The Epidemiology of Yersinia pestis and its Molecular Basis

by

Patrick Connolly

prepared for

Dr. Martin Mulligan

Memorial University BIOC4103April 13, 2006

The Epidemiology of Yersinia pestis and its Molecular Basis Though there have certainly been more devastating epidemics in the years since

the major plague outbreak of the 1300’s, few contagions have held such sinister connotations as that of Yersinia pestis, its causative agent. Though it can now be controlled quite easily with antibiotics, this bacterial agent claimed the lives of hundreds of millions during its major outbreaks, one of which ravaged Europe and Asia for over a century. Though the study of such an easily managed pathogen may now seem trivial, investigations of Y. pestis have led to a broader understanding of many biological processes, especially those involved in bacterial pathology and host immunity. Accordingly, this paper will discuss some of the important aspects of the virulence and pathology of Yersinia pestis.

Y. pestis, along with Y. pseudotuberculosis and Y. enterocolitica, are the three pathogenic members of the genus Yersinia. Though all of these small, Gram-negative coccobacilli are virulent, they don’t all infect in the same manner. Whereas Y. pestis causes the deadly systemic infection known as Black Death, the other two cause localized, non-fatal infections of the intestinal tract, known as gastroenteritis. They also differ in their modes of transmission, with the deadly Y. pestis transferring mainly through flea-bite, and the less virulent forms of Yersinia infecting by oral means, usually via contaminated food products. It was the evolution of this ability to transfer via flea vector that has been credited as one of the main reasons why Y. pestis has become so virulent in comparison to its close relatives.

Such variations in virulence characteristics have lead researchers to search for what genetic differences may have led to such drastic differences in pathology. Since all pathogenic Yersinia possess virtually the same 70-kbp virulence plasmid pCD1 (or pYV), it is speculated that this is not the element in which the crucial variation lies. The high virulence of Y. pestis was therefore theorized to reside within its two other virulence plasmids, the 9.6-kbp pPCP1 and the 101-kbp pMT1, which are unique to the species. Given the high homology between the chromosomes of pathogenic Yersinia, this seemed to be a fair assumption.

The aforementioned homology between pathogenic Yersinia species is greatest between Y. pseudotuberculosis and Y. pestis, owing to the latter’s recent branching from the Y. pseudotuberculosis. Since Y. pestis is theorized to have evolved only about 1500- 4000 years ago, the genomes are nearly clonal. For this reason, information from the two are very interchangeable, and Y. pseudotuberculosis is frequently used as a less virulent model system for the study of Y. pestis. This isn’t to say that Y. enterocolitica is not a suitable model, just that there is slightly more homology between the other two.

Before going any further, it is important to discuss the mode of infection of Y. pestis. As mentioned above, one of the key distinctions of this pathogen is its ability to be transferred via flea vector. This is facilitated in such a way that, following the feeding of a flea on an infected mammalian host, the bacteria multiply and form a clot in the flea gut. The clot forms in the spiny gut section called the proventriculus, blocking access of the blood meal to the midgut, where digestion would normally take place (See Fig. 1). In this fashion, the flea is forced to regurgitate at least part of the clot during its next feeding, or else it will starve. This leads to the continued spread of the disease, since, even after partial regurgitation, the bacteria will soon multiply to form another clot.

Once introduced into a peripheral site through the bite of a flea, the bacterial pathogen follows a defined course. Like the other virulent members of the genus Yersinia, Y. pestis displays an affinity for lymphoid tissue, in particular the lymph nodes of the groin and underarms. After multiplying and infecting these swollen lymph nodes, and in the process producing the characteristically painful buboes which give bubonic plague its moniker, the infection moves into the bloodstream. This results in the

systemic infection (septicaemia) which usually kills. Should the host survive into the later stages, the infection moves into the lungs, allowing for the even more deadly aerosol transmission characteristic of pneumonic plague (aerosol transmission is more deadly because the bacteria needs less time to acclimatize to the host). Any bites following the early septicaemic phase will ensure that any fleas moving on after host death will undoubtedly pass on the infection.

