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Home , Yersinia enterocolitica, Yersinia pestis

MECHANISMS OF PATHOGENIC ASPECTS OF TYPE III AND TWIN ARGININE TRANSLOCATION

MOA LAVANDER

DEPARTMENT OF MOLECULAR BIOLOGY, UMEÅ UNIVERSITY, SWEDEN DEPARTMENT OF MEDICAL COUNTERMEASURES, FOI NBC DEFENCE, UMEÅ, SWEDEN

UMEÅ 2005

Copyright © 2005 Moa Lavander ISBN: 91-7305-948-X

Printed in Sweden by Solfjädern Offset AB, Umeå.

TABLE OF CONTENTS

TABLE OF CONTENTS...... iii ABSTRACT...... v PAPERS IN THIS THESIS ...... vi 1. BACKGROUND ...... 1 1.1 GRAM-NEGATIVE IN THE PATHOGENIC NICHE ...... 1 1.2 A MOLECULAR VIEW ON YERSINIA INFECTIONS...... 2 1.2.1 : historic Reaper and potential tool of terror ...... 2 1.2.2 The enteropathogenic ancestry of Y. pestis...... 3 1.2.3 Plague for the mouse; a useful animal model...... 4 1.2.4 Yersinia reservoirs...... 4 1.2.5 First contact: adhere and invade...... 5 1.2.6 Multiplication within ...... 7 1.2.7 Extracellular persistence...... 8 1.2.8 Outcome of disease and strategies for transmission ...... 9 1.3 SECRETION SYSTEMS IN GRAM-NEGATIVE BACTERIA...... 10 1.3.1 Secretion across the plasma membrane ...... 10 1.3.1.1 The general secretion pathway (GSP/Sec)...... 11 1.3.2 Secretion across the outer membrane ...... 11 1.3.2.1 Autotransporters (AT) ...... 12 1.3.2.2 Two-partner secretion (TPS) ...... 12 1.3.2.3 Chaperon/usher pathways (CU)...... 12 1.3.2.4 Type I secretion (T1S)...... 13 1.3.2.5 Type II secretion (T2S) ...... 13 1.3.2.6 Type IV secretion (T4S)...... 14 1.4 TWIN ARGININE TRANSLOCATION (TAT)...... 15 1.4.1 The Tat translocase...... 15 1.4.2 The Tat signal peptide ...... 16 1.4.3 Faith and function of the Tat substrates...... 17 1.5 TYPE III SECRETION (T3S)...... 18 1.5.1 The flagellar T3SS and the substrate specificity switch ...... 19 1.5.2 Type III systems, tailored for their hosts ...... 21 1.5.3 Delivery of effector proteins by the type III secretion system....22 1.6 THE ENCODED YERSINIA TYPE III SECRETION SYSTEM...... 22 1.6.1 The common virulence plasmid ...... 22 1.6.2 The Ysc apparatus ...... 23 1.6.3 The Yop effectors...... 26 1.6.4 The Yersinia type III secretion chaperones...... 26 iii

1.6.5 A signal for the Yersinia type III secretion substrates ...... 27 1.6.6 The low calcium response, in vitro triggering of Yersinia T3S ..28 1.6.7 In vivo induction of Yersinia type III secretion ...... 28 2. AIMS ...... 30 3. RESULTS AND DISCUSSION ...... 31 3.1 CONCLUSIONS IN BRIEF FROM PAPERS I-IV...... 31 3.2 YERSINIA TWIN ARGININE TRANSLOCATION ...... 32 3.2.1 Yersinia Tat is functional ...... 32 3.2.2 A connection between Tat and Type III secretion? ...... 32 3.2.2 Tat is required for Yersinia virulence ...... 33 3.2.3 Yersinia Tat substrates in relation to virulence...... 34 3.2.4 Future perspectives of Tat ...... 34 3.3 ROLE OF YSCP AND YSCU IN YERSINIA TYPE III SECRETION...... 35 3.3.1 YscP ...... 35 3.3.1.1 YscP, a protein of dual functions...... 35 3.3.1.2 The role of YscP export...... 36 3.3.2 YscU ...... 37 3.3.2.1 YscU and the role of cleavage...... 37 3.3.3 The YscP and YscU connection ...... 38 3.3.4 Role of the needle in type III secretion...... 39 3.3.5 A model of substrate specificity switching by YscP and YscU..40 3.4 FINAL REMARKS ...... 42 ACKNOWLEDGEMENTS ...... 43 REFERENCES ...... 45 PAPERS I-IV

iv ABSTRACT

The and Y. pseudotuberculosis are related to the degree where the former is considered a subspecies of the latter, and still they cause disease of little resemblance in humans. Y. pestis is the causative agent of lethal bubonic and , while Y. pseudotuberculosis manifests itself as mild gastroenteritis. An important virulence determinant for these species is their ability to secrete and inject toxins (Yop effectors) into immune cells of the infected , in a bacterium-cell contact dependent manner. This ability depends on the extensively studied type III secretion system, a highly complex multicomponent structure resembling a needle. The induction of Yop secretion is a strictly controlled event. The two structural type III secretion components YscU and YscP are here shown to play a crucial role in this process, which is suggested to require an YscP mediated conformational change of the C-terminus of YscU. Proteolytic cleavage of YscU within this domain is further revealed to be a prerequisite for functional Yop secretion. The needle subcomponent itself, YscF, is recognised as a regulatory element that controls the induction of Yop effectors and their polarised delivery into target cells. Potentially, the needle might act as a sensor that transmits the inducing signal (i.e. target cell contact) to activate the type III secretion system. Secondly a, for Yersinia, previously unexplored system, the Twin arginine translocation (Tat) pathway, is shown to be functional and absolutely required for virulence of Y. pseudotuberculosis. A range of putative Yersinia Tat substrates were predicted in silico, which together with the Tat system itself may be interesting targets for future development of antimicrobial treatments. v

PAPERS IN THIS THESIS

This thesis is based on the following four articles, which are referred to in the text by their roman numerals.

I. Lavander, M., Ericsson, S. K., Bröms, J.E., and Forsberg, Å. The twin Arginine translocation system is essential for virulence of Yersinia pseudotuberculosis. Submitted manuscript.

II. Edqvist, P.J., Olsson, J., Lavander, M., Sundberg, L., Forsberg, Å., Wolf-Watz, H., and Lloyd, S.A. YscP and YscU regulate substrate specificity of the Yersinia type III secretion system. Journal of Bacteriology (2003)185: 2259- 2266.

III. Lavander, M., Sundberg, L., Edqvist, P.J., Lloyd, S.A., Wolf-Watz, H., and Forsberg, Å. Proteolytic cleavage of the FlhB homologue YscU of Yersinia pseudotuberculosis is essential for bacterial survival but not for type III secretion. Journal of Bacteriology (2002) 184: 4500-4509.

IV. Lavander, M. and Forsberg, Å. Proteolytic cleavage of YscU of Yersinia pseudotuberculosis is required for Yop effector secretion. Manuscript.

vi BACKGROUND

1. BACKGROUND

1.1 GRAM-NEGATIVE BACTERIA IN THE PATHOGENIC NICHE

Common to all bacteria are core functions, such as DNA replication, transcription and translation, synthesis of amino acids and nucleic acids. Moreover, bacteria utilise different strategies for survival depending on what ecological niche they occupy. An event that attracts particular interest from the scientific community is when the niche of choice is within the human body, especially when this host- bacterium relationship is harmful to the host. In this case the objectives for research are to understand, prevent and treat the infectious disease. To successfully invade and persist within a host, pathogenic bacteria depend on ‘virulence factors’, which includes elements important throughout the course of the infection. For instance, virulence factors enable bacteria to invade host tissues and protect the pathogens from the massive defence put up by the host . Virulence factors also aid in the survival and proliferation of the bacteria under unfavourable conditions such as low pH or under oxidative stress and permit efficient transmission to new hosts. Intriguing and highly varying infection strategies are employed by different pathogens, resulting in a wide range of symptoms of infectious diseases.

Figure 1. Schematic representation of a Gram-negative bacterial cell.

A characteristic feature of a Gram-negative bacterial cell is the presence of a double membrane, separated by a periplasmic space, protecting the integrity of the

1 BACKGROUND cytoplasm (fig. 1). Since pathogenic species largely rely on factors located on the bacterial surface, secreted into the extracellular milieu or translocated directly into the host cells during infection, specialised secretion systems are highly related to the ability of bacteria to cause disease. This has been the focus of the present study, where two secretion pathways – type III secretion (T3S) and twin arginine translocation (Tat) – have been analysed in pathogenic Yersinia species, causative agents of mild gastroenteritis or aggressive plague in humans. The different secretion systems of Gram-negative bacteria are reviewed below (chapter 1.3), with a more thorough description of T3S and Tat in chapters 1.4-1.6.

1.2 A MOLECULAR VIEW ON YERSINIA INFECTIONS

Characterised as Gram-negative, facultative anaerobic rod-shaped of the family Enterobacteriacea, the notoriety of one species of Yersinia hugely overshadows all others, i.e. Yersinia pestis the agent of Plague.

1.2.1 Plague: historic Reaper and potential tool of terror

No infectious disease has left such an imprint on history as the dreaded plague. Having occurred as devastating pandemics, reaping millions of lives, it has had profound impact on human civilisation. During the 14th century, plague alone claimed one third of the European population. Referred to as the , this is to present day the most dramatic pandemic in history. The culprit behind the disease is the bacterium Y. pestis that manifests itself as bubonic or pneumonic plague, aggressive infections that are lethal and relentless. Y. pestis inhabits populations, with transmission of the disease occurring via feeding off an infected animal (Brubaker, 1991; Smego et al., 1999). While plague today is endemic in Africa, Asia and the Americas, improved health and sanitation as well as the development of have dramatically reduced the impact of plague infections. Outbreaks of Y. pestis infections do however still occur, with the 2 BACKGROUND greatest risk of epidemics found in developing countries where plague is endemic and people live under insalubrious conditions (Duplantier et al., 2005; Gage and Kosoy, 2005; Perry and Fetherston, 1997). Further, with the rise of large scale terrorism, there is intensified awareness of the potential threat in use of Y. pestis as a agent (Inglesby et al., 2000).

1.2.2 The enteropathogenic ancestry of Y. pestis

Of the 11 members of the genus Yersinia, Y. pestis, Y. pseudotuberculosis and Y. enterocolitica are known to cause disease in humans (Cornelis et al., 1998; Sulakvelidze, 2000). The latter two cause comparatively mild gastrointestinal infections presented as fever, vomiting, acute diarrhoea and abdominal pains due to mesenteric lymphadenitis. These infections are usually cleared from the host within 7-14 days and do not require treatment, although septicaemia do occur in immunocompromised patients. The enteropathogenic yersiniae may further induce post infection complications such as reactive (Hannu et al., 2003; Smego et al., 1999). This thesis focuses on the remarkably closely related Y. pestis and Y. pseudotuberculosis. Having evolved from the much less malevolent ancestor Y. pseudotuberculosis as recently as 1,500-20,000 years ago, Y. pestis actually represents a clone within the Y. pseudotuberculosis species, making this an intriguing evolutionary event (Achtman et al., 1999; Achtman, 2004; Bercovier et al., 1980; Chain et al., 2004; Wren, 2003). The profoundly altered mode of transmission and pathogenesis of Y. pestis is reflected in dramatic changes within its (Achtman et al., 1999; Chain et al., 2004; Perry and Fetherston, 1997; Smego et al., 1999). Horisontal acquisition of two, for Y. pestis, unique pPla (~100 kb) and pFra (~9.6 kb) and a number of genomic islands presumably prompted the rapid evolution of the plague bacterium. Further, the Y. pestis contains a large number of pseudogenes, insertion sequences and extensive genomic rearrangements, corresponding to the abandonment of the 3 BACKGROUND enteropathogenic lifestyle and adaptation into a highly efficient killer (Chain et al., 2004; Deng et al., 2002; Parkhill et al., 2001).

1.2.3 Plague for the mouse; a useful animal model

The holy grail of Yersinia research has for a long time been, and still is, the invention of new cures for plague. To achieve this goal, studies of Yersinia virulence mechanisms are frequently carried out with the mouse as the animal model, and Y. pseudotuberculosis as a model organism for Y. pestis. The genetic similarities between the yersiniae, and the fact that Y. pseudotuberculosis causes systemic plague-like disease in mice, makes this a convenient system to study more severe infections (Gemski et al., 1980b). Even if deductions such as mouse = human and Y. pseudotuberculosis = plague, should be regarded with a healthy amount of scepticism, this model system has proven useful to learn more about the molecular mechanisms of the disease.

1.2.4 Yersinia reservoirs

Y. pseudotuberculosis is a resilient bacterium, well adapted to persist for long periods in low-nutrient conditions and at temperatures ranging from 4 to 45°C, thus increasing the opportunities of being taken up by a host (Smego et al., 1999). Humans are infected via contaminated food and water, or by contact with animals that can be asymptomatic carriers, suffering from prolonged or dying from the infection (Fukushima et al., 1989; Hodges et al., 1984; Laukkanen et al., 2003). In contrast to the enteric Yersinia species, Yersinia pestis is an obligate pathogen that will perish fairly promptly outside an infected host (Smego et al., 1999). The mammalian reservoir of Y. pestis is mainly represented by many different species of , with the most important arthropod being the Xenopsylla cheopsis (Hinnebusch, 2005; Lowell et al., 2005; Mencher et al., 2004). When the flea feeds on a mammal that has developed , the bacteria

4 BACKGROUND accompanying the engulfed blood will grow into a gelatinous that blocks the flea proventriculus (the valve that connects the oesophagus to the midgut). As blood is thus prevented from flowing into its stomach, the flea is starved, resulting in frantic but futile feeding attempts. Efficient regurgitation and inoculation of bacteria at the bite site will occur, thereby causing spread of the disease (Hinnebusch, 2005). Bacterial colonisation of the flea depends on the chromosomally encoded hemin storage system, Hms, which is suggested to synthesise extracellular polysaccharides required for biofilm formation (Hinnebusch et al., 1996; Jarrett et al., 2004). Further, the Yersinia murine toxin, Ymt, a encoded on the pFra plasmid is required for Y. pestis to persist within the flea (Du et al., 1995; Hinnebusch et al., 2002; Hinnebusch et al., 2000; Rudolph et al., 1999). Importantly, both Hms and Ymt are redundant for virulence in the mammalian host (Du et al., 1995; Kutyrev et al., 1992; Lillard et al., 1999). Interesting gene candidates with unresolved roles in vector colonisation are found within the Y. pestis genome, such as pathogenicity islands encoding products resembling insecticidal toxins (Chain et al., 2004; Deng et al., 2002; Parkhill et al., 2001).

1.2.5 First contact: adhere and invade

Shortly after ingestion, enteric Yersinia species leave the lumen of the gut to invade deep tissue sites as they traverse the M cells of the intestinal epithelium, a port of entry for many enteropathogenic microorganisms with the ability to adhere to and invade these cells (Autenrieth and Firsching, 1996; Boyle and Finlay, 2003; Clark et al., 1998; Neutra et al., 1996). Two adhesins identified for the gastrointestinal Yersinia species are invasin and YadA (Finlay and Caparon, 2005). Chromosomally encoded by the inv gene, Yersinia invasin is expressed at ambient temperature or at 37°C at pH 5.5 and under high Na2+ levels, suggesting that the conditions of the small intestine can maintain invasin expression after ingestion of 5 BACKGROUND the bacteria (Pepe et al., 1994). Anchored in the bacterial outer membrane with an elongated, rod-like protrusion on the surface, invasin binds with extraordinarily high affinity to β1 integrins exposed on the luminal side of the host M-cells to mediate entry into the cells (Clark et al., 1998; Hamburger et al., 1999; Isberg and Falkow, 1985; Isberg and Barnes, 2001; Marra and Isberg, 1997; Pepe and Miller, 1993; Van Nhieu and Isberg, 1991). Y. pseudotuberculosis inv mutants reveal defective colonisation of Peyer’s patches and the mesenteric lymph nodes, unable to transmigrate over the epithelium these strains show a tendency to colonise the luminal side of the intestine (Marra and Isberg, 1997). Encoded on the pYV plasmid, yadA expression is upregulated at 37°C. The autotransporter YadA then forms a multimeric lollipop-shaped protrusion from the outer bacterial membrane and suggestedly binds to extracellular matrix (ECM), thus contributing to adhesion and invasin independent uptake (Bolin et al., 1982; Eitel and Dersch, 2002; El Tahir and Skurnik, 2001; Hoiczyk et al., 2000; Roggenkamp et al., 2003; Yang and Isberg, 1993). Consistent with abandoning the enteropathogenic lifestyle, yadA and inv are merely present as pseudogenes in Y. pestis (Deng et al., 2002; Parkhill et al., 2001; Rosqvist et al., 1988; Simonet et al., 1996). Instead, the multifunctional Pla protein (plasminogen activator), encoded on the pPla plasmid, enables tissue invasion of Y. pestis from the initial site of the flea bite. Subcutaneous infections in mice with a pla mutant strain results in only localised infections, indicating a role for Pla in systemic dissemination of the bacterium (Cowan et al., 2000; Sodeinde et al., 1992; Welkos et al., 1997). Pla also mediates adherence of Y. pestis to ECM, and induces activation of plasminogen into resulting in uncontrolled proteolysis and damage to host tissues (Lahteenmaki et al., 1998; Lahteenmaki et al., 2005). Pla has also proved able to sponsor invasion of epithelial cells. The pH6 is a fibrillar putative adhesin found in both Y. pestis and Y. pseudotuberculosis. It is expressed at 37°C and below pH 6.7 (conditions similar to

6 BACKGROUND the phagolysosome) depends on the chaperone/usher pathway for assembly and has been shown to interact with eukaryotic cells and host lipoproteins (Ben-Efraim and Bichowsky-Slomnichi, 1964; Huang and Lindler, 2004; Lindler and Tall, 1993; Makoveichuk et al., 2003; Yang et al., 1996). A crucial skill for pathogenic bacteria is the acquisition of iron, which is sequestered by the animal host. (Ybt), found in all three human pathogenic yersiniae, expresses a system for secretion of a that scavenges ferric iron from the host prior to being transported back into the bacterial cytoplasm. This system is required for the initial colonisation of the host, as a Y. pestis Ybt defective mutant strain is unable to spread from the initial subcutaneous locus of infection. In contrast, Ybt is dispensable for later septicemic stages of plague, as intravenous injection of the same mutant strain will result in full-blown disease (Lesic and Carniel, 2004; Perry and Fetherston, 2004).

