THE PATHOGENIC YERSINIAE – ADVANCES IN THE UNDERSTANDING OF PHYSIOLOGY AND VIRULENCE Topic Editor Matthew S. Francis CELLULAR AND INFECTION MICROBIOLOGY Frontiers in Cellular and Infection Microbiology August 2014 | The pathogenic Yersiniae – advances in the understanding of physiology and virulence | 1 ABOUT FRONTIERS Frontiers is more than just an open-access publisher of scholarly articles: it is a pioneering approach to the world of academia, radically improving the way scholarly research is managed. The grand vision of Frontiers is a world where all people have an equal opportunity to seek, share and generate knowledge. Frontiers provides immediate and permanent online open access to all its publications, but this alone is not enough to realize our grand goals. 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ISSN 1664-8714 ISBN 978-2-88919-258-8 DOI 10.3389/978-2-88919-258-8 Frontiers in Cellular and Infection Microbiology August 2014 | The pathogenic Yersiniae – advances in the understanding of physiology and virulence | 2 For decades, pathogenic Yersinia have served as an inventive model organism for researchers seeking to understand the complexities of bacteria-host cell interactions. In fact, seminal studies on Yersinia virulence mechanisms contributed to the emergence and recognition of the research field - cellular microbiology. Researching Yersinia infection biology continues to identify and define fascinating virulence and survival mechanisms that advance and expand existing perceptions of bacterial-host encounters. This also includes research that defines how the pathogenic Yersiniae respond to diverse physicochemical stimuli to spatially and temporally control this armory of customized virulence and survival factors. Yet additional research demonstrates how the application of powerful whole genomic-based methodologies can open new frontiers that further facilitate understanding of bacterial evolution and pathogenicity. This Research Topic is therefore focused on presenting and summarizing new developments in Yersinia patho-physiology through highlighting cutting- edge studies on the Yersinia -host cell interaction and the network of regulatory control mechanisms that define this outcome. THE PATHOGENIC YERSINIAE – ADVANCES IN THE UNDERSTANDING OF PHYSIOLOGY AND VIRULENCE Scanning electron micrograph of Yersinia pseudotuberculosis bacteria recovered from a Luria Bertani agar plate after growth at room temperature for 48 hours. This image was generated by Dr Ikenna R. Obi formerly from the Department of Molecular Biology, Umeå University, with the technical assistance of Lenore Johansson and Per Hörstedt at the Electron Microscopy Platform at Umeå University. Topic Editor: Matthew S. Francis, Umeå University, Sweden Frontiers in Cellular and Infection Microbiology August 2014 | The pathogenic Yersiniae – advances in the understanding of physiology and virulence | 3 Table of Contents 05 The Pathogenic Yersiniae—Advances in the Understanding of Physiology and Virulence Matthew S. Francis 07 Yersinia Infection Tools—Characterization of Structure and Function of Adhesins Kornelia M. Mikula, Robert Kolodziejczyk and Adrian Goldman 21 Yersinia Pestis Ail: Multiple Roles of a Single Protein Anna M. Kolodziejek, Carolyn J. Hovde and Scott A. Minnich 31 Regulation of the Yersinia Type III Secretion System: Traffic Control Rebecca S. Dewoody, Peter M. Merritt and Melanie M. Marketon 44 The SycN/YscB Chaperone-Binding Domain of YopN is Required for the Calcium-Dependent Regulation of Yop Secretion by Yersinia Pestis Sabrina S. Joseph and Gregory V. Plano 56 Type II Secretion in Yersinia—A Secretion System for Pathogenicity and Environmental Fitness Dominik von Tils, Inga Blädel, M. Alexander Schmidt and Gerhard Heusipp 67 Toward a Molecular Pathogenic Pathway for Yersinia Pestis YopM Annette M. Uittenbogaard, R. Lakshman Chelvarajan, Tanya Myers-Morales, Amanda A. Gorman, W. June Brickey, Zhan Ye, Alan M. Kaplan, Donald A. Cohen, Jenny P .