Returning to the specific question regarding what might be one of the possible causes of the heightened virulence in Y. pestis, the pla (plasminogen activator) gene of pMT1 is likely important. Though it makes no difference during intravenous infection, the presence of Pla has been demonstrated to be vital to effective subcutaneous infection (REF!!!), such as that would occur during flea infection. This ability to infect via a subcutaneous introduction is one of the properties which differentiates Y. pestis from the enteropathogenic Yersinia. These findings with the pla gene has led researches to speculate that membrane-bound Pla plays an important role in helping Y. pestis establish an infection from a flea bite, though the mechanism is unclear.

Another important gene involved in Y. pestis’ increased transmission level is found on the pPCP plasmid. The ymt gene was initially incorrectly identitied as a Yersinia murine toxin when it was first characterized by Cherepanov et al. (2001), because it coded for a protein which was toxic to mice and rats, yet harmless to humans. It was later found that not only was it unnecessary for Y. pestis virulence in mice (Hinnebusch et al., 2000), but it was maximally expressed at 26ºC as opposed to the warm-blooded host temperature of 37ºC. Given that this was closer to the temperature at which fleas live, studies were done to determine whether this could be one of the factors that allow for transmission via flea vector. Hinnebusch et al. (2002) found that Ymt, which was actually a phospholipase D (PLD), was crucial in forming the cohesive bacterial aggregates which blocked flea digestion and forced regurgitation. The same study found that PLD activity of Ymt allowed Y. pestis to avoid cell lysis in the midgut of the flea, from where colonization normally begins. Blockage did not occur in ymt mutants because the bacteria could not survive and colonize the midgut, which is what would normally occur before moving up into the blockage-prone proventriculus (See Fig. 1).

FIG. 1: Cross-section of flea digestive tract.[From Salyers & Whitt, 2002]

Though the ymt gene was by no means the only reason why the plague had been able to transmit so easily, the evolution of this gene was an essential contributing virulence factor.

Another gene important to colonization and survival within the flea vector is the chromosomal hms gene of Y. pestis. Without the expression of Hms proteins, Jarrett et al. (2004) found that Y. pestis could not properly form biofilms in vitro. It is hypothesized that, in vivo, this ability to form biofilms on the acellular spines of the flea’s proventriculus is of primary importance in forming efficient clots. This theory is supported by the fact that fleas infected with hms mutants were still colonized, but only within the midgut, so no blockage of the proventriculus occurred. Without the accompanying regurgitation, transmission of Y. pestis via the flea vector would be much less effective.

Though there are most likely other genes which are expressed in the conditions present within the flea vector, an important set of genes are only expressed at host temperatures – a set of genes which code for a very interesting apparatus. Under permissive conditions at 37ºC, a whole slew of genes from the pCD1 plasmid are transcribed, and the type III secretion system (TTSS) is assembled. The TTSS is found in all pathogenic Yersinia (as well as many other plant and animal pathogens), but, though it is by no means exclusive to Y. pestis, it was within this organism that TTSS were first characterized.

The whole TTSS consists of about 27 components, including the secreted effector and translocator Yops (Yersinia outer proteins), as well as the different Ysc (Yersinia secretion) subunits which make up the injection apparatus (a.k.a. injectisome). It operates by providing a syringe-like, contact-dependant injection system with which to install a translocator pore in the host cell membrane. Cytotoxic effector Yops are then introduced into the host cell through this pore-injectisome system. The variation between species tends to reside mostly in the genes encoding the effector proteins, since different pathogenic organisms have different needs. For instance, some pathogens use the TTSS to promote their uptake while others, such as those of the genus Yersinia, use the system to inhibit phagocytosis by cells of the immune system, thus sustaining their extracellular lifestyle. The nature of the TTSS has allowed bacteria to customize it in order to cater to their specific needs as pathogens.

