m crobiology i & mmunology

2nd Student Retreat

Microbiology and Immunology PhD Program

Parpan 6th – 8th September 2009

Welcome

Dear Participants,

It is a great pleasure to welcome you to the 2009 Student Retreat of the Microbiology and Immunology PhD Program of the Lifescience Zurich Graduate School.

In September 2008, the first MIM PhD Student Retreat took place at Fiescheralp in the Wallis. Many thanks again to the organizers of last year’s meeting. We could profit a lot from your experience.

For the second MIM PhD Student Retreat this year we are going to another region of the Swiss Alps, to Parpan in the canton Graubünden. There will be time to discuss our own research projects during poster sessions and talks. In addition, we are very glad that guest speakers from different fields followed our invitation to participate in the meeting. And of course, there will be time to get to know each other better at the party on Sunday evening or during a hiking trip on Monday afternoon.

We thank you for participating in this meeting, and wish you a great time.

See you all in Parpan,

The 2009 Organizing Committee

- 1 - 2nd MIM PhD Student Retreat 2009 Thanks

Lenzerheide - Parpan The Organizing Committee 2009 www.lenzerheide.com Silvia Bleuler

Jörg Breitling Parpan is together with the villages of Malix, Churwalden, Valbella, Yao-Yun Fan Lenzerheide, Vaz/Obervaz (Lain, Muldain and Zorten), Lantsch/Lenz and Brienz/Brinzauls part of the Lenzerheide holiday region. Lenzerheide Tina Herbst is the holiday region in the canton of Graubünden which is most quickly Janin Hofmann reached from Zurich. No matter whether in deep snow or a lush green, the landscape of the Lenzerheide holiday region will tempt you to stay at any Wolfgang Kratky time of year. Enjoy the warm sun. Breathe the fresh mountain air. Thomas Ludersdorfer Andrea Mekker

Bettina Schulthess Gerhard Trunk Kerstin Wanke

The MIM-Program Logo was created by Luzia Reutimann

m crobiology i & mmunology

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Accommodation in Parpan

Thanks Grischalodge Hotel Post

Thank you all for coming and hopefully making this a great Hauptstrasse 34 experience for all of us. 7076 Parpan

We would like to thank the MIM PhD Program within the Life Science phone: +41(0)81 382 23 32 Zurich Graduate School for financing this event. fax: +41 (0)81 382 21 61 We are grateful to all the invited speakers for joining us in our student www.grischalodge.net retreat.

Thanks to Olympia Stefani for administrative support.

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Program

Sunday, 06 September 2009 08.15 Departure from main bus station Zurich (behind main train station Zurich HB) 10.30 Arrival in Parpan, check-in 11.30 – 12.00 Mounting of posters (odd numbers) 12.00 Lunch 13.30 – 15.00 Opening remarks, Student talks ST 01-04 15.00 – 15.30 Coffee break 15.30 – 16.50 Student talks ST 05-08 17.15 – 18.15 Lecture by Prof. P. Philippsen, “Control of polar growth and nuclear migration in fungal hyphae: Lessons learned from Ashbya gossypi” 18.30 Dinner 20.00 – 21.30 Poster session (odd numbers) 22.00 Party

Monday, 07 September 2009 08.00 – 08.50 Breakfast 08.50 – 09.30 Student talks ST 09-10 09.30 – 10.30 Lecture by Dr. J. Hombach, “Career opportunities in international health institutions” 10.30 – 11.00 Coffee break 11.00 – 12.00 Student talks ST 11-13 12.00 – 12.30 Change posters (even numbers) 12.30 – 13.30 Lunch 13.30 – 14.15 Lecture by Prof. U. Jenal, “Mechanisms of cell cycle progression and cell fate determination in Caulobacter crescentus” 14.45 Afternoon hiking trip 16.00 Zvieri at Alp Stätz 19.30 Dinner

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P 34 Tuesday, 08 September 2009 08.00 – 09.00 Breakfast

09.00 – 09.40 Student talks ST 14-15 Cellular helicase UAP56 interacts with both influenza A virus nucleoprotein and interferon induced antiviral 09.40 – 10.40 Poster session (even numbers) protein MxA 10.40 – 11.30 Coffee break, clear rooms 11.30 – 12.30 Lecture by Dr. K. Dumstrei Christian Wisskirchen, Jovan Pavlovic “Scientific writing and publishing” 13.00 Lunch Institute of Medical Virology, University of Zurich [email protected] 14.00 Final words of the organizers 15.00 Check-out, departure from Parpan

Influenza A Virus (IAV) is a segmented negativ RNA virus belonging to the 17.00 Arrival in Zurich family of Orthomyxoviridae. IAV causes influenza which is one of the major infectious diseases today. Replication takes place in the nucleus and during replication, several cellular functions are used by the virus. So far several proteins have been identified which interact with the Student Talks IAV Ribonucleoproteincomplex (RNP), especially with the nucleoprotein time-frame: 12 min talk, 3 min discussion (NP). IAV nucleoprotein has previously been shown to interact with UAP56 a cellular helicase, shown to be essential for mRNA export and spliceosome Sunday, 06 September 2009 assembly. Upon viral infection the interferon (IFN) induced antiviral MxA ST 01 13.45 by Dirk Blom protein is induced in humans, which was shown to strongly inhibit IAV ST 02 14.05 by Yao-Yun Fan replication. Until now the mechanism for MxA antiviral activity is still ST 03 14.25 by Tina Herbst unknown, as well as a possible target protein from IAV. We have discovered ST 04 14.45 by Koshika Yadava an interaction between UAP56 and MxA and were able to confirm the ST 05 15.30 by Janin Hofmann interaction in different cell lines, upon cotransfection as well as the ST 06 15.50 by Wolfgang Kratky endogenous interaction after IFN treatement. This interaction might be the ST 07 16.10 by Lysann Hesske missing link between MxA and IAV NP, because UAP56 interacts with both ST 08 16.30 by Anna Haas proteins and a sequestration of UAP56 from NP to MxA might be an explanation for a possible MxA mechanism. Unpublished data show a need Monday, 07 September 2009 for a cellular helicase for IAV replication, in absence of any helicases replication efficiency strongly decreased. We want to further confirm our ST 09 08.50 by Christian Lange results by checking viral titers in combination with an UAP56 siRNA ST 10 09.10 by N.N. knockdown, as well as a MxA titration experiment in which we want to ST 11 11.00 by Sarah Maria Dieckmann sequester UAP56 from IAV NP, trying to proof our hypothesized mechanism. ST 12 11.20 by Agnese Petrera ST 13 11.40 by Laura Morf

Tuesday, 08 September 2009 ST 14 09.00 by Andrea Mekker ST 15 09.20 by Kerstin Wanke

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P 33 Invited Speakers

Mechanistic insights into T help-dependent and T help- independent CD8+ T cell priming

Melanie Wiesel, Annette Oxenius

Institute of Microbiology, ETH Zurich [email protected]

Recent reports indicated that even in the setting of infections, which are thought to be largely independent of T help, CD4+ T cells play a crucial role in shaping the CD8+ T cell response, in particular with respect to maintenance and functionality of memory CD8+ T cells. As the requirement for T help seems to vary significantly with the experimental system, it is of importance to determine the mechanisms by which T helper cells confer help to CD8+ T cell responses. We used an experimental system consisting of CD8+ T cell priming with replication-incompetent virus like particles (VLPs) followed by secondary challenge either with vaccinia virus (VV), lymphocytic choriomeningitis virus (LCMV) or VLPs, and we found that CD4 T cell help is only crucial for secondary infections in case of VV challenge. Furthermore, also the primary CD8+ T cell response specific for VV was highly diminished in the absence of T help, indicating that the requirement for T help is rather linked to the type of infection rather than a general feature of secondary CD8+ T cell responses. We investigated whether a co-infection with VV and various other pathogens would overcome the T help requirement for induction of CD8+ T cell responses. To this end, antigen-specific CD8+ T cell were primed by Vaccinia virus in presence (“helped”) or absence (“helpless”) of T helper cells in combination with a heterologous LCMV, MCMV or Listeria monocytogenes infection. Interestingly, co-infection with LCMV completely restored the proliferation of helpless VV-specific CD8+ T cells to the level of helped VV- specific CD8+ T cells, whereas neither MCMV nor Listeria infection improved the proliferation capacity of helpless CD8+ T cells.

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P 32 Control of polar growth and nuclear migration in fungal hyphae: Lessons learned from Ashbya gossypii

A close molecular look on the ruffling process induced Prof. Peter Philippsen upon salmonella invasion in mammalian cells Institute of Applied Microbiology, Biozentrum, University of Basel, Basel, Pascale Vonäsch Switzerland

Institue of Microbiology, ETH Zurich [email protected] Over the past 20 years we have developed Ashbya gossypii as an experimental system to study two cell biological processes essential for fungal life style: Sustained polar surface expansion at tips of hyphae and Salmonella enterica subspecies I serovar Typhimurium are gram-negative, controlled dynamics of multiple nuclei within hyphae. Our experimental rod shaped facultative intracellular bacteria which cause severe approaches were influenced by many years of research experience in yeast gastroenteritis in human. Virulence of Salmonella spp. depends on two genetics and our unexpected discovery that the genome of A. gossypii is pathogenicity islands (SPI-1 and SPI-2) encoding two type III secretion highly syntenic with that of Saccharomyces cerevisiae. systems (TTSS) and different effector proteins which are injected trough After an introduction into A. gossypii biology, genomics and those systems into the host cell. SPI-1 mediates the entry into and SPI-2 transcriptomics the presentation will focus on new data obtained by ensures survival of the bacteria in the host cell. With the help of four SPI-1 monitoring GFP-labeled polarity factors, visualizing polar growth, and spindle effector proteins (SopE, SopE2, SopB, SipA) Salmonella induces its entry pole SPB) and microtubule (MT) proteins, visualizing nuclear dynamics. The into non- phagocytic, intestinal epithelial cells by acting on RhoGTPases and results can be summarized as follows. triggering the formation of actin- containing membranous protrusions Polar growth: In young, slowly growing hyphae all tested factors ("membrane ruffles") leading to internalization of the bacteria. SopE acts as a form a cortical layer at the tip front. These fluorescent caps comprise 40% to guanyl nucleotide exchange factor (GEF) for Rac1, Cdc42 and maybe other 50% of the total tip cortex area. During development to mature hyphae RhoGTPases. Although the function of SopE is known, its subcellular (growth speeds up to 3.5 um/min) exocyst and polarisome components localization and its interaction partners in the host cell are only partially (except AgBud6) gradually accumulate in the tip as a spheroid. This so- known. In the last years, an RNAi screen was performed in our lab to find called Spitzenkörper also accumulates secretory vesicles. Remarkably, new host genes potentially involved in the ruffling process. Many of the during hyphal maturation (up to 30 fold acceleration in growth) the cortical tip genes found are related to the endocytic pathway/vesicle formation. It was zone for vesicle fusions only slightly enlarges. Thus, in fast growing hyphae, recently described in the literature that Rac1 is activated on vesicles upon the Spitzenkörper is needed for increased vesicle fusion efficiency in a HGF stimulation of the cells. This raises the possibility, that SopE might be confined tip surface. This area overlaps with the distribution of the GTPase targeted to vesicles, were it activates Rac1 and/or other GTPases before AgCdc42 and shows spatial fluctuations centred around the growth axis. The they are transported to the plasma membrane. Preliminary result support this zone of surface growth is separated from sites of highly active endocytosis at hypothesis, as SopE can be found on small vesicles upon delivery in the host the rim of hyphal tips. Mechanistic implications will be discussed and cell. In my PhD thesis I will analyse the exact localization of SopE in the host compared with data from other fungi. cell at different timepoints of infection by using immunoflourescence Nuclear migration: We found three types of cytoplasmic MT- microscopy. I will further investigate the molecular mechanisms of SopE and dependent nuclear mobility: oscillation, rotation and bypassing. These are its (yet potential) co- players as well as their spatio-temporal regulation in the superimposed on one MT-independent mechanism, cotransport with the ruffling process. cytoplasmic streaming. A. gossypii SPBs can nucleate short (perpendicular) and very long (tangential) cytoplasmic MTs. The long MTs are important for nuclear bypassing and the short MTs for oscillation as concluded from phenotypes of SPB deletion mutants. The differences to nuclear migration in other fungi will be discussed.