-Y. Ting and Susan C. Straley 84 Early Sensing of Yersinia Pestis Airway Infection by Bone Marrow Cells Yaron Vagima, Yinon Levy, David Gur, Avital Tidhar, Moshe Aftalion, Hagar Abramovich, Eran Zahavy, Ayelet Zauberman, Yehuda Flashner, Avigdor Shafferman and Emanuelle Mamroud 94 Cell Death Programs in Yersinia Immunity and Pathogenesis Naomi H. Philip and Igor E. Brodsky 101 Fibrinolytic and Coagulative Activities of Yersinia Pestis Timo K. Korhonen, Johanna Haiko, Liisa Laakkonen, Hanna M. Järvinen and Benita Westerlund-Wikström 110 Crp Induces Switching of the CsrB and CsrC RNAs in Yersinia Pseudotuberculosis and Links Nutritional Status to Virulence Ann Kathrin Heroven, Maike Sest, Fabio Pisano, Matthias Scheb-Wetzel, Rebekka Steinmann, Katja Böhme, Johannes Klein, Richard Münch, Dietmar Schomburg and Petra Dersch 131 Hunger for Iron: The Alternative Siderophore Iron Scavenging Systems in Highly Virulent Yersinia Alexander Rakin, Lukas Schneider and Olga Podladchikova Frontiers in Cellular and Infection Microbiology August 2014 | The pathogenic Yersiniae – advances in the understanding of physiology and virulence | 4 138 Post-Transcriptional Regulation of Gene Expression in Yersinia Species Chelsea A. Schiano and Wyndham W. Lathem 154 Links Between Type III Secretion and Extracytoplasmic Stress Responses in Yersinia Josué Flores-Kim and Andrew J. Darwin 166 OmpR, A Response Regulator of the Two-Component Signal Transduction Pathway, Influences Inv Gene Expression in Yersinia Enterocolitica O9 Marta Brzóstkowska, Adrianna Raczkowska and Katarzyna Brzostek 180 Omics Strategies for Revealing Yersinia Pestis Virulence Ruifu Yang, Zongmin Du, Yanping Han, Lei Zhou, Yajun Song, Dongsheng Zhou and Yujun Cui 196 The Effect of Low Shear Force on the Virulence Potential of Yersinia Pestis: New Aspects that Space-Like Growth Conditions and the Final Frontier Can Teach us About a Formidable Pathogen Jason A. Rosenzweig and Ashok K. Chopra EDITORIAL published: 12 September 2013 doi: 10.3389/fcimb.2013.00051 The pathogenic Yersiniae—advances in the understanding of physiology and virulence Matthew S. Francis* Department of Molecular Biology and Umeå Centre for Microbial Research, Umeå University, Umeå, Sweden *Correspondence: matthew.francis@molbiol.umu.se Edited by: Yousef A. Kwaik, University of Louisville School of Medicine, USA Keywords: Yersinia , cellular physiology, metabolism, pathogenicity, host–pathogen interactions, immunity, host responses Of the ∼ 16 Yersinia species, only Y. pestis , Y. pseudotuberculosis , and Y. enterocolitica are pathogenic to humans (Koornhof et al., 1999; Smego et al., 1999). The zoonotic obligate pathogen Y. pestis is the causal agent of plague, a systemic disease that is usually fatal if left untreated. Free-living Y. enterocolitica and Y. pseudotubercu- losis are the agents of yersiniosis, a rarely systemic gastrointestinal disease. At the forefront of Yersinia research are studies of host cell contact, protein secretion, pathogenesis, immunity and the host response, nutrient sensing and sequestration and the control of gene expression. In this special research topic on the pathogenic Yersiniae is a compilation of reviews and research articles that highlight current knowledge and new developments in these areas of Yersinia pathophysiology. BACTERIA–HOST CELL CONTACT Yersinia is armored with diverse membrane anchored surface adhesins that each contribute to pathogen–host interactions. Mikula et al. (2013) explore the structure and virulence func- tion of the most prominent of these adhesins. They describe salient roles of select adhesins in intestinal pathogenesis by enteric Yersinia and give insight on how altered adhesive poten- tial through specific gene loss or gain may have contributed to Y. pestis lifestyle changes. Complementing this is Kolodziejek et al. (2012) that dissects specific physical properties and func- tional contributions of the esoteric Ail adhesin to Y. pestis infections. PROTEIN SECRETION The Ysc-Yop type III secretion system (T3SS) is encoded on a virulence plasmid common to all human pathogenic Yersinia (Cornelis et al., 1998). This injectisome is believed to provide a conduit through which Yop effector toxins can be delivered from the bacterial cytoplasm into the eukaryotic cell cytosol in one step or two (Edgren et al., 2012). Dewoody et al. (2013) provide insight into the regulatory mechanisms controlling injectisome assembly and the hierarchal coordination of substrate secretion. One important regulatory mechanism is the YopN secretion plug, and this is the focus of a study by Joseph and Plano (2013) that demonstrate a stretch of sequence within YopN - corre- sponding to a site for chaperone binding - is also required for environmental control of Ysc-Yop secretion. While Yersinia pathogenicity is correlated mostly to the plas- mid encoded Ysc-Yop T3SS, von Tils et al. (2012) discuss the impact of type II secretion systems (T2SS)—found in