Caspase-1 activation in macrophages infected with Yersinia pestis KIM requires the type III secretion system effector YopJ

Pathogenic Yersinia species utilize a type III secretion system (T3SS) to translocate effectors called Yersinia outer proteins (Yops) into infected host cells. Previous studies demonstrated a role for effector Yops in the inhibition of caspase-1-mediated cell death and secretion of interleukin-1beta (IL-1beta) in na?ve macrophages infected with Yersinia enterocolitica. Na?ve murine macrophages were infected with a panel of different Yersinia pestis and Yersinia pseudotuberculosis strains to determine whether Yops of these species inhibit caspase-1 activation. Cell death was measured by release of lactate dehydrogenase (LDH), and enzyme-linked immunosorbent assay for secreted IL-1beta was used to measure caspase-1 activation. Surprisingly, isolates derived from the Y. pestis KIM strain (e.g., KIM5) displayed an unusual ability to activate caspase-1 and kill infected macrophages compared to other Y. pestis and Y. pseudotuberculosis strains tested. Secretion of IL-1beta following KIM5 infection was reduced in caspase-1-deficient macrophages compared to wild-type macrophages. However, release of LDH was not reduced in caspase-1-deficient macrophages, indicating that cell death occurred independently of caspase-1. Analysis of KIM-derived strains defective for production of functional effector or translocator Yops indicated that translocation of catalytically active YopJ into macrophages was required for caspase-1 activation and cell death. Release of LDH and secretion of IL-1beta were not reduced when actin polymerization was inhibited in KIM5-infected macrophages, indicating that extracellular bacteria translocating YopJ could trigger cell death and caspase-1 activation. This study uncovered a novel role for YopJ in the activation of caspase-1 in macrophages.     

Arginine-143 of Yersinia enterocolitica YopP Crucially Determines Isotype-Related NF-κB Suppression and Apoptosis Induction in Macrophages

Pathogenic Yersinia spp. counteract host defense mechanisms by modulating the cellular signal relay in response to infection. Subversion of the antiapoptotic NF-kappaB signaling pathway by the Yersinia enterocolitica virulence protein YopP crucially determines the induction of apoptosis in Yersinia-infected macrophages. Here, we analyzed a panel of pathogenic, phylogenetically distinct Y. enterocolitica serotypes for their abilities to trigger macrophage apoptosis. Y. enterocolitica from the highly pathogenic serogroup O8 was substantially more effective in apoptosis induction than Yersinia from the serogroups O3 and O9. Complementation of yopP-knockout mutants revealed that this effect was specifically conferred by the serogroup O8 YopP. The amino acid sequences of YopPO8 and YopPO9 share 94% identity, and both YopP isotypes were found to interact with the NF-kappaB-activating kinase IKKbeta in macrophages. However, selectively, YopPO8 mediated efficient inhibition of IKKbeta activities, which led to substantial suppression of NF-kappaB activation. To localize the YopPO8-related effector domain, we interchanged stretches of amino acids and single amino acid residues between YopPO8 and YopPO9. Functional characterization of the resulting mutants revealed a major role of the arginine-143 residue in determining the inhibitory impact of YopP on IKKbeta activity and survival of macrophages.     

Reduced Apoptosis of Mouse Macrophages Induced by yscW Mutant of Yersinia pestis Results from the Reduced Secretion of YopJ and Relates to Caspase-3 Signal Pathway