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Prof. Peter Philippsen has an extensive experience in the area of functional P 31 genomics of yeast and filamentous fungi. His current research focuses on nuclear migration and polar growth control. He studied Chemistry at the University of Munich, Germany, where T cell response in Lpn infection he also did his PhD on the functional analysis of yeast tRNAs. After a position as scientific assistant in Munich, he was a postdoctoral fellow with Ron Davis at the Stanford University, USA. After having been associate Gerhard Trunk professor of Microbiology at the Biozentrum in Basel, Switzerland, and the University of Giessen, Germay, he became a full professor of Microbiology at Institute of Microbiology, ETH Zurich the University of Basel in 1991. [email protected] He is now the Head of Applied Microbiology at the Biozentrum of the University of Basel. The facultative intracellular pathogen Legionella pneumophila (Lpn) is the causative agent of Legionnaire’s disease, a severe pneumonia in immuno- compromised individuals. Lpn can be genetically manipulated, which makes it an attractive model organism to study the mechanisms of host resistance to bacteria which replicate in specialized non-degradative vacuoles. Studies in the guinea pig infection model and in mice suggest a role for an acquired cell-mediated immune response in the clearance of Lpn. Yet, the exact site and kinetics of T cell priming and differentiation during a respiratory Lpn infection has not been investigated so far. To examine this fundamental process, it would be useful to develop a mouse model for Lpn infections with traceable T cells of known specificities. However, up to now no Lpn-specific T cell epitopes have been identified, hampering the use of many standard immunological techniques usually applied in such studies. Therefore, we generated a Lpn-compatible plasmid encoding for ovalbumin (Ova) as a neoantigen. In order to be able to trace the expression of Ova and also follow the dissemination of Lpn in vivo, a GFP-fusion protein was designed. Lpn expressing the GFP-Ova fusion protein will allow to use existing T cell receptor (TCR) transgenic mice carrying TCRs specific for Ova to analyse the T cell response to Lpn infections in detail.

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P 30 Career opportunities in international health institutions

Dr. Joachim Hombach "Look closer" - Development of a novel microfluidics approach for single cell applications World Health Organization (WHO), Geneva, Switzerland

Philipp Stiefel, Julia Frunzke, Patrick Kiefer, Tomaso Zambelli, Julia Dr. Joachim Hombach started his career as a researcher in molecular and Vorholt cellular immunology, where he worked at the University of Zurich, Switzerland and the Max-Planck Institute for Immunology in Freiburg, Institute of Microbiology, ETH Zurich Germany. He holds a PhD from the University of Cologne, Germany. He also [email protected] holds a Master of Public Health from Johns Hopkins University, Baltimore, United States of America. Multiple peer-reviewed publications have emerged from his work. A typical culture of microorganisms looks quite homogenous to the human Before joining WHO, Dr. Hombach had a career in vaccine research eye, but a closer look on the level of gene expression, enzyme activity, or and implementation policy, with a focus on vaccines for the developing world. metabolism often reveals drastic cell-to-cell variation. Accordingly, biologists In this context, he had assignments as Director of vaccine policy at have realized that averaging data retrieved by analyzing bulks of cells can GlaxoSmithKline Biologicals S.A., and as Scientific Officer with the European obscure critical information about population heterogeneity. However, up to Commission. In the latter function, he was seminal in setting up the now most studies of cellular processes have been done using a whole European and Developing Countries Clinical Trials Partnership. He also bacterial population as starting material which results in an averaged readout served as board member of the European Malaria Vaccine Initiative. of these variations. This inaccuracy is primarily due to the lack of an easy He is now Coordinator for implementation research at the World method for single cell analysis. A novel instrument, called Fluidic Force Health Organization's Initiative for Vaccine Research (IVR). Microscope (FluidFM), could potentially be a great advantage in single cell research. The FluidFM combines the precise force-controlled positioning of an atomic force microscope (AFM) with nanofluidics. We plan to use a microchannel in the AFM cantilever to extract of cell material out of a single cell. Thereby, samples could be taken directly from the physiological environment avoiding negative influences caused by non-natural environments. The goal of my work is to apply the FluidFM to bacteria and couple it with ‘omics’ approaches such as transcriptomics in order to investigate cell-to-cell variability.

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Mechanisms of cell cycle progression and cell fate P 29 determination in Caulobacter crescentus

Prof. Urs Jenal Consequences of human Cytomegalovirus (HCMV) infection on the pro-coagulant activity of porcine Biozentrum, University of Basel, Basel, Switzerland Endothelial Cells (pEC)

Olmo Sonzogni, Lea Haeberli, Anne-Laure Millard, Nicolas J. Mueller The C. crescentus life cycle represents a paradigm for the developmental transition of bacteria from a motile to a sessile, surface‐attached life style. Division of Infectious Diseases and Hospital Epidemiology, Department of Each cell division produces a surface adherent stalked cell and a motile Medicine, University Hospital Zurich, University of Zurich swarmer cell. Whereas the newborn stalked cell immediately initiates a new [email protected] round of chromosome replication and cell division, the swarmer cell first differentiates into a stalked cell before it can start to divide. As a visual Due to the increasing demand of donor organs, xenotransplantation remains manifestation of this periodic cellular reprogramming, the cell poles the focus of intense research. In addition to immunological and physiological continuously change their identity. In the predivisional cell a flagellum and barriers, coagulation disorders have emerged as a main obstacle and have adhesive pili are assembled at the new cell pole originating from the last prompted great efforts in both deciphering cross-species incompatibilities round of division. Later, both organelles are lost during swarmer cell and in finding strategies to overcome them. differentiation and replaced with an adhesive holdfast and a stalk at the The success of transplantation is also frequently compromised by same pole. The seminar will introduce the audience to this bacterial model the occurrence of infections, among which the human cytomegalovirus system for cell differentiation and cell cycle control, and will then address (HCMV) most highly contributes to morbidity and mortality post transplant. some fundamental questions regarding the Caulobacter life cycle: What are After establishing a persistent latent infection in immunocompetent hosts, the mechanisms of Caulobacter cell fate determination and asymmetry, and this virus is frequently reactivated in immunosuppressed transplant recipients how is development coupled to cell cycle progression in this organism? and potentially triggers coagulation disorders and graft rejection. Recently, Recent findings suggest that the ubiquitous bacterial second messenger the capability of HCMV to infect porcine endothelial cells (pEC) has been cyclic di‐GMP plays a major role in orchestrating pole morphogenesis and in shown and well characterised. cell cycle control during the G1‐to‐S transition. The seminar will cover The aim of this ongoing in vitro study is to determine whether molecular, structural, and cellular aspects of key components involved in infection of pEC with HCMV can trigger coagulation in the context of c‐di‐GMP dependent regulation of C. crescentus development and cell cycle. xenotransplantation. First, the clotting times of human plasma after contact with HCMV-infected or non-infected pEC will be compared. Furthermore, we will thoroughly investigate both the expression and the activity of tissue factor Prof. Urs Jenal studied Experimental Biology at the ETH Zurich, (TF), the key factor in activation of the coagulation extrinsic pathway. Switzerland. He holds a PhD in Molecular Microbiology also from the ETH Expression of this protein will be characterized by western blot and by FACS Zurich. He then was a postdoctoral fellow at the ETH Zurich and at the analysis of infected and non-infected cultures. The activation level of TF will Stanford Medical School, USA. Since 1996 he is at the University of Basel, be investigated by measuring the ability of HCMV-infected and non-infected Switzerland, where he got his VENIA DOCENDI in Microbiology in the year pEC to activate Factor X (FX), a fundamental component downstream to TF 2000. in the coagulation pathway. He is now full professor at the University of Basel and Vice-director In the perspective of future clinical trials involving xenotransplants, it of the Biozentrum of the University of Basel. is crucial to achieve detailed understanding of potentially life-threatening His research focuses on signal transduction processes involving the post-transplant complications. Elucidating the effect of HCMV infection of the ubiquitous bacterial second messenger cyclic di-GMP. Using Caulobacter as porcine donor organs on coagulation mechanisms in the host will contribute a model he is interested in the molecular, structural, and cellular principles of to the development of new methods aiming to save lives by xeno- c-di-GMP signaling and its influence on cell differentiation and cell cycle transplantation. progression in bacteria.

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P 28 Scientific writing and publishing

Dr. Karin Dumstrei Fast screening of the pathogenicity of Burkholderia species by using non mammalian animal models The EMBO Journal, Heidelberg, Germany

Stephan Schwager, Susanne Uehlinger, Leo Eberl Dr. Karin Dumstrei received her PhD from the University of California Los

Angeles where she studied DE-cadherin mediated cell adhesion in Department of Microbiology, Institute of Plant Biology, University of Zurich Drosophila. She then went to the Max Planck Institute for Biophysical

Chemistry in Göttingen where she worked on primordial germ cell migration

in zebrafish. She joined The EMBO Journal as an editor in 2005. Species of the Burkholderia cepacia complex (Bcc) can cause diseases in plants, act as activator of opportunistic infection diseases in animals or in humans affected by immunodeficiency, chronically granulomatose or cystic fibrosis. However the mechanisms for virulence factors are poorly understood. To assess pathogenicity and virulence factors of Bcc strains and mutants the use of several animal models is required. In this study two new model organisms were used to understand and compare the results with the already existing model organism Caenorhabditis elegans. One was the greater wax moth larvae Galleria mellonella. The second organism is a close relative to C. elegans so called Panagrellus redivivus. This nematode has got the opportunity to be incubated at 37°C instead of 20-25°C which is the case for C. elegans. This is a big advantage to study human pathogens. There were found many correlations within the different models and also some host specific factors. Other ongoing studies are focused to understand the pathogenicity of Bcc in C. elegans. There, we try to understand how strains of the Bcc increase the mortality of C. elegans (by toxins, the amount of up taken bacteria, by the colonization of the gut and biofilm formation) and to find out, if C. elegans are able to distinguish between pathogenic and non pathogenic strains as food source. We showed that the use of different non mammalian model organisms are reliable, suitable, cost effective and fast tools to investigate virulence and pathogenic factors of different strains and mutants of the Bcc.

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P 27

Student Abstracts Effect of Sigma B and Sigma B controlled SpoVG on antibiotic resistance and capsule formation in Important notice: Staphylococcus aureus

This is a closed and private student meeting. None of the data Bettina Schulthess, Stefan Meier, Brigitte Berger-Bächi presented here or printed in this booklet are to be considered published. Institute of Medical Microbiology, University of Zurich [email protected]

Staphylococcus aureus is a leading cause of nosocomial and community- acquired infections. Its success as a pathogen is primarily due to a finely tuned time- and environment-dependent expression of virulence factors, and the ability to rapidly develop resistance to antibiotics. The alternative sigma factor Sigma B of S. aureus controls the expression of multiple genes, including virulence determinants and global regulators, promotes capsule production, and increases the resistance levels of methicillin-resistant (MRSA) and glycopeptide intermediate resistant S. aureus (GISA). Some of the genes influenced by Sigma B have a Sigma B promoter and can be regulated directly. However, many genes influenced by Sigma B are not preceeded by a Sigma B consensus sequence and must therefore be regulated indirectly. We show here that deletion of the Sigma B-controlled yabJ-spoVG operon, coding for potential downstream regulators of Sigma B, abolished capsule synthesis and reduced resistance in MRSA and GISA to the same extent as Sigma B inactivation. trans-complementation experiments with yabJ, spoVG and both yabJ-spoVG under the control of its own Sigma B promoter and a foreign constitutive promoter showed that SpoVG but not YabJ was required for complementation of yabJspoVG mutants. Neither of the constructs was able to restore the phenotypes of the sigB mutant, indicating that additional factors are involved. In conclusion, we suggest that the Sigma B controlled SpoVG is involved in capsule formation and antibiotic resistance in S. aureus. However, additional effectors are involved in the regulation of these phenomena via the alternative sigma factor Sigma B.