the genomes of all pathogenic and non-pathogenic Yersinia alike—to bacterial survival in the environment and in the host. IMMUNITY AND PATHOGENSIS Of the six known Ysc-Yop T3SS translocated effector tox- ins, YopM function remains enigmatic. As a consequence, Uittenbogaard et al. (2012) performed an exhaustive character- ization of early host cell responsiveness to YopM of Y. pestis . This revealed exciting new molecular pathways potentially targeted by YopM. Early host responsiveness was also the focus of Vagima et al. (2012), which presents a role for bone marrow derived cells in the early sensing of lung infections by Y. pestis . Moreover, rec- ognizing that cell death is a critical attribute of host immunity and microbial pathogenicity, Philip and Brodsky (2012) report on how cell death programs serve as both a Yersinia virulence strategy via T3SS-translocated YopJ effector function, as well as an elicitor of innate and adaptive immune responses designed to counteract Yersinia infections. Critical in the evolution of Y. pestis has been the acquisition of the gene pla encoding an omptin-like outer membrane plas- minogen activator protease (Pla). Korhonen et al. (2013) discuss this by summarizing features of controlled Pla protease activation, and its subsequent complex repertoire of interactions with host coagulation and fibrinolysis factors. NUTRIENT SENSING AND SEQUESTRATION A growing theme in infection biology research is the link between carbon metabolism and virulence (Poncet et al., 2009; Rohmer et al., 2011). Heroven et al. (2012) explore this concept in Yersinia pathogenicity using global omics-based profiling. They establish that the carbon storage regulator (Csr) system is controlled by the cAMP receptor protein (Crp), and together customize virulence gene expression according to the prevailing nutrient availability during Yersinia infections. A critical nutrient for growth of all life forms is iron. Rakin et al. (2012) describe an array of independent siderophore- mediated iron sequestration systems encoded by Y. pestis and the enteric Yersinia . They propose that by acquiring multiple alterna- tive endogenous siderophore systems with unique physiological properties, Yersinia has gained the capacity to adapt and thrive in diverse environmental niches. GENE EXPRESSION CONTROL Defining roles for small non-coding RNAs (ncRNAs) in regulation of gene expression dominates the infection biology landscape. Schiano and Lathem (2012) provide examples of small ncRNAs and various other post-transcriptional mechanisms in the regulation of virulence gene expression in Yersinia . They even propose that subtle sequence and/or regulatory differences found Frontiers in Cellular and Infection Microbiology www.frontiersin.org September 2013 | Volume 3 | Article 51 | CELLULAR AND INFECTION MICROBIOLOGY 5 Francis Yersinia physiology and virulence in certain small ncRNAs could account for some of the acquired lifestyle changes of Y. pestis Conditions that threaten the integrity of the bacterial envelope are collectively termed extracytoplasmic stresses (ECS). Bacteria encode distinct regulatory pathways designed to maintain bac- terial envelope integrity when challenged by ECS. Flores-Kim and Darwin (2012) describe how these ECS responsive pathways are also important for the control of Yersinia virulence determi- nants; particularly those embedded in the bacterial envelope such as integral membrane-spanning T3SSs and the well-known sur- face adhesin invasin. The environmental control of invasin gene expression in Y. enterocolitica is further explored by Brzostkowska et al. (2012). They demonstrate that OmpR, a response regulator of the EnvZ/OmpR two-component regulatory system, binds the inv promoter to directly negatively influence invasin expression. NEW FRONTIERS The systems biology era has revolutionized infectious biology research. Yang et al. (2012) illustrate the power of omics-based explorations in dissecting Yersinia -host cell interplay. In so doing, they provide a perspective on the future of omics-based research in benefitting our understanding of Yersinia cellular physiol- ogy and metabolism as well as host cellular responsiveness and immunity. Finally, manned space exploration demands an evaluation into the effect of such conditions on microbial virulence and infectious disease communicability. Rosenzweig and Chopra (2012) explain that low shear force conditions reduce the virulence capacity of Y. pestis . Thus, knowledge of the mechanisms behind these repres- sive effects could benefit understanding of Y. pestis pathogenicity both in space and on earth. REFERENCES Brzostkowska, M., Raczkowska, A., and Brzostek, K. (2012). OmpR, a response regulator of the two- component signal transduction pathway, influences inv gene expres- sion in Yersinia enterocolitica O9. Front. Cell. Infect. Microbiol . 