The virulence of the pathogenic Yersinia species depends on a plasmid-encoded type III secretion system (T3SS) that injects six Yersinia outer protein (Yop) effector proteins into the cytosol of macrophages, leading to disruption of host defence mechanisms. Here, we report that a T3SS structural protein YscW of Yersinia pestis contributed to the induction of apoptosis of murine macrophages. The apoptotic percentage of macrophages, from both mouse peritoneal cavity and spleen, and of RAW264.7 cell line, caused by the yscW mutant strain was significantly lower than that by wild type (WT) Y. pestis and yscW complemented strain. Meanwhile, detection of caspase-3 activity in macrophages, a key apoptosis-inducing protein, showed coincident results with the changes of macrophage apoptosis induced by WT, yscW mutant and complemented strains, indicating that macrophage apoptosis was related to caspase-3 signal pathways. However, ectopic expression of YscW in RAW264.7 cells cannot increase the macrophage apoptosis and death, suggesting that YscW itself could not induce macrophage apoptosis directly. To get insight into the mechanism of this phenomenon, we investigated the secretion of YopJ, which has been thought to be the only Yop effector related to apoptosis, in WT, mutant and complemented strains, respectively. Results showed that in yscW mutant strain, secretion of YopJ was decreased significantly in the supernatant than that in WT or complemented strain. This means although YscW does not induce apoptosis directly, it can indirectly affect apoptosis through reducing the secretion of YopJ.     

Yersinia pestis YopJ suppresses tumor necrosis factor alpha induction and contributes to apoptosis of immune cells in the lymph node but is not required for virulence in a rat model of bubonic plague

The virulence of the pathogenic Yersinia species depends on a plasmid-encoded type III secretion system that transfers six Yop effector proteins into host cells. One of these proteins, YopJ, has been shown to disrupt host cell signaling pathways involved in proinflammatory cytokine production and to induce macrophage apoptosis in vitro. YopJ-dependent apoptosis in mesenteric lymph nodes has also been demonstrated in a mouse model of Yersinia pseudotuberculosis infection. These results suggest that YopJ attenuates the host innate and adaptive immune response during infection, but the role of YopJ during bubonic plague has not been completely established. We evaluated the role of Yersinia pestis YopJ in a rat model of bubonic plague following intradermal infection with a fully virulent Y. pestis strain and an isogenic yopJ mutant. Deletion of yopJ resulted in a twofold decrease in the number of apoptotic immune cells in the bubo and a threefold increase in serum tumor necrosis factor alpha levels but did not result in decreased virulence, systemic spread, or colonization levels in the spleen and blood. Our results indicate that YopJ is not essential for bubonic plague pathogenesis, even after peripheral inoculation of low doses of Y. pestis. Instead, the effects of YopJ appear to overlap and augment the immunomodulatory effects of other Y. pestis virulence factors.     

Yersinia enterocolitica induces apoptosis and inhibits surface molecule expression and cytokine production in murine dendritic cells

Yersinia enterocolitica evades innate immunity by expression of a variety of pathogenicity factors. Therefore, adaptive immunity including CD4(+) T cells plays an important role in defense against Y. enterocolitica. We investigated whether Y. enterocolitica might target dendritic cells (DC) involved in adaptive T-cell responses. For this purpose, murine DC were infected with Y. enterocolitica wild-type and mutant strains prior to incubation with ovalbumin (OVA) as antigen and 5-(6)-carboxyfluorescein diacetate N-succinimidyl ester-labeled OVA-specific T cells from DO11.10 mice. While T-cell proliferation was partially affected by infection of DC with plasmid-cured and YopP-deficient Yersinia mutant strains, no T-cell proliferation occurred after infection of DC with wild-type Y. enterocolitica. Infection of DC with Y. enterocolitica wild type resulted in decreased up-regulation of major histocompatibility complex class II, CD54 (intercellular adhesion molecule 1), CD 80, and CD86 expression. Experiments with plasmid-cured Y. enterocolitica or a YopP-deficient mutant strain revealed that YopP accounts for inhibition of surface molecule expression. Wild-type Y. enterocolitica suppressed the release of KC, tumor necrosis factor alpha, interleukin-10 (IL-10), and IL-12 by DC, while infection of DC with plasmid-cured Y. enterocolitica or with the YopP-deficient mutant resulted in the production of these cytokines. Moreover, infection with wild-type Y. enterocolitica induced apoptosis in DC mediated by YopP. Apoptosis occurred despite translocation of NF-kappaB to the nucleus, as demonstrated by electromobility shift assays. Together, these data demonstrate that Y. enterocolitica targets functions of murine DC that are required for T-cell activation. This might contribute to evasion of adaptive immune responses by Y. enterocolitica.     