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P 26 Student Talks

Functional investigations of XoxF in M. extorquens AM1 using whole cell methanol conversion assays

Sabrina Schmidt, Patrick Kiefer, Philipp Christen, Nathanaël Delmotte, Julia Vorholt

Institute of Microbiology, ETH Zurich [email protected]

Methylotrophic bacteria possess the ability to use one-carbon substrates as sole source of carbon and energy. Methylobacterium extorquens AM1 belongs to the pink-pigmented facultative methylotrophs that colonizes plant surfaces where they benefit from methanol produced by plants. Methanol dehydrogenase is a well-characterized pyrroloquinoline quinone-dependent key enzyme for the utilization of methanol by M. extorquens AM1. The mxaFI genes encode the two-subunit methanol dehydrogenase in which the alpha subunit (MxaF) of the 2 2 tetramer has catalytic activity. XoxF is a MxaF paralog of unknown function in M. extorquens AM1. In order to obtain evidence for a potential role of XoxF, we performed growth analysis and methanol pulse experiments. A comparison of growth parameters of a xoxF deletion mutant and M. extorquens AM1 wild type during exponential growth phase, revealed similar yields during methanol consumption, but reduced specific methanol uptake rates and a reduced growth rate for the xoxF deletion mutant. In addition, pulse experiments in a bioreactor were performed with the xoxF deletion mutant and M. extorquens AM1 wild type. After growth on methanol and total substrate consumption, cells were starved and exposed to a methanol pulse. The specific methanol uptake rates were found to be lower for the xoxF deletion mutant than for the wild type. Taken together, these data suggest a role of XoxF in methanol oxidation in M. extorquens AM1.

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ST 01 P 25

Volatile-Mediated Impact of Bacteria on Plant Growth Characterization and Manipulation of Innate Immune Responses in the Domestic Cat Dirk Blom, Edward Connor, Florian Schiestl, Thomas Boller, Leo Eberl, Laure Weisskopf Céline Robert, Valentino Cattori, Marina Meli, Hans Lutz

Department of Microbiology, Institute of Plant Biology, University of Zurich Clinical Laboratory, Vetsuisse Faculty, University of Zurich [email protected] [email protected]

Bacteria are known to produce a wide spectrum of volatile organic In the prevention of pandemics due to emerging viral diseases, the compounds (VOCs). Recently, it has been discovered that these VOCs have manipulation of innate immunity holds the advantage of inducing in the host an influence on the growth of plants and fungi. In this study, we used a rapid defence mechanisms against a broad range of pathogens. The divided petri dish assay to screen 42 soil-borne bacterial strains for their domestic cat appears to be an ideal model to study innate immune volatile mediated effect on the growth of Arabidopsis thaliana and responses, as it is an out-bred species naturally affected by many viruses Rhizoctonia solani. The results show that the effects on plants for each strain that resemble in their biological properties those affecting humans. With the are depending on which medium they dare grown, ranging from six-fold objective to extend knowledge on the innate immune mechanisms in the growth promotion to killing. The effects observed in Rhizoctonia were much context of feline viral infection, several real-time PCR systems have been smaller. Additional tests revealed that the amount and density of bacteria developed for this species, to detect expression of genes already known to play an important role. Investigations are being done to assess the role of play an important role in early immune responses to viruses in mice and quorum sensing (QS, the ability of bacteria to sense their density and adapt humans. These novel tests enable to measure the implication of relevant their behavior accordingly) is involved in this density-dependent effects. The markers of innate immunity such as Toll-like receptors 3, 7, 8 and 9, various identity of the VOCs is being investigated using GC/MS analysis. The effect cytokines including interferon alpha and omega, as well as the intracellular of single compounds is being tested on the growth of Arabidopsis and the antiviral protein Mx. In a series of in vitro experiments using the cat as a changes in the plant’s physiology are currently under research. model, early immune responses to inoculation of feline primary immune cells and common cell lines with various viruses, namely the feline immuno- deficiency-, leukemia-, herpes-, calici-, and coronaviruses, are characterized. Furthermore, the immunologic responses elicited in these cells with a synthetic CpG-containing molecule are associated with the potential of this well-known immune response modifier to create resistance in vitro to the mentioned feline viral infections. Altogether, this work sheds light on early immune mechanisms to a broad range of viruses, and serves as basis for future experiments aimed at the prevention of viral infections not only in felids, but in humans and other species as well.

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P 24 ST 02

Viral env diversity and transmission clusters in primary Mass-spectrometric quantification of yeast N-linked HIV-infection: The Zurich Primary HIV infection Study glycoprotein site occupancy (ZPHI-study) Yao-Yun Fan Philip Rieder1, Beda Joos1, Viktor von Wyl1 , Herbert Kuster1, Christine Leemann1, Ulrich von Both1, Jürg Böni2, Sabine Yerly3, Institute of Microbiology, ETH Zurich Marek Fischer1, Huldrych Günthard1 and the Swiss HIV Cohort Study [email protected]

1 Univ Hosp Zurich, 2 NZR Univ Zurich, 3 Univ Hosp Geneva, Switzerland Asparagine-linked glycosylation is the most common post-translational modification of proteins catalyzed in eukaryotes by the multi-protein complex Background: To what extent viral heterogeneity can be transmitted during oligosaccharyltransferase (OST). However, database analysis showed PHI is a matter of debate. Here we aimed at analyzing HIV-env diversity in a approximately 70% of (NX(S/T); X ≠ P) sequons in proteins translocated into large number of PHI patients (pt). Moreover, we identified transmission the ER are actually glycosylated (1, 2), and the fundamental processes clusters by anonymously linking HIV-pol sequences from PHI pt with controlling site-specific N-glycosylation are poorly understood. We introduce participants of the Swiss HIV Cohort Study (SHCS). the MS-based site-occupancy analysis as a mean to measure OST activity in yeast genetic OST subunit mutant strains in order to clarify the functional role Methods: Cloning (16 clones/sample), and sequencing of the env C2V3C3 of each subunit in the aspect of enzyme activity and site-selectivity. domain was performed (total 1248 sequences). Pol sequences from 100 PHI For quantitative analysis of N-glycosylation site occupancy, stable pt were anonymously compared with those from the SHCS genotypic drug isotope labeling by amino acids (SILAC) and isobaric tag for relative and resistance database (4276 pt, 6500 sequences). Possible transmitters were absolute quantification (iTRAQ) can be applied for this study, which had identified by sequence clustering with bootstrap values >98% and genetic been widely used in proteomics field (3, 4). After endoglycosidase H distances <1.5% and confirmed by clonal analysis of the env region. digestion, glycosylated asparagines would be tagged with GlcNAc and the non-occupied ones were not. The site occupancy rate can be calculated as Results: Baseline env nucleotide sequence diversity from 78 pts ranged either the intensity / peak area of the occupied glycopeptides over the sum of from 0.11%-2.74% (median 0.48%) and neither correlated with viral load 5 2 7 all, or by the ratio reported from iTRAQ or SILAC results. (median 2.6*10 copies/ml; range 2.1*10 -4.0*10 ) nor with estimated time of We are aiming at the global analysis of site occupancy in yeast infection. Diversity of 10 pt (13%) was >1% despite short time after infection. glycoproteome and the glycoproteins covalently linked to the yeast Overall, pol sequences segregated into 28 distinct clusters which polysaccharide cell wall via glycosylphosphatidylinositol anchor remnants subsequently were confirmed by env sequences derived from plasma had been well studied by Endo H method along and by coupled with the samples from the SHCS repository. The identified transmission clusters quantitative MS labeling method. We are now focusing on microsomal ranged in size from 2-10 pt and comprised a total of 82 pt (42 PHI, 40 fractions so as to complete the yeast glycoproteome and quantitative SHCS). analysis of microsomal fractions can be further applied for ER-associated Conclusions: In the ZPHI-study we found 13% of pt with relatively high viral protein degradation dynamic studies. diversity shortly after HIV transmission. This suggests that transmission of multiple viral variants may occur more often than previously thought. A very 1. Ben-Dor, S et al., Glycobiology, 2004, 14, 95 high number of our PHI pt (42%) could be linked to possible transmitters. 2. Petrescu, A.-J et al., Glycobiology, 2004, 14, 103 3. Gingras, A-C et al., Nature Review, 2007, 8, 645 4. De Godoy LM et al., Genome Biology, 2006, 7, R50

- 54 - - 15 - 2nd MIM PhD Student Retreat 2009 Poster Abstracts

ST 03 P 23

Modulation of allergic airway inflammation by commensal The role of Antibody Dependent Cellular Cytotoxicity in bacteria HIV

1 1 2 2 Tina Herbst , Corinne Schaer ,Anke Sichelstiel , Ben Marsland , Kurt Lucy Reynell 3 4 1 Bürki , Kathy McCoy , Nicola Harris Institut of Medical Virologie, University Zurich 1 Environmental Biomedicine, ETH Zurich [email protected] 2 Global Health Institute, CHUV Lausanne 3 Institute for Laboratory Animal Science, University Zurich 4 Gastro-Intestinal Immunology, McMaster University, Hamilton, Canada Antibodies bound to viral proteins on the surface of infected cells, can, via [email protected] their constant regions, recruit immune effector cells bearing Fc receptors to destroy infected cells by a process termed antibody dependent cellular cytotoxicity (ADCC). Despite detection of ADCC mediating antibodies in Allergy is a debilitating disease mediated by exaggerated Th2-mediated patient sera, the impact of ADCC in controlling HIV infection is currently immune responses against common environmental antigens. The decline in unclear. the degree of exposure to microbes amongst industrialized populations has One major obstacle in studying ADCC activity has been the lack of been linked to the increasing prevalence of allergies in developed countries. suitable assay systems that allow detection of autologous ADCC activity and Epidemiological evidence indicates that intestinal microbes may provide the hence reveal the capacity of a given patient’s antibody response to trigger key to understanding this phenomenon. However, to-date no sound scientific ADCC lysis of cells infected with the patient’s own virus strain. During my evidence supporting their use exists, and the biological mechanisms thesis I aim to develop a suitable ADCC assay system, and to utilise this responsible for their proposed protective effect are still poorly understood. system to investigate autologous and heterologous ADCC activity elicited by We have developed an experimental system using gnotobiotic mice the patients’ immune system at different stages of the disease. By studying a by which we can investigate the biological mechanisms by which intestinal well characterized cohort of individuals with acute and chronic HIV infection, I bacteria impact on asthma and allergic diseases. aim to investigate if and to what extent ADCC activity may contribute to Our experiments demonstrate that intestinal bacteria provide viremia control. protection against experimental allergic asthma as evidenced by increased Natural Killer cells (NK) are the primary effectors of ADCC in viral allergic airway inflammation in OVA-challenged gnotobiotic mice compared infection. However, donor to donor variation in freshly isolated NK cytotoxic to SPF mice. Both CD4+ T cells and eosinophils were elevated in the activity is considerable, and development of a suitable FACS based ADCC airways of OVA-challenged gnotobiotic mice, and correlated with increased assay will involve standardising effector cell activity, and development of a local production of Th2 type cytokines and elevated serum IgE. These data staining regiment that allows clear distinction and quantification of target cell indicate that intestinal bacteria normally function to prevent exaggerated death in response to direct and antibody mediated cytotoxicity. Th2-mediated immunity and the development of allergic airway inflammation. We now aim to utilize this novel model to examine the mechanisms by which intestinal bacteria provide protection against allergy. The completion of these studies will provide valuable data that should help to refine therapies designed to boost immune competence and prevent allergic diseases.