2:153. doi: 10.3389/fcimb.2012.00153 Cornelis, G. R., Boland, A., Boyd, A. P., Geuijen, C., Iriarte, M., Neyt, C., et al. (1998). The virulence plasmid of Yersinia , an antihost genome. Microbiol. Mol. Biol. Rev. 62, 1315–1352. Dewoody, R. S., Merritt, P. M., and Marketon, M. M. (2013). Regulation of the Yersinia type III secretion system: traffic control. Front. Cell. Infect. Microbiol 3:4. doi: 10.3389/fcimb.2013.00004 Edgren, T., Forsberg, A., Rosqvist, R., and Wolf-Watz, H. (2012). Type III secretion in Yersinia: injectisome or not? PLoS Pathog. 8:e1002669. doi: 10.1371/journal.ppat.1002669 Flores-Kim, J., and Darwin, A. J. (2012). Links between type III secretion and extracytoplasmic stress responses in Yersinia Front. Cell. Infect. Microbiol . 2:125. doi: 10.3389/fcimb.2012.00125 Heroven, A. K., Sest, M., Pisano, F., Scheb-Wetzel, M., Steinmann, R., Bohme, K., et al. (2012). Crp induces switching of the CsrB and CsrC RNAs in Yersinia pseudotuberculosis and links nutri- tional status to virulence. Front. Cell. Infect. Microbiol . 2:158. doi: 10.3389/fcimb.2012.00158 Joseph, S. S., and Plano, G. V. (2013). The SycN/YscB chaperone-binding domain of YopN is required for the calcium-dependent regulation of Yop secretion by Yersinia pestis Front. Cell. Infect. Microbiol . 3:1. doi: 10.3389/fcimb.2013.00001 Kolodziejek, A. M., Hovde, C. J., and Minnich, S. A. (2012). Yersinia pestis Ail: multiple roles of a single protein. Front. Cell. Infect. Microbiol . 2:103. doi: 10.3389/fcimb.2012.00103 Koornhof, H. J., Smego, R. A. Jr., and Nicol, M. (1999). Yersiniosis. II: the pathogenesis of Yersinia infec- tions. Eur. J. Clin. Microbiol. Infect. Dis. 18, 87–112. doi: 10.1007/s100960050237 Korhonen, T. K., Haiko, J., Laakkonen, L., Jarvinen, H. M., and Westerlund- Wikstrom, B. (2013). Fibrinolytic and coagulative activities of Yersinia pestis Front. Cell. Infect. Microbiol . 3:35. doi: 10.3389/fcimb. 2013.00035 Mikula, K. M., Kolodziejczyk, R., and Goldman, A. (2013). Yersinia infec- tion tools-characterization of struc- ture and function of adhesins. Front. Cell. Infect. Microbiol . 2:169. doi: 10.3389/fcimb.2012.00169 Philip, N. H., and Brodsky, I. E. (2012). Cell death programs in Yersinia immunity and pathogenesis. Front. Cell. Infect. Microbiol . 2:149. doi: 10.3389/fcimb.2012.00149 Poncet, S., Milohanic, E., Maze, A., Nait Abdallah, J., Ake, F., Larribe, M., et al. (2009). Correlations between carbon metabolism and virulence in bacteria. Contrib. Microbiol. 16, 88–102. doi: 10.1159/000219374 Rakin, A., Schneider, L., and Podladchikova, O. (2012). Hunger for iron: the alternative siderophore iron scavenging systems in highly virulent Yersinia Front. Cell. Infect. Microbiol 2:151. doi: 10.3389/fcimb.2012.00151 Rohmer, L., Hocquet, D., and Miller, S. I. (2011). Are pathogenic bacteria just looking for food? Metabolism and microbial pathogenesis. Trends Microbiol. 19, 341–348. doi: 10.1016/j.tim.2011.04.003 Rosenzweig, J. A., and Chopra, A. K. (2012). The effect of low shear force on the virulence potential of Yersinia pestis : new aspects that space-like growth conditions and the final frontier can teach us about a formidable pathogen. Front. Cell. Infect. Microbiol . 2:107. doi: 10.3389/fcimb.2012.00107 Schiano, C. A., and Lathem, W. W. (2012). Post-transcriptional regulation of gene expression in Yersinia species. Front. Cell. Infect. Microbiol 2:129. doi: 10.3389/fcimb.2012.00129 Smego, R. A., Frean, J., and Koornhof, H. J. (1999). Yersiniosis I: microbi- ological and clinicoepidemiological aspects of plague and non- plague Yersinia infections. Eur. J. Clin. Microbiol. Infect. Dis. 18, 1–15. doi: 10.1007/s1009600 50219 Uittenbogaard, A. M., Chelvarajan, R. L., Myers-Morales, T., Gorman, A. A., Brickey, W. J., Ye, Z., et al. (2012). Toward a molecular pathogenic pathway for Yersinia pestis YopM. Front. Cell. Infect. Microbiol . 2:155. doi: 10.3389/fcimb.2012.00155 Vagima, Y., Levy, Y., Gur, D., Tidhar, A., Aftalion, M., Abramovich, H., et al. (2012). Early sensing of Yersinia pestis airway infec- tion by bone marrow cells. Front. Cell. Infect. Microbiol . 