1.2.6 Multiplication within macrophages

Although Yersinia infections primarily are extracellular, in vivo studies have revealed that all three human pathogenic species can be found within macrophages during early stages of colonisation (Pujol and Bliska, 2005). Y. pestis and Y. pseudotuberculosis share the ability to survive and multiply within macrophages, and in vitro assays place the pathogens within the phagolysosomes, where bacteria are challenged with a hostile environment of acidic pH, reactive oxygen and nitrogen species, antimicrobial peptides and (Pujol and Bliska, 2003; Straley and Harmon, 1984a, b). Y. pseudotuberculosis has been shown able to inhibit acidification of the phagosome and, as Y. pestis, the production of nitric oxide (Pujol and Bliska, 2005; Tsukano et al., 1999). PhoP/PhoQ, a pleiotropic regulatory two-component system of Gram-negative bacteria is important for intracellular survival of Yersinia within macrophages. In vitro stress assays with phoP mutant strains of Y. pestis and Y. pseudotuberculosis revealed the PhoP/PhoQ 7 BACKGROUND system to be important to resist acidic conditions, oxidative stress and Mg2+ starvation (Grabenstein et al., 2004; Oyston et al., 2000). The ability of Yersinia to subvert the macrophage functions from within is suggested to be a strategy for acclimatisation to the mammalian host, prior to facing the host immune defence, with a range of genes coding for virulence related proteins being upregulated at 37°C (Pujol and Bliska, 2005). One such , exclusive for Y. pestis and expressed from pFra, is the F1 antigen, which is exported and assembled via a chaperone/usher pathway to form a capsule-like structure that gives increased resistance against uptake by macrophages (Du et al., 2002).

1.2.7 Extracellular persistence

For colonisation of the extracellular compartments, the bacteria have to be able to thwart the different aspects of the immune defence, including recruitment of phagocytic cells, secretion of and complement mediated lysis. Even if route of infection and nature of the disease differ widely between the enteropathogenic Yersinia species and Y. pestis, they share tropism for lymphoid tissues where they resist the host innate immune response (Brubaker, 1991; Koornhof et al., 1999). This ability depends on the ~70kDa plasmid, pYV (plasmid of Yersinia virulence) shared exclusively between these three species (Ben-Gurion and Shafferman, 1981; Ferber and Brubaker, 1981; Gemski et al., 1980a; Gemski et al., 1980b). The plasmid codes for a secretion system, designated ‘type III’, and so called Yop effectors (Yersinia outer proteins), delivered by this system into eukaryotic cells (Rosqvist et al., 1994; Salmond and Reeves, 1993; Sory and Cornelis, 1994). Encoded on the plasmid are also the V-antigen (LcrV), and the adhesin YadA, which together with the Yops contribute to inhibition of and downregulation of the inflammatory response (Cornelis and Wolf-Watz, 1997; Cornelis et al., 1998; Nakajima et al., 1995; Sing et al., 2002).

8 BACKGROUND

Being a main focus of this thesis the type III secretion system will be discussed in more detail below (chapters 1.5-1.6). If yersiniae reach the bloodstream (an unusual event for the enteric species unless infecting an immunocompromised host) they have to evade the complement mediated lysis, also known as serum resistance. Factors that protect against the complement are generally located on the bacterial surface and the adhesin YadA (chapter 1.2.5) has been shown to contribute to serum resistance (Biedzka-Sarek et al., 2005). Lipopolysaccharides (LPS) are the major outer membrane components of Gram-negative bacteria, and an important virulence factor (Erridge et al., 2002). The LPS consists of the lipid A, which is anchored in the outer membrane, an oligosaccharide core and the O-antigenic polysaccharide. The O-antigen has been shown to be significant for virulence of enteric yersiniae, proposedly by contributing to serum resistance as seen for many other pathogens (Skurnik and Bengoechea, 2003). Interestingly, Y. pestis, which has to efficiently colonise the blood stream to promote transmission via the flea, has ‘rough’ LPS, i.e. it is devoid of the O-antigen (Bruneteau and Minka, 2003; Skurnik et al., 2000). However, this rough LPS is required for the activity of Pla (chapter 1.2.5) that has been shown to degrade components of the complement, thus contributing to Y. pestis serum resistance (Kukkonen et al., 2004).

1.2.8 Outcome of disease and strategies for transmission

Usually, pathogenic bacteria do not benefit from killing their hosts, but will increase the opportunities for multiplication and transmission by causing a prolonged balanced infection. This is the strategy employed by the entero- pathogenic Yersinia species, which cause self-limiting infections in healthy humans and can reside asymptomatically in other animal hosts for long periods of time (Smego et al., 1999). A very different pattern is seen for the dramatic progress of a Y. pestis infection. Here, the bacteria disseminate aggressively throughout the 9 BACKGROUND host’s lymphatic system, entering the bloodstream in 2-6 days, causing heavy septicaemia and infecting vital organs. Proliferation of Y. pestis within lymph nodes provokes a massive inflammatory response, resulting in characteristic swelling buboes in armpits, groin and neck; hence ‘’. Y. pestis can also spread to and colonise the lungs (pneumonic plague), facilitating aerosol transmission of the bacterium. While mortality for untreated bubonic plague is around 60%, the pneumonic form has a mortality rate close to 100%, most likely due to that the bacteria are already adapted to the mammalian host (Brubaker, 1991; Smego et al., 1999). The maintenance of the hypervirulent phenotype of Y. pestis is proposed to be a consequence of that even the most efficient flea vector, X. cheopsis, is difficult for the bacterium to colonise and also transmits the disease with low prevalence. This promotes extremely high numbers of bacteria within the blood stream of the host to improve the odds for transmission (Lorange et al., 2005).

1.3 SECRETION SYSTEMS IN GRAM-NEGATIVE BACTERIA

Protein secretion is not only a prerequisite for life, but also highly related to pathogenesis. An overview of secretion system used by Gram-negative bacteria is given below, with a more detailed description of type III secretion and twin arginine translocation in chapters 1.4-1.6.

1.3.1 Secretion across the plasma membrane

Two systems work in parallel for protein secretion across the inner membrane: the general secretion pathway (GSP or Sec, below) and twin arginine translocation (Tat, chapter 1.4). The autotransporters, two-partner secretion, chaperone/usher and type II secretion systems only account for secretion across the outer membrane and thus rely on Sec or Tat for the initial step of export from cytoplasm to periplasm.

10 BACKGROUND

1.3.1.1 The general secretion pathway (GSP/Sec) Sec is responsible for the major part of the export of proteins over the plasma membrane (de Keyzer et al., 2003; Muller et al., 2001). Found in bacteria, archaea and eukarya, the Sec pathway is the only secretion system shown to be a required for life, e.g. due to its essential role in membrane biogenesis (Dalbey and Chen, 2004; Pohlschroder et al., 1997). Substrates exported via Sec are defined by an N- terminal signal peptide, recognised by the chaperone SecB that targets the substrate to the SecYEG secretion channel. The secretion process is energised by ATP hydrolysis and the proton motive force, whereupon the signal peptide is cleaved off by a signal peptidase and the mature protein released into the periplasm (Dalbey and Von Heijne, 1992; de Keyzer et al., 2003; Driessen, 2001; Economou, 1999, 2000; Veenendaal et al., 2004).

1.3.2 Secretion across the outer membrane

Secretion across the outer membrane can occur via different systems as depicted in figure 2.

Figure 2. Secretion systems of Gram-negative bacteria. Schematic representation of secretion systems; T1S-T5S, denoting type I-V secretion. T2S and T5S depend on Sec or Tat for export over the inner membrane. AT = autotransporter, TPS = two partner secretion, CU = chaperon/usher. IM = inner membrane, PP = periplasm, OM = outer membrane and CM = host .

11 BACKGROUND

1.3.2.1 Autotransporters (AT) These minimalistic secretion systems consist of substrates that mediate their own transport across the outer membrane. AT proteins have an N-terminal Sec signal peptide followed by a passenger domain and a C-terminal translocation domain (Jacob-Dubuisson et al., 2004). The latter supposedly forms a β-barrel structure in the outer membrane, enabling delivery of the passenger to perform its exoprotein functions (Henderson et al., 2004). The first AT secretion system identified was the IgA of (Pohlner et al., 1987). Since this initial discovery, many AT proteins have been shown to play a role in pathogenesis of Gram-negative bacteria, contributing to e.g. serum resistance, attachment to eukaryotic cells and biofilm formation (Henderson et al., 1998; Henderson and Nataro, 2001; Newman and Stathopoulos, 2004).

1.3.2.2 Two-partner secretion (TPS) While autotransporter proteins provide for their own secretion, the TPS secreted proteins, commonly referred to as TpsA, depend on a dedicated partner protein, TpsB, to form the β-barrel conduit over the outer membrane (Guedin et al., 2000; Jacob-Dubuisson et al., 2004; Konninger et al., 1999). The typical TpsA is a large protein, 100-500kDa or more, involved in bacterial virulence (Jacob-Dubuisson et al., 2001). Haemolysins of (nosocomial infection) and (urinary tract infection) are part of a growing family of pore forming TpsA toxins (Hertle, 2005; Ondraczek et al., 1992). Further, many Gram-negative pathogens utilize this pathway to deliver adhesins to the bacterial surface (Julio and Cotter, 2005; Rojas et al., 2002; Smith et al., 2001).

1.3.2.3 Chaperon/usher pathways (CU) Chaperon/usher systems provide a means to assemble adhesins on the bacterial surface, as filamentous or afimbrial structures. A periplasmic chaperone is

12 BACKGROUND responsible for substrate recognition and interacts with the substrates to prevent premature aggregation and degradation while guiding them to the secretion machinery (Sauer et al., 2004). Examples of surface organelles assembled via the CU system include: the P pilus of uropahtogenic E. coli and the F1 and pH6 of Y. pestis (Chapman et al., 1999; Hull et al., 1981; Sauer et al., 2000; Zav'yalov et al., 1996).

1.3.2.4 Type I secretion (T1S) The T1S system consists of three subcomponents: i) an inner membrane ATP- binding cassette (ABC) that energises translocation and is responsible for substrate recognition and recruitment; ii) a pore forming outer membrane protein (OMP) and iii) a membrane fusion protein (MFP) that bridges the other two components (Delepelaire, 2004). The substrates are recognised by a C-terminal secretion signal and delivered from the cytoplasm to the exterior milieu without periplasmic intermediates (Remaut and Waksman, 2004). T1S has been shown to be involved in toxin delivery and can also constitute multidrug efflux pumps that contribute to bacterial multiresistance (Paulsen, 2003). Examples of type I secreted virulence determinants include haemolysin A from uropathogenic E. coli, RtxA toxin and adenylate cyclase toxin CyaA of () (Delepelaire, 2004; Thanassi and Hultgren, 2000).

1.3.2.5 Type II secretion (T2S) Type II secretion is responsible for the bulk export of Sec substrates destined for the extrabacterial compartment. Assembled from 12-15 components, the T2S structure spans the bacterial envelope, thus enabling use of the proton motive force of the inner membrane to energise substrate export (Pugsley et al., 1997; Sandkvist, 2001b). Type II exoprotein substrates carry a signal peptide for delivery over the plasmamembrane via either Sec or Tat, and are upon folding within the

13 BACKGROUND periplasmic space probably recognised by the T2S system via their highly specific three-dimensional structure (Nunn, 1999; Pugsley et al., 1997; Rossier and Cianciotto, 2005; Voulhoux et al., 2001). Many T2S components share homology with the type IV pathway for pilus biogenesis, and T2S systems have also been shown to assemble pilus-like structures (Durand et al., 2003; Sauvonnet et al., 2000; Vignon et al., 2003). These do however primarily span the periplasmic space and only form rudimentary structures on the bacterial surface. The role of the T2 pilus has not been determined, but may include gating of the T2S apparatus or a piston-like function in protein expulsion (Nunn, 1999; Sandkvist, 2001b). Many virulence factors are secreted via the T2 system, such as toxin from Vibrio cholerae, cell-wall degrading enzymes of Erwinia species (plant pathogens causing soft rot) and a range of virulence related exoproteins from the opportunistic pathogen , including exotoxin A and phospholipase C (Sandkvist, 2001a). A T2S system has been identified for some strains of Y. enterocolitica, denoted Yts1 (Yersinia type II secretion), that has been shown to be important for virulence in mice (Iwobi et al., 2003).

1.3.2.6 Type IV secretion (T4S) Type IV secretion machineries are assembled from at least 12 proteins forming a core complex anchoring the structure in the bacterial membranes, spanning the periplasmic space, with a pilus protruding from the bacterial surface. T4S systems support delivery of substrates in an uninterrupted route across both bacterial and host cell membranes (Remaut and Waksman, 2004). These organelles, related to bacterial conjugation systems, are utilised by Gram-negative bacteria for interbacterial DNA-transfer and adapted by pathogens for translocation of anti-host factors (proteins or DNA) into eukaryotic target cells (Christie, 1997, 2004; Remaut and Waksman, 2004). Virulence strategies that depend on T4S include delivery of oncogenic DNA into host cells by the plant pathogen Agrobacterium

14 BACKGROUND tumefaciens and pertussis toxin secretion by B. pertussis (Burns, 2003; Christie, 1997; Nagai and Roy, 2003). In a number of Y. pseudotuberculosis strains a type IV pilus gene cluster, that contributes to virulence via the oral route, has recently been identified (Collyn et al., 2002; Collyn et al., 2004).

1.4 TWIN ARGININE TRANSLOCATION (TAT)

The twin arginine translocation (Tat) system has attracted a vast amount of research interest during the past decade. A lot of the curiosity surrounding the Tat system stems from its unique ability to export proteins in an already folded state across the bacterial inner membrane. The Tat pathway was originally discovered in plant chloroplasts, where is supports protein transport from stroma into thylakoids. Energized by the transmembrane H+ gradient, this system was initially named the ∆pH pathway (Dalbey and Robinson, 1999; Settles et al., 1997; Settles and Martienssen, 1998). In bacteria, the Tat system was originally referred to as ‘Membrane targeting and translocation’ Mtt (Weiner et al., 1998) and shortly thereafter as ‘Twin arginine translocation’, a name that stuck (Sargent et al., 1998).

1.4.1 The Tat translocase

In Gram-negative bacteria the typical Tat translocase, as characterised in the model organism E. coli, is mainly built up from the components TatA, TatB and TatC encoded by the tatABCD operon. The remaining gene product, TatD, has been shown to be a cytoplasmic DNase redundant for Tat function, and without any known connection to the system apart from its co-localisation with the operon (Wexler et al., 2000). While TatABC are required for functional Tat, a fourth component, TatE, encoded by a monocistronic gene is redundant (Ize et al., 2002; Sargent et al., 1998; Yahr et al., 1995). TatA and TatB are both inserted into the bacterial inner membrane and share largely similar structures: an N-terminal

15 BACKGROUND membrane spanning hydrophobic α-helix and a C-terminal amphipatic α-helix protruding into the bacterial cytoplasm (Palmer and Berks, 2003; Porcelli et al., 2002; Sargent et al., 2001). TatC, the most conserved Tat component, has six membrane spanning domains, the N- and C-termini of the protein being located in the cytoplasm (Behrendt et al., 2004; Ki et al., 2004). The translocation of Tat substrates is hypothesized to occur in the following manner: TatC forms a 1:1 complex with TatB that may include low levels of TatA, although the bulk of TatA forms a homooligomeric channel in the inner membrane (Bolhuis et al., 2000; de Leeuw et al., 2002; Oates et al., 2003; Porcelli et al., 2002; Sargent et al., 2001). TatC is responsible for recognition of the Tat signal sequence (chapter 1.4.2) and when a substrate is present the TatA complex is transiently recruited to TatBC, providing a conduit for substrate export to the periplasmic space (Alami et al., 2003; Jongbloed et al., 2000). The translocation event occurs independently of ATP and is energised by the transmembrane proton gradient (Yahr and Wickner, 2001). Recent findings have shown that TatA forms different size complexes, probably assembled from modules of three or four TatA subunits. This would suggestedly allow the Tat translocation channel to adapt to the size of the substrate at hand (Gohlke et al., 2005; Oates et al., 2005).

1.4.2 The Tat signal peptide

Substrates exported via the Tat system are recognised by an N-terminal signal sequence, between 26 and 58 amino acids long, compared to 18-26 amino acids for the classical Sec signal peptides (Berks, 1996; von Heijne, 1985). Further, while the Sec signal peptide is devoid of consensus sequences, Tat substrates typically carry a (S/T)-R-R-x-FLK motif (Berks, 1996). This conserved sequence has a very strong amino acid bias, the twin arginines (RR) giving the system its name being the most invariable, but not completely irreplaceable for Tat function (Ize et al., 2002). Substrates with atypical signal sequences, including variations of the

16 BACKGROUND signature twin arginine have been found: TtrB has a motif, SKRQFLQ, where one of the two arginines is missing (Hinsley et al., 2001). Also, RNR instead of the typical RR is seen for the Rieske Fe/S protein, an essential component of the photosynthetic electron transport chain of chloroplasts in Spinacia oleracea (Molik et al., 2001). Similar to Sec, the Tat signal peptide is commonly cleaved off after successful export to the periplasmic space (Berks, 1996).

1.4.3 Faith and function of the Tat substrates

In contrast to Sec, Tat exports substrates that acquire their folding within the bacterial cytoplasm (Bogsch et al., 1998; Pugsley, 1993). The Tat pathway is believed to have evolved for delivery of cofactor-containing proteins across the plasmamembrane, and the vast majority of Tat substrates in E. coli undergo cytosolic cofactor incorporation (Berks et al., 2003; Halbig et al., 1999; Palmer et al., 2005). Cofactorless substrates also exist, as exemplified by halophilic archaea. These organisms have extremely high salt levels in their cytosol, and rapid folding is required to prevent protein damage under these conditions. As a result, the majority of exported proteins in these archaea take the Tat route (Hutcheon and Bolhuis, 2003; Rose et al., 2002). A third variant of Tat substrates are proteins that lack the typical signal peptide, but still are routed to Tat by a “piggy-back” mechanism. In other words, they form heteromeric complexes in the cytoplasm with Tat-signal containing partners (Palmer et al., 2005). Proteins translocated by Tat to the periplasm either remain there, are integrated into the outer or inner membrane or secreted to the extrabacterial milieu (Hatzixanthis et al., 2003; Ochsner et al., 2002; Sargent et al., 1998; Sargent et al., 2002). Twin arginine translocation has been shown to be connected to the ability to cause disease in a number of bacteria, for example phospholipase toxins exported via type II secretion in (Legionnaire’s disease) and P. 17 BACKGROUND aeruginosa have been shown to be Tat substrates (Rossier and Cianciotto, 2005; Voulhoux et al., 2001). Disruption of Tat by mutagenesis commonly results in strains displaying pleiotropic defects. Apart from impaired phospholipase delivery, a P. aeruginosa Tat mutant was also deficient for , biofilm formation, ability to endure osmotic stress, respiration under anaerobic conditions, iron- metabolism and was also shown to be attenuated for virulence in a rat lung model (Ochsner et al., 2002).