Contribution of the major secreted yops of Yersinia enterocolitica O:8 to pathogenicity in the mouse infection model

Pathogenic yersiniae (Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica) harbor a 70-kb virulence plasmid (pYV) that encodes a type III secretion system and a set of at least six effector proteins (YopH, YopO, YopP, YopE, YopM, and YopT) that are injected into the host cell cytoplasm. Yops (Yersinia outer proteins) disturb the dynamics of the cytoskeleton, inhibit phagocytosis by macrophages, and downregulate the production of proinflammatory cytokines, which makes it possible for yersiniae to multiply extracellularly in lymphoid tissue. Y. enterocolitica serotype O:8 belongs to the highly mouse-pathogenic group of yersiniae in contrast to Y. enterocolitica serotype O:9. However, there has been no systematic study of the contribution of Yops to the pathogenicity of Y. enterocolitica O:8 in mice. We generated a set of yop gene deletion mutants of Y. enterocolitica O:8 by using the novel Red cloning procedure. We subsequently analyzed the contribution of yopH, -O, -P, -E, -M, -T, and -Q deletions to pathogenicity after oral and intravenous infection of mice. Here we showed for the first time that a DeltayopT deletion mutant colonizes mouse tissues to a greater extent than the parental strain. The DeltayopO, DeltayopP, and DeltayopE mutants were only slightly attenuated after oral infection since they were still able to colonize the spleen and liver and cause systemic infection. The DeltayopO mutant was lethal for mice, whereas DeltayopP and DeltayopE mutants were successfully eliminated from the spleen and liver 2 weeks after infection. In contrast the DeltayopH, DeltayopM, and DeltayopQ mutants were highly attenuated and not able to colonize the spleen and liver on any of the days tested. The DeltayopH, DeltayopO, DeltayopP, DeltayopE, DeltayopM, and DeltayopQ mutants had only modest defects in the colonization of the small intestine and Peyer's patches. The DeltayopE mutant was eliminated from the small intestine 3 weeks after infection, whereas the DeltayopH, DeltayopP, DeltayopM, and DeltayopQ mutants continued to colonize the small intestine at this time.     

Growth of Yersinia pseudotuberculosis in mice occurs independently of Toll-like receptor 2 expression and induction of interleukin-10

Pathogenic Yersinia translocates effector proteins into target cells via a type III secretion system (TTSS), modulating the host immune response. A component of the TTSS translocon, LcrV, has been implicated in preventing inflammation through Toll-like receptor 2 (TLR2) by inducing expression of the anti-inflammatory cytokine interleukin-10 (IL-10). TLR2(-/-) mice were reported to be less susceptible to the enteropathogen Yersinia enterocolitica. To determine whether TLR2 also plays a role in recognition of the enteropathogen Yersinia pseudotuberculosis and whether this results in an immune response that is detrimental to the host, we evaluated the macrophage cytokine response to live Y. pseudotuberculosis and analyzed the susceptibility of TLR2(-/-) mice to enteropathogenic Yersinia. We find that Yersinia induction of macrophage IL-10 occurs independently of TLR2 and LcrV and is blocked by the TTSS. In particular, the TTSS effector protein YopJ, which inhibits production of the inflammatory cytokine tumor necrosis factor alpha (TNF-alpha), also inhibits IL-10 expression. Consistent with these results, IL-10 is undetectable in Y. pseudotuberculosis-infected mouse tissues until advanced stages of infection. In addition, we find that TLR2(-/-) mice (derived independently from those used in previous studies) do not display altered susceptibility to enteropathogenic Yersinia compared to wild-type mice. Tissue levels of IL-10, as well as the inflammatory cytokines TNF-alpha, IL-6, and gamma interferon and the chemokine macrophage chemotactic protein 1, are similar in TLR2(+/+) and TLR2(-/-) mice during enteropathogenic Yersinia infection. Therefore, the absence of TLR2 alone does not affect the cytokine response of macrophages to, or the in vivo growth and survival of, enteropathogenic Yersinia.     

