Yersinia: Molecular and Cellular Biology Chapter Abstracts

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Chapter 1
The Yersinia pestis Chromosome
Nicholas R. Thomson and Julian Parkhill

The whole genome sequences of two Y. pestis biovars, Mediaevalis and Orientalis, have recently been published. Each isolate sequenced is thought to represent the predominant strain from different plague pandemics: The Black Death and Modern Plague. Comparison of the two sequenced strains and other members of the Enterobacteriaceae reveal that Y. pestis exhibits many of the characteristics of an organism that has undergone a recent dramatic change in lifestyle. This chapter will focus on the global architecture of the Y. pestis chromosome as well as looking at the fine detail contained within the genome that gives some insights into how this pathogen has developed into such a potent foe.

Chapter 2
Age, Descent and Genetic Diversity within Yersinia pestis
Mark Achtman

This article was originally intended as a review of the literature on the population genetics of Yersinia pestis but, unfortunately, there is essentially no such literature to review. Therefore, in lieu of a literature review, I shall summarize molecular evidence from which the source and age of these bacteria can be deduced. I shall attempt to also explain some of the population genetic concepts on the basis of which such data can be interpreted.

Chapter 3
The Yersinia pestis-Specific Plasmids pFra and pPla
Luther E. Lindler

In addition to the common Yersinia virulence plasmid that is ~ 70-kb, typical strains of Yersinia pestis harbor two unique plasmids. These two plasmids are generally referred to as the "Murine Toxin" plasmid, pFra, and the "pesticin" plasmid, pPla. The Y. pestis-specific plasmids are approximately 100 and 9.6-kb in size, respectively Both of these molecules encode DNA sequences similar to those found on plasmids from Gram-negative enteric organisms with which Y. pestis may have exchanged genetic material. Thus, these plasmids contain the molecular fingerprints left behind during the evolution and emergence of Y. pestis as a unique pathogen. Furthermore, the detailed analysis of pFra at the genomic-level revealed how virulence plasmids can be assembled in nature as a mosaic of multiple genetic elements. Taken together, the study of these Y. pestis-specific plasmids has yielded valuable lessons in how serial coalescence of multiple genetic elements in a bacterium can contribute to the emergence of new bacterial diseases. This chapter presents the salient features of Y. pestis pathogenesis in relation to the factors encoded by the pFra and pPla plasmids as well as the genetics of the virulence factors encoded by these extrachromosomal elements.

Chapter 4
The Evolution of Flea-Borne Transmission in Yersinia pestis
B. Joseph Hinnebusch

Transmission by fleabite is a recent evolutionary adaptation that distinguishes Yersinia pestis, the agent of plague, from Yersinia pseudotuberculosis and all other enteric bacteria. The very close genetic relationship between Y. pestis and Y. pseudotuberculosis indicates that just a few discrete genetic changes were sufficient to give rise to flea-borne transmission. Y. pestis exhibits a distinct infection phenotype in its flea vector, and a transmissible infection depends on genes that are specifically required in the flea, but not the mammal. Transmission factors identified to date suggest that the rapid evolutionary transition of Y. pestis to flea-borne transmission within the last 1,500 to 20,000 years involved at least three steps: acquisition of the two Y. pestis-specific plasmids by horizontal gene transfer; and recruitment of endogenous chromosomal genes for new functions. Perhaps reflective of the recent adaptation, transmission of Y. pestis by fleas is inefficient, and this likely imposed selective pressure favoring the evolution of increased virulence in this pathogen.

Section II. Cellular Biology of Yersinia.

Chapter 5
N-Acylhomoserine Lactone-Mediated Quorum Sensing in Yersinia
Steven Atkinson, R. Elizabeth Sockett, Miguel Cámara and Paul Williams

Bacterial cell-to-cell communication ('quorum sensing') is mediated by structurally diverse, small diffusible signal molecules which regulate gene expression as a function of cell population densitY. Many different Gram-negative animal, plant and fish pathogens employ N-acylhomoserine lactones (AHLs) as quorum sensing signal molecules which control a variety of physiological processes including bioluminescence, swarming, antibiotic biosynthesis, plasmid conjugal transfer, biofilm development and virulence. AHL-dependent quorum sensing is highly conserved in both pathogenic and non-pathogenic members of the genus Yersinia. Yersinia pseudotuberculosis for example, produces at least six different AHLs and possesses two homologues of the LuxI family of AHL synthases and two members of the LuxR family of AHL-dependent response regulators. In all Yersinia species so far examined, the genes coding for LuxR and LuxI homologues are characteristically arranged convergently and overlapping. In Y. pseudotuberculosis AHL-dependent quorum sensing is involved in the control of cell aggregation and swimming motility, the latter via the flagellar regulatory cascade. This is also the case for swimming and also swarming motility in Yersinia enterocolitica. However the role of AHL-dependent quorum sensing in Yersinia pestis remains to be determined.