1.5 TYPE III SECRETION (T3S)

A general overview of type III secretion is given here, with a more in depth review of the Yersinia type III secretion system (T3SS) in chapter 1.6. Providing a means for delivery of numerous structurally and functionally diverse virulence proteins into eukaryotic cells, the T3SS is an important tool for interkingdom communication utilised by both pathogenic and symbiotic bacteria to establish a relationship with their eukaryotic hosts (Alfano and Collmer, 2004; Dale et al., 2001; Dale et al., 2002; Francis et al., 2004; He et al., 2004; Viprey et al., 1998). Since the initial discovery of T3S, a range of infectious diseases have been correlated to bacteria that employ this secretion system as a virulence strategy (Aizawa, 2001; Hueck, 1998; Pallen et al., 2003). With the number of sequenced rapidly increasing, it is also evident that T3S systems are encoded by many pathogens for which its role in virulence remains to be determined (Pallen et al., 2005). Many infections in animals, including humans, are caused by T3SS carrying pathogens, such as: the previously mentioned Yersinia spp, Salmonella spp (food poisoning and ), Shigella spp (), P. aeruginosa (opportunistic infections), Burkholderia pseudomallei and B. mallei ( and ), Bordetella spp. (respiratory tract infections) and pathogenic species of E. coli that cause gastrointestinal disease of varying severity (Hueck, 1998;

18 BACKGROUND

Pallen et al., 2003). Also plants suffer from the attacks of bacterial pathogens, such as Pseudomonas syringae, Ralstonia solanacearum or species of Erwinia and Xanthomonas, which utilise T3S to induce wilt, rot, necroses, galls and blight (Buttner and Bonas, 2002).

1.5.1 The flagellar T3SS and the substrate specificity switch

The components of the bacterial flagellum are secreted by a type III secretion system that largely is regarded as the mother of all T3S systems (Macnab, 1999; Saier, 2004). The flagellum is a tripartite organelle consisting of: the inner membrane basal body (containing a central rod), the hook, and the helical filament protruding from the bacterial cell like a rotary tail used for propulsion (fig. 3 and fig. 4, left panel). Reflecting their evolutionary relatedness, similarities between the flagellar T3SS and the injectisome-type T3SS is seen on the levels of sequence, structure as well as function (Aizawa, 2001; Blocker et al., 2003; Hueck, 1998; Macnab, 1999). Electron microscope studies of type III injectisomes from E. coli, Salmonella enterica and reveals basal bodies highly resembling those of the flagellum (fig. 4, inset and fig 5) (Blocker Figure 3. The bacterial flagellum. Schematic representation of the flagellum et al., 2001; Kimbrough and Miller, 2000; with the T3S apparatus (within the basal body) anchored in the bacterial Kubori et al., 1998; Kubori et al., 2000; membranes (Macnab, 1996). Sekiya et al., 2001). 19 BACKGROUND

Figure 4. The flagellar substrate specificity switch. Electron micrographs of flagella from S. enterica. Left panel: wild type flagellum and purified hook-basal body complexes (inset). Middle: polyhook phenotype of fliK mutant strain. Right: partial suppression of the fliK mutant phenotype by 2nd site mutation within the cytoplasmic domain of FlhB resulting in a polyhook filament phenotype. Bar = 105 nm. Reprinted with permission from (Williams et al., 1996). Copyright 1996 ASM.

Flagellar assembly is a strictly controlled process. The organelle is built from base to tip; new components are exported through the conduit formed by the flagellum itself and added on the growing end only after the earlier parts have been completed (Aizawa, 1996; Macnab, 1996, 2004). One critical event is the so called substrate specificity switch that occurs when assembly of the hook is completed and the flagellar T3S system ceases to export and assemble hook/rod type substrates in favour of the filament subcomponent FliC. Strains mutated in FliK are deficient for this switch and can not initiate FliC secretion but instead assemble hook after hook, a so called polyhook (fig. 4, middle) (Patterson-Delafield et al., 1973; Suzuki and Iino, 1981). Further investigation of this phenomenon revealed that FliK interacts with the inner membrane component FlhB to control the switch, since the polyhook phenotype of the fliK mutant was partially suppressed by second site mutations in the FlhB. As a result a polyhook-filament phenotype is seen, i.e. the strain is still impaired for termination of hook assembly but able to form a filament (fig. 4, right) (Hirano et al., 1994; Kutsukake et al., 1994; Williams et al., 1996). The T3S injectisomes of Shigella, Salmonella and Yersinia have all

20 BACKGROUND revealed a corresponding switch deficiency when mutated in the FliK analogues Spa32, InvJ and YscP respectively. These mutants can not terminate assembly of the needle structure, resulting in superlong needles, and are also unable to initiate secretion of effector proteins (Paper II) (Agrain et al., 2005a; Journet et al., 2003; Kubori et al., 2000; Magdalena et al., 2002).

1.5.2 Type III systems, tailored for their hosts

While the basal bodies of secretion systems are homogenous in appearance, not only the delivered effectors but also the surface structures vary depending on the nature of the host-pathogen interaction. For organisms that come into close contact with the host cell membranes, the T3S systems form comparatively small needle- like protrusions, for Shigella (~45 nm), Salmonella (~80nm) and Yersinia (varying results of ~42nm or 60-80nm) (fig. 5) (Hoiczyk and Blobel, 2001; Journet et al., 2003; Kubori et al., 2000; Tamano et al., 2000; Tamano et al., 2002).

Figure 5. Electron micrograph of isolated Figure 6. Electron micrograph of the type III secretion injectisomes of S. EspA-filament assembled by the T3SS of enterica. Notice resemblance of basal body to enteropathogenic E. coli. Arrow indicating the flagellar system, fig 4, inset. Bar = 100 filaments interacting with surface of HEL nm. Reprinted with permission from (Kubori cells. Bar = 0.25µm. Reprinted with et al., 1998). Copyright 1998 AAAS. permission from (Knutton et al., 1998). Copyright 1998 EMBO.

Enteropathogenic E. coli, on the other hand, utilises T3S to come into close contact with cells of the intestinal epithelium and accordingly supports a long filamentous type III protrusion that spans the mucus layer surrounding these cells (fig. 6) 21 BACKGROUND

(Knutton et al., 1998). Similarly, the T3S system of the plant pathogen P. syringae assembles a sturdy pilus for effector delivery, corresponding to the thick plant cell wall (fig. 7A) (He and Jin, 2003; Roine et al., 1997).

1.5.3 Delivery of effector proteins by the type III secretion system

The exact mechanism by which the effectors reach the host cell cytosol has not been resolved, but the common hypothesis is that the delivery occurs via a hollow channel formed by the secretion apparatus itself (Ghosh, 2004). In P. syringae, secretion of effectors has in deed been shown to occur through the Hrp-pilus type III apparatus, as shown in figure 7 (Jin and He, 2001).

Figure 7. Evidence of secretion through the T3S Hrp pilus of P. syringae. Electron micrographs of P. syringae with immuno gold labelling of the effector protein AvrPto. Using a system with expression of the AvrPto effector under control of an inducible promoter, the authors grew the bacteria under Hrp-inducing conditions to get assembly of the Hrp pili (indicated by arrows) on the bacterial surface and (A) did not induce AvrPto (B) induced Hrp and AvrPto simultaneously or (C) induced AvrPto after assembly of the pilus. This shows that newly synthesised effector proteins are secreted through the Hrp pilus as they emerege at its distal tip. Bars = 100 nm. Reprinted with permission from (Jin and He, 2001). Copyright 2001 AAAS.

1.6 THE PLASMID ENCODED YERSINIA TYPE III SECRETION SYSTEM

1.6.1 The common virulence plasmid

The ~70 kb plasmid pYV (plasmid of Yersinia virulence) encodes a type III secretion system shared by Y. pestis, Y. pseudotuberculosis and Y. enterocolitica

22 BACKGROUND

(Ben-Gurion and Shafferman, 1981; Portnoy et al., 1984). The plasmid contains genes for: the Ysc (Yersinia secretion) proteins that build up the secretion organelle, effector Yops (Yersinia outer proteins) that interfere with host cell signalling, the translocators responsible for delivery of the effectors across the host cell membrane, chaperones for the effectors and the translocators, and also regulatory components required for the delicate regulation of the system (fig. 8)

Figure 8. The plasmid encoded Yersinia T3SS. The common virulence plasmid is here represented by the map of pIB1 of Y. pseudo- tuberculosis. Based on unpublished data (P. Cherepanov and T. Svensson). Reprinted with permission from (Schesser et al., 2000). Copyright 2004 ASM Press. .

1.6.2 The Ysc apparatus

The Ysc proteins (table 1) represent the most highly conserved group of T3S proteins, and are predicted to build up the Yersinia T3S organelle as depicted in figure 9. While electron microscopy has been used to visualise the external parts of the organelle, including the YscF needle structures and the ring-shaped outer membrane pores formed by YscC, the membrane spanning basal body has never been isolated or visualised for Yersinia (Hoiczyk and Blobel, 2001; Koster et al., 1997). Thus, the model in fig 9 is based on observations made for related T3SSs (chapter 1.5.1) including the flagellar.

23 BACKGROUND

Table 1. Ysc proteins encoded by the virA, virB, virC and virG operons of pYV. Size Required for Protein Comments Localisation* (kDa) Yop secretion virC YscA 3.8 Hypothetical protein. Unknown N YscB 15.4 Together with SycN forms a CP yscB mutant heterodimeric chaperone for YopN. derepressed YscC 67.3 Forms oligomeric secretin OM ring of Ysc OM Y apparatus. Secreted via Sec. YscD 46.9 IM/PP Y YscE 7.4 Interacts with YscG. CP/IM Y YscF 9.4 Structural needle subcomponent. Surface Y YscG 13.0 Chaperone-like properties, binds YscE. CP/IM Y YscH 18.4 Secreted N YscI 12.6 Chaperone-like properties. CP Y YscJ 27.0 Lipoprotein, possible membrane linker. IM/PP/CP Y Secreted via Sec. YscK 23.9 Interacts with YscQ (Yeast 2/3 hybrid CP Y data). YscL 24.9 Interacts with YscN and YscQ (Yeast 2/3 CP Y Hybrid data). virB YscN 47.8 Predicted ATPase. IM/CP Y YscO 19.0 Secreted Y YscP 50.4 Controls needle length and substrate Secreted Y specificity switch. YscQ 34.4 Interacts with YscL and YscK (Yeast 2/3 CP Y hybrid data). YscR 24.4 Structural component, protrudes into CP. IM Y YscS 9.6 Structural component. IM Y YscT 28.4 Structural component. IM ? YscU 40.4 Structural component, protrudes into CP. IM Y Regulates substrate specificity switch with YscP. virA YscX 13.6 Interacts with YscY. Secreted Y YscY 13.1 Possible chaperone for YscX, interacts CP Y with LcrH. YscV/LcrD 77.8 Structural component, protrudes into CP. IM Y virG YscW 14.6 Lipoprotein piloting YscC to OM and OM Y stabilising the secretin structure. Secreted via Sec. * For some proteins, location is predicted but not proven. OM = outer membrane, PP = periplasm, IM = inner membrane, CP = cytoplasm. References: Allaoui et al., 1994; Allaoui et al., 1995; Bergman et al., 1994; Burghout et al., 2004; Day and Plano, 1998; Day et al., 2000; Day and Plano, 2000; Edqvist et al., 2003; Fields et al., 1994; Francis et al., 2001; Goguen et al., 1984; Haddix and Straley, 1992; Hoiczyk and Blobel, 2001; Iriarte and Cornelis, 1999; Jackson et al., 1998; Jackson and Plano, 2000; Journet et al., 2003; Koster et al., 1997; Michiels et al., 1991; Payne and Straley, 1998, 1999; Plano et al., 1991; Plano and Straley, 1993, 1995; Woestyn et al., 1994.

24 BACKGROUND

The localisation of different Yscs is predicted by a combination of in silico structure analysis, experimental data as well as by homology to known components from other TTS systems. A large number of the Ysc proteins are assembled in the inner membrane, and their exact roles not resolved but may be primarily structural. YscN is a peripheral inner membrane protein, and suggested to be the ATPase that energises the Ysc machinery (Woestyn et al., 1994).

Figure 9. The Ysc secretion apparatus. Schematic Some inner membrane Yscs possess representation of the predicted Yersinia type III large soluble domains that protrude secretion machinery spanning the bacterial double membrane, with a needle-like protrusion and the into the cytoplasm, suggesting a translocator pore in the host cell membrane. possible function for them within this compartment (table 1, fig. 9). As for the flagellar T3SS (chapter 1.5.1), assembly of the Ysc apparatus has to occur in an ordered manner. This is apparent as some of the components of the machinery are exported via the Sec system, while others rely on the T3S system itself for their delivery. The outer membrane secretin protein, YscC, and its pilot, YscW, are examples of Sec dependent substrates while the needle component YscF, as well as the Ysc proteins released into the extrabacterial milieu, depends on a functional T3S machinery for export (table 1, fig. 9). Apart from being building blocks of the secretion apparatus, some Yscs have regulatory roles. One example is the soluble exported protein YscP that, like its homologue

25 BACKGROUND

FliK of the flagellar T3SS, is involved in the T3S substrate specificity switch (chapter 1.5.1, Paper II).

1.6.3 The Yop effectors

The Yop effectors delivered by the Ysc machinery work in concert to inhibit the phagocytic ability of host cells and to repress the inflammatory response, thus enabling extracellular colonisation (Juris et al., 2002). An overview of the Yop effectors and their biological roles is given in table 2.

Table 2. The Yersinia Yop effectors. Effector Chaperone Activity Cellular effect

Disruption of focal complexes, inhibition Anti-phagocytosis, anti- YopH SycH PTPase of pro-inflammatory signalling

YopE SycE GAP Disruption of actin Anti-phagocytosis microfilaments

Serine- Disruption of actin YpkA - Anti-phagocytosis Threonine microfilaments kinase

YopT SycT Cysteine Disruption of actin Anti-phagocytosis protease microfilaments

Cysteine Inhibition of MAPK Induction of of YopJ - protease and NF-κB signalling macrophages, anti- pathways inflammation

Leucin-rich YopM - Locates to nucleus Interferes with eukaryotic repeat protein cell cycle Review: Bliska, 2000; Cornelis et al., 1998; Juris et al., 2002; Navarro et al., 2005.

Virulence studies in mice suggest that YopH, YopE, YpkA and YopM are essential for virulence while YopT is not, and YopJ data are contradictory (Bolin and Wolf- Watz, 1988; Forsberg and Wolf-Watz, 1988; Galyov et al., 1993; Galyov et al., 1994; Leung et al., 1990; Monack et al., 1998; Trulzsch et al., 2004).

1.6.4 The Yersinia type III secretion chaperones

Syc proteins (specific Yop chaperones) bind to one or more specific partner with high affinity and are characteristically small, acidic proteins with a C-terminal 26 BACKGROUND amphiphilic α-helix structure (Feldman and Cornelis, 2003; Marenne et al., 2004). There are multiple suggested roles for the type III chaperones, including stabilisation, anti-aggregation, prevention of premature folding, establishing a hierarchy for secretion and regulation of the T3SS (Feldman and Cornelis, 2003; Marenne et al., 2004). Consistent with their important roles, several substrates depend on chaperones, including the effector proteins YopE, YopH and YopT (table 2). The secreted negative regulators YopN and LcrQ are also chaperoned, by YscB+SycN and SycH respectively, while the translocators YopB and YopD require the LcrH (SycD) chaperone (Day and Plano, 1998; Jackson et al., 1998; Neyt and Cornelis, 1999; Wattiau et al., 1994; Wulff-Strobel et al., 2002). Further, there are Ysc proteins with chaperone-like properties, such as YscI and YscY, which are both encoded adjacent to genes for secreted proteins, YscH and YscX respectively, a common pattern seen for chaperones and their partners (table 1).

1.6.5 A signal for the Yersinia type III secretion substrates

Not only the effector Yops but also the translocators, some Ysc components and regulatory proteins depend on the T3S for export. While primarily studied for the effector Yops, the nature of the secretion signal is not clear. Residing within the N- terminus it has been proposed to lie either within the mRNA or the amino acid sequence and may be characterised by a specific pattern of amphipaticity. Recognition could occur directly by the Ysc apparatus itself, or via chaperones that will recognise and target the substrate to the machinery for delivery (Anderson and Schneewind, 1997; Lloyd et al., 2001a; Lloyd et al., 2001b; Lloyd et al., 2002; Michiels et al., 1990; Sorg et al., 2005; Sory et al., 1995). Consensus remains to be reached, and quite possibly alternative secretion signals might exist for different substrates.

27 BACKGROUND

1.6.6 The low calcium response, in vitro triggering of Yersinia T3S

While inhibited by mM levels of Ca2+, in vitro expression and secretion of Yop proteins can be triggered by cultivation of Yersinia at 37°C in calcium depleted medium (Forsberg et al., 1987; Heesemann et al., 1986; Michiels et al., 1990). The induction of type III secretion in the absence of Ca2+ is accompanied by growth arrest, a phenomenon referred to as ‘the low calcium response’ (LCR) (Goguen et al., 1984; Straley and Bowmer, 1986). Due to this phenotype, the growth of yersiniae at 37°C is denoted calcium dependent (CD). Mutants defective for LCR are classified as either calcium independent (CI) or temperature sensitive (TS). CI strains never enter growth restriction and are usually impaired for Yop expression and secretion. This is the phenotype for most ysc mutant strains and mutants defective for positive regulation. TS, or ‘calcium blind’, strains are restricted for growth at 37°C irrespective of calcium and commonly unable to repress both Yop expression and secretion in the presence of Ca2+. This phenotype reflects mutation in the negative regulation of Yop expression and, usually, secretion (Perry and Fetherston, 1997). It is difficult to appreciate the biological importance of the calcium response, which may even be an artifactual in vitro effect, but it has nonetheless been very valuable for in vitro investigation of the Yersinia T3SS.

1.6.7 In vivo induction of Yersinia type III secretion

A simplified model for the in vivo regulation of Yersinia type III secretion is presented below, without dwelling on controversies. At 37°C, transcription of the genes encoded by the pYV plasmid is induced due to i) release of the histone-like repressor YmoA from the DNA, leading to conformational changes that favours transcription ii) expression of the positive regulator LcrF/VirF (Cornelis et al., 1991; Hoe and Goguen, 1993; Jackson et al., 2004; Lambert de Rouvroit et al., 1992; Yother et al., 1986). The Ysc machinery is assembled and basal levels of Yop proteins are expressed, but prior to contact with 28 BACKGROUND the target cell a negative feed-back mechanism prevents secretion as well as elevated expression of the effectors. This mechanism relies on LcrG and a complex formed by YopN, TyeA and the heterodimeric YopN chaperone YscB+SycN, which obstructs the secretion channel prior to cell contact (Cheng and Schneewind, 2000; Cheng et al., 2001; Ferracci et al., 2005; Schubot et al., 2005). During these conditions negative regulators will be contained within the bacterium; a complex of LcrQ, YopD and LcrH, together repress Yop synthesis by an unknown mechanism. Upon contact with the host cell, the obstruction of the Ysc apparatus is relieved, allowing LcrQ and YopD to be secreted. Thus, bacterium-cell contact lead to derepression of the Yersinia type III secretion system, followed by subsequential translocation of Yop effectors into the host cells (Francis et al., 2001; Pettersson et al., 1996; Rimpilainen et al., 1992; Williams and Straley, 1998). The translocation depends on the three translocator proteins LcrV, YopB and YopD to form a pore in the host cell membrane, thus supporting directed delivery of the effectors into the cytosol of the eukaryotic cell to exert their functions (Forsberg et al., 1994; Holmstrom et al., 2001; Marenne et al., 2003; Persson et al., 1995; Pettersson et al., 1999; Rosqvist et al., 1994; Rosqvist et al., 1995; Sarker et al., 1998; Sory and Cornelis, 1994).