Type III secretion system-dependent translocation of ectopically expressed Yop effectors into macrophages by intracellular Yersinia pseudotuberculosis

Yersinia pseudotuberculosis is a Gram-negative bacterial pathogen. Virulence in Y. pseudotuberculosis requires the plasmid-encoded Ysc type III secretion system (T3SS), which functions to translocate a set of effectors called Yops into infected host cells. The effectors function to antagonize phagocytosis (e.g., YopH) or to induce apoptosis (YopJ) in macrophages infected with Y. pseudotuberculosis. Additionally, when antiphagocytosis is incomplete and Y. pseudotuberculosis is internalized by macrophages, the bacterium can survive in phagosomes. Previous studies have shown that delivery of effectors into host cells occurs efficiently when Yersinia is extracellular. However, it is not clear whether the T3SS can be utilized by intracellular Y. pseudotuberculosis to translocate Yops. This possibility was investigated here using Y. pseudotuberculosis strains that express YopJ or YopH under the control of an inducible promoter. Bone marrow-derived murine macrophages were infected with these strains under conditions that prevented the survival of extracellular bacteria. Effector translocation was detected by measuring apoptosis or the activities of Yop-β-lactamase fusion proteins. Results showed that macrophages underwent apoptosis when YopJ expression was induced prior to phagocytosis, confirming that delivery of this effector prior to or during uptake is sufficient to cause cell death. However, macrophages also underwent apoptosis when YopJ was ectopically expressed after phagocytosis; furthermore, expression of the translocator YopB from intracellular bacteria also resulted in increased cell death. Analysis by microscopy showed that translocation of ectopically expressed YopH- or YopJ-β-lactamase fusions could be correlated with the presence of viable Y. pseudotuberculosis in macrophages. Collectively, our results suggest that the Ysc T3SS of Y. pseudotuberculosis can function within macrophage phagosomes to translocate Yops into the host cytosol.     

Factors promoting acute and chronic diseases caused by yersiniae.

The experimental system constructed with the medically significant yersiniae provides a powerful basic model for comparative study of factors required for expression of acute versus chronic disease. The system exploits the close genetic similarity between Yersinia pestis, the etiological agent of bubonic plague, and enteropathogenic Yersinia pseudotuberculosis and Yersinia enterocolitica. Y. pestis possesses three plasmids, of which one, shared by the enteropathogenic species, mediates a number of virulence factors that directly or indirectly promote survival within macrophages and immunosuppression. The two remaining plasmids are unique and encode functions that promote acute disease by enhancing bacterial dissemination in tissues and resistance to phagocytosis by neutrophils and monocytes. These properties are replaced in the enteropathogenic yersiniae by host cell invasins and an adhesin which promote chronic disease; the latter are cryptic in Y. pestis. Additional distinctions include specific mutational losses in Y. pestis which result in loss of fitness in natural environments plus gain of properties that facilitate transmission and infection via fleabite.     

Mitogen-activated protein kinase-dependent interleukin-1α intracrine signaling is modulated by YopP during Yersinia enterocolitica infection

Yersinia enterocolitica is a food-borne pathogen that preferentially infects the Peyer's patches and mesenteric lymph nodes, causing an acute inflammatory reaction. Even though Y. enterocolitica induces a robust inflammatory response during infection, the bacterium has evolved a number of virulence factors to limit the extent of this response. We previously demonstrated that interleukin-1α (IL-1α) was critical for the induction of gut inflammation characteristic of Y. enterocolitica infection. More recently, the known actions of IL-1α are becoming more complex because IL-1α can function both as a proinflammatory cytokine and as a nuclear factor. In this study, we tested the ability of Y. enterocolitica to modulate intracellular IL-1α-dependent IL-8 production in epithelial cells. Nuclear translocation of pre-IL-1α protein and IL-1α-dependent secretion of IL-8 into the culture supernatant were increased during infection with a strain lacking the 70-kDa virulence plasmid compared to the case during infection with the wild type, suggesting that Yersinia outer proteins (Yops) might be involved in modulating intracellular IL-1α signaling. Infection of HeLa cells with a strain lacking the yopP gene resulted in increased nuclear translocation of pre-IL-1α and IL-1α-dependent secretion of IL-8 similar to what is observed with bacteria lacking the virulence plasmid. YopP is a protein acetylase that inhibits mitogen-activated protein kinase (MAP kinase)- and NF-κB-dependent signal transduction pathways. Nuclear translocation of pre-IL-1α and IL-1α-dependent secretion of IL-8 in response to Yersinia enterocolitica infection were dependent on extracellular signal-regulated kinase (ERK) and p38 MAP kinase signaling but independent of NF-κB. These data suggest that Y. enterocolitica inhibits intracellular pre-IL-1α signaling and subsequent proinflammatory responses through inhibition of MAP kinase pathways.     