Chapter 6
The Invasin Protein of Enteropathogenic Yersinia Species: Integrin Binding and Role in Gastrointestinal Diseases
Ka-Wing Wong, Penelope Barnes and Ralph R. Isberg

The invasin of enteropathogenic Yersinia is an outer membrane protein that promotes the attachment of bacteria to mammalian cells via binding to multiple members of the integrin superfamily of cell adhesion molecules. The protein is an example of a group of proteins of high sequence similarity involved in mediating attachment to host cell surfaces that are found on the surface of many Gram-negative organisms. Attachment of the bacteria via invasin can result in promoting internalization of the bacterium into a phagosome, in an event that is antagonized by the Yersinia Yop proteins. After oral inoculation of model host animals with enteropathogenic Yersinia, this uptake process appears to occur after attachment to M cells, which are located over Peyer's patches and interface with the lumen of the small intestine. Efficient colonization of local lymph nodes, therefore, requires the presence of the invasin protein. Studies on the molecular mechanism of invasin-promoted uptake indicate that high affinity binding is a critical determinant of uptake, and clustering of integrin receptors results in activation of Rho family members, which is a necessary prelude to the formation of a phagosome encompassing the bacterium.

Chapter 7
Transcriptional Regulation in Yersinia: An Update
Michael Marceau

In response to the ever-present need to adapt to environmental stress, bacteria have evolved complex (and often overlapping) regulatory networks that respond to various changes in growth conditions, including entry into the host. The expression of most bacterial virulence factors is regulated; thus the question of how bacteria orchestrate this process has become a recurrent research theme for every bacterial pathogen, and the three pathogenic Yersinia species are no exception. The earliest studies of regulation in these species were prompted by the characterization of plasmid-encoded virulence determinants, and those conducted since have continued to focus on the principal aspects of virulence in these pathogens. Most Yersinia virulence factors are thermally regulated, and are active at either 28^oC (the optimal growth temperature) or 37^oC (the host temperature). However, regulation by this omnipresent thermal stimulus occurs through a wide variety of mechanisms, which generally act in conjunction with (or are modulated by) additional controls for other environmental cues such as pH, ion concentration, nutrient availability, osmolarity, oxygen tension and DNA damage. Yersinia's recent entry into the genome sequencing era has given scientists the opportunity to study these regulators on a genome-wide basis. This has prompted the first attempts to establish links between the presence or absence of regulatory elements and the three pathogenic species' respective lifestyles and degrees of virulence.

Chapter 8
Identification of Yersinia Genes Expressed During Host Infection
Andrew J. Darwin

One of the obvious goals during the study of bacterial pathogenesis is to identify the bacterial genes required for growth within the host. Historically, this has presented a significant technological challenge. However, with this goal in mind, the in vivo expression technology (IVET) and signature-tagged mutagenesis (STM) techniques were developed during the 1990s. Both techniques have been used to identify virulence genes in Y. enterocolitica and Y. pseudotuberculosis, using variations of their mouse models of infection. In this chapter, each of these studies is described individually, including the pertinent details of how each was done, and a brief discussion of the genes identified. In addition, the results of these IVET and STM screens are compared, and the striking lack of overlap between the genes identified is discussed. Most of these studies were only recently published, which means that there have been few follow-up studies on some of the novel virulence genes identified. However, the Y. enterocolitica hreP, rscR and psp genes have become the subject of further publications, which are also summarized here. Finally, I briefly describe the use of the genome-wide (but not in vivo) technology, subtractive hybridization, to identify Yersinia virulence genes.