29 AIMS

2. AIMS

The aim of this thesis was to determine the roles played by the components YscF, YscP and YscU in Yersinia type III secretion.

A second objective was investigation of the putative twin arginine translocation system of pathogenic Yersinia and its potential role in virulence.

30 RESULTS AND DISCUSSION

3. RESULTS AND DISCUSSION

3.1 CONCLUSIONS IN BRIEF FROM PAPERS I-IV

I. Both Yersinia pestis and Y. pseudotuberculosis express a functional twin arginine translocation system that in the latter is shown to be required for motility, acid resistance and virulence. Several putative Tat substrates can be identified within the genome sequences of both species.

. II. A Y. pseudotuberculosis yscP mutant strain secretes excess amount of the needle subcomponent YscF and is impaired for Yop secretion. This phenotype can be suppressed by 2nd site mutations in the cytoplasmic domain of YscU, suggesting that these two proteins together control the type III secretion substrate specificity switch.

III. YscU is cleaved within a conserved site (Asn-Pro-Thr-His) in the cytoplasmic domain. Introduction of mutations at this site results in uncleavable YscU variants. When expressed in trans, these proteins restore Yop secretion to an yscU mutant strain, suggesting that cleavage is redundant for YscU function.

IV. Expression of the uncleavabe yscU variants in cis, however, reveal that cleavage is indeed required for Yersinia type III secretion. This illustrates the importance of the strictly conserved site as well as that the complex regulation of the components of the T3SS depends on expression of YscU in its native context.

31 RESULTS AND DISCUSSION

3.2 YERSINIA TWIN ARGININE TRANSLOCATION

3.2.1 Yersinia Tat is functional

Facilitated by the ever increasing number of sequenced genomes, putative twin arginine translocation systems have been identified in a wide range of bacteria (Dilks et al., 2003). As shown in Paper I, the presence of a fully functional Tat system could be confirmed for Y. pestis and Y. pseudotuberculosis. Not surprisingly, the closely related Y. enterocolitica also encodes a complete Tat system (Sanger institute; www.sanger.ac.uk/Projects/Y_enterocolitica), although its functionality remains to be confirmed.

3.2.2 A connection between Tat and Type III secretion?

My first incentive to study the Yersinia Tat system was due to an intriguing observation of a connection between Tat and the type III secretion system as reported by Michael Vasil and coworkers (unpublished results). Their studies on the opportunistic pathogen P. aeruginosa revealed that type III secretion requires a functional Tat system. Despite being pathogens of dissimilar life styles, with very different outcomes of infection, the virulence plasmid encoded T3SS of Yersinia is remarkably similar to the chromosomally encoded system of P. aeruginosa, with a number of components being functionally interchangeable between the two systems (Bröms et al., 2003a, b; Frithz-Lindsten et al., 1998). With Tat in the inner membrane and the T3S apparatus spanning the bacterial envelope, crosstalk between the two systems also in Yersinia was a distinct possibility, in particular since PscO, the YscO homologue (table 1), of P. aeruginosa was considered a putative Tat substrate (M. Vasil, unpublished results). As seen in table 3 both these proteins possess a twin arginine (bold) motif in the N-terminus.

32 RESULTS AND DISCUSSION

Table 3. N-terminal amino acid sequences of PscO and YscO Protein Residue 1-36 PscO 1-MSLALLLRVRRLRLDRAERAQGRQLLRVRAAAQEHT--- YscO 1-MIRRLHRVKVLRVERAEKAIKTQQACLQAAHRRHQE--- Genebank accession numbers: PscO: NP_250387; YscO: CAF25411.

However, with increased understanding of the Tat signal peptide, more accurate predictions of putative substrates can now be made. Analysis of these two putative signal sequences with the TatP software, or with Pedant pattern search (methods described in Paper I) suggests that they are not true Tat substrates. This implies that the Tat system of P. aeruginosa must affect the function of the T3SS in some other way than by impairing secretion of PscO. Moreover, the search for a Tat-T3SS connection in Yersinia turned out negative, since the Yersinia tatC mutant strain proved indistinguishable from wild type Yersinia in all aspects of T3S analysed (Paper I).

3.2.2 Tat is required for Yersinia virulence

Oral infection of mice revealed the Y. pseudotuberculosis tatC mutant to be highly attenuated for virulence and impaired for colonisation of lymphoid organs like the Peyer’s patches and (Paper I). Since the mutant also exhibited increased in vitro sensitivity to acid, a possible explanation to the severe attenuation could be that the bacteria were eradicated by the low pH within the mouse gastrointestinal tract. To resolve this issue, the virulence of the tatC strain was studied by infection via the intraperitoneal route. The results showed that, also for this route, the Tat system is essential for virulence. Fascinatingly, the tatC mutant was equally attenuated as the virulence plasmid cured strain, i.e. lethal at a dose of 107 bacteria/ml compared to 103 for wild type infections (S.K. Ericsson and M. Lavander, unpublished). The acid sensitivity is an interesting phenotype also for this mode of infection, since it may deprive Y. pseudotuberculosis of its ability to survive and multiply within macrophages (chapter 1.2.6). Still, the tatC mutant showed survival within J774 macrophage-like cells equally well as the wild type 33 RESULTS AND DISCUSSION strain, suggesting that this is not the case, although the outcome might be different if activated macrophages were used.

3.2.3 Yersinia Tat substrates in relation to virulence

In silico methods were used to identify putative Tat substrates for Y. pestis and Y. pseudotuberculosis (table 3 in Paper I). Some of these are promising candidates that could explain of the phenotypes of the tatC mutant. The resistance to acidic conditions could possibly be mediated by the carbonic anhydrase (CA) identified in the genomes of both Y. pestis and Y. pseudotuberculosis that may constitute an important virulence factor. CAs are zinc metalloenzymes, found in both pro- and eukaryotes, that catalyze the reversible hydration of CO2 [CO2+H2O ↔ − + HCO3 +H ]. They are known to be involved in many physiological processes and have also been proposed to contribute to intracellular survival of bacteria. Interestingly, a recent publication on demonstrated a periplasmic CA that was required for in vitro acid resistance (Marcus et al., 2005; Smith and Ferry, 2000). Other interesting putative Tat substrates included YbtP and YbtQ (table 3 in Paper I), two proteins involved in iron acquisition, an ability absolutely crucial for colonisation of a host (chapter 1.2.5). Both YbtP and YbtQ have been shown to be essential for iron uptake by Yersiniabactin, and a Y. pestis ybtP mutant strain was avirulent via the subcutaneous route (Fetherston et al., 1999). Further, proteins involved in transport and a number of proteins of unknown functions are interesting for future investigations of their potential roles in Yersinia virulence (table 3 in Paper I).

3.2.4 Future perspectives of Tat

The in silico screening for putative Tat substrates turned up a number of interesting proteins that may contribute to the ability of Yersinia to cause disease. Proteomic comparison of fractions of the extracytoplasmic bacterial compartments derived from the tatC mutant and the isogenic wild type strain will contribute to the search 34 RESULTS AND DISCUSSION for Tat substrates. Verification and complementation of these screens will determine which proteins are i) true substrates of Yersinia Tat, and ii) important for virulence. The severe attenuation of the tatC mutant strain makes Tat an interesting target for development of novel antimicrobial therapeutics. One could envisage two different approaches: either targeting of a Tat substrate identified as crucial for virulence or, alternatively, to block the Tat system directly. The latter approach would be the method of choice, since the substrates may have human homologues (for example the carbonic anhydrase), while the system itself is found in prokaryotes, archaea and plants but, importantly, not in mammals. Utilisation of compound libraries has previously been successfully employed to locate substances that suppress the T3SS of Yersinia and could constitute a useful method also in the present case (Kauppi et al., 2003; Nordfelth et al., 2005).

3.3 ROLE OF YSCP AND YSCU IN YERSINIA TYPE III SECRETION

3.3.1 YscP

YscP is a 50 kDa soluble protein found both intrabacterially and secreted in a type III dependent manner (Payne and Straley, 1999; Stainier et al., 2000). YscP homologues exist in several T3SSs. While their overall sequence conservation is low, they possess a globular domain, essential for function, within the C-terminus. This structure is conserved and interchangeable between the closest related YscP homologues (Agrain et al., 2005a).

3.3.1.1 YscP, a protein of dual functions Paper II shows that YscP is required for substrate specificity switching (chapter 1.5.1), as an yscP mutant exports excessive amounts of the needle component YscF and is impaired for secretion of Yop effectors. The function for the switch is located within the C-terminus of YscP, as revealed by introduction of deletions in 35 RESULTS AND DISCUSSION this domain resulting in a non-functional protein (M. Lavander, unpublished). This is consistent with results from Agrain and coworkers, where function for Yop secretion was mapped between amino acids 385-500 of the 515 amino acid YscP protein of Y. enterocolitica coinciding with the conserved globular domain (Agrain et al., 2005a). A second function of YscP is regulation of the length of the YscF needle. Apparently, the length of the needle is in direct relation to the length of the YscP peptide, suggesting that YscP acts as a molecular ruler (Journet et al., 2003).

3.3.1.2 The role of YscP export The very role of YscP export has not been determined. The signal for YscP export was hypothesised to be located within the extreme N-terminus, since amino acid 4- 7 in InvJ (the YscP homologue from the S. enterica SPI-1 encoded T3SS), had been shown to be necessary and sufficient for T3S dependent export of this protein (Russmann et al., 2002). To determine the minimal secretion signal of YscP, small deletions were introduced within the N-terminus of Y. pseudotuberculosis YscP

(table 4). The YscP∆2-34 variant, although stably expressed, was not secreted and could not restore Yop secretion to an yscP deletion mutant.

Table 4. Effect of deletions within the YscP N-terminusa

Construct N-terminal amino acid sequence Yop secretion YscP expression YscP secretion

Wild-type YscP MNKITTRSPLEPEYQPLGKPHALDACVDFEQALLHNNKGNCHPK ++ ++ ++ YscP (∆2-34) M------HNNKGNCHPK - +++ - YscP (∆6-34) MNKIT------HNNKGNCHPK ++ +++ ++ YscP (∆9-34) MNKITTRS------HNNKGNCHPK ++ +++ ++ YscP (∆11-34) MNKITTRSPL------HNNKGNCHPK ++ +++ ++ a) YscP variants with the described deletions were expressed in trans under control of the tac promoter of pMMB66HE in the yscP mutant strain pIB69 (Paper II). The number of plus (+) indicates levels of protein in supernatant (secretion) or total samples (expression) as detected with α-Yop and α-YscP antisera.

In contrast, by keeping the first 5,8 or 10 residues, the same deletion variant could now be secreted as well as support Yop secretion, suggesting that the first five 36 RESULTS AND DISCUSSION amino acids of YscP constitute a minimal secretion signal. Curiously, Agrain and coworkers recently used sequential deletions to reveal that YscP of Y. enterocolitica may actually contain two secretion signals, within amino acid 1-35 and 97-137 respectively. To prevent YscP secretion both signals had to be disrupted, resulting in a protein able to support Yop secretion but not to control needle length (Agrain et al., 2005b). This proposed second secretion signal seems to be missing in Y. pseudotuberculosis YscP, where deletion of residues 2-34 efficiently prohibits export of the protein (table 4). Even if my results indicate that the ability to export YscP is required for Yop secretion, it is conceivable that the export may be a side effect of localising in the type III secretion apparatus during induction of the system, rather than a prerequisite for YscP function. YscP is secreted to the extracellular milieu, primarily under type III secretion inducing conditions (minus Ca2+). However, overexpression of the protein results in high levels of YscP export also under non- inducing conditions (plus Ca2+). This shows that there is a hierarchy of secretion, as the type III dependent delivery of YscP across the bacterial double membrane occurs under conditions when Yops are still restrained from secretion (M. Lavander, unpublished).

3.3.2 YscU

YscU is a 40 kDa inner membrane protein, with four transmembrane domains and a C-terminal tail protruding into the cytoplasm. YscU belongs to a highly conserved family of core components that are absolutely essential for type III secretion (Allaoui et al., 1994; Hueck, 1998). Schematic representations of YscU can be found in Paper III, fig. 1 and Paper IV, fig. 6.

3.3.2.1 YscU and the role of cleavage An early observation when expressing YscU in trans was that the protein is cleaved between Asn-263 and Pro-264 within a strictly conserved four amino acid 37 RESULTS AND DISCUSSION site (Asparagine-Proline-Threonine-Histidine) in the C-terminus, and that mutation within this site abolished proteolysis (Papers III-IV). The conserved nature of the site, together with the observation of cleavage, indicated that the proteolysis may be of importance for YscU function. While initial studies in trans suggested that uncleavable variants of YscU could still promote Yop secretion, identical mutants expressed in cis could not (Papers III-IV). This is a clear illustration of the importance of working in the native context to avoid misleading high-copy effects, even more so when studying a highly complex apparatus where ratios between individual components may be crucial. If cleavage is indeed coupled to functional Yop-secretion, how does this occur? One possibility is that YscU generally adopts a conformation that is required for its function, but makes the protein prone to cleavage, i.e. cleavage as a secondary effect. The proteolysis may on the other hand play a more direct role, as a primary event for functional T3S. For example, the role of proteolysis could be to set the YscUCC (amino acid 264-354, i.e. the C-terminal cleavage product) domain free to perform a function at a different locus of the T3S system. In flagellar T3SSs a protein,

FlhX, homologous to the YscUCC domain, has been identified in many bacteria, suggesting a biological role for this peptide (Pallen et al., 2005). Alternatively, the parting of YscUCC from YscUCN (amino acid 211-263, extending from the transmembrane domain of YscU) could allow the latter to promote Yop secretion from within the apparatus. In this model, YscUCC would function as an inhibitor of

YscUCN.

3.3.3 The YscP and YscU connection

Previous studies have indicated a direct interaction between YscU and YscP. For example, the observation of an interaction between the analogous proteins FlhB and FliK from the flagellar T3SS as seen in protein overlay assays supports the notion of a physical interaction also between YscU and YscP (Minamino and Macnab, 2000). Additionally, 2nd site mutations within the C-terminal domain of 38 RESULTS AND DISCUSSION

YscU allow for substrate specificity switching even without the presence of YscP (Paper II). However, attempts to confirm this interaction with various methods, including yeast two-hybrid, Flag pull-downs, and protein overlays, have been unsuccessful (M. Lavander, unpublished). This may suggest that the interaction for some reason is difficult to capture: either it is very transient, easily disassociates under less-than-optimal in vitro conditions or possibly that some third component is needed to stabilise of the interaction between YscU and YscP. Then again, there is the scenario where the two proteins actually do not physically interact, and that another yet unidentified component may be responsible for the crosstalk between YscU and YscP in the in vivo situation. My belief is however that the two do indeed interact and that YscP thereby under permissive conditions induces a conformational change in the C-terminus of YscU that allows it to support Yop secretion. The hypothesis presented in Paper II suggests that the YscP dependent conformational change may be simulated by 2nd site mutations in YscU.

3.3.4 Role of the needle in type III secretion

A key element in T3S is the needle component YscF. The exact role of the needle in T3S is not clear, although the general belief is that it acts as a conduit through which the Yop effectors reach the target cell. To study the role of the needle, variants of YscF mutagenised with alanine substitutions were engineered and investigated for their ability to complement an yscF mutant strain. While a number of substitutions at conserved residues resulted in non-functional YscF variants

(data not shown), the substitution mutant YscFD46A gave a more intriguing phenotype. Whereas wild type YscF supported Yop secretion only under inducing 2+ in vitro conditions (minus Ca ), the YscFD46A mutant showed constitutive expression and secretion of Yop effectors and translocators (fig. 10 A). Furthermore, the mutant did not display the polarised translocation observed for wild type Yersinia but instead delivered high amounts of Yops to the culture

39 RESULTS AND DISCUSSION medium during cell infection assays (data not shown). Consequently, the mutant strain was severely delayed in YopE mediated cytotoxicity (fig. 10B).

Fig. 10. YscFD46A confers constitutive Yop secretion to the Yersinia T3SS. Wild type Y. pseudotuberculosis YPIII(pIB102) and an yscF mutant strain YPIII(pIB202) (Paper II) were utilised in Yop expression and secretion assay and infection of HeLa cells. YscF variants were expressed in trans under control of the tac promoter of pMMB66EH. A) Total samples (T) and supernatants (S), showing expression and secretion respectively, in bacterial cultures grown at 37°C, +/- Ca2+ as implied. Proteins were detected with specific polyclonal antisera. B) Cytotoxic response, seen as rounding up of the cells due to disruption of the cytoskeleton, in HeLa cells after 3h of infection.

Taken together, these observations suggest that the needle component is somehow involved in regulation of the T3SS, possibly by acting as a sensor of the extra- bacterial milieu transmitting the inducing signal (cell contact in vivo, calcium depletion in vitro) to the T3SS. The role of YscF as a sensor was also recently suggested in a publication on Y. pestis (Torruellas et al., 2005).

3.3.5 A model of substrate specificity switching by YscP and YscU

A speculative model for how YscP and YscU could contribute to induction of Yop secretion is presented here. During non secretion permissive conditions at 37°C (i.e. in presence of calcium or prior to target cell contact) Yop secretion is physically prohibited: A complex formed by YopN, its heterodimeric chaperon YscB+SycN and TyeA, which 40 RESULTS AND DISCUSSION anchors the complex at the inner membrane, is responsible for obstructing the secretion apparatus (Ferracci et al., 2005; Schubot et al., 2005). Upon cell contact, or depletion of calcium, the needle acts as a sensor and transmits the signal to the system (Torruellas et al., 2005). This signal is conferred to YscP, which alters the conformation of the C-terminal domain of YscU to a secretion-permissive state. YscU then interacts, directly or indirectly, with the ATPase YscN to alter the substrate specificity of the system. This interaction enables docking of chaperones to YscN, followed by dissociation of the chaperone and ATPase mediated unfolding and secretion of the substrate (Akeda and Galan, 2005; Gauthier and Finlay, 2003; Thomas et al., 2004). The first substrate to be secreted is YopN, thus unblocking the channel, which allows for secretion of negative regulatory elements (LcrQ and YopD) that normally suppress expression of the Yop effectors. This results in elevated Yop expression and Yop secretion completing the substrate specificity switch.

3.3.5 Future perspectives for YscU and YscP One interesting and unidentified factor, that potentially could be a regulator of type III secretion, is the protease responsible for YscU cleavage. The ClpXP and Lon proteases have been shown to be involved in the regulation of type III secretion. More precisely, these proteases have been shown to be required for degradation of the histone-like protein YmoA, which represses transcription of T3S genes (chapter 1.6.7) (Jackson et al., 2004). The observation of YscU cleavage occurring in T3SS negative backgrounds (Paper III) implies that the responsible protease is ubiquitously expressed, or possibly that YscU might induce autolysis. However, no domains suggesting a proteolytic activity are found within YscU. Further, interactions between YscP, YscU and other components of the Yersinia T3SS would be very valuable for an understanding of the substrate specificity switch. This would facilitate future work with placing the two proteins in a larger

41 RESULTS AND DISCUSSION context and designing experiments to establish the functions of the interactions involved in induction of Yop secretion.