It is interesting to note that the TTSS has been shown to be a descendant of the flagellar export system that is used to build the flagella. This genealogy is mostly based on the fact that motility and chemotaxis, processes in which flagella are integral, are ancient mechanisms. It is therefore assumed that TTSS is the newer of the two. They are known to be related processes in part because 11 core components, mostly those associated with the bacterial cytoplasmic membrane, are conserved in all TTSS and flagellar export systems. These conserved proteins, given their location in the inner bacterial membrane, are likely involved in recognition and targeting, two important parts of the TTSS which will be discussed later.

There were several important observations which led to the establishment of the TTSS concept. Firstly, Rosqvist et al. (1991) confirmed that when purified effector Yops were supplied in the medium of the host cell, no toxicity resulted. It wasn’t until the effector Yops were manualled microinjected that cytotoxicity resulted. Sory and Cornelis (1994) went one step further by building a recombinant Y. entercolitica strain which

possessed a YopE-adenylate cyclase hybrid. They used a calmodulin-dependant adenylate cyclase, since the calmodulin would only be present in the eukaryotic host cells. This allowed them to track the accumulation of cAMP and to state with confidence that the YopE hybrid was being injected into the host cell through some mechanism. Before the discovery of this system, it was believed that all toxins were excreted into the extracellular medium, from which they attached to hosts and the active portion entered. Studies such as these gave clear indication that a bacterial system was being used to translocate the cytotoxic effector Yops across the host cell plasma membrane.

The main piece of machinery involved in toxin transfer is the injection apparatus, or injectisome. Fully assembled, it consists of two pairs of rings, one pair in each of the inner and outer bacterial membranes, which are connected by a central shaft extending through the peptidoglycan layer. Attached to this membrane-anchored structure is a needle complex, a helical formation made from YscF subunits, which extends out into the extracellular space. There are also a few subunits attached to the cytoplasmic portion of the bacterial injectisome which, as mentioned before, are involved in recognition and targeting of elements to be secreted. Though the function of most injectisome subunits have not been fully characterized, they are gradually becoming more well-understood. For instance, there is the YscL subunit which is an ATPase necessary for the ATP-dependant secretion process, and which also happens to show significant homology with a portion of the FoF1-ATPase of mitochondrial oxidative phosphorylation (Pallen at al., 2006).

It has recently been shown by Journet et al. (2003) that the needle length of the injectisome is genetically determined. In studying yscP, they found that a truncated variant of the gene produced needles of indeterminate length. It seemed that that YscF subunits that formed the needle complex just kept polymerizing and elongating without regulation. Further investigation led to the discovery that adjustment of the length of an internal segment of YscP resulted in needles which were shorter or longer than those of wild-type Yersinia. By removing or adding repeats of the sequence, they could control the length of the assembled needles using the strictly linear formula of 1.9 Å per residue. Since the C- and N-termini were necessary for YscP function, it was hypothesized that the two ends acted as separate anchors to both the growing tip and the basal body. Furthermore, it was proposed that as YscP was being stretched out by the polymerization of secreted YscF (needle subunit) onto the growing needle end, YscP would undergo a change in conformation that would signal, via the internal anchor, to stop secreting YscF.

So why would the TTSS evolve so that the needle length was under hereditary control? A subsequent study by Mota et al. (2005) found that needle length needs to span a minimal length in order to be fully functional. This minimal length seems to be determined by the topology of the cell surfaces, both the bacterial and host, as influenced by structures such as those involved in adhesion. It would make sense for the pathogenic bacteria to be able to change its needle length to suit possible future conditions, through random deletions and insertions as opposed to whole protein reconfigurations.

The next of the three main categories of TTSS components are the translocator Yops. Their identities are YopB, YopD, and LcrV, and they are the proteins involved in the pore formation that allows for translocation of the effector Yops. YopB and YopD have hydrophobic domains, which suggests that they probably act as transmembrane proteins. Some of the first evidence supporting their role was provided by Neyt and

Cornelis (1999), who worked with Y. enterocolitica mutants which expressed translocator Yops, but not effector Yops. They found that macrophages “infected” with these bacteria became permeable to small dyes, suggesting the formation of a small pore. They also found that when the macrophages were preloaded with small fluorescent markers, the marker leaked into the extracellular space following “infection”. Since all were needed to produce the above observations, YopB, YopD and LcrV were proposed to be the translocator proteins involved directly in pore formation on the host membrane.

