time and part time occupation. Additi- onally, 251 guests worked in various projects, receiving their payment from third parties. Along with 188 senior scientists, almost 300 PhD-students, and 22 engineers were working at the HZI.

Boards and Assemblies of the HZI The boards and assemblies of the HZI are the Board of Trustees, the Super- visory Board, the Scientifi c Committee and the Managing Directors.

Board of Trustees The Board of Trustees is formed by the three trustees of the HZI, the Federal Republic of Germa- ny, the State of Niedersachsen, and the State of Saarland, represented by their respective departments, the Federal Ministry of Education and Research (BMBF), the Finance The Scientifi c and Adminstrative Directors of the HZI, Ministry of Niedersachsen, and the Ministry of Economics Prof. Dr. Dirk Heinz (right) and Ulf Richter (left). Photo: HZI of the Saarland.

Supervisory Board The Supervisory Board (SB) oversees the legality, expedience and economy of the management. Scientists Council The Scientists Council of the HZI It decides on general research goals, the principal research advises the Management in scientifi c matters. It consists by policy and fi nancial affairs of the centre. It consists of a one half of the heads of the departments and by the other maximum of 11 members. half of elected research group leaders, project leaders or junior research group leaders. The Managing Directors, Scientifi c Advisory Committee The Scientifi c Advisory the members of the Steering Committee, one member of Committee (SC) consists of external scientifi c experts. It the work council and one member of the administration, advises the Supervisory Board with regard to the R&D pro- appointed by the Administrative Director, are guests of the gramme as well as the general research strategy of the HZI. assembly.

Managing Directors The Managing Directors of the HZI: Steering Committee The Steering Committee advises the Research & Development: Prof. Dr. Dirk Heinz (acting) Managing Directors of the HZI in all important questions Administration: Ulf Richter, MBA of the Centre. Members are the Managing Directors, the Programme Speaker, and the Topic Speakers.

Staff Council The Staff Council has certain consultation and co-determination rights in personnel and social que- stions. It consists of 11 members, elected by the HZI staff. Chairman is John Aubert.

Equal Opportunities Offi cer is Evelyn Rohn-Stenzel. FACTS AND FIGURES 177

Members of the Supervisory Board (SB) and the Scientifi c Advisory Committee (SC), Status: 19.01.2011

Function Name, Title Organisation Locality Chairman SB Brumme-Bothe, MinDir’in Bärbel BMBF Berlin Vice-Chairman SB Gevers, MinDirig Dr. Heiko NMWK Hannover SB Köpke, OR Heinz-Hermann Ministry of Finances, Hannover Niedersachsen SB + SC Baum, Prof. Dr. Christopher MHH Hannover SB Dersch, Prof. Dr. Petra HZI Braunschweig SB Weiß, Dr. Siegfried HZI Braunschweig SB + SC Zettlmeissl, Dr. Gerd Intercell AG Wien SB + SC Müller-Goymann, Prof. Dr. Christel TU Braunschweig SB + SC Vice-Chairman SC Schendel, Prof. Dr. Dolores HMGU München SB + SC Kurth, Dr. Bärbel-Maria Robert-Koch-Institut Berlin SB + SC Daniel, Prof. Dr. Hannelore Wissenschaftszentrum Freising Weihenstephan SB + SC Chairman SC Pfeffer, Prof. Dr. med. Klaus Universitätsklinikum Düsseldorf SC Hacker, Prof. Dr. Jörg Nationale Akademie Halle der Wissenschaften SC Rosenthal, Prof. Dr. Walter MDC Berlin SC Brakhage, Prof. Dr. Axel HKI Jena SC Apweiler, Dr. Rolf EBI Cambridge SC Wilmanns, Dr. Matthias EMBL Hamburg SC Hakenbeck, Prof. Dr. Regine TU Kaiserslautern SC Hämmerling, Prof. Dr. Günter DKFZ Heidelberg SC Di Santo, Prof. Dr. James Institut Pasteur Paris 178 FACTS AND FIGURES FACTS AND FIGURES 179

Chart of Organisation, Status February 01, 2011

Scientifi c Advisory Commitee (SC) Supervisory Board (SB) Executive Board (DR) Staff Council (BR) Representative Body for Prof. Dr. med. K. Pfeffer, Chair MinDir‘in B. Brumme-Bothe, Chair J. Aubert, Chair Disabled Employees MinDirig H. Gevers, Nieders. MWK, Vice Chair B. Dojka (VPS)

Scientifi c Director (GFW) Prof. Dr. D. Heinz (acting) Assembly of Equal Opportunity Administrative Director (GFA) U. Richter Scientists (WV) Commissioner (GB) Dr. W.-R. Abraham, Chair E. Rohn-Stenzel

Dept. of Molecular Dept. of Gene Regulation Dept. of Molecular Dept. of Medical Dept. of Infection Dept. of Medicinal Infrastructure Infection Biology (MIBI) and Differentiation (RDIF) Structural Biology (MOSB) Microbiology (MMIK) Genetics (INFG) Chemistry (MCH) Prof. Dr. Petra Dersch Dr. H. Hauser Prof. Dr. D. Heinz Prof. Dr. G.S. Chhatwal Prof. Dr. K. Schughart Prof. Dr. M. Kalesse

RG Chronic Pseudo- RG Molecular RG Biophysical RG Infection RG Experimental RG Microbial Drugs Administration and Staff Units monas Infections (CPI) Immunology (MOLI) Analysis (BA) Immunology (INI) Animal Unit (TEE) (MWIS) Infrastructure (VIN) U. Richter / Prof. Dr. S. Häußler Dr. S. Weiß Dr. V. Wray PD Dr. E. Medina Dr. H. Riedesel Prof. Dr. R. Müller U. Richter Prof. Dr. D. Heinz