3.4 FINAL REMARKS

As the discovery of antibiotics offered a cure also for plague, the threat from Y. pestis may seem like ancient history. However, pathogens evolve ways to overcome the obstacles in their way and, consequently, multiresistant strains of Y. pestis have been isolated from patients in Madagascar, where plague is endemic (Galimand et al., 1997; Guiyoule et al., 2001). Currently, a sub-unit based on the Y. pestis V- and F1-antigens is raising the hopes for protection against both bubonic and pneumonic plague (Heath et al., 1998; Titball and Williamson, 2001, 2004; Williamson et al., 1995). While the V-antigen (LcrV) is present in all virulent Yersinia strains, there is evidence of fully virulent naturally occurring Y. pestis strains devoid of the F1-antigen (Davis et al., 1996). Efficiency of the new sub-component vaccine against these isolates is an unresolved issue. The type III secretion system and, as shown in this thesis, the twin arginine translocation system are required for Yersinia cause disease. Since both T3S and Tat are essential virulence determinants for a number of pathogens, these secretion systems are interesting targets for the development of treatments that may be active against a wide range of bacteria. While progress has already been made for chemical suppression of T3S (Kauppi et al., 2003; Nordfelth et al., 2005), the Tat system is unexplored as an antimicrobial target. Future investigations to identify key drugs that block the Tat system would be a valuable contribution to the development of novel therapies, which potentially could be efficient against a diversity of infectious diseases caused by bacterial pathogens.

42 TACK!

ACKNOWLEDGEMENTS

Jag skulle vilja säga TACK till alla som varit med på färden. Till handledar- mannen, Åke, för att ha erbjudit motiv och tillfälle att begå detta dåd. Alla prima fina bakterier på medicinskt skydd (utan hänsyn till tidens gång): Anna-Lena, Anna M, Elisabet, Eva Lu, Jay-Tee, Kerstin, Laila, Pia och Tina. Ännu mer blandade bakterier (och en parasit) från molekylärbiologen i form av forna och nuvarande medlemmar i grupper Francis, Fällman, Milton, Persson, Rosqvist, Shingler och Wolf-Watz. Bonustack till Petra E, Janne, Sara, Margareta, RolleR, Hasse, Vicky och Matt för ovärderlig hjälp med Yscar, jäst, antisera, celler, EM-tips och dylika ting. Alla mina talangfulla småtjejer i kronologisk ordning: Ann-Louise, Lisa, Ritu och Britta. BarbaSolan som, tillsammans med Marléne (darkness!), trollat med Tat- mössen. Lenóre för magisk hand med elektroniskt mikroskop. Lena L för jästhjälp. Lotta, Sonja och Katarina för oslagbar markservice. Jonas och Göran för celler i sista sekunden. Ulrika D-H, för visat förtroende. Kjerstin som ordnat tjogvis med artiklar. Marie L som tagit hand om struliga resebokningar och massa annat fix. Eva-Christine som fyllt i ALLA mina aktivitetsrapporter, du är en pärla! Rumskompisen, Charlotta, som fått timmar att flyga till surr när vi borde ha gjort något vettigt. Mästerkocken och spådamen, Lena S. Emelie som introducerade irish coffee på menyn. Sandra som så gärna ville smaka… Min favvotjej Jeanette, som det på något konstigt sätt är helt omöjligt att ha tråkigt tillsammans med. Ok, kanske lite småsegt på O’Hare, men i övrigt kommer jag inte på ett enda tillfälle.

Stort tack till alla andra tomtar, änglar, nördar, sångfåglar, ölbryggare och coola katter som fixat, trixat, flyttat, knattrat, hämtat, gycklat, jazzat, bjussat och skrönat.

Rötterna: norra provinsernas allra bästa föräldrar Mommy och Lill-Paapei, superduperFarmor, lillbrorsan Björn med Frida och Isse (han är då för fantastisk

43 TACK! den pojken), allra minsta brorsan Mats och Lotta. Tack för allt. Samt övriga Lundinare och Lavandrar (inklusive Westins), ni är en förträfflig skara! Kompisar från förr, T&K, TBAFC, Anna-Karin, Ulf, Jocke, Anders, Henrik och Bröllarna, samt gästspelarna Anna och Frida. Resesällskapet och konspiratörerna Semmelmeister och Sellle. Och sist men störst, han som får mitt hjärta att slå ett extra slag: Anders, ti-tick!

______här kan du skriva ditt namn om jag glömde bort dig, vilket naturligtvis absolut inte var min mening och jag ber genuint om ursäkt för min tankspriddhet. (Ring 090-106639 så bjuder jag på en öl (leg. 18 år) som plåster på såren).

44 REFERENCES

REFERENCES

1. Achtman, M., Zurth, K., Morelli, G., Torrea, G., Guiyoule, A., and Carniel, E. (1999) Yersinia pestis, the cause of plague, is a recently emerged clone of Yersinia pseudotuberculosis. Proc Natl Acad Sci U S A 96: 14043-14048. 2. Achtman, M. (2004) Age, Descent and Genetic Diversity within Yersinia pestis. In Yersinia: Molecular and Cellular Biology. Carniel, E. and Hinnebusch, B.J. (eds). Wymondham, U.K.: Horizon Bioscience, pp. 17-29. 3. Agrain, C., Callebaut, I., Journet, L., Sorg, I., Paroz, C., Mota, L.J., and Cornelis, G.R. (2005a) Characterization of a Type III secretion substrate specificity switch (T3S4) domain in YscP from . Mol Microbiol 56: 54-67. 4. Agrain, C., Sorg, I., Paroz, C., and Cornelis, G.R. (2005b) Secretion of YscP from Yersinia enterocolitica is essential to control the length of the injectisome needle but not to change the type III secretion substrate specificity. Mol Microbiol 57: 1415-1427. 5. Aizawa, S.I. (1996) Flagellar assembly in Salmonella typhimurium. Mol Microbiol 19: 1-5. 6. Aizawa, S.I. (2001) Bacterial flagella and type III secretion systems. FEMS Microbiol Lett 202: 157-164. 7. Akeda, Y., and Galan, J.E. (2005) Chaperone release and unfolding of substrates in type III secretion. Nature 437: 911-915. 8. Alami, M., Luke, I., Deitermann, S., Eisner, G., Koch, H.G., Brunner, J., and Muller, M. (2003) Differential interactions between a twin-arginine signal peptide and its translocase in . Mol Cell 12: 937-946. 9. Alfano, J.R., and Collmer, A. (2004) Type III secretion system effector proteins: double agents in bacterial disease and plant defense. Annu Rev Phytopathol 42: 385-414. 10. Allaoui, A., Woestyn, S., Sluiters, C., and Cornelis, G.R. (1994) YscU, a Yersinia enterocolitica inner membrane protein involved in Yop secretion. J Bacteriol 176: 4534-4542. 11. Allaoui, A., Schulte, R., and Cornelis, G.R. (1995) Mutational analysis of the Yersinia enterocolitica virC operon: characterization of yscE, F, G, I, J, K required for Yop secretion and yscH encoding YopR. Mol Microbiol 18: 343-355. 12. Anderson, D.M., and Schneewind, O. (1997) A mRNA signal for the type III secretion of Yop proteins by Yersinia enterocolitica. Science 278: 1140-1143. 13. Autenrieth, I.B., and Firsching, R. (1996) Penetration of M cells and destruction of Peyer's patches by Yersinia enterocolitica: an ultrastructural and histological study. J Med Microbiol 44: 285-294. 14. Behrendt, J., Standar, K., Lindenstrauss, U., and Bruser, T. (2004) Topological studies on the twin-arginine translocase component TatC. FEMS Microbiol Lett 234: 303-308. 15. Ben-Efraim, S., and Bichowsky-Slomnichi, L. (1964) The Ph 6 Antigen in Strains of Pseudotuberculosis and Its Relation to Biological Activities. J Bacteriol 87: 236-237. 16. Ben-Gurion, R., and Shafferman, A. (1981) Essential virulence determinants of different Yersinia species are carried on a common plasmid. Plasmid 5: 183-187. 17. Bercovier, H., Mollaret, H., Alonso, J., Brault, J., Fanning, G.R., Steigerwalt, A., and Brenner, D. (1980) Intra- and interspecies relatedness of Yersinia pestis by DNA hybridization and its relationship to Yersinia pseudotuberculosis. Current Microbiology 4: 225-229. 18. Bergman, T., Erickson, K., Galyov, E., Persson, C., and Wolf-Watz, H. (1994) The lcrB (yscN/U) gene cluster of Yersinia pseudotuberculosis is involved in Yop secretion and shows 45 REFERENCES

high homology to the spa gene clusters of Shigella flexneri and Salmonella typhimurium. J Bacteriol 176: 2619-2626. 19. Berks, B.C. (1996) A common export pathway for proteins binding complex redox cofactors? Mol Microbiol 22: 393-404. 20. Berks, B.C., Palmer, T., and Sargent, F. (2003) The Tat protein translocation pathway and its role in microbial physiology. Adv Microb Physiol 47: 187-254. 21. Biedzka-Sarek, M., Venho, R., and Skurnik, M. (2005) Role of YadA, Ail, and Lipopolysaccharide in Serum Resistance of Yersinia enterocolitica Serotype O:3. Infect Immun 73: 2232-2244. 22. Bliska, J.B. (2000) Yop effectors of Yersinia spp. and actin rearrangements. Trends Microbiol 8: 205-208. 23. Blocker, A., Jouihri, N., Larquet, E., Gounon, P., Ebel, F., Parsot, C., Sansonetti, P., and Allaoui, A. (2001) Structure and composition of the Shigella flexneri "needle complex", a part of its type III secreton. Mol Microbiol 39: 652-663. 24. Blocker, A., Komoriya, K., and Aizawa, S. (2003) Type III secretion systems and bacterial flagella: insights into their function from structural similarities. Proc Natl Acad Sci U S A 100: 3027-3030. Epub 2003 Mar 3011. 25. Bogsch, E.G., Sargent, F., Stanley, N.R., Berks, B.C., Robinson, C., and Palmer, T. (1998) An essential component of a novel bacterial protein export system with homologues in plastids and mitochondria. J Biol Chem 273: 18003-18006. 26. Bolhuis, A., Bogsch, E.G., and Robinson, C. (2000) Subunit interactions in the twin-arginine translocase complex of Escherichia coli. FEBS Lett 472: 88-92. 27. Bolin, I., Norlander, L., and Wolf-Watz, H. (1982) Temperature-inducible outer membrane protein of Yersinia pseudotuberculosis and Yersinia enterocolitica is associated with the virulence plasmid. Infect Immun 37: 506-512. 28. Bolin, I., and Wolf-Watz, H. (1988) The plasmid-encoded Yop2b protein of Yersinia pseudotuberculosis is a virulence determinant regulated by calcium and temperature at the level of transcription. Mol Microbiol 2: 237-245. 29. Boyle, E.C., and Finlay, B.B. (2003) Bacterial pathogenesis: exploiting cellular adherence. Curr Opin Cell Biol 15: 633-639. 30. Bröms, J.E., Forslund, A.L., Forsberg, A., and Francis, M.S. (2003a) Dissection of homologous translocon operons reveals a distinct role for YopD in type III secretion by Yersinia pseudotuberculosis. Microbiology 149: 2615-2626. 31. Bröms, J.E., Sundin, C., Francis, M.S., and Forsberg, A. (2003b) Comparative analysis of type III effector translocation by Yersinia pseudotuberculosis expressing native LcrV or PcrV from Pseudomonas aeruginosa. J Infect Dis 188: 239-249. Epub 2003 Jul 2001.. 32. Brubaker, R.R. (1991) Factors promoting acute and chronic diseases caused by yersiniae. Clin Microbiol Rev 4: 309-324. 33. Bruneteau, M., and Minka, S. (2003) Lipopolysaccharides of bacterial pathogens from the genus Yersinia: a mini-review. Biochimie 85: 145-152. 34. Burghout, P., van Boxtel, R., Van Gelder, P., Ringler, P., Muller, S.A., Tommassen, J., and Koster, M. (2004) Structure and electrophysiological properties of the YscC secretin from the type III secretion system of Yersinia enterocolitica. J Bacteriol 186: 4645-4654. 35. Burns, D.L. (2003) Type IV transporters of pathogenic bacteria. Curr Opin Microbiol 6: 29-34. 36. Buttner, D., and Bonas, U. (2002) Getting across--bacterial type III effector proteins on their way to the plant cell. Embo J 21: 5313-5322.

46 REFERENCES

37. Chain, P.S., Carniel, E., Larimer, F.W., Lamerdin, J., Stoutland, P.O., Regala, W.M., Georgescu, A.M., Vergez, L.M., Land, M.L., Motin, V.L., Brubaker, R.R., Fowler, J., Hinnebusch, J., Marceau, M., Medigue, C., Simonet, M., Chenal-Francisque, V., Souza, B., Dacheux, D., Elliott, J.M., Derbise, A., Hauser, L.J., and Garcia, E. (2004) Insights into the evolution of Yersinia pestis through whole-genome comparison with Yersinia pseudotuberculosis. Proc Natl Acad Sci U S A 101: 13826-13831. Epub 12004 Sep 13829. 38. Chapman, D.A., Zavialov, A.V., Chernovskaya, T.V., Karlyshev, A.V., Zav'yalova, G.A., Vasiliev, A.M., Dudich, I.V., Abramov, V.M., Zav'yalov, V.P., and MacIntyre, S. (1999) Structural and functional significance of the FGL sequence of the periplasmic chaperone Caf1M of Yersinia pestis. J Bacteriol 181: 2422-2429. 39. Cheng, L.W., and Schneewind, O. (2000) Yersinia enterocolitica TyeA, an intracellular regulator of the type III machinery, is required for specific targeting of YopE, YopH, YopM, and YopN into the cytosol of eukaryotic cells. J Bacteriol 182: 3183-3190. 40. Cheng, L.W., Kay, O., and Schneewind, O. (2001) Regulated secretion of YopN by the type III machinery of Yersinia enterocolitica. J Bacteriol 183: 5293-5301. 41. Christie, P.J. (1997) Agrobacterium tumefaciens T-complex transport apparatus: a paradigm for a new family of multifunctional transporters in eubacteria. J Bacteriol 179: 3085-3094. 42. Christie, P.J. (2004) Type IV secretion: the Agrobacterium VirB/D4 and related conjugation systems. Biochim Biophys Acta 1694: 219-234. 43. Clark, M.A., Hirst, B.H., and Jepson, M.A. (1998) M-cell surface beta1 integrin expression and invasin-mediated targeting of Yersinia pseudotuberculosis to mouse Peyer's patch M cells. Infect Immun 66: 1237-1243. 44. Collyn, F., Lety, M.A., Nair, S., Escuyer, V., Ben Younes, A., Simonet, M., and Marceau, M. (2002) Yersinia pseudotuberculosis harbors a type IV pilus gene cluster that contributes to pathogenicity. Infect Immun 70: 6196-6205. 45. Collyn, F., Billault, A., Mullet, C., Simonet, M., and Marceau, M. (2004) YAPI, a new Yersinia pseudotuberculosis . Infect Immun 72: 4784-4790. 46. Cornelis, G.R., Sluiters, C., Delor, I., Geib, D., Kaniga, K., Lambert de Rouvroit, C., Sory, M.P., Vanooteghem, J.C., and Michiels, T. (1991) ymoA, a Yersinia enterocolitica chromosomal gene modulating the expression of virulence functions. Mol Microbiol 5: 1023-1034. 47. Cornelis, G.R., and Wolf-Watz, H. (1997) The Yersinia Yop virulon: a bacterial system for subverting eukaryotic cells. Mol Microbiol 23: 861-867. 48. Cornelis, G.R., Boland, A., Boyd, A.P., Geuijen, C., Iriarte, M., Neyt, C., Sory, M.P., and Stainier, I. (1998) The virulence plasmid of Yersinia, an antihost genome. Microbiol Mol Biol Rev 62: 1315-1352. 49. Cowan, C., Jones, H.A., Kaya, Y.H., Perry, R.D., and Straley, S.C. (2000) Invasion of epithelial cells by Yersinia pestis: evidence for a Y. pestis-specific invasin. Infect Immun 68: 4523-4530. 50. Dalbey, R.E., and Von Heijne, G. (1992) Signal peptidases in prokaryotes and eukaryotes--a new protease family. Trends Biochem Sci 17: 474-478. 51. Dalbey, R.E., and Robinson, C. (1999) Protein translocation into and across the bacterial plasma membrane and the plant thylakoid membrane. Trends Biochem Sci 24: 17-22. 52. Dalbey, R.E., and Chen, M. (2004) Sec-translocase mediated membrane protein biogenesis. Biochim Biophys Acta 1694: 37-53. 53. Dale, C., Young, S.A., Haydon, D.T., and Welburn, S.C. (2001) The insect endosymbiont Sodalis glossinidius utilizes a type III secretion system for cell invasion. Proc Natl Acad Sci U S A 98: 1883-1888.