- 16 - - 53 - 2nd MIM PhD Student Retreat 2009 Student Talks

P 22 ST 04

Role of two ECF sigma factors in Bradyrhizobium The role of NOD1 signaling in the immune response japonicum against Leishmania major

Luzia Reutimann, P. Stiefel, H. Hennecke, S. Mesa Koshika Yadava, R. Reissmann, B. J. Marsland

Institute of Microbiology, ETH Zurich Institute of Molecular Biomedicine, ETH Zurich [email protected] [email protected]

Oxidative stress plays an important role in the context of the plant infection The specificity of the innate immune response is determined by the process by bacteria. One important reason is that reactive oxygen species recognition of pathogen associated molecular patterns including bacterial cell belong to the plant defense system against intruders (1). Rhizobia must wall components such as peptidoglycan and lipopolysaccharide by Pattern therefore have special means to overcome this threat in order to establish Recognition Receptors. Nod like receptors are newly described cytosolic, root-nodule symbiosis. Lately, it has been shown in the Medicago sativa/ innate receptors composed of over 20 members including NOD1 and NOD2. Sinorhizobium meliloti symbiosis that H2O2 is not merely harmful for infecting NOD1 recognizes bacterial peptidoglycan moieties containing D-glutamyl- bacteria, but is actually essential for an optimal development of symbiosis meso-diaminopimelic acid (iE-DAP). It is an important regulator of immune (2). Bacteria possess a wide set of regulatory genes. One possibility to adapt responses against gram negative and some gram-positive bacteria. We to changes in the environment is differential gene expression by the use of assessed the importance of NLR signaling in protective responses against alternative σ factors, to which the extracytoplasmic function (ECF) σ factors the protozoan, Leishmania major using NOD1 knockout mice on the belong. ECF σ factors are known to be necessary for in the transcription of C57BL/6 background. We found that the deficiency in NOD1 resulted in a genes involved in many different tasks, including stress responses (3). delayed neutrophil and monocyte recruitment to the site of primary infection In Bradyrhizobium japonicum, the nitrogen-fixing soybean and consequently the control of parasite replication was compromised and it endosymbiont, an ECF sigma factor-coding gene was found to be highly disseminated systemically. Paradoxically, we detected increased induced in response to oxidative stress (4). Therefore, the idea was born that inflammatory cytokines and nitric oxide production in NOD1 knockout this σ factor might be involved in the regulation of the oxidative stress animals, presumably due to the enhanced parasite load. Overall the adaptive response. Additionally, a close homologue of that σ factor gene exists in the immune response of NOD1 knockout mice was unaffected and ultimately genome of B. japonicum. Mutants of both σ factor genes as well as a double they were able to control the infection. In L. major infection signaling through mutant were constructed. Stress tests with these mutants revealed a NOD1 appears to be important for orchestrating the recruitment of the early phenotypic difference to the wild type. The higher sensitivities of the mutants innate cells and its absence results in a dysregulated innate immune support the hypothesis that these σ factors play a role in the oxidative stress response and enhanced inflammation. response. Furthermore, an analysis of the regulon of these two σ factors with microarrays may yield information about the regulatory mechanisms and scope in cells.

[1] Apel and Hirt (2004) Annu. Rev. Plant Biol. 55:373–99. [2] Jamet et al. (2007) J. Bacteriol. 189:8741-5. [3] Helmann et al. (2002) Adv. Microb. Physiol. 46:47-110. [4] Mesa et al. (2009) in preparation

- 52 - - 17 - 2nd MIM PhD Student Retreat 2009 Poster Abstracts

ST 05 P 21

Loss of non-canonical NFkB signaling in immune cells Stromal cell specific transgenic mouse models for a aborts adaptive immunity. deepened understanding of the T cell zone stroma

Janin Hofmann, Melanie Greter, Burkhard Becher Lucas Onder1, Elke Scandella1, Christian Mayer2, Tim Sparwasser2 , Burkhard Ludewig1 Institute of Experimental Immunology, Deptartment of Pathology, University of Zurich 1 Institute of Immunobiology, Cantonal Hospital St. Gallen, St. Gallen [email protected] 2 Inst. für Mikrobiologie, Immunologie und Hygiene, Technische Universität München, München, Germany [email protected] The inflammatory canonical NFkB pathway is critically involved in virtually all aspects of inflammation in general. Yet, the role of the alternative, non- canonical NFkB pathway in inflammation and adaptive immunity remains The stromal cell network plays a key role in the development of lymphoid largely elusive. The alternative pathway is primarily mediated through the tissues and in lifelong maintenance of secondary lymphoid organ structure. NFkappa-B inducing kinase (NIK) which in turn leads to the phosphorylation Stromal cell- specific expression of the constitutive chemokines CCL13, and the cleavage of p100 to p52. Among the receptors engaging NIK is the CCL19 and CCL21 ensures controlled lymphocyte attraction and therefore LTbR, which is also required to form the anlage for secondary lympoid organizes the microenvironment of lymphoid organs. Many viral infections, tissues (SLTs). Due to a point mutation within NIK, alymphoplasia (aly) mice such as human immunodeficiency virus or measles virus infections are do not develop SLTs and are highly immunodeficient. However, while the associated with an immunopathological destruction of the lymphoid organ immunodeficiency of aly mice is widely held to stem from their developmental microenvironment. A recent study from our laboratory has shown that malformation, it has been overlooked, that the mutation of NIK itself could infection of mice with the lymphocytic choriomengitis virus leads to potentially lesion the development of immune responses. To verify this destruction of secondary lymphoid organ structure that is mediated by virus- notion, we generated a series of bone marrow chimeric mice (BMC) in which specific cytotoxic T cells. Furthermore, stromal cell - lymphoid tissue inducer the absence of SLTs was disconnected from the hematopoietic loss of NIK (LTi) cell interaction was shown to be crucial for restoration of adult function. We generated mice, which lack all SLTs, but are equipped with a secondary lymphoid organ integrity after acute LCMV infection (Scandella et normal systemic immune system (wt?aly), and conversely, mice with normal al, Nature Immunology, 2008). However, the cellular and molecular SLTs, but lacking NIK in all leukocytes (aly?wt). Surprisingly, we discovered mechanisms underlying LTi cell - stromal cell interaction and stromal cell that NIK is vital for the development of autoimmune disease, while SLTs (ie. function in general remain to be characterized in more detail. The aim of this LNs, spleen etc.) are essentially dispensable for cell-mediated immunity. We project is to analyze stromal cell functions in vivo using novel transgenic found that NIK is required for the polarization of effector T cells and that mouse models. To this end, the gene of the Cyclization Recombinase (Cre) TH17 and TH1 cells cannot be generated in the absence of NIK. Preliminary will be expressed under the control of constitutive promoters (ELC/CCL19 data implicate the involvement of NIK in a discrete and novel pathway and podoplanin/gp38) that are active in stromal cells. Future experiments will required for the formation of cell-mediated immune responses. involve Cre inducible reporter gene expression, targeted stromal cell depletion and stromal cell- specific antigen presentation in order to further characterize the role of stromal cells during viral infections.

- 18 - - 51 - 2nd MIM PhD Student Retreat 2009 Student Talks

P 20 ST 06

Modulation of coronavirus vector-induced immune response Pattern recognition versus inflammation in CD8+ T cell priming by dendritic cells. 1 1 1 2 1 Monika Nussbacher , Divine Makia , Luisa Cervantes , , Roland Züst , Klara 1 1 3 3 Kristin Eriksson , Reinhard Maier , Josef Janda , Frederic Levy , Daniel Wolfgang Kratky 3 3 4 1 Speiser , Piedro Romero , Pierre-Simon Rohrlich , Burkhard Ludewig , 1 Volker Thiel Institute of Microbiology, ETH Zurich [email protected] 1 2 Insitute of Immunobiology, Cantonal Hospital St. Gallen, Unidad de Investigatición Médica en Immunoquímica, Hospital de Especialidades, 3 Centro Médico Nacional Siglo XXI. IMSS. México. Ludwig Institute for Efficient priming of naïve T cells by dendritic cells (DCs) requires three 4 Cancer Research Lausanne, Université de Besançon, Besançon, France. signals: 1. The cognate peptide:MHC, 2. costimulation, and 3. immuno- [email protected] modulatory cytokines/etc. Only fully mature DCs which have recognized conserved microbial structures (PAMPs) via pattern recognition receptors The success of immunotherapeutical approaches strongly relies on specific (such as toll-like receptors) can provide all these signals. However, DCs can antigen targeting to dendritic cells (DCs) in an environment that provides also be activated in trans by inflammatory mediators secreted by PAMP- optimal immunostimulatory signals. In our research group a bio-safe triggered cells. Although DCs which were activated by this indirect mode coronavirus-based vaccine vector platform that delivers multiple antigens to deliver signal 1 & 2, they fail to secrete immunomodulatory cytokines such as professional antigen-presenting cells was developed. Murine coronavirus- IL-12. In the context of CD4+ T cell responses it has been shown that trans- based virus-like particles encoding epitopes from the lymphocytic activated DCs do not promote the differentiation of fully functional T helper choriomeningitis virus glycoprotein or human Melan-A, in combination with cells, even in the presence of mature non-presenting DCs. the immunostimulatory cytokine GM-CSF, selective targeted DCs in vitro and The aim of this project is to investigate the consequences of DC in vivo resulting in vector-mediated antigen expression, and efficient activation by inflammatory mediators for subsequent T cell priming. In maturation of DCs. In mice, a single application of only low doses elicited particular, we want to test whether the findings from experiments with CD4+ strong and long-lasting cytotoxic T-cell responses which provided protective T cells also apply for CD8+ T cells. antiviral and antitumor immunity. Furthermore, the efficient activation of In in vivo and in vitro experiments, we show that inflammatory human tumor-specific CD8+ T cells by mature DCs transduced with Melan-A- mediators are sufficient for DCs to upregulation of molecules which are recombinant human coronavirus 229E indicates that this novel vaccine crucial for migration and subsequent CD8 T cell priming. However, in sharp platform mediates the delivery of antigens and immunostimulatory cytokines contrast to directly activated (PAMP- triggered) DCs, indirectly-activated DCs to those cellular components of the immune system that initiate and maintain fail to produce proinflammatory cytokines, a hallmark of full DC activation. protective immunity. In preliminary studies we could show that these differences in DC As the application of GM-CSF already enhanced immunogenicity, activation seem to have major impact on CD8 T cell priming. Indirectly- we are now trying to further modulate the coronavirus vector-induced activated DCs prime CD8+ T cells insufficiently with respect to expansion, immune response with the reverse genetic setup of recombinant cytokine production and effector function. Coronavirus-based vectors expressing different immunostimulatory In ongoing studies we aim to study the differences between direct cytokines, namely Interleukin 15 (IL15), Interleukin 2 (IL2) and fms-like and indirect DC activation in more detail. We are specially interested in tyrosin kinase 3 ligand (Flt3L). aspects involved in positive and negative regulation of T cell function which With these approaches modulation of the immune response may explain the differential outcome on the level of CD8+ T cells. generated by this novel vaccine platform will be examined in viral and tumour models to get insight on the antigen specific CTL response, synergistic effects of the cytokines and protective as well as prophylactic vaccination approaches.