One interesting line of research revolves around the soluble LcrV (low-calcium response V) protein (translocator Yop, despite the nomenclature) involved in facilitating translocation across the host cell plasma membrane. It had previously been established that LcrV was necessary for the formation of the translocation pore, but recent reports by Mueller et al. (2005) have better characterized this T3SS component. By employing visual evidence from scanning transmission electron microscopy (STEM), they have determined that wild-type LcrV forms a well-defined tip structure at the end of the needle (See arrow in Fig. 2A). Knockout bacteria deprived of their lcrV gene lack the ability to form pores, and also show a distinctly different pointed needle-tip, with the tip complex apparently missing (See star in Fig. 2B). Given that only LcrV is present in the wild-type tip structure, and that it is essential for pore assembly, it has been proposed that LcrV acts as an assembly platform on which the host membrane pore is constructed from YopB and YopD.

The above assembly platform hypothesis is supported by the work of Holmström et al. (2001), which revealed that LcrV seemed to be involved in the determination of pore size. It was found that a central region of the protein determined the channel size, and therefore the amount of effector Yops which were translocated into the host. This proposal was supported by the fact that expression of different homologs of LcrV (from T3SS of other species) in

lcrV mutants, formed pores of differing sizes. It would seem that, while acting as an assembly platform, LcrV plays a role in determining the size of the pore being formed. It seems worth noting, that to the best of this reader’s knowledge, neither of the aforementioned studies seem to have been connected before.

The final class of type III secretion proteins, the secreted effector Yops, are the most important with respect to the actual functionality of the system. Though there isn’t much variation in their function within the genus Yersinia, the effector Yops are where TTSS-utilizing bacteria differ according to the goals of their pathogenicity. Yersinia

FIG. 2: STEM imagery of needles with and without LcrV.[From Mueller et al., 2005]

pestis utilizes at least 6 main secreted effector Yops – YopH, YopE, YopT, YpkA, YopM, and YopJ. Effector Yops which are very similar, if not the same, are present in all pathogenic Yersinia, though sometimes with different names. Their antiphagocytic effects are manifested through their targeting and disruption of key molecules involved in bacterial uptake.

The secretion regulation of the effector Yops is an important part of their functioning, making sure that they are delivered in the right order and at the right time. One way in which the bacterial TTSS assures that its effectors are secreted only on contact with the host cell, is through the low-calcium response phenomenon. This helps to avoid premature injection after assembly at 37ºC, by keeping the injectisome closed with a protein plug while free calcium levels are high. Since intracellular free calcium levels are low due to complexing with calmodulin, and extracellular calcium levels are high, effector Yops presumeably won’t be secreted until needle contact is made with low-calcium intracellular environment. The mechanism of this process is not entirely clear, but by doing things such as studying different secretion patterns with various mutations to key proteins, a rough hypothesis has been put forward.

This rough hypothesis (Cheng et al., 2001) involves a cytosolic YopN–SycN–YscB–TyeA complex (See Fig. 3). Though it may seem confusing at first, it is actually fairly simple. Under high calcium conditions, YopN (neither translocator nor effector Yop) is targeted for secretion via the TTSS. YopN on its own carries a sequence which inhibits its own secretion, but the YscB–SycN complex binds to YopN, masking this preventative sequence. In addition, TyeA binds to a separate YopN sequence, preventing its secretion once more. YopN, targeted to the secretion apparatus yet not able to be secreted, functions to block secretion of any effector Yops. When the needle enters the low-calcium environment of the host cell, a signal is somehow relayed to TyeA, which dissociates from YopN. This allows YopN to be secreted, freeing up the injectisome for secretion of effector Yops.