47 REFERENCES

54. Dale, C., Plague, G.R., Wang, B., Ochman, H., and Moran, N.A. (2002) Type III secretion systems and the evolution of mutualistic endosymbiosis. Proc Natl Acad Sci U S A 99: 12397- 12402. Epub 12002 Sep 12394. 55. Davis, K.J., Fritz, D.L., Pitt, M.L., Welkos, S.L., Worsham, P.L., and Friedlander, A.M. (1996) Pathology of experimental pneumonic plague produced by fraction 1-positive and fraction 1- negative Yersinia pestis in African green monkeys (Cercopithecus aethiops). Arch Pathol Lab Med 120: 156-163. 56. Day, J.B., and Plano, G.V. (1998) A complex composed of SycN and YscB functions as a specific chaperone for YopN in Yersinia pestis. Mol Microbiol 30: 777-788. 57. Day, J.B., Guller, I., and Plano, G.V. (2000) Yersinia pestis YscG protein is a Syc-like chaperone that directly binds yscE. Infect Immun 68: 6466-6471. 58. Day, J.B., and Plano, G.V. (2000) The Yersinia pestis YscY protein directly binds YscX, a secreted component of the type III secretion machinery. J Bacteriol 182: 1834-1843. 59. de Keyzer, J., van der Does, C., and Driessen, A.J. (2003) The bacterial translocase: a dynamic protein channel complex. Cell Mol Life Sci 60: 2034-2052. 60. de Leeuw, E., Granjon, T., Porcelli, I., Alami, M., Carr, S.B., Muller, M., Sargent, F., Palmer, T., and Berks, B.C. (2002) Oligomeric properties and signal peptide binding by Escherichia coli Tat protein transport complexes. J Mol Biol 322: 1135-1146. 61. Delepelaire, P. (2004) Type I secretion in gram-negative bacteria. Biochim Biophys Acta 1694: 149-161. 62. Deng, W., Burland, V., Plunkett, G., 3rd, Boutin, A., Mayhew, G.F., Liss, P., Perna, N.T., Rose, D.J., Mau, B., Zhou, S., Schwartz, D.C., Fetherston, J.D., Lindler, L.E., Brubaker, R.R., Plano, G.V., Straley, S.C., McDonough, K.A., Nilles, M.L., Matson, J.S., Blattner, F.R., and Perry, R.D. (2002) Genome sequence of Yersinia pestis KIM. J Bacteriol 184: 4601-4611. 63. Dilks, K., Rose, R.W., Hartmann, E., and Pohlschroder, M. (2003) Prokaryotic utilization of the twin-arginine translocation pathway: a genomic survey. J Bacteriol 185: 1478-1483. 64. Driessen, A.J. (2001) SecB, a molecular chaperone with two faces. Trends Microbiol 9: 193-196. 65. Du, Y., Galyov, E., and Forsberg, A. (1995) Genetic analysis of virulence determinants unique to Yersinia pestis. Contrib Microbiol Immunol 13: 321-324. 66. Du, Y., Rosqvist, R., and Forsberg, A. (2002) Role of fraction 1 antigen of Yersinia pestis in inhibition of phagocytosis. Infect Immun 70: 1453-1460. 67. Duplantier, J.M., Duchemin, J.B., Chanteau, S., and Carniel, E. (2005) From the recent lessons of the Malagasy foci towards a global understanding of the factors involved in plague reemergence. Vet Res 36: 437-453. 68. Durand, E., Bernadac, A., Ball, G., Lazdunski, A., Sturgis, J.N., and Filloux, A. (2003) Type II protein secretion in Pseudomonas aeruginosa: the pseudopilus is a multifibrillar and adhesive structure. J Bacteriol 185: 2749-2758. 69. Economou, A. (1999) Following the leader: bacterial protein export through the Sec pathway. Trends Microbiol 7: 315-320. 70. Economou, A. (2000) Bacterial protein translocase: a unique molecular machine with an army of substrates. FEBS Lett 476: 18-21. 71. Edqvist, P.J., Olsson, J., Lavander, M., Sundberg, L., Forsberg, A., Wolf-Watz, H., and Lloyd, S.A. (2003) YscP and YscU regulate substrate specificity of the Yersinia type III secretion system. J Bacteriol 185: 2259-2266. 72. Eitel, J., and Dersch, P. (2002) The YadA protein of Yersinia pseudotuberculosis mediates high- efficiency uptake into human cells under environmental conditions in which invasin is repressed. Infect Immun 70: 4880-4891. 48 REFERENCES

73. El Tahir, Y., and Skurnik, M. (2001) YadA, the multifaceted Yersinia adhesin. Int J Med Microbiol 291: 209-218. 74. Erridge, C., Bennett-Guerrero, E., and Poxton, I.R. (2002) Structure and function of lipopolysaccharides. Microbes Infect 4: 837-851. 75. Feldman, M.F., and Cornelis, G.R. (2003) The multitalented type III chaperones: all you can do with 15 kDa. FEMS Microbiol Lett 219: 151-158. 76. Ferber, D.M., and Brubaker, R.R. (1981) Plasmids in Yersinia pestis. Infect Immun 31: 839-841. 77. Ferracci, F., Schubot, F.D., Waugh, D.S., and Plano, G.V. (2005) Selection and characterization of Yersinia pestis YopN mutants that constitutively block Yop secretion. Mol Microbiol 57: 970- 987. 78. Fetherston, J.D., Bertolino, V.J., and Perry, R.D. (1999) YbtP and YbtQ: two ABC transporters required for iron uptake in Yersinia pestis. Mol Microbiol 32: 289-299. 79. Fields, K.A., Plano, G.V., and Straley, S.C. (1994) A low-Ca2+ response (LCR) secretion (ysc) locus lies within the lcrB region of the LCR plasmid in Yersinia pestis. J Bacteriol 176: 569-579. 80. Finlay, B.B., and Caparon, M. (2005) Bacterial Adherence to Cell Surfaces and Extracellular Matrix. In Cellular Microbiology, 2nd edition. Cossart, P., Boquet, P., Normark, S. and Rappuoli, R. (eds). Washington, DC: ASM Press, pp. 105-120. 81. Forsberg, A., Bolin, I., Norlander, L., and Wolf-Watz, H. (1987) Molecular cloning and expression of calcium-regulated, plasmid-coded proteins of Y. pseudotuberculosis. Microb Pathog 2: 123-137. 82. Forsberg, A., and Wolf-Watz, H. (1988) The virulence protein Yop5 of Yersinia pseudotuberculosis is regulated at transcriptional level by plasmid-plB1-encoded trans-acting elements controlled by temperature and calcium. Mol Microbiol 2: 121-133. 83. Forsberg, A., Rosqvist, R., and Wolf-Watz, H. (1994) Regulation and polarized transfer of the Yersinia outer proteins (Yops) involved in antiphagocytosis. Trends Microbiol 2: 14-19. 84. Francis, M.S., Lloyd, S.A., and Wolf-Watz, H. (2001) The type III secretion chaperone LcrH co- operates with YopD to establish a negative, regulatory loop for control of Yop synthesis in Yersinia pseudotuberculosis. Mol Microbiol 42: 1075-1093. 85. Francis, M.S., Schesser, K., Forsberg, A., and Wolf-Watz, H. (2004) Type III Secretion Systems in Animal- and Plant-Interacting Bacteria. In Cellular Microbiology, 2nd Edition. Cossart, P., Normark, S. and Rappuoli, R. (eds). Washington, DC: ASM Press, pp. 362-392. 86. Frithz-Lindsten, E., Holmstrom, A., Jacobsson, L., Soltani, M., Olsson, J., Rosqvist, R., and Forsberg, A. (1998) Functional conservation of the effector protein translocators PopB/YopB and PopD/YopD of Pseudomonas aeruginosa and Yersinia pseudotuberculosis. Mol Microbiol 29: 1155-1165. 87. Fukushima, H., Gomyoda, M., Ishikura, S., Nishio, T., Moriki, S., Endo, J., Kaneko, S., and Tsubokura, M. (1989) Cat-contaminated environmental substances lead to Yersinia pseudotuberculosis infection in children. J Clin Microbiol 27: 2706-2709. 88. Gage, K.L., and Kosoy, M.Y. (2005) Natural history of plague: perspectives from more than a century of research. Annu Rev Entomol 50: 505-528. 89. Galimand, M., Guiyoule, A., Gerbaud, G., Rasoamanana, B., Chanteau, S., Carniel, E., and Courvalin, P. (1997) Multidrug resistance in Yersinia pestis mediated by a transferable plasmid. N Engl J Med 337: 677-680. 90. Galyov, E.E., Hakansson, S., Forsberg, A., and Wolf-Watz, H. (1993) A secreted protein kinase of Yersinia pseudotuberculosis is an indispensable virulence determinant. Nature 361: 730-732.

49 REFERENCES

91. Galyov, E.E., Hakansson, S., and Wolf-Watz, H. (1994) Characterization of the operon encoding the YpkA Ser/Thr protein kinase and the YopJ protein of Yersinia pseudotuberculosis. J Bacteriol 176: 4543-4548. 92. Gauthier, A., and Finlay, B.B. (2003) Translocated receptor and its chaperone interact with ATPase of the type III secretion apparatus of enteropathogenic Escherichia coli. J Bacteriol 185: 6747-6755. 93. Gemski, P., Lazere, J.R., and Casey, T. (1980a) Plasmid associated with pathogenicity and calcium dependency of Yersinia enterocolitica. Infect Immun 27: 682-685. 94. Gemski, P., Lazere, J.R., Casey, T., and Wohlhieter, J.A. (1980b) Presence of a virulence- associated plasmid in Yersinia pseudotuberculosis. Infect Immun 28: 1044-1047. 95. Ghosh, P. (2004) Process of protein transport by the type III secretion system. Microbiol Mol Biol Rev 68: 771-795. 96. Goguen, J.D., Yother, J., and Straley, S.C. (1984) Genetic analysis of the low calcium response in Yersinia pestis mu d1(Ap lac) insertion mutants. J Bacteriol 160: 842-848. 97. Gohlke, U., Pullan, L., McDevitt, C.A., Porcelli, I., de Leeuw, E., Palmer, T., Saibil, H.R., and Berks, B.C. (2005) The TatA component of the twin-arginine protein transport system forms channel complexes of variable diameter. Proc Natl Acad Sci U S A 102: 10482-10486. Epub 12005 Jul 10418. 98. Grabenstein, J.P., Marceau, M., Pujol, C., Simonet, M., and Bliska, J.B. (2004) The response regulator PhoP of Yersinia pseudotuberculosis is important for replication in macrophages and for virulence. Infect Immun 72: 4973-4984. 99. Guedin, S., Willery, E., Tommassen, J., Fort, E., Drobecq, H., Locht, C., and Jacob-Dubuisson, F. (2000) Novel topological features of FhaC, the outer membrane transporter involved in the secretion of the Bordetella pertussis filamentous hemagglutinin. J Biol Chem 275: 30202-30210. 100. Guiyoule, A., Gerbaud, G., Buchrieser, C., Galimand, M., Rahalison, L., Chanteau, S., Courvalin, P., and Carniel, E. (2001) Transferable plasmid-mediated resistance to in a clinical isolate of Yersinia pestis. Emerg Infect Dis 7: 43-48. 101. Haddix, P.L., and Straley, S.C. (1992) Structure and regulation of the Yersinia pestis yscBCDEF operon. J Bacteriol 174: 4820-4828. 102. Halbig, D., Wiegert, T., Blaudeck, N., Freudl, R., and Sprenger, G.A. (1999) The efficient export of NADP-containing glucose-fructose oxidoreductase to the periplasm of Zymomonas mobilis depends both on an intact twin-arginine motif in the signal peptide and on the generation of a structural export signal induced by cofactor binding. Eur J Biochem 263: 543-551. 103. Hamburger, Z.A., Brown, M.S., Isberg, R.R., and Bjorkman, P.J. (1999) Crystal structure of invasin: a bacterial integrin-binding protein. Science 286: 291-295. 104. Hannu, T., Mattila, L., Nuorti, J.P., Ruutu, P., Mikkola, J., Siitonen, A., and Leirisalo-Repo, M. (2003) Reactive arthritis after an outbreak of Yersinia pseudotuberculosis serotype O:3 infection. Ann Rheum Dis 62: 866-869. 105. Hatzixanthis, K., Palmer, T., and Sargent, F. (2003) A subset of bacterial inner membrane proteins integrated by the twin-arginine translocase. Mol Microbiol 49: 1377-1390. 106. He, S.Y., and Jin, Q. (2003) The Hrp pilus: learning from flagella. Curr Opin Microbiol 6: 15- 19. 107. He, S.Y., Nomura, K., and Whittam, T.S. (2004) Type III protein secretion mechanism in mammalian and plant pathogens. Biochim Biophys Acta 1694: 181-206. 108. Heath, D.G., Anderson, G.W., Jr., Mauro, J.M., Welkos, S.L., Andrews, G.P., Adamovicz, J., and Friedlander, A.M. (1998) Protection against experimental bubonic and pneumonic plague by a recombinant capsular F1-V antigen fusion protein vaccine. Vaccine 16: 1131-1137.

50 REFERENCES

109. Heesemann, J., Gross, U., Schmidt, N., and Laufs, R. (1986) Immunochemical analysis of plasmid-encoded proteins released by enteropathogenic Yersinia sp. grown in calcium-deficient media. Infect Immun 54: 561-567. 110. Henderson, I.R., Navarro-Garcia, F., and Nataro, J.P. (1998) The great escape: structure and function of the autotransporter proteins. Trends Microbiol 6: 370-378. 111. Henderson, I.R., and Nataro, J.P. (2001) Virulence functions of autotransporter proteins. Infect Immun 69: 1231-1243. 112. Henderson, I.R., Navarro-Garcia, F., Desvaux, M., Fernandez, R.C., and Ala'Aldeen, D. (2004) Type V protein secretion pathway: the autotransporter story. Microbiol Mol Biol Rev 68: 692- 744. 113. Hertle, R. (2005) The family of serratia type pore forming toxins. Curr Protein Pept Sci 6: 313- 325. 114. Hinnebusch, B.J., Perry, R.D., and Schwan, T.G. (1996) Role of the Yersinia pestis hemin storage (hms) locus in the transmission of plague by fleas. Science 273: 367-370. 115. Hinnebusch, B.J., Rudolph, A.E., Cherepanov, P., Dixon, J.E., Schwan, T.G., and Forsberg, A. (2002) Role of Yersinia murine toxin in survival of Yersinia pestis in the midgut of the flea vector. Science 296: 733-735. 116. Hinnebusch, B.J. (2005) The evolution of flea-borne transmission in Yersinia pestis. Curr Issues Mol Biol 7: 197-212. 117. Hinnebusch, J., Cherepanov, P., Du, Y., Rudolph, A., Dixon, J.D., Schwan, T., and Forsberg, A. (2000) Murine toxin of Yersinia pestis shows phospholipase D activity but is not required for virulence in mice. Int J Med Microbiol 290: 483-487. 118. Hinsley, A.P., Stanley, N.R., Palmer, T., and Berks, B.C. (2001) A naturally occurring bacterial Tat signal peptide lacking one of the 'invariant' arginine residues of the consensus targeting motif. FEBS Lett 497: 45-49. 119. Hirano, T., Yamaguchi, S., Oosawa, K., and Aizawa, S. (1994) Roles of FliK and FlhB in determination of flagellar hook length in Salmonella typhimurium. J Bacteriol 176: 5439-5449. 120. Hodges, R.T., Carman, M.G., and Mortimer, W.J. (1984) Serotypes of Yersinia pseudotuberculosis recovered from domestic livestock. N Z Vet J 32: 11-13. 121. Hoe, N.P., and Goguen, J.D. (1993) Temperature sensing in Yersinia pestis: translation of the LcrF activator protein is thermally regulated. J Bacteriol 175: 7901-7909. 122. Hoiczyk, E., Roggenkamp, A., Reichenbecher, M., Lupas, A., and Heesemann, J. (2000) Structure and sequence analysis of Yersinia YadA and Moraxella UspAs reveal a novel class of adhesins. Embo J 19: 5989-5999. 123. Hoiczyk, E., and Blobel, G. (2001) Polymerization of a single protein of the pathogen Yersinia enterocolitica into needles punctures eukaryotic cells. Proc Natl Acad Sci U S A 98: 4669-4674. 124. Holmstrom, A., Olsson, J., Cherepanov, P., Maier, E., Nordfelth, R., Pettersson, J., Benz, R., Wolf-Watz, H., and Forsberg, A. (2001) LcrV is a channel size-determining component of the Yop effector translocon of Yersinia. Mol Microbiol 39: 620-632. 125. Huang, X.Z., and Lindler, L.E. (2004) The pH 6 antigen is an antiphagocytic factor produced by Yersinia pestis independent of Yersinia outer proteins and capsule antigen. Infect Immun 72: 7212-7219. 126. Hueck, C.J. (1998) Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev 62: 379-433. 127. Hull, R.A., Gill, R.E., Hsu, P., Minshew, B.H., and Falkow, S. (1981) Construction and expression of recombinant plasmids encoding type 1 or D-mannose-resistant pili from a urinary tract infection Escherichia coli isolate. Infect Immun 33: 933-938. 51 REFERENCES

128. Hutcheon, G.W., and Bolhuis, A. (2003) The archaeal twin-arginine translocation pathway. Biochem Soc Trans 31: 686-689. 129. Inglesby, T.V., Dennis, D.T., Henderson, D.A., Bartlett, J.G., Ascher, M.S., Eitzen, E., Fine, A.D., Friedlander, A.M., Hauer, J., Koerner, J.F., Layton, M., McDade, J., Osterholm, M.T., O'Toole, T., Parker, G., Perl, T.M., Russell, P.K., Schoch-Spana, M., and Tonat, K. (2000) Plague as a biological weapon: medical and public health management. Working Group on Civilian Biodefense. Jama 283: 2281-2290. 130. Iriarte, M., and Cornelis, G.R. (1999) Identification of SycN, YscX, and YscY, three new elements of the Yersinia yop virulon. J Bacteriol 181: 675-680. 131. Isberg, R.R., and Falkow, S. (1985) A single genetic locus encoded by Yersinia pseudotuberculosis permits invasion of cultured animal cells by Escherichia coli K-12. Nature 317: 262-264. 132. Isberg, R.R., and Barnes, P. (2001) Subversion of integrins by enteropathogenic Yersinia. J Cell Sci 114: 21-28. 133. Iwobi, A., Heesemann, J., Garcia, E., Igwe, E., Noelting, C., and Rakin, A. (2003) Novel virulence-associated type II secretion system unique to high-pathogenicity Yersinia enterocolitica. Infect Immun 71: 1872-1879. 134. Ize, B., Gerard, F., Zhang, M., Chanal, A., Voulhoux, R., Palmer, T., Filloux, A., and Wu, L.F. (2002) In vivo dissection of the Tat translocation pathway in Escherichia coli. J Mol Biol 317: 327-335. 135. Jackson, M.W., Day, J.B., and Plano, G.V. (1998) YscB of Yersinia pestis functions as a specific chaperone for YopN. J Bacteriol 180: 4912-4921. 136. Jackson, M.W., and Plano, G.V. (2000) Interactions between type III secretion apparatus components from Yersinia pestis detected using the yeast two-hybrid system. FEMS Microbiol Lett 186: 85-90. 137. Jackson, M.W., Silva-Herzog, E., and Plano, G.V. (2004) The ATP-dependent ClpXP and Lon proteases regulate expression of the Yersinia pestis type III secretion system via regulated proteolysis of YmoA, a small histone-like protein. Mol Microbiol 54: 1364-1378. 138. Jacob-Dubuisson, F., Locht, C., and Antoine, R. (2001) Two-partner secretion in Gram-negative bacteria: a thrifty, specific pathway for large virulence proteins. Mol Microbiol 40: 306-313. 139. Jacob-Dubuisson, F., Fernandez, R., and Coutte, L. (2004) Protein secretion through autotransporter and two-partner pathways. Biochim Biophys Acta 1694: 235-257. 140. Jarrett, C.O., Deak, E., Isherwood, K.E., Oyston, P.C., Fischer, E.R., Whitney, A.R., Kobayashi, S.D., DeLeo, F.R., and Hinnebusch, B.J. (2004) Transmission of Yersinia pestis from an infectious biofilm in the flea vector. J Infect Dis 190: 783-792. Epub 2004 Jul 2012. 141. Jin, Q., and He, S.Y. (2001) Role of the Hrp pilus in type III protein secretion in Pseudomonas syringae. Science 294: 2556-2558. 142. Jongbloed, J.D., Martin, U., Antelmann, H., Hecker, M., Tjalsma, H., Venema, G., Bron, S., van Dijl, J.M., and Muller, J. (2000) TatC is a specificity determinant for protein secretion via the twin-arginine translocation pathway. J Biol Chem 275: 41350-41357. 143. Journet, L., Agrain, C., Broz, P., and Cornelis, G.R. (2003) The needle length of bacterial injectisomes is determined by a molecular ruler. Science 302: 1757-1760. 144. Julio, S.M., and Cotter, P.A. (2005) Characterization of the filamentous hemagglutinin-like protein FhaS in Bordetella bronchiseptica. Infect Immun 73: 4960-4971. 145. Juris, S.J., Shao, F., and Dixon, J.E. (2002) Yersinia effectors target mammalian signalling pathways. Cell Microbiol 4: 201-211.