RG Microbial Communi- RG Model Systems for RG Recombinant Protein RG Microbial Interactions Dept. of Immune Control RG Environmental Human Resources Public Relations (ÖA) cation (KOM) Infection and Immunity Expression (RPEX) and Processes (MINP) (IMMK) Microbiology (UWM) (PA) M. Braun Prof. Dr. I. Wagner-Döbler (MSYS) Dr. D. Wirth Dr. J. van den Heuvel Prof. Dr. D. Pieper Prof. Dr. B. Schraven Prof. Dr. K. Timmis J. Schinkel

RG Genom Analytics RG Cellular Proteom Dept. of Vaccinology and RG System-oriented Immunology Finance Department Controlling (CO) - Dr. M. Strätz (DMC) (Deputy) (GMAK) Research (CPRO) Applied Microbiology (VAC) and Infl ammation Research (FA) (SIME) Prof. Dr. I. Schmitz - NN (BCO) Dr. R. Geffers Dr. L. Jänsch Prof. Dr. Dr. C.A. Guzmán D.-M. Reinhardt - Dr. R.-J. Müller (PWC)

JRG Chronic Infections RG Microbial Proteomics RG Chemical Micro- Dept. of Experimental Purchasing Internal Auditing (IR) and Cancer (CHIK) (MPRO) biology (CMIK) Immunology (EXIM) Department (EM) R. Lomberg – Anti-cor- Prof. Dr. L. Zender Prof. Dr. K. Riedel Dr. W.-R. Abraham Prof. Dr. J. Hühn B. Balster ruption-commissioner

JRG Macromolecular JRG Phagosome Biology RG Immunoregulation Legal Affairs (JUR) Occupational Safety Interactions (MMIA) (PHAB) (IREG) Dr. C. Kügler- Specialist (FaSi) Prof. Dr. C. Ritter Dr. M. Gutierrez PD Dr. D. Bruder HIPS Walkemeyer Dr. E. Grund

JRG Structure-based JRG Immune Aging and JRG Viral Immune Dept. of Microbial Natural Patents (PS) Scientifi c Information & Infection Biology (SBIB) Chronic Infections (IMCI) Modulation (VIMM) Products (MINS) D. Meseke Internationalization (WI) Dr. T. Lührs Dr. Dr. L. Cicin-Sain Dr. M. Brinkmann Prof. Dr. R. Müller Prof. Dr. R. Jonas

Scientifi c Groups JRG at Centre for Struc- Dept. of Systems JRG Actinobacteria Meta- Technical Library (BIB) tural Systems Biology – Immuno logy (SIMM) bolic Engineering Group Services (TB) A. Plähn DESY, Prof. Dr. I. Kursula Prof. Dr. M. Meyer-Hermann (AMEG) Dr. A. Luzhetskyy O. Rabe

Abbreviations: Dept. of Chemical Dept. of Drug Delivery Computer Centre Dept Department Biology (CBIO) (DDEL) (RZ) RG Research Group Dr. F. Sasse (acting head) Prof. Dr. C.-M. Lehr Dr. N. Bedorf (CIO) JRG Junior Research Group PWC Scientifi c Controlling RG Biologic Systems Dept. of Drug Design and Safety and Environ- Analysis (BISA) Prof. Dr. Optimization (DDOP) mental Affairs (SU) BCO Financial Controlling U. Bilitewski (acting head) Prof. Dr. R. Hartmann Dr. E. Grund DMC External Funding Controlling DSB Data Security Organisation/Admi- HIPS Helmholtz-Institute for nistration-IT (ORG) Pharma ceutical Research Saarland, H. Ohrdorf - DSB Saarbrücken CIO Chief Information Offi cer 180 IMPRINT

Research Report 2010/11

Published by Helmholtz Centre for Infection Research GmbH (HZI) Inhoffenstraße 7 D-38124 Braunschweig, Germany Telephone +49 (0)531-61 81-0 Telefax +49 (0)531-61 81-2655 [email protected] | www.helmholtz-hzi.de

Member of Helmholtz Association of National Research Centres

Editor-in-Chief: Prof. Dr. Rainer Jonas (V.i.S.d.P.) | [email protected]

Editoral Assistance: Monica Kirchner | Dr. Bastian Dornbach | Dr. Clemens Ostrowicz

Text Editing and Realisation: Dr. Jo Schilling | [email protected]

Translations: ADREM Sprachdienstleistungen sowie Ronning Übersetzungsdienst (Twincore)

© 2011 HZI Braunschweig

Photographs The portraits and further photos were taken by Bierstedt – pages: 48, 71, 80, 90, 91, 98, 106, 111, 113, 127-129, 172 Dornbach – pages: 30, 35, 39, 118 Gramann – pages: 7, 9, 19, 22, 28, 42, 44, 64, 69, 73, 74, 77, 84, 97, 102, 103, 108, 110, 119, 132, 138, 175, 176 Hans – pages: 79, 86, 89 Hübner – pages: 114, 175 Krämer – pages: 15, 30, 34, 40, 74, 76, 85, 88, 94-96, 99, 104, 112, 115, 117, 126 Sondermann – page: 5 HZI collection – pages: 2, 52, 59, 60, 67, 70, 72, 78, 81, 87, 93, 105, 107, 112, 122, 124, 130, 131 HIPS collection – pages: U2, U4, 2, 36, 39, 40, 125 Twincore collection – page: U1, U2, 2 The following portraits were made available by the authors – pages: 28 (Ciesek), 28 (Steinmann), 40 (Schäfer), 75 (Dersch), 92 (Hühn), 105 (Zender), 116 (Meyer-Hermann), 120 (Riedel), 123 (Kursula) For all other photographs, the authors and/or institutions are mentioned below the legends.

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