Interaction of Yersinia enterocolitica with macrophages leads to macrophage cell death through apoptosis.

Suppression of the host defense is one of the hallmarks of Yersinia enterocolitica infection. This enteric pathogen resists phagocytosis and interferes with macrophage functions from an extracellular localization (oxidative-burst generation and tumor necrosis factor alpha production). In this study, we investigated the fate of the Y. enterocolitica-infected macrophage. We found that murine J774A.1 macrophages and macrophages derived from human monocytes were killed by infection with Y. enterocolitica. Analysis of cellular morphology and DNA fragmentation revealed that macrophage cell death occurs through the induction of apoptosis. A total of 92% +/- 5% (mean +/- standard deviation) of murine J774A.1 macrophages and 74% +/- 6% of human monocyte-derived macrophages underwent apoptosis upon Yersinia infection after 4 and 20 h, respectively. The broad-spectrum caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone blocked completion of the Yersinia-induced apoptotic program but not the surface exposure of phosphatidylserine as an early-stage apoptotic event. Analysis of different Yersinia mutants showed that macrophage apoptosis depends on a functional Y. enterocolitica type III protein secretion system. Apoptotic cell death of macrophages was not related to the YopE-mediated cytotoxic effect of Yersinia, since disruption of actin microfilaments by a Y. enterocolitica strain expressing a restricted repertoire of yop genes, including YopE, did not result in macrophage apoptosis. Furthermore, Yersinia-induced cytotoxic alterations in epithelial HeLa cells, which are conferred by YopE, did not lead to apoptosis. Our data demonstrate for the first time that Y. enterocolitica promotes the apoptosis of macrophages, an effect which is clearly distinct from the morphological alterations mediated by Yersinia on epithelial HeLa cells.     

Effect of low- and high-virulence Yersinia enterocolitica strains on the inflammatory response of human umbilical vein endothelial cells

Pathogenic strains of Yersinia spp. inject a set of Yop effector proteins into eukaryotic cells by using a plasmid-encoded type III secretion system. In this study, we analyzed the inflammatory response of human umbilical vein endothelial cells (HUVECs) after infection with different Yersinia enterocolitica strains. We found that both expression of intercellular adhesion molecule 1 and release of the cytokines interleukin-6 (IL-6) and IL-8 by HUVECs are downregulated in a YopP-dependent way, demonstrating that YopP plays a major role in the inflammatory response of these cells. Infection of HUVECs with several low-virulence (biotype 2, 3, and 4) and high-virulence (biotype 1B) Y. enterocolitica strains showed that biotype 1B isolates are more efficient in inhibiting the inflammatory response than low-virulence Y. enterocolitica strains and that this effect depends on the time of contact. We extended the results of Ruckdeschel et al. and found that on the basis of the presence or absence of arginine-143 of YopP (K. Ruckdeschel, K. Richter, O. Mannel, and J. Heesemann, Infect. Immun. 69:7652-7662, 2001) all the Y. enterocolitica strains used fell into two groups, which correlate with the low- and high-virulence phenotypes. In addition, we found that high-virulence strains inject more YopP into the cytosol of eukaryotic target cells than do low-virulence strains.     