- 50 - - 19 - 2nd MIM PhD Student Retreat 2009 Poster Abstracts

ST 07 P 19

Induction of inhibitory CNS-derived and stimulatory blood- Mutational analysis of promising Bradyrhizobium derived DCs suggest a dual role for GM-CSF in the CNS japonicum genes in the context of a possible function in the soybean root-nodule symbiosis

Lysann Hesske, Christine Cretton, Adriano Fontana, Tobias Suter Valerie Murset, M. Koch, G. Pessi, H. Hennecke Department of Clinical Immunology, University Hospital of Zurich, University of Zurich Institute of Microbiology, ETH, Zurich [email protected] [email protected]

Dendritic cells (DCs) play a pivotal role in regulating beneficial immune Bradyrhizobium japonicum is a nitrogen-fixing bacterium which lives either in responses and in the initiation of autoimmune diseases such as multiple free-living conditions or in symbiosis with its soybean host plant. In order to sclerosis (MS) and its animal model experimental autoimmune find yet unknown genes important in symbiosis, a recently described whole- encephalomyelitis (EAE). We previously found that bulk DCs isolated from genome B. japonicum Affymetrix GeneChip was used [1]. A transcriptome the EAE-diseased central nervous system (CNS) inhibit T cell proliferation. In comparison of bacterial cells from soybean nodules with free-living cells this study, we analyzed closer the composition and function of CNS-DCs and shows that the expression of over 692 genes are induced in symbiosis [2]. the role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in Among the genes specifically induced in symbiosis is a gene which the development of these DCs. The major DC population at peak EAE is predicted to code for an ACC deaminase (blr0241). This enzyme degrades consists of a CD11b+ F4/80+ inflammation-associated DC subtype (inflDC) a precursor of ethylene. Thus, it could antagonize the inhibitory influence of which inhibited T cell proliferation whereas myeloid (myDCs) and lymphoid the phytohormone ethylene on nodule formation, development or function DCs (lyDCs) stimulated T cell expansion. However, only inflDCs derived from and therefore directly influence a signal coming from the plant. ACC CNS resident precursor cells showed the inhibitory phenotype. Interestingly, deaminase mutants of other rhizobia are known to be affected in their ability DCs isolated from the CNS of mice treated i.c. with GM-CSF showed the to nodulate [3,4]. same phenotypes. CNS-derived inflDCs prevented T cell activation and Another example is an operon of five genes (blr2131-2136). The cytokine production whereas blood-derived inflDCs and myDCs supported T preliminary annotation of these genes promises an interesting function, cell activation. Moreover, the inhibitory inflDCs are likely to be microglia- possibly the biosynthesis of a secondary compound. Mutants of both the derived, since microglia cells were able to inhibit T cell proliferation upon operon blr2131-2136 and the putative ACC deaminase are under treatment with GM-CSF. Despite the capacity of GM-CSF to induce CNS- construction. They will be tested for their growth behavior and for their ability derived inhibitory DCs, EAE-diseased mice treated with GM-CSF developed to infect plants and fix nitrogen. more severe EAE. GM-CSF thus has a dual role in the CNS: it directs DCs of an immune privileged site like the CNS towards an inhibitory phenotype and [1] Hauser et al., Mol. Genet. Genomics, 278:255-71, 2007 recruits peripheral DCs exhibiting pro-inflammatory functions. However, the [2] Pessi et al., MPMI 20: 1353–63, 2007 results indicate that the pro-inflammatory effect of GM-CSF dominates over [3] Uchiumi et al., J Bac, 186: 2439-2448, 2004 the GM-CSF-mediated induction of inhibitory inflDCs. [4] Ma et al., Can J Microbiol. 48:947-54, 2002

- 20 - - 49 - 2nd MIM PhD Student Retreat 2009 Student Talks

P 18 ST 08

New insights into chronic immune activation during HIV-1 Role of extracytoplasmic function (ECF) sigma factor σecfG infection: HIV-1 replication activates CD4+ T cells with in stress response and symbiotic efficiency in specificities for persistent Herpes viruses Bradyrhizobium japonicum Anna Haas1, Manuela Rehr1, Frederik Graw2, Peter Rusert3, Walter Bossart3, 4 3 4 1 Nadezda Masloboeva, Benjamin Gourion, Julia A. Vorholt, Hauke Herbert Kuster , Alexandra Trkola , Huldrych F. Günthard , Annette Oxenius Hennecke, Hans-Martin Fischer 1 Institute of Microbiology, ETH Zurich, 2 Institute of Integrative Biology, ETH 3 4 Institute of Microbiology, ETH Zurich Zurich, Institute of Medical Virology, University of Zurich, Division of [email protected] Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, University of Zurich [email protected] PhyR was recently described as a master regulator of general stress response in Methylobacterium extorquens. It is an unusual type of response BACKGROUND: Chronic immune activation is suggested to be a main regulator consisting of a receiver domain and an extracytoplasmic function cause of CD4+ T cell depletion during chronic HIV-1 infection. T cells (ECF) sigma factor-like domain. In the soybean symbiont Bradyrhizobium exhibiting an hyperactivated phenotype are not necessarily HIV-infected or japonicum, the phyR homologue (bll7795) is divergently expressed from an HIV-specific, however little is known about their antigenic specificities and operon encoding the sigma factor σecfG (Blr7797) and its putative anti-sigma the mechanism(s) by which HIV causes activation of CD4+ T cells during factor NepR (Bsr7796). chronic infection. Using deletion mutants and phenotypic assays, it was shown that METHODS AND FINDINGS: We analyzed the dynamics of HIV- and non- ecfG HIV-specific CD4+ T cell responses during HIV rebound after interruption of PhyR and the σ are involved in the general stress response of B. japonicum. In addition, both mutants had symbiotic defects on the plant hosts antiretroviral therapy (ART) by IFNgamma-ELISpot assay. In a cohort of 32 Glycine max (soybean) and Vigna radiata (mungbean). patients we longitudinally quantified CD4+ T cell responses to HIV-, to four persistent Herpes viral antigens (CMV, EBV, HSV1+2, VZV) and to two non- Microarray analysis revealed that PhyR and σecfG control highly persistent antigens (Streptokinase-Streptodornase (SKSD) and Tetanus congruent regulons suggesting both regulators are part of the same Toxoid (TT)) before and during viral rebound. We observed that the signalling cascade. Currently, selected EcfG/PhyR target genes are dynamics of the HIV-specific CD4+ T cell response were significantly mutationally analyzed to study their potential involvement in the general correlated to the dynamics of the responses specific for persistent Herpes stress response and/or symbiotic interaction with host plants. To this end, a viral antigens. This was seen in the absence of detectable reactivation of cluster of five functionally undefined genes which are organized in two herpes viral replication. In contrast, no correlation was detected between divergently oriented operons, was deleted. The resulting mutant strain is heat HIV-specific CD4+ T cell responses and responses directed towards non- sensitive, yet symbiotically proficient. Thus the EcfG/PhyR regulon persistent antigens. Notably, the dynamics of HIV- and Herpes viral antigen- comprises genes whose products are crucial for free-living stress conditions, specific CD4+ T cells responses correlated with expression levels of the symbiosis or both. activation marker CD40 on dendritic cells (DCs). Finally, we are also studying a putative histidine kinase which might CONCLUSIONS: These data suggest that HIV replication costimulates be involved in PhyR-/NepR-mediated signalling to EcfG. Preliminary results activation of CD4+ T cells specific for persistent Herpes viral antigens via indicate that this protein is important for free-living growth as deduced from activation of DCs. CD4+ T cells specific for antigens of the Herpes virus the highly impaired growth of a respective mutant strain. family are abundant in latently infected individuals. We propose that a large

proportion of activated T cells during untreated HIV infection may be specific Reference: for Herpes viral antigens and identify a novel mechanism contributing to Gourion et al., Mol. Microbiol., 73:291-305, 2009 chronic immune activation in untreated HIV-1 infection.

- 48 - - 21 - 2nd MIM PhD Student Retreat 2009 Poster Abstracts

ST 09 P 17

Antibodies against Epidermodysplasia Verruciformis- HIV-1 entry and its inhibition associated Canine papillomavirus 3 detected by ELISA in sera of dogs from Europe and Africa Axel Mann

Christian. E. Lange1,2, K. Tobler2, C. Favrot1, M. Müller3, J.O. Institut of Medical Virology, University of Zurich 4 2 [email protected] Nöthling , M. Ackermann

1 Clinic for Small Animal Internal Medicine, Dermatology Unit, Vetsuisse Virus entry into target cells is the first step in the viral life-cycle and hence an Faculty, University of Zurich important checkpoint for interference with viral infection by the host immune 2 Institute of Virology, Vetsuisse Faculty, University of Zurich defense, vaccines and pharmaceutical agents. 3 Applied Tumor Virology, Deutsches Krebsforschungszentrum, Heidelberg, Human Immunodeficiency Virus 1 (HIV-1) entry is mediated by the Germany viral envelope glyco-proteins gp41 and gp120 and is initiated through the 4 Faculty of Veterinary Science, University of Pretoria, South Africa interaction of gp120 with the target cell CD4 receptor. A following cascade of [email protected] conformational changes within the envelope glyco-proteins, involving

engagement of a co-receptor, leads to fusion of the viral and cellular

membranes and productive infection. The role of Papillomaviruses (PVs) in the development of canine cancers is The overall aim of this thesis project is to dissect the modes by controversial. However, recently a novel canine PV (CPV3) was detected in which antibodies and antiviral agents can inhibit viral entry. To this end a a dog affected with a condition reminiscent of Epidermodysplasia more detailed understanding of the cellular and viral moieties involved in viral Verruciformis (EV). entry is required, in order to gain new insights into the viral entry mechanism The aim of the present study was to investigate into the itself and to define new epitopes for neutralizing antibodies and entry seroprevalence of CPV3 by using generic ELISAs for the detection of inhibitors. antibodies against either canine oral papilloma virus (COPV) or CPV3. Critical components that determine the efficacy of viral entry, both Therefore, the capsid proteins of both PV types were expressed as GST- within the viral particle as well as properties of the target cell, are studied in fusion protein antigens and adsorbed to glutathione casein-coated ELISA various in vitro infection systems. plates. With the aim of gaining a more detailed concept of virus surface After showing that PV type-specific antibodies could be detected in glycoprotein function, DARPin binding proteins specific for the viral envelope the sera from dogs with confirmed COPV or CPV3 infection, CPV3- as well proteins are selected using ribosome display. Identified binders will be used as COPV-seropositive dogs were detected in two sets of canine sera to map epitopes on surface glyco-proteins that are critical for virus infectivity collected in Switzerland and South Africa, respectively. We found specific and are thus targets for future vaccines and drugs. antibodies against COPV as well as against CPV3 among the tested sera and also a large number being positive for both antigens. The seropre- valence for PV antibodies was hereby with 21.9% (COPV) and 26.9% (CPV3) among the tested dogs from South Africa higher than among the dogs from Switzerland with 10.5% (COPV) and 1.3% (CPV3). Our data suggest a need for further CPV-related seroepidemiological surveys in different countries, especially in the context of clinical manifestations and possible breed predispositions. For this purpose, the newly developed ELISAs can be a useful tool.