Chapter 9
Immune Responses to Yersinia
Erwin Bohn and Ingo B. Autenrieth

Most of the data available on immunity to enteric Yersinia come from experimental studies in rodents, particularly murine studies with Yersinia enterocolitica. Yersinia virulence factors (e.g., YadA, Yops) function to evade innate defense mechanisms such as complement or phagocytosis, which allows a certain degree of bacterial replication and dissemination in host tissue. Enteric Yersinia transmigrate the intestinal epithelial barrier via M cells into the Peyer's patches. Infection of the Peyer's patches stimulates the recruitment of CD11b+ cells such as granulocytes and macrophages. These events and the secretion of cytokines such as IL-12, IFN-g, IL-18 and TNF-a are crucial for clearance of the Yersinia infection. Besides these innate immune responses, adaptive immune defense mechanisms provided by T cells are necessary for clearance of Yersinia infections. Thus, because the virulence factors of enteric Yersinia partially subvert innate immune responses, extracellularly located enteric Yersinia need to be controlled by adaptive immune defense mechanisms that are typically required for resolution of intracellularly located pathogens. Production of immunglobulins by B cells directed against Yersinia virulence factors such as YadA, LcrV and, exclusively for Y. pestis, F1 provide a protective immunity upon a secondary challenge with Yersinia.

Chapter 10
Superantigens of Yersinia pseudotuberculosis
Jun Abe

Bacterial superantigens have been implicated in the pathogenesis of several human diseases. Among them, toxic shock syndrome is a prototypic acute intoxication caused by the pyrogenic exotoxin family of superantigens. Yersinia pseudotuberculosis produces a superantigen, YPM, which is a 14.5-kDa protein able to stimulate human T cells bearing Vb3, 9, 13.1, and 13.2 segments on the T cell receptor (TCR). There are three variant proteins of YPM (YPM-a, -b, -c) and genetic studies suggest that these variant genes came from an ancestral ypm gene transferred from another species into the genome of Y. pseudotuberculosis. The ypm genes are not present in all strains, and ypm positive strains are much more frequently isolated in Far East Asia, including Japan, than in Europe. In Japan, Y. pseudotuberculosis infection is frequently associated with systemic symptoms such as erythematous skin rash, cervical lymphadenopathy, and the late onset of interstitial nephritis and coronary aneurysms. The high incidence of the YPM-producing strains in Japan, together with the higher anti-YPM antibody titers in patients with the systemic manifestations, implicatesYPM in the pathogenesis of these symptoms.

Section IIIA. Molecular Biology of Yersinia. Chromosome-Encoded Factors.

Chapter 11
Lipopolysaccharides of Yersinia
Mikael Skurnik

All Gram-negative bacteria including the members of the genus Yersinia possess an outer membrane (OM). Most of the outer leaflet of the OM is occupied by lipopolysaccharide (LPS), which is composed of two biosynthetic entities: (i) lipid A-core oligosaccharide; and attached to it (ii) O-specific polysaccharide, also known as O-antigen. This biosynthetic division has relevance for the understanding of the biology and genetics of LPS; therefore, the biosynthesis of these two LPS entities will be first briefly discussed in this chapter. The O-antigens are highly variable and form the basis for serotyping of strains. Y. pseudotuberculosis strains have been assigned to 21 and Y. enterocolitica and Y. enterocolitica-like strains to more than 70 serotypes, while Y. pestis strains lack O-antigen and are therefore serologically homogeneous. Chemical structures of LPS and the genetic basis of their biosynthesis have been determined for a number of Yersinia strains representing different species and seotypes, and an overall picture of the relationship between genetics and structure is emerging.

Chapter 12
Flagella: Organelles for Motility and Protein Secretion
Glenn M. Young

Flagella represent an excellent trait to study the evolution of the genus Yersinia and have provided insight on the function of bacterial type III secretion systems. Flagella are organelles produced by enteropathogenic species of the genus Yersinia that provide a means for motilitY. This bacterial organelle also forms a type III secretion system for the export of non-flagellar proteins to the outside of the bacterium. Recent studies of flagellar functions have provided remarkable insight on how both flagellar-dependent motility and flagellar-dependent protein secretion contribute to the biology of the flagellated Yersinia species. The ability to migrate to a favorable environment certainly contributes to the survival of enteropathogenic Yersinia during free-living stages of their life cycle, but this attribute also can affect interactions with host organisms. Flagella promote the invasion of mammalian cells and, in some cases, appear to affect the pathogenic outcomes. Interactions with the host may also be influenced by the proteins delivered into the host by the flagellar type III secretion system.