Destabilization of YopE by the ubiquitin-proteasome pathway fine-tunes Yop delivery into host cells and facilitates systemic spread of Yersinia enterocolitica in host lymphoid tissue

Pathogenic Yersinia species inject a panel of Yop virulence proteins by type III protein secretion into host cells to modulate cellular defense responses. This enables the survival and dissemination of the bacteria in the host lymphoid tissue. We have previously shown that YopE of the Y. enterocolitica serogroup O8 is degraded in the host cell through the ubiquitin-proteasome pathway. YopE normally manipulates rearrangements of the actin cytoskeleton and triggers phagocytosis resistance. To shed light into the physiological role of YopE inactivation, we mutagenized the lysine polyubiquitin acceptor sites of YopE in the Y. enterocolitica serogroup O8 virulence plasmid. The resulting mutant strain escaped polyubiquitination and degradation of YopE and displayed increased intracellular YopE levels, which was accompanied by a pronounced cytotoxic effect on infected cells. Despite its intensified activity on cultured cells, the Yersinia mutant with stabilized YopE showed reduced dissemination into liver and spleen following enteral infection of mice. Furthermore, the accumulation of degradation-resistant YopE was accompanied by the diminished delivery of YopP and YopH into cultured, Yersinia-infected cells. A role of YopE in the regulation of Yop translocation has already been described. Our results imply that the inactivation of YopE by the proteasome could be a tool to ensure intermediate intracellular YopE levels, which may effectuate optimized Yop injection into host cells. In this regard, Y. enterocolitica O8 appears to exploit the host ubiquitin proteasome system to destabilize YopE and to fine-tune the activities of the Yop virulence arsenal on the infected host organism.     

Polymorphisms in the lcrV gene of Yersinia enterocolitica and their effect on plague protective immunity

Current efforts to develop plague vaccines focus on LcrV, a polypeptide that resides at the tip of type III secretion needles. LcrV-specific antibodies block Yersinia pestis type III injection of Yop effectors into host immune cells, thereby enabling phagocytes to kill the invading pathogen. Earlier work reported that antibodies against Y. pestis LcrV cannot block type III injection by Yersinia enterocolitica strains and suggested that lcrV polymorphisms may provide for escape from LcrV-mediated plague immunity. We show here that polyclonal or monoclonal antibodies raised against Y. pestis KIM D27 LcrV (LcrV(D27)) bind LcrV from Y. enterocolitica O:9 strain W22703 (LcrV(W22703)) or O:8 strain WA-314 (LcrV(WA-314)) but are otherwise unable to block type III injection by Y. enterocolitica strains. Replacing the lcrV gene on the pCD1 virulence plasmid of Y. pestis KIM D27 with either lcrV(W22703) or lcrV(WA-314) does not affect the ability of plague bacteria to secrete proteins via the type III pathway, to inject Yops into macrophages, or to cause lethal plague infections in mice. LcrV(D27)-specific antibodies blocked type III injection by Y. pestis expressing lcrV(W22703) or lcrV(WA-314) and protected mice against intravenous lethal plague challenge with these strains. Thus, although antibodies raised against LcrV(D27) are unable to block the type III injection of Y. enterocolitica strains, expression of lcrV(W22703) or lcrV(WA-314) in Y. pestis did not allow these strains to escape LcrV-mediated plague protective immunity in the intravenous challenge model.     

Analysis of chaperone-dependent Yop secretion/translocation and effector function using a mini-virulence plasmid of Yersinia enterocolitica