2:143. doi: 10.3389/fcimb.2012.00143 von Tils, D., Bladel, I., Schmidt, M. A., and Heusipp, G. (2012). Type II secretion in Yersinia -a secretion system for pathogenicity and environmental fitness. Front. Cell. Infect. Microbiol . 2:160. doi: 10.3389/fcimb.2012.00160 Yang, R., Du, Z., Han, Y., Zhou, L., Song, Y., Zhou, D., et al. (2012). Omics strategies for revealing Yersinia pestis virulence. Front. Cell. Infect. Microbiol . 2:157. doi: 10.3389/fcimb.2012.00157 Received: 26 August 2013; accepted: 26 August 2013; published online: 12 September 2013. Citation: Francis MS (2013) The pathogenic Yersiniae—advances in the understanding of physiology and virulence. Front. Cell. Infect. Microbiol. 3 :51. doi: 10.3389/fcimb.2013.00051 This article was submitted to the jour- nal Frontiers in Cellular and Infection Microbiology. Copyright © 2013 Francis. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the origi- nal author(s) or licensor are credited and that the original publication in this jour- nal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Cellular and Infection Microbiology www.frontiersin.org September 2013 | Volume 3 | Article 51 | 6 REVIEW ARTICLE published: 08 January 2013 doi: 10.3389/fcimb.2012.00169 Yersinia infection tools—characterization of structure and function of adhesins Kornelia M. Mikula 1,2 † , Robert Kolodziejczyk 1† and Adrian Goldman 1 * 1 Macromolecular X-Ray Crystallography Group, Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, Helsinki, Finland 2 The National Doctoral Program in Informational and Structural Biology, Åbo Academy, Turku, Finland Edited by: Matthew Francis, Umeå University, Sweden Reviewed by: Volkhard A. Kempf, Hospital of the Goethe University Frankfurt, Germany Juan A. Hermoso, Spanish National Research Council - CSIC, Spain *Correspondence: Adrian Goldman, Macromolecular X-Ray Crystallography Group, Institute of Biotechnology, University of Helsinki, PO Box 65, FIN-00014, Finland. e-mail: adrian.goldman@helsinki.fi † These authors equally contributed to this work. Among the seventeen species of the Gram-negative genus Yersinia , three have been shown to be virulent and pathogenic to humans and animals— Y. enterocolitica , Y. pseudotuberculosis , and Y. pestis . In order to be so, they are armoured with various factors that help them adhere to tissues and organelles, cross the cellular barrier and escape the immune system during host invasion. The group of proteins that mediate pathogen–host interactions constitute adhesins. Invasin, Ail, YadA, YadB, YadC, Pla, and pH 6 antigen belong to the most prominent and best-known Yersinia adhesins. They act at different times and stages of infection complementing each other by their ability to bind a variety of host molecules such as collagen, fibronectin, laminin, β 1 integrins, and complement regulators. All the proteins are anchored in the bacterial outer membrane (OM), often forming rod-like or fimbrial-like structures that protrude to the extracellular milieu. Structural studies have shown that the anchor region forms a β -barrel composed of 8, 10, or 12 antiparallel β -strands. Depending on the protein, the extracellular part can be composed of several domains belonging to the immunoglobulin fold superfamily, or form a coiled-coil structure with globular head domain at the end, or just constitute several loops connecting individual β -strands in the β -barrel. Those extracellular regions define the activity of each adhesin. This review focuses on the structure and function of these important molecules, and their role in pathogenesis. Keywords: adhesins, bacterial, Yersinia enterocolitica , Yersinia pseudotuberculosis , Yersinia pestis , outer membrane proteins, X-ray structure, structure–function relationship INTRODUCTION Yersiniae belong to the Enterobacteriaceae family; they are Gram- negative, facultative anaerobes. Seventeen different species of Yersinia genus have so far been reported, of which three have been shown to be pathogenic to humans and animals. These are the enteropathogens Y. enterocolitica and Y. pseudotuberculosis, and one zoonotic pathogen Y. pestis . Pathogenicity is correlated mostly with 70-kb virulence plasmid pYV carried by all three of them and with additional chromosomally encoded proteins (Cornelis, 1994). Other Yersinia species are either avirulent or their pathogenicity has not been reported. Y. enterocolitica and Y. pseudotuberculosis are responsible for a wide range of diseases from