Chapter 13
Iron and Heme Uptake Systems
Robert D. Perry and Jacqueline D. Fetherston

The pathogenic Yersiniae contain a number of different iron transport systems. The ability to use exogenous siderophores probably allows the enteropathogens to more effectively compete with other microbes in the intestinal lumen and in the environment outside the host. In Y. pestis, there appears to be a hierarchy of iron transport systems with some being more important than others during different stages of mammalian infection. Thus, the Ybt siderophore-dependent system is absolutely required during the initial stages of bubonic plague while the Yfe transporter is important during the later stages of disease. The other inorganic and heme transport systems do not seem to play a role in disease in mice by a subcutaneous route of infection. Perhaps one or more of these systems is relevant in pneumonic plague or is important for disease in other animals. Alternatively, some of the iron transport systems (the aerobactin/Fhu system, for example) may simply reflect the Enterobacteriaceae lineage of Y. pestis.

Chapter 14
The High-Pathogenicity Island: A Broad-Host-Range Pathogenicity Island
Biliana Lesic and Elisabeth Carniel

Highly pathogenic Yersinia (Y. enterocolitica 1B, Y. pseudotuberculosis and Y. pestis) harbor a pathogenicity island termed the High-Pathogenicity Island (HPI). The Yersinia HPI carries genes involved in the biosynthesis, transport and regulation of the siderophore Yersiniabactin and can thus be regarded as an iron-uptake island. Its presence confers to the bacterium the ability to disseminate in its host and cause a systemic infection. The Yersinia HPI has kept the potential to be mobile within the chromosome of Y. pseudotuberculosis. A unique characteristic of this island is its wide distribution among different enterobacteria genera such as E. coli, Klebsiella, Citrobacter, Enterobacter, Serratia and Salmonella. In E. coli, the island is prevalent in extraintestinal isolates and is associated with a higher virulence. A second and degenerate HPI is present on the chromosomes of Y. pestis and Y. pseudotuberculosis. HPI-like elements have also been recently identified in the insect pathogen Photorhabdus luminescens and the Gram-positive species Corynebacterium diphtheriae. These HPI-like elements potentially encode iron-uptake systems, but whether they are still functional remains to be determined.

Chapter 15
YAPI, a New Pathogenicity Island in Enteropathogenic Yersiniae
François Collyn, Michael Marceau and Michel Simonet

We describe a large (98 kilobases) DNA segment found in enteropathogenic species Y. pseudotuberculosis. Baptised "YAPI" for Yersinia Adhesion Pathogenicity Island because it codes for a type IV pilus that promotes bacterial adherence to the intestinal mucosa, this chromosomal fragment fulfills all the pathogenicity island (PAI) criteria: (i) it bears at least one gene cluster involved in bacterial pathogenicity (the pil locus, encoding a type IV pilus); (ii) it is associated with a tRNA locus (tRNA-phe); (iii) is flanked by directly-repeated sequences; (iv) it comprises a variety of mobility genes (whether intact or not), especially an ORF encoding a prophage integrase and situated upstream of the flanking tRNA gene; and finally (v) its sequence has a heterogenous GC content, reflecting a genetic mosaic. An homologous YAPI is present in the Y. enterocolitica chromosome while the PAI is absent in Y. pestis.

Section IIIB. Molecular Biology of Yersinia. Plasmid-Encoded Factors.

Chapter 16
The pYV Plasmid and the Ysc-Yop Type III Secretion System
Marie-Noëlle Marenne, Luís Jaime Mota and Guy R. Cornelis

The three pathogenic Yersinia spp. (Y. pestis, Y. pseudotuberculosis and Y. enterocolitica) harbor a 70-kb pYV plasmid that is essential for their virulence. The pYV plasmid encodes the Yop virulon consisting of a complete type III secretion system, called Ysc-Yop. This highly sophisticated virulence system allows extracellular Yersinia bacteria to inject "effector" Yop proteins directly into the cytosol of the eukaryotic host cells. The proteins are secreted across the two bacterial membranes and are also translocated across the eukaryotic cell membrane. This is achieved in a tightly regulated manner by a complex protein secretion machinery, the Ysc injectisome, and by "translocator" Yop proteins that are secreted by the Ysc machinery and presumably insert in the eukaryotic cell membrane. The proper functioning of the system also requires the assistance in the bacterial cytoplasm of the Syc chaperones, which bind and assist secretion of Yop proteins. Once inside the eukaryotic cell, the Yop effector proteins will subvert and disrupt host cell signaling pathways, incapacitating the host innate immune system, in particular inhibiting phagocytosis and downregulating the anti-inflammatory response.