We have constructed a mini-pYV plasmid (pTTSS) harboring the Yersinia type three secretion system (TTSS) and the adhesin yadA on a low-copy vector. Using this system we could demonstrate for the first time that YopO, YopP, YopM, and YopQ do not require any of the known or orphan chaperones for efficient secretion/translocation. Y. enterocolitica harboring pTTSS, (WA-C(pTTSS)) was able to secrete and translocate single Yop effector proteins in trans. WA-C(pTTSS) proved to be stable and secretion of Yops was Ca2+ and temperature dependent as is the case for the parental strain. This shows that all genes necessary for translocation and expression of the Ca(2+)-dependent phenotype are contained within the cloned region. In contrast to previously published multiple yop mutants which were constructed by sequential deletion of yops, our system which harbors only the TTSS region without yops, chaperones, and unknown ORFs can be sequentially complemented with yops and sycs of choice. WA-C(pTTSS) was able to translocate YopE, YopM and YopT into HeLa cells as demonstrated by Western blotting. Translocation of YopE and YopT was strictly dependent on the presence of their respective chaperones, whereas YopM did not require a chaperone for translocation. WA-C(pTTSS) harboring yopT and sycT was shown to translocate active YopT by demonstrating modification of the small GTP-binding protein RhoA. This shows for the first time that RhoA modification is strictly dependent on YopT and does not require additional effector Yops. WA-C(pTTSS) harboring YopP was shown to induce apoptosis. This system is ideal to study chaperone-dependent Yop secretion/ translocation without the background of other effector Yops (YopE, YopM, YopO, YopP, YopT, YopH), chaperones (SycE, SycH, SycT) and unknown ORFs. In addition this system can secrete heterologous proteins fused to the N-terminal secretion/translocation domain of YopE.     

Turning Yersinia pathogenesis outside in: subversion of macrophage function by intracellular yersiniae

Three bacterial species within the genus Yersinia are causative agents of human disease. Yersinia pestis is transmitted by fleas or in aerosols, infects regional lymph nodes or lungs, and causes the highly lethal disease known as plague. Yersinia enterocolitica and Yersinia pseudotuberculosis are enteric pathogens most commonly associated with self-limiting infections of the mesenteric lymph nodes. Although Y. pestis and the enteropathogenic Yersinia species utilize different modes of transmission and cause different diseases, they rely on a common set of "core" virulence determinants to successfully infect a mammalian host. These virulence factors are encoded on the bacterial chromosome and on an approximately 70-kb plasmid. Once established in lymphoid tissue, all three Yersinia species replicate as aggregates of extracellular bacteria within necrotic lesions or abscesses. At this stage of the infectious process, the bacteria resist phagocytosis by neutrophils, which are able to destroy the bacteria if they are internalized. A type III secretion system encoded on the 70-kb plasmid functions to export multiple proteins (the Yops and LcrV) that are delivered to the extracellular milieu, the plasma membrane, or the cytosol of a host target cell. The Yops and LcrV act in concert to inhibit phagocytosis and downregulate inflammation. Although it is clear that the bulk of bacterial multiplication occurs in an extracellular phase, there is also evidence that all three pathogenic Yersinia survive and multiply in macrophages. Survival and replication of Yersinia in macrophages may occur throughout the infection, but is likely to be of greatest importance at early stages of colonization. That macrophages can serve as permissive sites for bacterial replication in vivo is supported by in vitro experiments, which demonstrate that Y. pestis, Y. peudotuberculosis, and Y. enterocolitica share the ability to survive and multiply in macrophage phagosomes. There is also evidence that the bacteria can subvert the functions of macrophages from within, by inhibiting phagosome acidification (Y. pseudotuberculosis) and the production of nitric oxide (Y. pestis and Y. pseudotuberculosis). Although considerable attention has been focused on how Yersinia subverts the functions of phagocytes from the outside, the study of how these bacteria subvert macrophage functions from the inside will lead to a better overall understanding of Yersinia pathogenesis.     

Discordance in the effects of Yersinia pestis on the dendritic cell functions manifested by induction of maturation and paralysis of migration