mild diarrhoea, enterocolitis, septica, and mesenteric lymphadenitis to reactive arthritis and iritis (Cover and Aber, 1989). They are transmitted by inges- tion of contaminated food. The most frequent outbreaks of Y. enterocolitica have had their origin in infected, undercooked pork meat, but bacteria have also been found in other mam- malian hosts (Bottone, 1997). The most common reservoirs for Y. pseudotuberculosis , on the other hand, have been reported to be carrots and lettuce (Jalava et al., 2006). Immediately after oral uptake of contaminated food or water (Mazigh et al., 1984), bacteria traverse through the gastrointestinal tract until they reach the terminal ileum. At this point bacteria already have present on their surface the outer membrane (OM) protein invasin, which is expressed in stationary phase at low temper- atures (e.g., in stored food) (Pepe and Miller, 1993). It plays a crucial role during the first phases of infection by facilitating effi- cient translocation across the intestinal epithelial barrier. During this phase, a second protein, Ail, is also important ( Figure 1A ). Bacteria traverse the epithelial barrier through the M cells (micro- fold cells) that are associated with Payer’s patches (Grassl et al., 2003). After translocation to the basolateral side of the Peyer’s patches, invasin binds to β 1 integrins, which induce production of chemokines like IL-8. In the Payer’s patches, bacteria replicate and express another adhesin, YadA ( Figure 1A ), which protects bacte- ria against phagocytosis of recruited polymorphonuclear leuko- cytes (PMN) and monocytes, as well as downregulates expression of invasin. PMN and monocytes may lead to tissue disruption and bacterial transport to gut associated lymphoid tissues. YadA and Ail enable bacterial dissemination to mesenteric lymph nodes by protecting against the host immune system. Moreover, YadA facil- itates adhesion to collagen, which is crucial for Y. enterocolitica in causing reactive arthritis, a sterile inflammation of the joints. This iritis and erythema nodosum are post-infection sequelae of Y. enterocolitica mediated by YadA (Cover and Aber, 1989). In more severe infections bacteria can further colonize other Frontiers in Cellular and Infection Microbiology www.frontiersin.org January 2013 | Volume 2 | Article 169 | CELLULAR AND INFECTION MICROBIOLOGY 7 Mikula et al. Yersinia adhesins—structure and function FIGURE 1 | Schematic overview of proteins expressed in Yersiniae outer membrane during infection. Bacterial outer membrane (OM) with outer core of LPS (OC) in purple and adhesins expressed at different stages of infection. (A) Adhesisn of Y. enterocolitica and Y. pseudotuberculosis : invasin in yellow, YadA in dark green, Ail in red, and O-Antigen in light grey; (B) Adhesins of Y. pestis : Pla in green, YadB in blue, YadC in orange, Ail in red. ECM stands for extracellular matrix. All the molecules are on approximately the same scale. organs like liver, spleen, kidney, or lungs, but infection is usually self-limiting (Grassl et al., 2003). The third human pathogenic Yersnia species, Y. pestis , evolved around 1500–20,000 years ago from Y. pseudotuberculosis by lat- eral gene transfer and gene inactivation (Achtman et al., 1999). Y. pestis lost non-essential housekeeping genes, has inactivated genes encoding for proteins needed for intestinal pathogenesis, like invasin and YadA, although it is not known if inactivation of yadA and inv increased Y. pestis virulence (Achtman et al., 1999). The key step in the evolution of Y. pestis was the acquisition of the pFra plasmid. This, together with the ability to express chro- mosomally encoded proteins that Y. pseudotuberculosis was not enables Y. pestis to be transmitted by fleas from one mammalian host to another. Y. pestis expresses unique proteins associated with virulence, Pla (Achtman et al., 1999) and the recently discovered YadA and YadC ( Figure 1B ) (Forman et al., 2008). Y. pestis also expresses Ail and pH 6 antigens that are present in other Yersinia species ( Figure 1B ). Y. pestis is the most virulent and invasive of the three species, causing highly fatal pneumonic, bubonic, and septicemic plague (Perry and Fetherston, 1997). Pneumonic plague is the least com- mon but most deadly form, progressing very rapidly from flu-like symptoms to overwhelming pneumonia. It spreads by inhalation of respiratory droplets during contact with the infected person (Perry and Fetherston, 1997). It