52 REFERENCES

146. Kauppi, A.M., Nordfelth, R., Uvell, H., Wolf-Watz, H., and Elofsson, M. (2003) Targeting bacterial virulence: inhibitors of type III secretion in Yersinia. Chem Biol 10: 241-249. 147. Ki, J.J., Kawarasaki, Y., Gam, J., Harvey, B.R., Iverson, B.L., and Georgiou, G. (2004) A periplasmic fluorescent reporter protein and its application in high-throughput membrane protein topology analysis. J Mol Biol 341: 901-909. 148. Kimbrough, T.G., and Miller, S.I. (2000) Contribution of Salmonella typhimurium type III secretion components to needle complex formation. Proc Natl Acad Sci U S A 97: 11008-11013. 149. Knutton, S., Rosenshine, I., Pallen, M.J., Nisan, I., Neves, B.C., Bain, C., Wolff, C., Dougan, G., and Frankel, G. (1998) A novel EspA-associated surface organelle of enteropathogenic Escherichia coli involved in protein translocation into epithelial cells. Embo J 17: 2166-2176. 150. Konninger, U.W., Hobbie, S., Benz, R., and Braun, V. (1999) The haemolysin-secreting ShlB protein of the outer membrane of Serratia marcescens: determination of surface-exposed residues and formation of ion-permeable pores by ShlB mutants in artificial lipid bilayer membranes. Mol Microbiol 32: 1212-1225. 151. Koornhof, H.J., Smego, R.A., Jr., and Nicol, M. (1999) . II: The pathogenesis of Yersinia infections. Eur J Clin Microbiol Infect Dis 18: 87-112. 152. Koster, M., Bitter, W., de Cock, H., Allaoui, A., Cornelis, G.R., and Tommassen, J. (1997) The outer membrane component, YscC, of the Yop secretion machinery of Yersinia enterocolitica forms a ring-shaped multimeric complex. Mol Microbiol 26: 789-797. 153. Kubori, T., Matsushima, Y., Nakamura, D., Uralil, J., Lara-Tejero, M., Sukhan, A., Galan, J.E., and Aizawa, S.I. (1998) Supramolecular structure of the Salmonella typhimurium type III protein secretion system. Science 280: 602-605. 154. Kubori, T., Sukhan, A., Aizawa, S.I., and Galan, J.E. (2000) Molecular characterization and assembly of the needle complex of the Salmonella typhimurium type III protein secretion system. Proc Natl Acad Sci U S A 97: 10225-10230. 155. Kukkonen, M., Suomalainen, M., Kyllonen, P., Lahteenmaki, K., Lang, H., Virkola, R., Helander, I.M., Holst, O., and Korhonen, T.K. (2004) Lack of O-antigen is essential for plasminogen activation by Yersinia pestis and Salmonella enterica. Mol Microbiol 51: 215-225. 156. Kutsukake, K., Minamino, T., and Yokoseki, T. (1994) Isolation and characterization of FliK- independent flagellation mutants from Salmonella typhimurium. J Bacteriol 176: 7625-7629. 157. Kutyrev, V.V., Filippov, A.A., Oparina, O.S., and Protsenko, O.A. (1992) Analysis of Yersinia pestis chromosomal determinants Pgm+ and Psts associated with virulence. Microb Pathog 12: 177-186. 158. Lahteenmaki, K., Virkola, R., Saren, A., Emody, L., and Korhonen, T.K. (1998) Expression of plasminogen activator pla of Yersinia pestis enhances bacterial attachment to the mammalian extracellular matrix. Infect Immun 66: 5755-5762. 159. Lahteenmaki, K., Edelman, S., and Korhonen, T.K. (2005) Bacterial metastasis: the host plasminogen system in bacterial invasion. Trends Microbiol 13: 79-85. 160. Lambert de Rouvroit, C., Sluiters, C., and Cornelis, G.R. (1992) Role of the transcriptional activator, VirF, and temperature in the expression of the pYV plasmid genes of Yersinia enterocolitica. Mol Microbiol 6: 395-409. 161. Laukkanen, R., Niskanen, T., Fredriksson-Ahomaa, M., and Korkeala, H. (2003) Yersinia pseudotuberculosis in pigs and pig houses in Finland. Adv Exp Med Biol 529: 371-373. 162. Lesic, B., and Carniel, E. (2004) The high-pathogenicity island: a broad-host-range pathogenicity island. In Yersinia: molecular and cellular biology. Carniel, E. and Hinnebusch, B.J. (eds). Wymondham: Horizon Bioscience, pp. 285-306.

53 REFERENCES

163. Leung, K.Y., Reisner, B.S., and Straley, S.C. (1990) YopM inhibits platelet aggregation and is necessary for virulence of Yersinia pestis in mice. Infect Immun 58: 3262-3271. 164. Lillard, J.W., Jr., Bearden, S.W., Fetherston, J.D., and Perry, R.D. (1999) The haemin storage (Hms+) phenotype of Yersinia pestis is not essential for the pathogenesis of bubonic plague in mammals. Microbiology 145: 197-209. 165. Lindler, L.E., and Tall, B.D. (1993) Yersinia pestis pH 6 antigen forms fimbriae and is induced by intracellular association with macrophages. Mol Microbiol 8: 311-324. 166. Lloyd, S.A., Forsberg, A., Wolf-Watz, H., and Francis, M.S. (2001a) Targeting exported substrates to the Yersinia TTSS: different functions for different signals? Trends Microbiol 9: 367-371. 167. Lloyd, S.A., Norman, M., Rosqvist, R., and Wolf-Watz, H. (2001b) Yersinia YopE is targeted for type III secretion by N-terminal, not mRNA, signals. Mol Microbiol 39: 520-531. 168. Lloyd, S.A., Sjostrom, M., Andersson, S., and Wolf-Watz, H. (2002) Molecular characterization of type III secretion signals via analysis of synthetic N-terminal amino acid sequences. Mol Microbiol 43: 51-59. 169. Lorange, E.A., Race, B.L., Sebbane, F., and Joseph Hinnebusch, B. (2005) Poor vector competence of fleas and the evolution of hypervirulence in Yersinia pestis. J Infect Dis 191: 1907-1912. Epub 2005 Apr 1929. 170. Lowell, J.L., Wagner, D.M., Atshabar, B., Antolin, M.F., Vogler, A.J., Keim, P., Chu, M.C., and Gage, K.L. (2005) Identifying sources of human exposure to plague. J Clin Microbiol 43: 650- 656. 171. Macnab, R.M. (1996) Flagella and motility. In Escherichia coli and Salmonella: cellular and molecular biology. Vol. 1. Neidhardt, F.C., Curtiss, R., Ingraham, J.L., Lin, E.C.C., Low, K.B., Magasanik, B., Reznikoff, W.S., Riley, M., Schaechter, M. and Umbarger, H.E. (eds). Washington, D. C.: ASM Press, pp. 123-145. 172. Macnab, R.M. (1999) The bacterial flagellum: reversible rotary propellor and type III export apparatus. J Bacteriol 181: 7149-7153. 173. Macnab, R.M. (2004) Type III flagellar protein export and flagellar assembly. Biochim Biophys Acta 1694: 207-217. 174. Magdalena, J., Hachani, A., Chamekh, M., Jouihri, N., Gounon, P., Blocker, A., and Allaoui, A. (2002) Spa32 regulates a switch in substrate specificity of the type III secreton of Shigella flexneri from needle components to Ipa proteins. J Bacteriol 184: 3433-3441. 175. Makoveichuk, E., Cherepanov, P., Lundberg, S., Forsberg, A., and Olivecrona, G. (2003) pH6 antigen of Yersinia pestis interacts with plasma lipoproteins and cell membranes. J Lipid Res 44: 320-330. 176. Marcus, E.A., Moshfegh, A.P., Sachs, G., and Scott, D.R. (2005) The periplasmic alpha-carbonic anhydrase activity of Helicobacter pylori is essential for acid acclimation. J Bacteriol 187: 729- 738. 177. Marenne, M.N., Journet, L., Mota, L.J., and Cornelis, G.R. (2003) Genetic analysis of the formation of the Ysc-Yop translocation pore in macrophages by Yersinia enterocolitica: role of LcrV, YscF and YopN. Microb Pathog 35: 243-258. 178. Marenne, M.N., Mota, L.J., and Cornelis, G.R. (2004) The pYV plasmid and the Ysc-Yop type III secreitno system. In Yersinia: molecular and cellular biology. Carniel, E. and Hinnebusch, B.J. (eds). Wymondham: Horizon Bioscience, pp. 319-348. 179. Marra, A., and Isberg, R.R. (1997) Invasin-dependent and invasin-independent pathways for translocation of Yersinia pseudotuberculosis across the Peyer's patch intestinal epithelium. Infect Immun 65: 3412-3421.

54 REFERENCES

180. Mencher, J.S., Smith, S.R., Powell, T.D., Stinchcomb, D.T., Osorio, J.E., and Rocke, T.E. (2004) Protection of black-tailed prairie dogs (Cynomys ludovicianus) against plague after voluntary consumption of baits containing recombinant raccoon poxvirus vaccine. Infect Immun 72: 5502- 5505. 181. Michiels, T., Wattiau, P., Brasseur, R., Ruysschaert, J.M., and Cornelis, G. (1990) Secretion of Yop proteins by Yersiniae. Infect Immun 58: 2840-2849. 182. Michiels, T., Vanooteghem, J.C., Lambert de Rouvroit, C., China, B., Gustin, A., Boudry, P., and Cornelis, G.R. (1991) Analysis of virC, an operon involved in the secretion of Yop proteins by Yersinia enterocolitica. J Bacteriol 173: 4994-5009. 183. Minamino, T., and Macnab, R.M. (2000) Domain structure of Salmonella FlhB, a flagellar export component responsible for substrate specificity switching. J Bacteriol 182: 4906-4914. 184. Molik, S., Karnauchov, I., Weidlich, C., Herrmann, R.G., and Klosgen, R.B. (2001) The Rieske Fe/S protein of the cytochrome b6/f complex in chloroplasts: missing link in the evolution of protein transport pathways in chloroplasts? J Biol Chem 276: 42761-42766. Epub 42001 Aug 42728. 185. Monack, D.M., Mecsas, J., Bouley, D., and Falkow, S. (1998) Yersinia-induced apoptosis in vivo aids in the establishment of a systemic infection of mice. J Exp Med 188: 2127-2137. 186. Muller, M., Koch, H.G., Beck, K., and Schafer, U. (2001) Protein traffic in bacteria: multiple routes from the ribosome to and across the membrane. Prog Nucleic Acid Res Mol Biol 66: 107- 157. 187. Nagai, H., and Roy, C.R. (2003) Show me the substrates: modulation of host cell function by type IV secretion systems. Cell Microbiol 5: 373-383. 188. Nakajima, R., Motin, V.L., and Brubaker, R.R. (1995) Suppression of cytokines in mice by protein A-V antigen fusion peptide and restoration of synthesis by active immunization. Infect Immun 63: 3021-3029. 189. Navarro, L., Alto, N.M., and Dixon, J.E. (2005) Functions of the Yersinia effector proteins in inhibiting host immune responses. Curr Opin Microbiol 8: 21-27. 190. Neutra, M.R., Frey, A., and Kraehenbuhl, J.P. (1996) Epithelial M cells: gateways for mucosal infection and immunization. Cell 86: 345-348. 191. Newman, C.L., and Stathopoulos, C. (2004) Autotransporter and two-partner secretion: delivery of large-size virulence factors by gram-negative bacterial pathogens. Crit Rev Microbiol 30: 275- 286. 192. Neyt, C., and Cornelis, G.R. (1999) Role of SycD, the chaperone of the Yersinia Yop translocators YopB and YopD. Mol Microbiol 31: 143-156. 193. Nordfelth, R., Kauppi, A.M., Norberg, H.A., Wolf-Watz, H., and Elofsson, M. (2005) Small- molecule inhibitors specifically targeting type III secretion. Infect Immun 73: 3104-3114. 194. Nunn, D. (1999) Bacterial type II protein export and pilus biogenesis: more than just homologies? Trends Cell Biol 9: 402-408. 195. Oates, J., Mathers, J., Mangels, D., Kuhlbrandt, W., Robinson, C., and Model, K. (2003) Consensus structural features of purified bacterial TatABC complexes. J Mol Biol 330: 277-286. 196. Oates, J., Barrett, C.M., Barnett, J.P., Byrne, K.G., Bolhuis, A., and Robinson, C. (2005) The Escherichia coli twin-arginine translocation apparatus incorporates a distinct form of TatABC complex, spectrum of modular TatA complexes and minor TatAB complex. J Mol Biol 346: 295- 305. Epub 2004 Dec 2013. 197. Ochsner, U.A., Snyder, A., Vasil, A.I., and Vasil, M.L. (2002) Effects of the twin-arginine translocase on secretion of virulence factors, stress response, and pathogenesis. Proc Natl Acad Sci U S A 99: 8312-8317. Epub 2002 May 8328. 55 REFERENCES

198. Ondraczek, R., Hobbie, S., and Braun, V. (1992) In vitro activation of the Serratia marcescens hemolysin through modification and complementation. J Bacteriol 174: 5086-5094. 199. Oyston, P.C., Dorrell, N., Williams, K., Li, S.R., Green, M., Titball, R.W., and Wren, B.W. (2000) The response regulator PhoP is important for survival under conditions of macrophage- induced stress and virulence in Yersinia pestis. Infect Immun 68: 3419-3425. 200. Pallen, M.J., Chaudhuri, R.R., and Henderson, I.R. (2003) Genomic analysis of secretion systems. Curr Opin Microbiol 6: 519-527. 201. Pallen, M.J., Beatson, S.A., and Bailey, C.M. (2005) Bioinformatics, genomics and evolution of non-flagellar type-III secretion systems: a Darwinian perspective. FEMS Microbiol Rev 29: 201- 229. 202. Palmer, T., and Berks, B.C. (2003) Moving folded proteins across the bacterial cell membrane. Microbiology 149: 547-556. 203. Palmer, T., Sargent, F., and Berks, B.C. (2005) Export of complex cofactor-containing proteins by the bacterial Tat pathway. Trends Microbiol 13: 175-180. 204. Parkhill, J., Wren, B.W., Thomson, N.R., Titball, R.W., Holden, M.T., Prentice, M.B., Sebaihia, M., James, K.D., Churcher, C., Mungall, K.L., Baker, S., Basham, D., Bentley, S.D., Brooks, K., Cerdeno-Tarraga, A.M., Chillingworth, T., Cronin, A., Davies, R.M., Davis, P., Dougan, G., Feltwell, T., Hamlin, N., Holroyd, S., Jagels, K., Karlyshev, A.V., Leather, S., Moule, S., Oyston, P.C., Quail, M., Rutherford, K., Simmonds, M., Skelton, J., Stevens, K., Whitehead, S., and Barrell, B.G. (2001) Genome sequence of Yersinia pestis, the causative agent of plague. Nature 413: 523-527. 205. Patterson-Delafield, J., Martinez, R.J., Stocker, B.A., and Yamaguchi, S. (1973) A new fla gene in Salmonella typhimurium--flaR--and its mutant phenotype-superhooks. Arch Mikrobiol 90: 107-120. 206. Paulsen, I.T. (2003) Multidrug efflux pumps and resistance: regulation and evolution. Curr Opin Microbiol 6: 446-451. 207. Payne, P.L., and Straley, S.C. (1998) YscO of Yersinia pestis is a mobile core component of the Yop secretion system. J Bacteriol 180: 3882-3890. 208. Payne, P.L., and Straley, S.C. (1999) YscP of Yersinia pestis is a secreted component of the Yop secretion system. J Bacteriol 181: 2852-2862. 209. Pepe, J.C., and Miller, V.L. (1993) Yersinia enterocolitica invasin: a primary role in the initiation of infection. Proc Natl Acad Sci U S A 90: 6473-6477. 210. Pepe, J.C., Badger, J.L., and Miller, V.L. (1994) Growth phase and low pH affect the thermal regulation of the Yersinia enterocolitica inv gene. Mol Microbiol 11: 123-135. 211. Perry, R.D., and Fetherston, J.D. (1997) Yersinia pestis--etiologic agent of plague. Clin Microbiol Rev 10: 35-66. 212. Perry, R.D., and Fetherston, J.D. (2004) Iron and Heme uptake systems. In Yersinia: molecular and cellular biology. Carniel, E. and Hinnebusch, B.J. (eds). Wymondham: Horizon Bioscience, pp. 257-283. 213. Persson, C., Nordfelth, R., Holmstrom, A., Hakansson, S., Rosqvist, R., and Wolf-Watz, H. (1995) Cell-surface-bound Yersinia translocate the protein tyrosine phosphatase YopH by a polarized mechanism into the target cell. Mol Microbiol 18: 135-150. 214. Pettersson, J., Nordfelth, R., Dubinina, E., Bergman, T., Gustafsson, M., Magnusson, K.E., and Wolf-Watz, H. (1996) Modulation of virulence factor expression by pathogen target cell contact. Science 273: 1231-1233. 215. Pettersson, J., Holmstrom, A., Hill, J., Leary, S., Frithz-Lindsten, E., von Euler-Matell, A., Carlsson, E., Titball, R., Forsberg, A., and Wolf-Watz, H. (1999) The V-antigen of Yersinia is