- 22 - - 47 - 2nd MIM PhD Student Retreat 2009 Student Talks

P 16 ST 10

Altered NK cell Development and Enhanced NK Cell- Mediated Resistance to MCMV in NKG2D-Deficient Mice

Biljana Zafirova1, Sanja Mandarić1,*, Ronald Antulov1, Astrid N.N. Krmpotić1, Helena Jonsson2, Wayne M. Yokoyama2, Stipan Jonjić1, Bojan Polić1

1 Department of Histology and Embryology, University of Rijeka School of Medicine, B. Branchetta 20, HR-51000 Rijeka, Croatia 2 Howard Hughes Medical Institute, Rheumatology Division, Washington University Medical Centre, 660 S. Euclid Ave, Box 8045, St. Louis, MO 63110 U.S.A. * Present address : Institute of Microbiology, ETH Zurich, Wolfgang-Pauli Strasse 10, 8093 Zurich, Switzerland [email protected]

NKG2D is a potent activating receptor on NK cells which acts as a molecular sensor for stress exposed cells expressing NKG2D ligands such as infected or tumor transformed cells. Although NKG2D is expressed on NK cell precursors, its role in NK cell development is still not known. We have generated NKG2D-deficient mice by targeting the Klrk-1 locus. Here we provide evidence for dual role of NKG2D in the physiology of NK cells: one, rather regulatory, implicated in their development, homeostasis and survival, and, other, important for exerting their effector functions. We observed the following consequences of NKG2D deficiency on the NK cell development and homeostasis: a) faster division of NK cells ; b) perturbation in size of NK cell subpopulations and, c) their augmented sensitivity to apoptosis. As expected, NKG2D-/- NK cells were considerably less responsive to tumor cell targets expressing NKG2D ligands, thus confirming the important role of NKG2D for exertion of their effector functions NKG2D-/- mice however showed an enhanced NK cell-mediated resistance to MCMV infection as a consequence of NK cell dysregulation. Altogether, these findings provide evidence for yet unknown regulatory function of NKG2D in NK cell physiology.

- 46 - - 23 - 2nd MIM PhD Student Retreat 2009 Poster Abstracts

ST 11 P 15

Phylogeny of hemotrophic mycoplasmas – main focus on Negative Regulation of the IFN-α signaling by the hemotrophic mycoplasmas in horses Influenza Matrix Protein (M1)

Sarah Maria Dieckmann1, Katrin Groebel1, Michael Dieckmann2, Thomas Ludersdorfer, Jovan Pavlovic Ludwig E. Hoelzle1 Institute of Medical Virology, University of Zurich 1 Institute of Veterinary Bacteriology, University of Zurich [email protected] 2 Equine Practice Beekenhof, Bommelsen, Germany [email protected] Complex organisms have evolved sophisticated mechanisms to prevent and control infection by various pathogens or viruses. Among these mechanisms Hemotrophic mycoplasmas (HM) are specialized bacteria, which usually the interferon (IFN) system, which is part of the innate immune system, parasitize on the surface of erythrocytes, but they can also occur represents the first step of defense against viral infection in vertebrates. intracellularly. Thus, they cause damages and deformations of erythrocytes Secretion of IFNs prepares uninfected cells for combating oncoming virus. resulting in various symptoms (e.g. hemolytic anemia, icterus and infertility). Products of IFN-stimulating genes (ISGs) mediate an antiviral action. The Up to now there are at least 14 HM isolates described, which were in some invading virus will be impaired in infection and replication, which provides extent formerly classified as Eperythrozoon or Haemobartonella within the time for an adaptive immune response. Influenza A is counteracting the IFN group of Rickettsia. Due to 16S rRNA analyses they have been reclassified system by inhibiting the type I IFN production through the non-strucural viral as Mycoplasma species within the group of Mollicutes. protein 1 (NS1). Paradoxically the NS1 deletion mutant of influenza virus In horses, infections with HM have not been observed so far, could still propagate in the host cell. This observation suggested a second although this kind of infection has been described for various animal species mechanism by which the virus was able to circumvent the cellular defense (e.g. pig, cattle, sheep, dog and cat). Horses with anemia of unknown mechanisms. etiology were presented to equine practices in Northern Germany. They This study demonstrates an antagonistic activity of the M1-protein of showed unspecific clinical signs, which also have been reported in Influenza A in the IFN-α/ß signaling pathway. An IFN-stimulated response combination with HM infections in other animal species (e.g. reduced element (ISRE)-promoter-driven reporter system was used to detect the condition, lack of performance, loss of weight and apathy). negative regulation of the IFN signaling cascade by the M1-protein. This Bacterial structures, similar to known HM infecting other animals, effect was independently confirmed by direct measurement of MxA levels in were observed by fluorescence and scanning electron microscopy. Influenza A infected cells. These data identified the IFN-α/ß signaling Sequencing of 16S rRNA PCR products resulted in 16S rRNA fragments cascade as a target of the virus. The M1-protein did not exert an effect on showing homologies of 83-94% homologies to well known HM species. A IFN transcription, suggesting that in contrast to the Influenza NS1 protein, it phylogenetic analysis was performed. Two subclusters could be clearly interfered with the action rather than the production of IFN-α/ß. Furthermore observed within the cluster of HM. Interestingly, the first cluster comprises M1 was shown to inhibit the phosphorylation of several IFN-α/ß signaling Mycoplasma species formerly known as Eperythrozoon species and the proteins. In conclusion, this study elicited an alternative mechanism of second cluster comprises Mycoplasma species formerly known as influenza A to counteract the innate immune response. Haemobartonella species. The novel infective agent isolated from horses belongs to the second cluster.

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P 14 ST 12

CD40 expression is required for cytotoxicity of alloreactive Investigation of Mycobacterium tuberculosis zinc CTLs against murine and human renal tubular epithelial metalloprotease Zmp1 cells Agnese Petrera, Peter Sander, Janine Hähnlein, Beat Amstutz Anna Katharina Kraus, Astrid Starke, Jin Chen, Rudolf Wüthrich, Thomas Fehr, Ying Wäckerle-Men Institute for Medical Microbiology, University of Zurich [email protected] Division for Nephrology and Institute of Physiology, University of Zurich [email protected] Despite five decades of control programmes and the availability of efficacious drugs, tuberculosis (TB) is still a major cause of mortality in the Allo-reactive cytotoxic T lymphocytes (Allo-CTLs) induce tubulointerstitial world. M. tuberculosis, the causative agent of this disease, parasitizes injury during kidney allograft rejection. Renal tubular epithelial cells (RTECs) macrophages by arresting maturation of phagosomes into phagolysosomes, do not express B7, but constitutively express CD40. The aim of this study modulating host-cell apoptotic pathways and suppressing the host immune was to investigate the role of CD40 for mouse and human allo-CTL-RTEC response. interaction. Recently, a new strategy of mycobacterial phagosome maturation Murine responder cells (either whole splenocytes or purified CD8+ T arrest relying on the M. tuberculosis -zinc metalloprotease Zmp1 has been cells) were restimulated for 5 days in vitro with either autologous, allogenic described (Master et al., 2008). M. tuberculosis zmp1 deletion mutants (B6, H-2b) or third party (B10.RIII, H-2r) splenocytes and then tested against display reduced survival in mice. Zmp1 is required for optimal mycobacterial primary RTECs from B6 or CD40-/- mice (both H-2b) in a standard 51Cr survival in infected macrophages, as phagosomes harboring Zmp1 deficient release assay. RTECs were prestimulated with IFN-β and IFN-γ to induce mycobacteria undergo enhanced maturation into phagolysosomes. This high surface expression of MHC class I and II. suggests that Zmp1 is required to maintain mycobacterial phagosome CBA-splenocytes showed a high cytolytic activity against B6 RTECs maturation arrest. Most importantly, vaccination of mice with BCG zmp1 when restimulated with B6 splenocytes. In contrast, no killing was detected deletion mutants decreases lung pathology and increases survival compared by responders restimulated with autologous or third party splenocytes. to the protective efficacy of standard BCG (Böttger, submitted for Addition of a partially activating anti-CD40 mAb (FGK4.5) did not significantly publication). increase killing. To ensure that the observed cytotoxicity was indeed CD8+ T The aim of this PhD project is the characterization of M. tuberculosis cell-mediated and CD40-dependent, we repeated this experiment using Zmp1 and the identification of substrates of this metalloprotease. In order to MACS-purified CD8+ T cells and compared wild-type with CD40-/- target clarify whether Zmp1 acts directly on the host proteins or if it affects the host cells. We did not see any killing on CD40-/- target cells, and adding the same immune system indirectly by modulating endogenous substrates, a anti-CD40 mAb again did not increase killing of WT targets. proteomics approach has been chosen. Putative endogenous or host protein We then investigated the role of CD40 for cytotoxicity of human allo- substrates may be detected with a 2D-gel experiment and iTRAQ approach. CTLs against a human RTEC cell line (HK-2). No killing was seen under These results, along with the characterization of enzymatic properties and standard conditions. However, when an activating anti-CD40 mAb was the crystallographic structure of Zmp1, will complete the understanding of added, substantial killing of HK-2 cells was observed. this key virulence factor, making it a potential target for vaccine design or Thus, CD40 is an important costimulatory molecule for the drug intervention. interaction between allo-CTLs and RTECs in mouse and man. Strategies to block or down-regulate CD40 on RTECs might be therapeutically useful to prevent kidney allograft rejection.

- 44 - - 25 - 2nd MIM PhD Student Retreat 2009 Poster Abstracts

ST 13 P 13

Triggers and Tricks of Encystation in Giardia lamblia Isolation and characterization of new cross-subtype neutralizing antibodies against Influenza A virus Laura Morf1, C. Spycher1, H. Rehrauer2, C. Aquino Fournier2, H. G. 3 1 Morrison , A. B. Hehl Ines Kohler, Raphael Hafen, Lars Hangartner

1 Institute of Parasitology, University of Zurich, Institute of Medical Virology, University of Zurich 2 Functional Genomics Center Zurich, [email protected] 3 Marine Biological Laboratory, Woods Hole, MA, USA [email protected] Influenza viruses still represent a significant and worldwide health threat. Their ability to escape the immune system by antigenetic variability makes Giardia lamblia is protozoan parasite causing sever and often persistent the development of vaccines eliciting immunity to multiple influenza strains diarrhea worldwide. It has a simple two stage life cycle consisting of the and subtypes very challenging. Influenza A viruses are subclassified by its dividing, flagellated trophozoite causing giardiasis and the highly infectious two major surface proteins: first hemagglutinin, which mediates cell entry by cyst. Trophozoite colonize and reproduce in the upper and middle part of the binding to sialic acid receptors on the host cell and second neuroaminidase small intestine and while descending into the ileus cyst formation is induced promoting cell exit. The variability of the influenza surface proteins arises and cysts are shed into the environment by feces. from domains that can easily be altered without affecting the virus’ ability for The triggers of encystation in vivo are widely unknown but several cell attachment, entry, or release. specific conditions are described to trigger differentiation in vitro. Encystation The need for a vaccination strategy that induces long-lasting and entails synthesis, maturation and regulated secretion of the cyst wall (CW) cross-reactive immunity is evident. In this work, we aim at defining and material to form the CW, which is highly environmental resistant and protects characterizing epitopes in influenza hemagglutinin that can elicit protective the parasite within. This highly effective biopolymer appears to have a very but strain- and subtype independent antibody responses. To this end, we will simple composition and the hypothesis is that regulation of few genes is isolate and characterize new cross-subtype neutralizing monoclonal involved in the complex process of encystation. In this work we aim to better antibodies against influenza A from healthy donors. In order to assess the understand the structural components of the CW, the mechanism and width of the neutralizing activity of these monoclonal antibodies, we will kinetics of transcriptional induction and the molecular triggers of encystation generate recombinant attenuated viruses including all influenza A in vitro and in vivo. hemagglutinin subtypes. Antibodies emerging from this screen will enable us We induced encystation in vitro and used whole-genome to define more conserved epitopes within the hemagglutinin that can confer microarrays to monitor the transcriptional profile at 45min, 3h and 7h post heterotypic and heterosubtypic protection. This knowledge will then be used induction. Our results reveal a two step transcriptional response with around to develop immunogens that target those heterosubtypic epitopes. 50 genes that are upregulated during the process. Because all in vitro Our strategy is a promising approach for broad-spectrum protection protocols for induction likely produce significant off-target effects we against seasonal and pandemic influenza viruses and is therefore of great repeated the 7 hour analysis using a different protocol. Comparative global public health interest. analyses of these datasets revealed a core group of about 13 genes that are induced independently of the trigger used and were classified as bona fide encystation genes. Within this group, bioinformatic analysis confirmed the overrepresentation of an upstream transcription factor binding element (Myb binding element).