may also occur as a compli- cation after bubonic or septicaemic plague. If treatment is not started during the first 24 h after the first symptoms, it is usually fatal within 48 h (Felek et al., 2010). Bubonic plague (the most common form) occurs a few days after an infected fleabite or wound exposure to contaminated material. It causes swollenness and tenderness of lymph nodes, as well as gastrointestinal com- plaints. Very often secondary plague septicemia or bacteremia can occur, which is also highly fatal if untreated (40–60% mortality rates) (Perry and Fetherston, 1997). Finally, primary septicemic plague is present mainly in the bloodstream. Infection occurs by fleabites, contact with infectious material via open wounds or spread from the lymphatic system as a result of advanced stages of bubonic plague (Felek et al., 2010). The mortality rate of this forms 30–50% if left untreated (Perry and Fetherston, 1997). In bubonic plague, Y. pestis travels from the initial site of infec- tion to lymph nodes, most likely inside macrophages. When bac- teria reach the lymph nodes, they escape from the macrophages and start to grow extracellularly to high numbers, leading to the formation of bubos (swollen lymph nodes). Bacteria resistant to phagocytosis spread into the bloodstream causing septicaemic plague. Moreover, infection can continue and bacteria further col- onize blood, liver, spleen, or even lungs, which leads to secondary pneumonic plague (Perry and Fetherston, 1997). Expression of Pla enables Y. pestis to disseminate from the initial side of infec- tion to lymph nodes and travel in the bloodstream. Pla also facilitates serum resistance. Moreover, while bacteria travel in the macrophages, expression of pH 6 antigen, another adhesin, is induced. This prevents plague bacteria from phagocytosis by those macrophages and after escape, from later phagocytosis (Perry and Fetherston, 1997). Expression of Ail helps bacteria to survive in blood as it is involved in serum resistance and medi- ates adhesion to epithelial cells and extracellular matrix (ECM) proteins (Miller et al., 2001). Of all the adhesins expressed by Y. pestis , Ail is the most important for Yop delivery ( Yersinia outer proteins, secreted by the Yersinia III type secretion system) (Felek et al., 2010). In this review, we focus on structural and functional aspects of the adhesins Invasin, YadA, YadB, YadC, Ail, Pla, and pH 6 antigen ( Tables 1 , 2 ), which are expressed during host invasion by Yersinia species. INVASIN–THE FIRST ADHESIN EXPRESSED DURING INVASION OF ENTEROPATHOGENIC Yersinia Invasin is an adhesin expressed by enteropathogenic (EPEC) species of Yersinia, Y. enterocolitica , and Y. pseudotuberculosis Frontiers in Cellular and Infection Microbiology www.frontiersin.org January 2013 | Volume 2 | Article 169 | 8 Mikula et al. Yersinia adhesins—structure and function Table 1 | Summary of functions of Yersinia adhesins. Protein Function Organism References CHROMOSOMALLY ENCODED PROTEINS Invasin Invasion of epithelial cells Ye a , Yp a Isberg et al., 1987 β 1 integrin binding Ye, Yp Clark et al., 1998 Induction of cytokine production Ye, Yp Grassl et al., 2003 YadB, YadC Invasion of epithelial cells Ype a Forman et al., 2008 Not known Yp Forman et al., 2008 Ail Adhesion to epithelial cells Ye, Ype Miller et al., 2001 Binding to laminin and fibronectin Ye, Ype Yamashita et al., 2011 Serum resistance Ye, Yp, Ype Biedzka-Sarek et al., 2008b Yop delivery b Ye, Yp, Ype Felek et al., 2010 pH 6 Resistance to phagocytosis Ye, Ype Yang et al., 1996; Huang and Lindler, 2004 Escape from macrophages Ype Lindler and Tall, 1993 Haemagglutination Yp, Ype Yang et al., 1996 Interaction with lipoproteins Ype Makoveichuk et al., 2003 Interaction with Fc of IgG Ype Zav’yalov et al., 1996 Yop delivery Ype Felek et al., 2010 Tissue adhesion Yp Yang et al., 1996 Adhesion Ye Yang et al., 1996 PLASMID ENCODED PROTEINS YadA Essential for virulence Ye Roggenkamp et al., 1995 Invasion of epithelial cells Yp Eitel and Dersch, 2002 Binding to ECM molecules collagen, fibronectin, and laminin Ye, Yp Schulze-Koops et al., 1992; Flügel et al., 1994; Heise and Dersch, 2006 Adhesion to epithelial cells, neutrophils, and macrophages Ye, Yp Heesemann et al., 1987; Roggenkamp et al., 1996 Serum resistance Ye, Yp Lambris et al., 2008 Autoagglutination Ye, Yp Hoiczyk et al., 2000 Yop delivery Ye, Yp Visser et al., 1995 Pla Plasminogen activation Ype Beesley et al., 1967 Adherence and invasion