The encounter between invading microorganisms and dendritic cells (DC) triggers a series of events which include uptake and degradation of the microorganism, induction of a maturation process, and enhancement of DC migration to the draining lymph nodes. Various pathogens have developed strategies to counteract these events as a measure to evade the host defense. In the present study we found that interaction of the Yersinia pestis EV76 strain with DC has no effect on cell viability and is characterized by compliance with effective maturation, which is manifested by surface display of major histocompatibility complex class II, of costimulatory markers, and of the chemokine receptor CCR7. This is in contrast to maturation inhibition and cell death induction exerted by the related species Yersinia enterocolitica WA O:8. Y. pestis interactions with DC were found, however, to impair functions related to cytoskeleton rearrangement. DC pulsed with Y. pestis failed to adhere to solid surfaces and to migrate toward the chemokine CCL19 in an in vitro transmembrane assay. Both effects were dependent on the presence of the pCD1 virulence plasmid and on a bacterial growth shift to 37 degrees C prior to infection. Moreover, while instillation of a pCD1-cured Y. pestis strain into mouse airways triggered effective transport of alveolar DC to the mediastinal lymph node, instillation of Y. pestis harboring the plasmid failed to do so. Taken together, these results suggest that virulence plasmid-dependent impairment of DC migration is the major mechanism utilized by Y. pestis to subvert DC function.     

Modulation of the low-calcium response in Yersinia pestis via plasmid-plasmid interaction

Virulent cells of Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica are known to exhibit a low-calcium response in vitro characterized by restriction of growth and induction of V antigen at 37 degrees C in Ca2+-deficient media (Lcr+). A shared Lcr plasmid mediates these properties and encodes yersiniae outer membrane peptides (Yops) that are expressed in Lcr+ Y. pseudotuberculosis and Y. enterocolitica but not Y. pestis. We present direct evidence here verifying that synthesis of major Yops in the former two species is, like V, repressed by Ca2+ and that these structures are located primarily in the outer membrane. We also verified that rabbits infected with live Lcr+ Y. pestis can raise antibodies against V and Yops. Similar antisera, however, were recovered after immunization with sterile extracts of Ca2+-starved Lcr+ cells of Y. pestis. Results of immunoblots obtained with these antisera showed that restricted Y. pestis produced Yops of about 46 kDa (YopB) and 44 kDa (YopC) after shiftup by addition of Ca2+ at 37 degrees C or reduction of temperature to 26 degrees C. It is established that virulent cells of Y. pestis also possess a unique plasmid known to mediate pesticinogeny (Pst+). Restricted Lcr+, Pst- Y. pestis expressed YopB and YopC plus additional 76 kDa (YopF), 48 kDa (YopH), 36 kDa (YopD), 32.5 kDa (YopJ), and 27 kDa (YopE) outer membrane structures at concentrations comparable to those in Ca2+-starved Y. pseudotuberculosis and Y. enterocolitica. These findings indicate that carriage of the Pst plasmid prevents expression of the Lcr plasmid-mediated Yops in wild type Y. pestis.     

Serogroup-related escape of Yersinia enterocolitica YopE from degradation by the ubiquitin-proteasome pathway

Pathogenic Yersinia spp. employ a type III protein secretion system that translocates several Yersinia outer proteins (Yops) into the host cell to modify the host immune response. One strategy of the infected host cell to resist the bacterial attack is degradation and inactivation of injected bacterial virulence proteins through the ubiquitin-proteasome pathway. The cytotoxin YopE is a known target protein of this major proteolytic system in eukaryotic cells. Here, we investigated the sensitivity of YopE belonging to different enteropathogenic Yersinia enterocolitica serogroups to ubiquitination and proteasomal degradation. Analysis of the YopE protein levels in proteasome inhibitor-treated versus untreated cells revealed that YopE from the highly pathogenic Y. enterocolitica serotype O8 was subjected to proteasomal destabilization, whereas the YopE isotypes from serogroups O3 and O9 evaded degradation. Accumulation of YopE from serotypes O3 and O9 was accompanied by an enhanced cytotoxic effect. Using Yersinia strains that specifically produced YopE from either Y. enterocolitica O8 or O9, we found that only the YopE protein from serogroup O8 was modified by polyubiquitination, although both YopE isotypes were highly homologous. We determined two unique N-terminal lysines (K62 and K75) in serogroup O8 YopE, not present in serogroup O9 YopE, that served as polyubiquitin acceptor sites. Insertion of either lysine in serotype O9 YopE enabled its ubiquitination and destabilization. These results define a serotype-dependent difference in the stability and activity of the Yersinia effector protein YopE that could influence Y. enterocolitica pathogenesis.