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P 12 ST 14

The use of insects in flu vaccination The role of persistent viral infections in immune senescence Raphael Hafen, Ines Kohler, Lars Hangartner Andrea Mekker, Lea Haeberli, Vincent Tchang, Urs Karrer Institute of Medical Virology, University of Zurich

[email protected] Division of Infectious Diseases and Hospital Epidemiology, Department of

Internal Medicine, University Hospital Zurich, University of Zurich

[email protected] The general goal of our group is to find antigens on the flu virus Influenza surface protein Hemaglutinin. These antigens should be conserved in all influenza serotypes to allow a subtype unspecific vaccination strategy. In Aging of the immune system, commonly termed immune senescence, is order to discover such antigens we need to produce reasonable amounts of clinically associated with an increased frequency and severity of infectious the different Hemaglutinins from each virus serotype. For this purpose we diseases and an increased incidence of cancer, chronic inflammatory rely on the Baculovirus expression system. This system is based on the disorders and autoimmunity. Immune senescence is also linked to age- infection of Spodoptera frugiperda insect cells (Sf9) with the Autographa associated accumulation of memory and effector T cells reducing the californica nuclear polyhedrosis virus (AcNPV). This virus is engineered in a `immunological space`. Upon infection with Cytomegaloviruses (CMV) in way that the gene of interest can be inserted in its genome. Upon viral humans and mice permanent immune surveillance by virus-specific T cells is infection of a cell the protein is expressed. AcNPV infection of Sf9 cells mandatory for the control of the viral infection. Memory inflation, i.e. a pattern results in the shut down of host gene expression allowing for a high rate of + of longitudinal accumulation of certain epitope-specific CD8 T cells, is a recombinant mRNA and protein production. Recombinant proteins can be peculiarity of the T cell response against these persistent viruses. To produced at levels ranging between 0.1% and 50% of the total insect cell maintain viral control the host has to allocate substantial immunologic protein. We test two different systems and want to establish the one that resources and this effort seems to increase with age. To test the hypothesis suits our needs for large-scale protein production the best. whether and how persistent viral infections might propagate immune

senescence we are establishing a mouse model for infection enhanced immune senescence (IEIS). Subsequent infections with different persistent viruses and prime-boost approaches were preformed to enhance the pressure on the immune system to control persistent viruses resulting hypothetical in an early immune senescence. Removal of the murine thymus abolished the recruitment of new naïve T cells resulting in an imbalance between the naïve and memory T cell pool, a characteristic of immune senescence. To test the different approaches for the IEIS-model proving our hypothesis, MCMV-infected mice will be challenged with several pathogens and the immune response, especially the protection, will be investigated. According to our hypothesis young persistent infected mice will induce less protection to a new infection similar to aged mice.

- 42 - - 27 - 2nd MIM PhD Student Retreat 2009 Poster Abstracts

ST 15 P 11

Cytomegalovirus and immune senescence: Vaccination efficacy is impaired in CMV-infected elderly individuals The role of PD-1 during chronic LCMV infection:

Kerstin Wanke1, A. von Braun1, L. Häberli1, W. Bosshart2, F.X. Helge Frebel Heinz3, L. Held4, R. Steffen4, U. Karrer1 Institute of Microbiology, ETH Zurich 1 Division of Infectious Diseases, University Hospital of Zurich, 2 Institute of [email protected] Medical Virology, University of Zurich, 3 Institute of Virology, Medical University, Vienna, Austria, 4 Institute for Social and Preventive Medicine, University of Zurich, [email protected] Though the immune system is capable of clearing most types of viral infections, there are numerous viral pathogens that cause infections being Due to the age associated decline in immune function also called immune slowly or never resolved and therefore leading to a protracted or chronic senescence, elderly individuals are more susceptible to infectious diseases infection of the host. While the causes of the immune system’s inability to and show a poor response to vaccination. Recent evidence implicates that resolve distinct viral infections still are matter of intense investigation, it is persistent infection with Herpesviruses in general and human known that endogenous immunoregulatory mechanisms can contribute to the Cytomegalovirus (CMV) in particular might propagate earlier onset of failure of the immune system to control these infections. immune senescence. By infecting mice with the arenavirus LCMV (Lymphocytic To directly investigate whether and how persistent CMV infection choriomeningitis virus), a resolved or chronic infection can be induced, influences immune senescence, we performed a prospective controlled depending on the respective viral strain and the dose of injection. Chronic vaccination trial in a group of CMV-positive (N=68) and CMV-negative LCMV infection is marked by the functional exhaustion of CD8 and CD4 T (N=69) healthy elderly individuals using the vaccine against Tick Borne cells which is characterized by a strong reduction of cytokine secretion (IL-2, Encephalitis Virus (TBEV). The vaccination efficacy was used as surrogate TNF-α and IFN-γ), a reduced capability to proliferate and an impaired marker for immune senescence. Vaccine induced TBEV-specific immunity development of T cell memory. As T cell responses are pivotal for the control was analysed longitudinally by ELISA and neutralisation assay for antibodies of LCMV infection, the observation of functional exhaustion of T cells in and by IFNγ-EliSpot for T cells. T cell immunity against CMV and other chronic infection might be intimately linked to the failure of antiviral control. human Herpesviruses was quantified by IFNγ-EliSpot as well and correlated This study aims at investigating the impact of different with the vaccine induced immune response against TBEV. Polychromatic immunoregulatory pathways on the development and maintenance of T cell flow cytometry was used for a detailed phenotypic and functional analysis of exhaustion during chronic LCMV infection with a special focus on the differences in the T cell and dendritic cell compartment. inhibitory PD-1/PD-L1 pathway. An LCMV infection which normally leads to Persistent infection with CMV leads to a significant reduction in chronicity in wt mice is lethal in PD-1 ko mice within 7 days. The question is antibody as well as antigen-specific T cell response against TBEV vaccine. addressed which cellular mechanisms account for this lethality. Preliminary There is a negative correlation between protective immunity after TBEV- data suggest an essential role of CD8 T cells, as antibody-mediated vaccination and the frequency of T cells directed against common depletion prior to infection results in a prolonged survival. However, on the Herpesviruses and against CMV, implicating a T cell based mechanism of day of decease, the frequency and the functionaltiy of LCMV-specific CD8 T interference. In CMV-positive individuals naïve T cell numbers are reduced cells in chronically infected PD-1 ko mice is comparable to that in wild type while memory T cells with a late differentiation phenotype are increased, thus mice, questioning a direct CD8 T cell-mediated immunopathology. showing a trend towards further differentiation. However, T cell functionality is similar in both groups. In conclusion, our data demonstrate that CMV-infection impairs protective immunity after TBEV vaccination in healthy elderly, potentially by a T cell based mechanism that propagates immune senescence.

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P 10 Poster Abstracts

Regulatory T cells control CNS-infiltrating of autoreactive T cells during viral infection without affection the antiviral immune response

Sonja Firner1, Luisa Cervantes-Barragan1, Ingo Bechmann2, Tim Sparwasser3, Ari Waisman4, Volker Thiel1, Burkhard Ludewig1

1 Research Department, Kantonsspital St. Gallen, St. Gallen, 2 Institute of Clinical Neuroanatomy, Johann Wolfgang Goethe-University, Frankfurt am Main, Germany, 3 Institute for Medical Microbiology, Immunology, and Hygiene Technische Universität München, Munich, Germany, 4 Medical Department, Johannes Gutenberg-University Mainz, Mainz, Germany. [email protected]

Regulatory T cells are essential for suppressing immune responses to autoantigens and therefore help to prevent autoimmunity. During viral infections in the CNS, an indiscriminate regulation of T cells can prevent autoimmune diseases but could also impair the control of viral replication. We analyzed here the impact of regulatory T cells in the control of T cell infiltration to the CNS, virus-induced CNS pathology, and viral clearance using the mouse hepatitis virus (MHV) A59 intranasal infection model; a virus infection that leads to encephalitis and demyelination. MHV infection of “depletion of regulatory T cell” (DEREG) mice, where regulatory T cells can be transiently depleted by diphtheria toxin injection, revealed that the lack of regulatory T cells during MHV infection leads to an increased T cell infiltration and pathology in the CNS. However, antiviral T cells response were not affected by the depletion of FoxP3+CD4+ T cells indicating that regulatory T cells control infiltration of T cells to the CNS without impairing or delaying the antiviral immune response. Moreover, in MHV infected mice, adoptively transferred-myelin oligodendrocyte glycoprotein (MOG35-50)- specific CD4+ T cells proliferated in cervical lymph nodes and migrated to the CNS, suggesting that MHV infection in the CNS induces the activation of self-reactive T cells which are controlled by regulatory T cells to reduce the risk of developing inflammatory CNS disease.

- 40 - - 29 - 2nd MIM PhD Student Retreat 2009 Poster Abstracts

P 01 P 09

Legionella Vacuole Proteome of Bacterial Mutant Strains

Ivo Finsel, Hubert Hilbi Aebischer David Division of Cell Biology, Institute of Zoology, University of Zurich [email protected] [email protected]

Legionella pneumophila, the causative agent of the pneumonia Legionnaire's disease, is an opportunistic human pathogen. Intracellular growth of the Gram-negative bacterium within phagocytic cells depends on formation of a unique "Legionella containing vacuole" (LCV). LCVs avoid fusion with lysosomes, acquire endosomal markers and associate with mitochondria, early secretory vesicles and the endoplasmic reticulum. Uptake of the bacteria, LCV formation and intracellular growth require the bacterial Icm/Dot type IV secretion system (T4SS), which translocates more than 120 different "effector" proteins into the host cells. The role of Icm/Dot substrates has been difficult to define, because the effectors seem to be functionally redundant, and even the deletion of whole effector families did not show a phenotype in terms of intracellular growth. Recently, we established a method, which allows the isolation of LCVs from Dictyostelium discoideum, a social soil amoeba. The purification scheme involves immunomagnetic separation with an antibody against SidC, an effector that selectively binds to phosphatidylinositol-4-phosphate (PtdIns4P) exclusively on LCVs, and a subsequent gradient centrifugation step. The proteome of enriched LCVs was determined by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). We currently purify LCVs harbouring L. pneumophila strains lacking effector proteins and determine their proteome. A comparison with LCVs harbouring wildtype L. pneumophila should provide insights into the role of the deleted effector protein. Furthermore, using quantitative proteomics, we will analyze the changes in host protein levels on the vacuoles. To verify the findings of the MS analysis, the different protein profiles of LCVs harbouring mutant or wildtype L. pneumophila will be confirmed by fluorescence microscopy.