to epithelial cells Ype Sodeinde et al., 1992 Degradation of laminin and fibrin Ype Haiko et al., 2009 Serum resistance Ype Sodeinde et al., 1992 Yop delivery Ype Felek et al., 2010 a Abbreviations: Yp, Yersinia pseudotuberculosis; Ye, Yersinia entercolitica; Ype, Yersinia pestis. b Indirect, via laminin and fibronectin binding. Table 2 | Summary of structures of Yersinia adhesins discussed. Protein Region PDB Localization Organism References Invasin D1-D5 domains 1CWV extracellular Yp a Hamburger et al., 1999 β -barrel 4E1T outer membrane Yp Fairman et al., 2012 YadA Head + neck 1P9H extracellular Ye a Nummelin et al., 2004 Ail β -barrel 3QRA outer membrane Ype a Yamashita et al., 2011 Pla β -barrel 2X55 outer membrane Ype Eren et al., 2010 a Abbreviations: Yp, Yersinia pseudotuberculosis; Ye, Yersinia entercolitica; Ype, Yersinia pestis. (Isberg et al., 1987). It acts during the first phase of infection, and is responsible for initial colonization and internalization with host cells. Invasin is chromosomally encoded by the inv gen, which is maximally expressed at 25 ◦ C, pH 8 or at 37 ◦ C, pH 5.5 but poorly at 37 ◦ C, pH 8. This indicates that invasin is expressed prior to oral uptake (i.e., in stored food), which may be beneficial for rapid transcytosis through the epithelial layer, or in the intestinal tissue (Grassl et al., 2003; Uliczka et al., 2011). Difference in expres- sion efficiency of invasin depends on Y. enterocolitica serotype and the regulatory factors present in the serotypes. Invasin expres- sion is repressed at 37 ◦ C in the O:8 and O:9 serotypes by Frontiers in Cellular and Infection Microbiology www.frontiersin.org January 2013 | Volume 2 | Article 169 | 9 Mikula et al. Yersinia adhesins—structure and function rapid degradation of the invA activator RovA (transcriptional activator), and silencing mediated by negative regulator H-NS (nucleoid structuring protein), which forms higher order com- plexes in a concentration-dependent manner and causes gene silencing (Wyborn et al., 2004; Uliczka et al., 2011). Constitutive expression of invasin at 25 ◦ C, as well as at 37 ◦ C in the O:3 serotype, results from insertion of the IS1667 element into the regulatory region of the invA gene. This disrupts the inhibitory region that binds H-NS (Uliczka et al., 2011). Moreover, it has been shown that the single amino acid substitution P98S increases the thermostability of RovA, leading to higher expression of invasin (Uliczka et al., 2011). The nature of this stabilization is uncertain as no major structural changes have been observed, but it leads to greatly decreased proteolysis of RovA (Uliczka et al., 2011). Nonetheless, despite the large amount of invasin expressed at 25 and 37 ◦ C by the O:3 strain, cell invasion is sig- nificantly reduced or does not occur at all for bacteria pregrown at 25 ◦ C (Bialas et al., 2012). This appears to be because high expression of the O-antigen in the LPS (lipopolysaccharide) cre- ates steric hindrance that prevents interactions between invasin and the host cell surface whereas at 37 ◦ C expression of O-antigen is repressed allowing better access of invasin to host cells. This is unlike other Y. enterocolitica and Y. pseudotubrculosis serotypes, where the highest level of invasin expression occurs when they are cultured at moderate temperatures (Uliczka et al., 2011). Invasins (about 92 kDa) are closely related sequentially and structurally to the intimins, OM proteins from the EPEC and enterohemorrhagic (EHEC) E. coli strains. A topology model (Tsai et al., 2010) suggested that the invasin/intimin family has a conserved modular architecture, composed of: (1) signal sequence, (2) hydrophilic α -domain, (3) β -barrel domain, (4) hydrophilic α ∗ -domain, and (5) extracellular domain ( Figure 2A ). The structures of the invasin extracellular domain (1CWV) ( Figure 2B ) (Hamburger et al., 1999) and β -barrel (PDB: 4E1T) ( Figure 2C ) (Fairman et al., 2012) reflect a modu- lar architecture. The signal sequence allows translocation through the inner membrane (IM) via the Sec secretion mechanism and is cleaved off afterwards. There are two hydrophilic α -domains that reside in the periplasm and are separated in sequence by the β -barrel. By having a β -barrel at the N-terminus and the extracellular domain at the C-terminus ( Figure 1A ), Invasin has an inverse arrangement to the classical autotransporter (AT) system (type V secretion system), and so has been proposed to constitute a