56 REFERENCES

surface exposed before target cell contact and involved in virulence protein translocation. Mol Microbiol 32: 961-976. 216. Plano, G.V., Barve, S.S., and Straley, S.C. (1991) LcrD, a membrane-bound regulator of the Yersinia pestis low-calcium response. J Bacteriol 173: 7293-7303. 217. Plano, G.V., and Straley, S.C. (1993) Multiple effects of lcrD mutations in Yersinia pestis. J Bacteriol 175: 3536-3545. 218. Plano, G.V., and Straley, S.C. (1995) Mutations in yscC, yscD, and yscG prevent high-level expression and secretion of V antigen and Yops in Yersinia pestis. J Bacteriol 177: 3843-3854. 219. Pohlner, J., Halter, R., Beyreuther, K., and Meyer, T.F. (1987) Gene structure and extracellular secretion of Neisseria gonorrhoeae IgA protease. Nature 325: 458-462. 220. Pohlschroder, M., Prinz, W.A., Hartmann, E., and Beckwith, J. (1997) Protein translocation in the three domains of life: variations on a theme. Cell 91: 563-566. 221. Porcelli, I., de Leeuw, E., Wallis, R., van den Brink-van der Laan, E., de Kruijff, B., Wallace, B.A., Palmer, T., and Berks, B.C. (2002) Characterization and membrane assembly of the TatA component of the Escherichia coli twin-arginine protein transport system. Biochemistry 41: 13690-13697. 222. Portnoy, D.A., Wolf-Watz, H., Bolin, I., Beeder, A.B., and Falkow, S. (1984) Characterization of common virulence plasmids in Yersinia species and their role in the expression of outer membrane proteins. Infect Immun 43: 108-114. 223. Pugsley, A.P. (1993) The complete general secretory pathway in gram-negative bacteria. Microbiol Rev 57: 50-108. 224. Pugsley, A.P., Francetic, O., Possot, O.M., Sauvonnet, N., and Hardie, K.R. (1997) Recent progress and future directions in studies of the main terminal branch of the general secretory pathway in Gram-negative bacteria--a review. Gene 192: 13-19. 225. Pujol, C., and Bliska, J.B. (2003) The ability to replicate in macrophages is conserved between Yersinia pestis and Yersinia pseudotuberculosis. Infect Immun 71: 5892-5899. 226. Pujol, C., and Bliska, J.B. (2005) Turning Yersinia pathogenesis outside in: subversion of macrophage function by intracellular yersiniae. Clin Immunol 114: 216-226. 227. Remaut, H., and Waksman, G. (2004) Structural biology of bacterial pathogenesis. Curr Opin Struct Biol 14: 161-170. 228. Rimpilainen, M., Forsberg, A., and Wolf-Watz, H. (1992) A novel protein, LcrQ, involved in the low-calcium response of Yersinia pseudotuberculosis shows extensive homology to YopH. J Bacteriol 174: 3355-3363. 229. Roggenkamp, A., Ackermann, N., Jacobi, C.A., Truelzsch, K., Hoffmann, H., and Heesemann, J. (2003) Molecular analysis of transport and oligomerization of the Yersinia enterocolitica adhesin YadA. J Bacteriol 185: 3735-3744. 230. Roine, E., Wei, W., Yuan, J., Nurmiaho-Lassila, E.L., Kalkkinen, N., Romantschuk, M., and He, S.Y. (1997) Hrp pilus: an hrp-dependent bacterial surface appendage produced by Pseudomonas syringae pv. tomato DC3000. Proc Natl Acad Sci U S A 94: 3459-3464. 231. Rojas, C.M., Ham, J.H., Deng, W.L., Doyle, J.J., and Collmer, A. (2002) HecA, a member of a class of adhesins produced by diverse pathogenic bacteria, contributes to the attachment, aggregation, epidermal cell killing, and virulence phenotypes of Erwinia chrysanthemi EC16 on Nicotiana clevelandii seedlings. Proc Natl Acad Sci U S A 99: 13142-13147. Epub 12002 Sep 13123. 232. Rose, R.W., Bruser, T., Kissinger, J.C., and Pohlschroder, M. (2002) Adaptation of protein secretion to extremely high-salt conditions by extensive use of the twin-arginine translocation pathway. Mol Microbiol 45: 943-950. 57 REFERENCES

233. Rosqvist, R., Skurnik, M., and Wolf-Watz, H. (1988) Increased virulence of Yersinia pseudotuberculosis by two independent mutations. Nature 334: 522-524. 234. Rosqvist, R., Magnusson, K.E., and Wolf-Watz, H. (1994) Target cell contact triggers expression and polarized transfer of Yersinia YopE cytotoxin into mammalian cells. Embo J 13: 964-972. 235. Rosqvist, R., Persson, C., Hakansson, S., Nordfeldt, R., and Wolf-Watz, H. (1995) Translocation of the Yersinia YopE and YopH virulence proteins into target cells is mediated by YopB and YopD. Contrib Microbiol Immunol 13: 230-234. 236. Rossier, O., and Cianciotto, N.P. (2005) The Legionella pneumophila tatB gene facilitates secretion of phospholipase C, growth under iron-limiting conditions, and intracellular infection. Infect Immun 73: 2020-2032. 237. Rudolph, A.E., Stuckey, J.A., Zhao, Y., Matthews, H.R., Patton, W.A., Moss, J., and Dixon, J.E. (1999) Expression, characterization, and mutagenesis of the Yersinia pestis murine toxin, a phospholipase D superfamily member. J Biol Chem 274: 11824-11831. 238. Russmann, H., Kubori, T., Sauer, J., and Galan, J.E. (2002) Molecular and functional analysis of the type III secretion signal of the Salmonella enterica InvJ protein. Mol Microbiol 46: 769-779. 239. Saier, M.H., Jr. (2004) Evolution of bacterial type III protein secretion systems. Trends Microbiol 12: 113-115. 240. Salmond, G.P., and Reeves, P.J. (1993) Membrane traffic wardens and protein secretion in gram- negative bacteria. Trends Biochem Sci 18: 7-12. 241. Sandkvist, M. (2001a) Type II secretion and pathogenesis. Infect Immun 69: 3523-3535. 242. Sandkvist, M. (2001b) Biology of type II secretion. Mol Microbiol 40: 271-283. 243. Sargent, F., Bogsch, E.G., Stanley, N.R., Wexler, M., Robinson, C., Berks, B.C., and Palmer, T. (1998) Overlapping functions of components of a bacterial Sec-independent protein export pathway. Embo J 17: 3640-3650. 244. Sargent, F., Gohlke, U., De Leeuw, E., Stanley, N.R., Palmer, T., Saibil, H.R., and Berks, B.C. (2001) Purified components of the Escherichia coli Tat protein transport system form a double- layered ring structure. Eur J Biochem 268: 3361-3367. 245. Sargent, F., Berks, B.C., and Palmer, T. (2002) Assembly of membrane-bound respiratory complexes by the Tat protein-transport system. Arch Microbiol 178: 77-84. Epub 2002 May 2022. 246. Sarker, M.R., Neyt, C., Stainier, I., and Cornelis, G.R. (1998) The Yersinia Yop virulon: LcrV is required for extrusion of the translocators YopB and YopD. J Bacteriol 180: 1207-1214. 247. Sauer, F.G., Knight, S.D., Waksman, Gj, and Hultgren, S.J. (2000) PapD-like chaperones and pilus biogenesis. Semin Cell Dev Biol 11: 27-34. 248. Sauer, F.G., Remaut, H., Hultgren, S.J., and Waksman, G. (2004) Fiber assembly by the chaperone-usher pathway. Biochim Biophys Acta 1694: 259-267. 249. Sauvonnet, N., Vignon, G., Pugsley, A.P., and Gounon, P. (2000) Pilus formation and protein secretion by the same machinery in Escherichia coli. Embo J 19: 2221-2228. 250. Schesser, K., Francis, M.S., Forsberg, A., and Wolf-Watz, H. (2000) Type III secretion systems in animal- and plant-interacting bacteria. In Cellular Microbiology. Cossart, P., Boquet, P., Normark, S. and Rappuoli, P. (eds). Washington: ASM Press, pp. 239-263. 251. Schubot, F.D., Jackson, M.W., Penrose, K.J., Cherry, S., Tropea, J.E., Plano, G.V., and Waugh, D.S. (2005) Three-dimensional structure of a macromolecular assembly that regulates type III secretion in Yersinia pestis. J Mol Biol 346: 1147-1161. Epub 2005 Jan 1120. 252. Sekiya, K., Ohishi, M., Ogino, T., Tamano, K., Sasakawa, C., and Abe, A. (2001) Supermolecular structure of the enteropathogenic Escherichia coli type III secretion system and 58 REFERENCES

its direct interaction with the EspA-sheath-like structure. Proc Natl Acad Sci U S A 98: 11638- 11643. 253. Settles, A.M., Yonetani, A., Baron, A., Bush, D.R., Cline, K., and Martienssen, R. (1997) Sec- independent protein translocation by the maize Hcf106 protein. Science 278: 1467-1470. 254. Settles, A.M., and Martienssen, R. (1998) Old and new pathways of protein export in chloroplasts and bacteria. Trends Cell Biol 8: 494-501. 255. Simonet, M., Riot, B., Fortineau, N., and Berche, P. (1996) Invasin production by Yersinia pestis is abolished by insertion of an IS200-like element within the inv gene. Infect Immun 64: 375- 379. 256. Sing, A., Rost, D., Tvardovskaia, N., Roggenkamp, A., Wiedemann, A., Kirschning, C.J., Aepfelbacher, M., and Heesemann, J. (2002) Yersinia V-antigen exploits toll-like receptor 2 and CD14 for interleukin 10-mediated immunosuppression. J Exp Med 196: 1017-1024. 257. Skurnik, M., Peippo, A., and Ervela, E. (2000) Characterization of the O-antigen gene clusters of Yersinia pseudotuberculosis and the cryptic O-antigen gene cluster of Yersinia pestis shows that the plague is most closely related to and has evolved from Y. pseudotuberculosis serotype O:1b. Mol Microbiol 37: 316-330. 258. Skurnik, M., and Bengoechea, J.A. (2003) The biosynthesis and biological role of lipopolysaccharide O-antigens of pathogenic Yersiniae. Carbohydr Res 338: 2521-2529. 259. Smego, R.A., Frean, J., and Koornhof, H.J. (1999) Yersiniosis I: microbiological and clinicoepidemiological aspects of plague and non-plague Yersinia infections. Eur J Clin Microbiol Infect Dis 18: 1-15. 260. Smith, A.M., Guzman, C.A., and Walker, M.J. (2001) The virulence factors of Bordetella pertussis: a matter of control. FEMS Microbiol Rev 25: 309-333. 261. Smith, K.S., and Ferry, J.G. (2000) Prokaryotic carbonic anhydrases. FEMS Microbiol Rev 24: 335-366. 262. Sodeinde, O.A., Subrahmanyam, Y.V., Stark, K., Quan, T., Bao, Y., and Goguen, J.D. (1992) A surface protease and the invasive character of plague. Science 258: 1004-1007. 263. Sorg, J.A., Miller, N.C., and Schneewind, O. (2005) Substrate recognition of type III secretion machines--testing the RNA signal hypothesis. Cell Microbiol 7: 1217-1225. 264. Sory, M.P., and Cornelis, G.R. (1994) Translocation of a hybrid YopE-adenylate cyclase from Yersinia enterocolitica into HeLa cells. Mol Microbiol 14: 583-594. 265. Sory, M.P., Boland, A., Lambermont, I., and Cornelis, G.R. (1995) Identification of the YopE and YopH domains required for secretion and internalization into the cytosol of macrophages, using the cyaA gene fusion approach. Proc Natl Acad Sci U S A 92: 11998-12002. 266. Stainier, I., Bleves, S., Josenhans, C., Karmani, L., Kerbourch, C., Lambermont, I., Totemeyer, S., Boyd, A., and Cornelis, G.R. (2000) YscP, a Yersinia protein required for Yop secretion that is surface exposed, and released in low Ca2+. Mol Microbiol 37: 1005-1018. 267. Straley, S.C., and Harmon, P.A. (1984a) Growth in mouse peritoneal macrophages of Yersinia pestis lacking established virulence determinants. Infect Immun 45: 649-654. 268. Straley, S.C., and Harmon, P.A. (1984b) Yersinia pestis grows within phagolysosomes in mouse peritoneal macrophages. Infect Immun 45: 655-659. 269. Straley, S.C., and Bowmer, W.S. (1986) Virulence genes regulated at the transcriptional level by Ca2+ in Yersinia pestis include structural genes for outer membrane proteins. Infect Immun 51: 445-454. 270. Sulakvelidze, A. (2000) Yersiniae other than Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis: the ignored species. Microbes Infect 2: 497-513.

59 REFERENCES

271. Suzuki, T., and Iino, T. (1981) Role of the flaR gene in flagellar hook formation in Salmonella spp. J Bacteriol 148: 973-979. 272. Tamano, K., Aizawa, S., Katayama, E., Nonaka, T., Imajoh-Ohmi, S., Kuwae, A., Nagai, S., and Sasakawa, C. (2000) Supramolecular structure of the Shigella type III secretion machinery: the needle part is changeable in length and essential for delivery of effectors. Embo J 19: 3876-3887. 273. Tamano, K., Katayama, E., Toyotome, T., and Sasakawa, C. (2002) Shigella Spa32 is an essential secretory protein for functional type III secretion machinery and uniformity of its needle length. J Bacteriol 184: 1244-1252. 274. Thanassi, D.G., and Hultgren, S.J. (2000) Multiple pathways allow protein secretion across the bacterial outer membrane. Curr Opin Cell Biol 12: 420-430. 275. Thomas, J., Stafford, G.P., and Hughes, C. (2004) Docking of cytosolic chaperone-substrate complexes at the membrane ATPase during flagellar type III protein export. Proc Natl Acad Sci U S A 101: 3945-3950. Epub 2004 Mar 3944. 276. Titball, R.W., and Williamson, E.D. (2001) Vaccination against bubonic and pneumonic plague. Vaccine 19: 4175-4184. 277. Titball, R.W., and Williamson, E.D. (2004) Yersinia pestis (plague) . Expert Opin Biol Ther 4: 965-973. 278. Torruellas, J., Jackson, M.W., Pennock, J.W., and Plano, G.V. (2005) The Yersinia pestis type III secretion needle plays a role in the regulation of Yop secretion. Mol Microbiol 57: 1719- 1733. 279. Trulzsch, K., Sporleder, T., Igwe, E.I., Russmann, H., and Heesemann, J. (2004) Contribution of the Major Secreted Yops of Yersinia enterocolitica O:8 to Pathogenicity in the Mouse Infection Model. Infect Immun 72: 5227-5234. 280. Tsukano, H., Kura, F., Inoue, S., Sato, S., Izumiya, H., Yasuda, T., and Watanabe, H. (1999) Yersinia pseudotuberculosis blocks the phagosomal acidification of B10.A mouse macrophages through the inhibition of vacuolar H(+)-ATPase activity. Microb Pathog 27: 253-263. 281. Van Nhieu, G.T., and Isberg, R.R. (1991) The Yersinia pseudotuberculosis invasin protein and human fibronectin bind to mutually exclusive sites on the alpha 5 beta 1 integrin receptor. J Biol Chem 266: 24367-24375. 282. Wattiau, P., Bernier, B., Deslee, P., Michiels, T., and Cornelis, G.R. (1994) Individual chaperones required for Yop secretion by Yersinia. Proc Natl Acad Sci U S A 91: 10493-10497. 283. Veenendaal, A.K., van der Does, C., and Driessen, A.J. (2004) The protein-conducting channel SecYEG. Biochim Biophys Acta 1694: 81-95. 284. Weiner, J.H., Bilous, P.T., Shaw, G.M., Lubitz, S.P., Frost, L., Thomas, G.H., Cole, J.A., and Turner, R.J. (1998) A novel and ubiquitous system for membrane targeting and secretion of cofactor-containing proteins. Cell 93: 93-101. 285. Welkos, S.L., Friedlander, A.M., and Davis, K.J. (1997) Studies on the role of plasminogen activator in systemic infection by virulent Yersinia pestis strain C092. Microb Pathog 23: 211- 223. 286. Wexler, M., Sargent, F., Jack, R.L., Stanley, N.R., Bogsch, E.G., Robinson, C., Berks, B.C., and Palmer, T. (2000) TatD is a cytoplasmic protein with DNase activity. No requirement for TatD family proteins in sec-independent protein export. J Biol Chem 275: 16717-16722. 287. Vignon, G., Kohler, R., Larquet, E., Giroux, S., Prevost, M.C., Roux, P., and Pugsley, A.P. (2003) Type IV-like pili formed by the type II secreton: specificity, composition, bundling, polar localization, and surface presentation of peptides. J Bacteriol 185: 3416-3428.

60 REFERENCES

288. Williams, A.W., Yamaguchi, S., Togashi, F., Aizawa, S.I., Kawagishi, I., and Macnab, R.M. (1996) Mutations in fliK and flhB affecting flagellar hook and filament assembly in Salmonella typhimurium. J Bacteriol 178: 2960-2970. 289. Williams, A.W., and Straley, S.C. (1998) YopD of Yersinia pestis plays a role in negative regulation of the low-calcium response in addition to its role in translocation of Yops. J Bacteriol 180: 350-358. 290. Williamson, E.D., Eley, S.M., Griffin, K.F., Green, M., Russell, P., Leary, S.E., Oyston, P.C., Easterbrook, T., Reddin, K.M., Robinson, A., and Titball, R.W. (1995) A new improved sub-unit vaccine for plague: the basis of protection. FEMS Immunol Med Microbiol 12: 223-230. 291. Viprey, V., Del Greco, A., Golinowski, W., Broughton, W.J., and Perret, X. (1998) Symbiotic implications of type III protein secretion machinery in Rhizobium. Mol Microbiol 28: 1381- 1389. 292. Woestyn, S., Allaoui, A., Wattiau, P., and Cornelis, G.R. (1994) YscN, the putative energizer of the Yersinia Yop secretion machinery. J Bacteriol 176: 1561-1569. 293. von Heijne, G. (1985) Signal sequences. The limits of variation. J Mol Biol 184: 99-105. 294. Voulhoux, R., Ball, G., Ize, B., Vasil, M.L., Lazdunski, A., Wu, L.F., and Filloux, A. (2001) Involvement of the twin-arginine translocation system in protein secretion via the type II pathway. Embo J 20: 6735-6741. 295. Wren, B.W. (2003) The yersiniae--a model genus to study the rapid evolution of bacterial pathogens. Nat Rev Microbiol 1: 55-64. 296. Wulff-Strobel, C.R., Williams, A.W., and Straley, S.C. (2002) LcrQ and SycH function together at the Ysc type III secretion system in Yersinia pestis to impose a hierarchy of secretion. Mol Microbiol 43: 411-423. 297. Yahr, T.L., Hovey, A.K., Kulich, S.M., and Frank, D.W. (1995) Transcriptional analysis of the Pseudomonas aeruginosa exoenzyme S structural gene. J Bacteriol 177: 1169-1178. 298. Yahr, T.L., and Wickner, W.T. (2001) Functional reconstitution of bacterial Tat translocation in vitro. Embo J 20: 2472-2479. 299. Yang, Y., and Isberg, R.R. (1993) Cellular internalization in the absence of invasin expression is promoted by the Yersinia pseudotuberculosis yadA product. Infect Immun 61: 3907-3913. 300. Yang, Y., Merriam, J.J., Mueller, J.P., and Isberg, R.R. (1996) The psa locus is responsible for thermoinducible binding of Yersinia pseudotuberculosis to cultured cells. Infect Immun 64: 2483-2489. 301. Yother, J., Chamness, T.W., and Goguen, J.D. (1986) Temperature-controlled plasmid regulon associated with low calcium response in Yersinia pestis. J Bacteriol 165: 443-447. 302. Zav'yalov, V.P., Abramov, V.M., Cherepanov, P.G., Spirina, G.V., Chernovskaya, T.V., Vasiliev, A.M., and Zav'yalova, G.A. (1996) pH6 antigen (PsaA protein) of Yersinia pestis, a novel bacterial Fc-receptor. FEMS Immunol Med Microbiol 14: 53-57.

61