- 30 - - 39 - 2nd MIM PhD Student Retreat 2009 Poster Abstracts

P 08 P 02

IL-1a expression and secretion Streptococcus mutans pknB is involved in regulation of genetic competence, bacteriocin production and cell-wall Antonia Fettelschoss metabolism

Unit for Experimental Immunotherapy, Department of Dermatology, Liliana Danusia Banu1, Georg Conrads2, Hubert Rehrauer3, Elaine University Hospital of Zurich, University of Zurich 4 1 Allan and Jan R. van der Ploeg [email protected]

1 Institute of Oral Biology, University of Zurich, 2 Division of Oral Microbiology

and Immunology, RWTH Aachen University Hospital, RWTH Aachen, IL-1a and -b are important proinflammatory cytokines. In contrast to IL-1b, IL- Germany, 3 Functional Genomics Center Zurich, UZH/ETH Zurich, 4 Eastman 1a can be expressed on the cell surface and does not require cleavage to be Dental Institute, London, United Kingdom, [email protected] biologically active. However, the exact mechanisms of IL-1a cell surface expression and of IL-1a secretion are unknown. Dental caries is caused by microorganisms that metabolize carbohydrates We are investigating which factors influence and regulate IL-1a present in our diet to lactic acid. Streptococcus mutans, the major etiological surface expression and secretion using an in vitro model involving LPS cause of this disease, thrives in an environment which is constantly stimulated monocytes and macrophages. At different time points the amount changing. To detect and to adapt to these changes, bacteria employ different of intracellular IL-1a, surface IL-1a, and IL-1a secretion are measured by signal transduction systems. Two-component systems (TCS) comprise a FACS analysis, and ELISA, respectively. membrane-located sensor histidine kinase and a response regulator which is IL-1a is expressed in various tumor types, and has been shown to localized in the cytoplasm. Serine/threonine protein kinases (STPKs) consist correlate with tumor progression. Therefore we are evaluating the relative of an N-terminal kinase domain in the cytoplasm, a central membrane- biological importance of the different IL-1a forms in tumor cells, i.e. the located domain and a C-terminal sensory domain located extracellularly. intracellular, the cell surface, and the secreted form. We have generated Upon detection of a signal outside of the cell, STPKs are auto- different EL4 cell lines expressing either intracellular IL-1a, surface IL-1a, or phosphorylated. The phosphate moiety is subsequently transferred to serine only secreted IL-1a. These different tumor cell lines shall be compared in or threonine residues of target proteins, resulting in a change of activity of tumor mouse models using either C57BL/6 or IL-1RI-/- mice. We will quantify these proteins. Dephosphorylation of an STPK is thought to be carried out by tumor growth, angiogenesis and metastasis formation. their cognate serine-threonine protein phosphatase (STPP). S. mutans contains 13 TCS and one pair of STPK/STPP (PknB and PppL respectively). Mutation of pknB results in a pleiotropic phenotype: it causes defects in the development of genetic competence, in the synthesis of bacteriocins, in the ability to form biofilms and in tolerance to low pH. Transcriptome analysis revealed that expression of several genes likely involved in cell-wall biosynthesis had changed in a pknB knock-out mutant. The expression of genes that code for the synthesis of bacteriocins and genetic competence was decreased in the pknB mutant. These genes are also controlled by one of the TCS, ComDE. The expression of two other genes, bsmH and SMU.2146, was also downregulated in the pknB mutant. These genes were shown to be controlled by VicK, the histidine kinase of the VicKR TCS. The results suggest that PknB indirectly regulates gene expression by modulation the activity of the two-component signal transduction systems VicKR and ComDE through phosphorylation.

- 38 - - 31 - 2nd MIM PhD Student Retreat 2009 Poster Abstracts

P 02 P 07

Specificity of CD8 T-Cell Response in HIV Infection Role of the putative lipid phosphatase LppA in the replication of Legionella pneumophila Sonia Bastidas, Annette Oxenius Sabrina Engelhardt, Hubert Hilbi Institute for Microbiology, ETH Zurich [email protected] Division of Cell Biology, Institute of Zoology, University of Zurich [email protected]

Continuous loss of CD4+ T lymphocytes and systemic immune activation are hallmarks of chronic HIV-1 infection. Chronic immune activation is Legionella pneumophila, an intracellular parasite of freshwater protozoa, is characterized by an elevated expression of activation markers on T abundant in aquatic environments all over the world. In man-made systems, lymphocytes. It is suggested that apoptosis (activation induced cell death) of such as cooling towers or drinking water systems, the bacterium gained these abnormally activated cells is one of the main causes of T-cell decline. relevance as human pathogen. Inhalation of Legionella-containing aerosols Interestingly, T-cells with an activated phenotype are neither necessarily leads to the infection of alveolar macrophages, possibly causing a severe HIV-specific nor HIV-infected. The mechanisms underlying this phenomenon pneumonia termed Legionnaires` disease. To ensure survival inside the host known as bystander activation are not well defined. Additionally, little to cell, Legionella pneumophila evades endosomal degradation by subverting nothing is known about the antigen-specificity of bystander activated T-cells. signaling and vesicle trafficking pathways of its host and forms a replication- We will study this bystander activation of T-cells and their antigen permissive Legionella-containing vacuole (LCV). specificities by studying CD8+ and CD4+ T cell responses via IFN-γ Phosphoinositides (PIs) of the host cells represent a prominent ELISPOT at different time points before and after interruption ART. target for pathogenic bacteria, because PIs play a central role in phagocytosis, signaling pathways, vesicle trafficking, and cytoskeleton rearrangements. L. pneumophila employs several selectively translocated effector proteins, which specifically bind to PIs, thus subverting host processes. LppA (Legionella protein/phosphoinositide phosphatase A) is a putative protein/lipid phosphatase of L. pneumophila. Sequence alignment of the P-loop motif of LppA and different protein tyrosine/ PI-phosphatases revealed high similarity. Therefore, we hypothesize that LppA is a PI phosphatase, which alters the PI levels of the LCV to disturb host signaling pathways and to allow replication of the bacteria inside the host cells. To gain insights into the function of LppA we analyze: (i) the growth- phase dependent expression of LppA, (ii) the presence of LppA in different Legionella spp., (iii) the localization of LppA on the LCV, (iv) phosphatase activity of the purified protein, and (v) phenotypes of a L. pneumophila ΔLppA mutant strain.

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P 06 P 03

TlpX: a Bradyrhizobium japonicum protein with a dual role Fungal lectins: evidence for a defense role against predators and parasites Doris Bühler, S. Landolt, H. Hennecke, H.-M. Fischer Silvia Bleuler-Martínez1, Mattia Garbani1, Alex Butschi2, Markus Institute of Microbiology, ETH Zurich Aebi1, Markus Künzler1 [email protected] 1 Institute of Microbiology. Department of Biology, ETH Zurich 2 Institute of Molecular Biology, University of Zurich Bradyrhizobium japonicum is a Gram-negativ bacterium capable of [email protected] undergoing symbiosis with soybean or to exist as a free-living soil bacterium. When in symbiosis, B. japonicum must adapt to a totally different environment. Among other factors, it must cope with the 10’000-fold lower Lectins are widely present in higher fungi, being up-regulated in specific oxygen partial pressure in the nodules compared to the atmosphere outside organs such as fruiting bodies and sclerotia, where they represent an nodules. To efficiently respire under these different oxygen conditions, B. important fraction of soluble, cytoplasmic proteins. However, no experimental japonicum can synthesize several terminal oxidases with different affinities evidence supports any defined role of fungal lectins, and a lack of phenotype for O2. Among those, we find the low-affinity aa3–type cytochrome c oxidase in strains with silenced or knockout lectin genes suggest that are not involved relevant for respiration under free-living oxic conditions and the high-affinity in sexual development. We tested a panel of characterized fungal lectins cbb3–type cytochrome c oxidase which enables respiration under symbiotic from different Basidiomycetes and Ascomycetes for toxicity towards three conditions. model organisms: Caenorhabditis elegans, Aedes aegypti and TlpX is a periplasmic thioredoxin-like protein playing a role in both Acanthamoeba castellanii. These organisms were fed on E. coli cells the symbiotic and the free-living lifestyle of B. japonicum. In the latter, the recombinantly expressing the fungal lectins in the cytoplasm. We found that tlpX mutant phenotype suggests a function similar to what was observed for in this type of assay, most of the fungal lectins are toxic to at least one of the Sco proteins (synthesis of cytochrome c oxidase) which are well studied in three target organisms. Furthermore, we demonstrated that the toxicity is several eukaryotes (e.g. yeast, human) and prokaryotes (e.g. B. subtilis). dependent on the specific lectin-carbohydrate interaction, by impairing the Sco proteins are needed for maturation of the aa3–type cytochrome c binding of the lectin to the exposed glycans in the target organisms. oxidase (COX), probably by assisting metallation of the CuA site. The Aditionally, we established laboratory co-cultures between the fungivore successful complementation of a tlpX mutant by supplementing copper to the nematode Aphelenchus avenae and Coprinopsis cinerea in order to evaluate medium and copper binding studies with purified TlpX indicate that it might the ecological significance of the fungal nematotoxic lectins towards a natural act as a copper donor for aa3 COX also in B. japonicum. Yet, this function predator. We observed that predation by the nematode causes a four-fold alone cannot explain the symbiotic phenotype of a tlpX mutant which is the induction of the galectin (CGL2) in the vegetative mycelium of Coprinopsis. lack of efficient nitrogen fixation. The reason for this is that aa3 COX is not cinerea. We propose that many multicellular fungi have an inducible innate essential for symbiosis, as inferred from the wild-type symbiotic phenotype of immune system that includes cytoplasmic lectins as defense molecules a mutant lacking aa3 COX. Therefore, TlpX assumes another function in against predators and parasites. symbiosis. In this poster, I present evidences that exclude that the symbiosis- relevant cbb3 COX gets metallated by TlpX, as well as some work that has been done on other potential TlpX targets, namely NosZ, an enzyme involved in denitrification and another putative oxidase.

- 36 - - 33 - 2nd MIM PhD Student Retreat 2009 Poster Abstracts

P 04 P 05

In vitro characterization of the FixK2 regulatory protein of Functional characterisation of essential oligosaccharyl- Bradyrhizobium japonicum transferase subunits in Saccharomyces cerevisiae

Mariette Bonnet, H. M. Fischer, H. Hennecke, S. Mesa Jörg Breitling, Markus Aebi

Institute of Microbiology, ETH Zurich Institute of Microbiology, ETH Zurich [email protected] [email protected]

In the soybean endosymbiont Bradyrhizobium japonicum, two linked N-linked glycosylation of proteins in the endoplasmic reticulum (ER) is one of cascades are involved in the regulation of its different lifestyles. In the FixLJ- the most abundant posttranslational modifications. The transfer of the lipid FixK2 cascade, the FixLJ two-component regulatory system activates linked oligosaccharide precursors to specific asparagine residues of a expression of the transcription factor gene fixK2 at a concentration of ≤5 % nascent polypeptide chain is catalysed by the oligosaccharyltransferase of O2 in the gas phase. FixK2 is a member of the CRP/FNR superfamily, but (OTase). In higher eukaryotes the OTase is a oligomeric complex composed unlike its homologs, it neither possesses the CRP-specific residues involved of up to eight subunits. Although the catalytic subunit has been identified the in cAMP binding nor contains the FNR-specific cysteine residues necessary function of most of the other subunits of the OTase complex is still unclear. to bind [4Fe-4S]2+ clusters. Previous experiments in our laboratory showed This study is aimed to elucidate the function of the essential OTase subunits that FixK2 acts as homodimer without any cofactor [1]. Recently, we have OST1 and WBP1 in the yeast Saccharomyces cerevisiae. To this end a noticed that FixK2 can be inactivated by the formation of an intermolecular screening of a mutant library is performed and the resulting mutants are disulfide bridge via C183 in the DNA-binding domain [2]. Replacement of analysed with regards to their influence on glycosylation of various target C183 by a serine residue led to a non-redox-responsive protein derivative proteins and OTase complex formation. which is even more active than the wild-type protein. In addition to this type of regulation, we observed that a truncated FixK2 derivative is always co- purified together with the full-length protein from FixK2-overproducing E. coli cells. Mass spectrometry analysis revealed that this secondary form is a C- terminally cleaved FixK2 derivative which lacks the last twelve amino acids (FixK2 1-220). Different strategies have been applied to get a more homogenous population and to biochemically characterize FixK2. Especially, the construction of a tag-free derivative allowed us to purify a full-length active FixK2 via the IMPACT system (NEB). In order to get insights into the molecular mechanism of FixK2, we will focus on the crystallization trials with the (C183S) FixK2 protein variant.

[1] Mesa et al., J. Bacteriol. 187:3229-3388, 2005. [2] Mesa et al., unpublished.

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