Chapter 17
The Plasminogen Activator Pla of Yersinia pestis: Localized Proteolysis and Systemic Spread
Timo K. Korhonen, Maini Kukkonen, Ritva Virkola, Hannu Lang, Marjo Suomalainen, Pöivi Kyllönen and Kaarina Lähteenmäki

The plasminogen activator (Pla) of the plague bacterium Yersinia pestis is a cell-surface protease that belongs to the omptin family of enterobacterial aspartic proteases. Pla is a critical virulence factor in the pathogenesis of plague and specifically enables the spread of the bacterium from the subcutaneous site of a flea bite. Pla may enhance the invasiveness of Y. pestis by multiple mechanisms. It proteolytically activates human plasminogen to plasmin and inactivates a2-antiplasmin, the major plasmin inhibitor in human serum, which would be predicted to result in uncontrolled, highly potent proteolysis at the infection site. Pla also is an adhesin with affinity for laminin of basement membranes, and we hypothesize that bacterial adhesiveness localizes the formed plasmin activity onto susceptible targets, such as laminin and basement membranes. The combined activities of Pla are known to damage basement membrane and extracellular matrix and impair their barrier function in vitro, a behaviour that is reminiscent of metastatic tumor cell migration. Pla also degrades the complement component C3, which may increase resistance of Y. pestis to killing by phagocytes. Finally, Pla is an invasin that promotes the in-vitro invasion of Y. pestis into human epithelial and endothelial cells. The adhesive and the invasive functions of Pla can be genetically dissected from the proteolytic activitY. Pla is predicted to have a 10-stranded antiparallel b-barrel conformation, with five short surface-exposed loops. The active site is located in a groove at the outermost surface of the barrel. Residues and regions in the surface loops that are important for the functions of Pla have been identified. Pla has a dual interaction with lipopolysaccharide: it requires rough lipopolysaccharide to be functionally active but its functions are sterically hindered by an O-side chain. Y. pestis lacks an O-antigen, and an advantage for Y. pestis of having a rough lipopolysaccharide may be its ability to fully utilize Pla functions.

Chapter 18
Structure Assembly and Applications of the Polymeric F1 Antigen of Yersinia pestis
Sheila MacIntyre, Stefan D. Knight and Laura J. Fooks

F1 polymer is a fine, fibrillar, surface structure with a capsule-like appearance. It is unique to Yersinia pestis, is produced in large amounts early in infection and is a powerful immunogen. It stimulates production of protective antibodies which complement V antigen induced protection. Thus, despite the fact that it is not essential for virulence in rodent models, it remains a major component of newer generation subunit and DNA vaccines and a target of diagnostic kits. The polymer is assembled by the periplasmic chaperone: outer membrane usher pathwaY. The polymer is a series of Ig-like modules formed via donor strand complementation between neighbouring Caf1 subunits. The high resolution crystal structure of two assembly intermediates has revealed the structure of F1 and provided the basis for a novel model of chaperone driven polymer assemblY. As the nature of the assembly of F1 unfolds, information critical to recombinant vaccine development is being revealed.

Chapter 19
pVM82: A Conjugative Plasmid of the Enteric Yersinia that Contributes to Pathogenicity
George B. Smirnov

The 123-kb conjugative plasmid pVM82 is often present in Yersinia pseudotuberculosis serotype I strains. Strains possessing pVM82 have been isolated from patients during large-scale outbreaks of the disease, but not from sporadic cases. Y. pseudotuberculosis strains that contain pVM82 cause more acute, severe, and generalized infections; and survive longer in infected experimental animals than do pVM82-free strains. The presence of pVM82 correlated with suppression of the antibody response of rabbits against specific Y. pseudotuberculosis antigens and with suppression of the cellular immune response in a mouse model of pseudotuberculosis infection. pVM82 also conferred increased resistance to the bactericidal effect of normal rabbit serum and certain basic dyes.

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