Knowing what prevents effector Yops from being secreted prematurely is only part of the concern of secretion regulation, since now it is a question of how these Yops were targeted for secretion in the first place. The effector Yops don’t seem to have a

FIG. 3: Hypothesis for the low-calcium response[From Cheng et al., 1991]

classic secretion signal in common, but they do all have different polypeptide sequences of about 15 required N-terminal residues. During their initial characterization, there was some confusion over what type of secretion signal was needed -- mRNA or peptide. Anderson and Schneewind (1997) found that no point mutation was identified which satisfactorily eliminated secretion of YopE and YopN, in contrast to what would be expected with most other types of export signals. Also, secretion ability for these Yops persisted with certain large frameshift mutations, further indicating that regulation probably involved mRNA signal sequences. The next problem arose when Lloyd et al. (2001), demonstrated that YopE secretion remained, even when almost every possible base-pair in the signal region had been altered in such a way that wobble allowed for the peptide sequence to remain the same. This result seemed to indicate that the signal was surely not mRNA in nature. Though the signal sequence is still controversial, it is now theorized that the common motif recognized for secretion in an amphipathic peptide helix (Forsberg et al., 2003). This was supported by the fact that a synthetic serine-isoleucine repeat sequence acted as a functional target sequence for secretion (Lloyd et al., 2001). Though these N-terminal signal sequences integrated within the effector Yop are surely important to secretion regulation, there may be other factors as well.

One of these other factors which may play a multifaceted role in secretion is the small acidic Syc (specific Yop chaperone) protein which resides in the cytosol of Y. pestis in secretion-permissive conditions. There is little or no homology between different Syc proteins, and each one is specific for its partner Yop effector. Only some effectors, such as YopE and YopH, have these complimentary chaperones, but the ones which do require them in order to be secreted efficiently.

There have been two proposed functions which these chaperones may fulfill in relation to secretion -- targeting or maintenance of conformation. The targeting function may work simply by getting the partner Yops to the secretion apparatus, or perhaps by indicating the sequence in which their partner Yops should be secreted with respect to other Yops. As we shall see, YopH and YopE have specific antiphagocytic functions which might require them to be the first Yops injected into the host. The second potential function of the Syc might be to maintain the conformation of their partner Yops. Given that the injectisome needle is so narraw, Yops cannot travel through in their native conformation, and therefore the chaperones may be crucial for the secretion of certain Yops which have a greater tendency to fold into these globular conformations. It has been shown (Lloyd et al., 2001) that YopE’s chaperone, SycE, is necessary for the rapid secretion of stored YopE, but not for its gradual cotranslational secretion. This provides evidence that Sycs can function in maintaining stores of their partner Yops in a secretion-ready conformation, allowing for rapid injection on contact with the host cell.

Before exploring the activities of the individual effector Yops, a short review of the human immune system would be in order (Wikipedia, 2006). The two primary branches of the human immune system are the innate and the adaptive immune systems. The innate immune system works as the immediate first line of defence, working in a non-specific manner to eliminate any generically-recognizable invading pathogens as soon as they are encountered. This system is made up primarily of professional phagocytes such as macrophages, monocytes and neutrophils -- cells which express a variety of pathogen-binding receptors which induce phagocytosis on contact. When activated, these cells secrete important pro-inflammatory cytokines which stimulate other

immune cells from both the innate and adaptive immune systems. Antibody-secreting B cells and helper T cells make up part of the adaptive immune system, and provide a second line of denfense. This secondary defense is aided by stimulation from and communication with the innate immune system. In this fashion, the innate immune system not only provides immediate protection from bacterial infections, but also helps prepare the adaptive immune response for future action.

Following contact of phagocytic cells with recognized pathogens, certain cellular processes begin in order to facilitate engulfment of the bacteria. One such phagocytic process is the formation of focal adhesions and similar structures within the defending host cell. They are formed when, following integrin binding to extracellular ligands, the cytoplasmic side of the integrins become associated with the actin cytoskeleton. The integrins are then, along with many other membrane proteins, clustered together to form a focal adhesion. Structure like these are necessary to “gain traction”, linking integrins or other adhesion molecules to the cytoskeleton, in order to enable cellular mobilization or engulfment of pathogens.

A common phagocytic regulatory feature is the phosphorylation of tyrosine residues on specific regulatory molecules of the signal transduction pathways. Many of these important molecules are Rho GTPases. While functional in various other cellular processes, these GTPases are of specific importance to phagocytosis, due to the part they play in cytoskeletal rearrangement. Because of this need for de/re-phosphorylation of regulatory molecules, tyrosine kinases and phosphatases play a key role in the receptor-mediated cytoskeletal changes which are required for phagocyte migration and adhesion, and thus which are necessary for phagocytosis.

As indicated by their nomenclature, all Rho GTPases are coupled to a guanine which designates whether the GTPase is in active conformation, and capable of inducing the cellular response via its designated effector (different from effector Yop) (See Fig. 4). A Rho GTPase with an attached GTP is active, while one with a hydrolyzed GDP is inactive. The cycle between active and inactive GTPase is regulated by two main types of enzymes. Guanine-nucleotide exchange factors (GEFs) replace a GDP with a GTP, activating the Rho GTPase, while GTPase activating proteins (GAPs) stimulate the inherent GTP activity of the the Rho GTPase, resulting in its inactivation as it hydrolyzes its GTP to GDP. Rho GTPases involved in actin reorganization, such as RhoA, Rac1 and Cdc42, become major targets of effector Yops during infection by Y. pestis’ TTSS.

FIG. 4: Interference with Rho GTPase activation/deactivation cycle.[Edited from Aepfelbacher, 2004]

These monomeric Rho GTPases (RhoA, Rac1, and Cdc42) are all important to phagocytosis, and all contribute to reorganization of the actin cytoskeleton. For instance, RhoA promotes assembly of the contractile actins stress fibres which are anchored to the aforementioned focal adhesion structures. In fact, phagocytosis via complement receptors requires RhoA. Rac1 and Cdc42 are required in the formation of the protrusive actin structures which extend from the cell body, attaching to substrates during migration and bacterial engulfment. Though these Rho GTPases have many other cellular functions, only those especially important to cytoskeletal reorganization have been addressed.

Now that the background pertaining to the process of phagocytosis has been set forth, discussion of the individual effector Yops’ modes of action can begin. The 6 TTSS effectors appear to work synergistically to prevent their elimination via the host immune system, especially considering that the loss of even one can result in reduction or nullification in the virulence of Y. enterocolitica (Grosdent et al., 2002). Though all of the effectors somehow work to quickly prevent engulfment of their bacterial Yersinia injector, three in particular (YopE, YopH and YpkA) act directly on the Rho GTPases (See Fig. 4). Though they do all work on the same molecules, they go about their business in different ways.

The effector YopE, one of the aforementioned GAPs, increases the hydrolysis of RhoA, Rac1 and Cdc42, thereby inactivating them. Its immediate effect is a blockage of phagocytosis, the rapidity of which might be owing to it’s rapid injection via chaperone- (SycE-)dependent secretion (see above). The GAP activity of YopE has proved essential for the effect of Y. pestis on the cytoskeleton, and for its virulence in general.

YopT, despite have similar effects as YopE, works in a very different manner which has recently been elucidated. Shao and Dixon (2003) determined that, though the amounts of host Rho GTPase remained constant, YopT was a cysteine protease which was cleaving their C-terminal cysteines. The problem lay in the fact that there was that a post-translational modification of these Rho GTPases which was responsible for adding a hydrophobic prenyl group onto this cysteine. This prenyl group was used to anchor the GTPase on the inner plasma membrane, and when that anchor was lost, the GTPase activity of these enzymes was abolished. Given that the end result, the inactivation of RhoA, Rac1 and Cdc42, is essentially the same as that of YopE, the similar morphological and antiphagocytic effects make perfect sense.

The mode of action of YpkA is less clear than those of the previously mentioned Yops, though it is assuredly necessary for virulence (Forsberg et al., 2003). It possesses three functional domains with distinct activities -- an autophosphorylating serine/threonine kinase site, an actin-binging site which activates the kinase activity, and a RhoA/Rac1 binding site (Aepfelbacher, 2004). YpkA is clearly relevant to pathogenesis, but not much is known about its actual mechanism aside from the fact that its Rho GTPase-binding seems to disrupt the cytoskeleton. These disruptions, however, appear to occur relatively late in infection, making their antiphagocytic effect questionable (Forsberg et al., 2003). YpkA remains one of the more mysterious of the effector Yops involved in the TTSS.

YopH, which has been proposed to account for up to 50% of the antiphagocytic effect on neutrophils and macrophages (Aepfelbacher, 2004), is one by far the most active protein tyrosine phosphatases known (Forsberg et al., 2003). This effector Yop functions in deregulating the interactions between the actin cytoskeleton and the

membrane-bound integrins of the focal adhesion complexes. It does this by dephosphorylating a number of substrates, specifically p130Cas, focal adhesion kinase (FAK), paxillin, Fyn-binding protein (FyB) and SKAP-HOM, in a number of cells such as neutrophils and macrophages. By disrupting the formation of focal complexes and similar structures, the phagocytic cell is prevented from engulfing the Y. pestis bacterium. YopH seems to mediate many other effects, such as the suppression of the macrophage oxidative burst (Aepfelbacher, 2004), and the blockage of early Ca++ signalling in neutrophils, the latter of which is important to the secretion of antibacterial granules (Andersson et al., 1999). Yop H also prevents the release of chemokines that attract other monocytes, and impairs the ability of T and B cells to be activated through their antigen receptors (Cornelis, 2005). All these effects help Yersinia throw many cells of the immune system into disarray, preventing them from mounting a proper immune response.

The strongly acidic YopM effector, whose function had previously been unknown, has recently been implicated in a different type of attack on the innate immune system. It was observed by Kerschen et al. (2004), that YopM appears to play a role in the systemic depletion of NK cells, nonphagocytic participants in the innate immune response. It is possible that this reduction is related to the vesicle-mediated migration of YopM into the host nucleus, where it alters expression of many genes products, including various interleukins (Kerschen et al., 2004). Though YopM has no known antiphagocytic function, it is still indispensable to the virulence of Yersinia.

The effector YopJ, a cysteine protease, has long been known to have a role in disrupting key signalling cascades involved in the inflammatory response and cell survival. In fact, it has just recently been observed by Zhou et al. (2006), that this disruption is caused by YopJ’s deubiquitinase activity on critical pathway components. Its principal effect appeared to be the inhibition of nuclear factor kappa-B (NF-κB), which is not only the key regulator of the protective inflammatory response (Ruckdeschel et al., 2003), but also a preventative factor against apoptosis. The onset of apoptosis is rightly regulated, with the end-result being determined by an integration of various death-inducing and death-preventing signals. It turns out that NF-κB is involved in a death-preventing pathway which upregulates the synthesis of proteins which counteract apoptosis. It seems that YopJ induces, along with some unknown pro-apoptotic factor, inhibition of NF-κB which results in macrophage-specific apoptosis (Ruckdeschel et al., 2003). Given that this effector’s inhibition of both the NF-κB and mitogen-activated protein kinase (MAPK) kinase pathways also disrupt the overall inflammatory response of the immune system, YopJ acts as yet another fundamental effector in Y. pestis virulence.

Having discussed the molecular basis of much of the epidemiology and pathology associated with Yersinia pestis, it should be apparent that a number of factors come into play when discussing virulence. The study of many of these factors have led to discoveries in the various fields, such as bacterial pathology, just as much as it has given us better insight into many aspects of our own immune system. Hopefully it has become obvious that the study of a bacterium, not matter how deadly it may be, is never just about the bacterium. In the characterization of these virulent pathogens, knowledge can be gleaned -- be it about the universal workings of the type III secretion system, the interactions between cells of the human immune system, or the better understanding of a prominent event in world history.

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