Yersinia Outer Proteins Research Articles

Alternative Endogenous Protein Processing via an Autophagy-Dependent Pathway Compensates forYersinia-Mediated Inhibition of Endosomal Major Histocompatibility Complex Class II Antigen Presentation

Extracellular Yersinia pseudotuberculosis employs a type III secretion system (T3SS) for translocating virulence factors (Yersinia outer proteins [Yops]) directly into the cytosol of eukaryotic cells. Recently, we used YopE as a carrier molecule for T3SS-dependent secretion and translocation of listeriolysin O (LLO) from Listeria monocytogenes. We demonstrated that translocation of chimeric YopE/LLO into the cytosol of macrophages by Yersinia results in the induction of a codominant antigen-specific CD4 and CD8 T-cell response in orally immunized mice. In this study, we addressed the requirements for processing and major histocompatibility complex (MHC) class II presentation of chimeric YopE proteins translocated into the cytosol of macrophages by the Yersinia T3SS. Our data demonstrate the ability of Yersinia to counteract exogenous MHC class II antigen presentation of secreted hybrid YopE by the action of wild-type YopE and YopH. In the absence of exogenous MHC class II antigen presentation, an alternative pathway was identified for YopE fusion proteins originating in the cytosol. This endogenous antigen-processing pathway was sensitive to inhibitors of phagolysosomal acidification and macroautophagy, but it did not require the function either of the proteasome or of transporters associated with antigen processing. Thus, by an autophagy-dependent mechanism, macrophages are able to compensate for the YopE/YopH-mediated inhibition of the endosomal MHC class II antigen presentation pathway for exogenous antigens. This is the first report demonstrating that autophagy might enable the host to mount an MHC class II-restricted CD4 T-cell response against translocated bacterial virulence factors. We provide critical new insights into the interaction between the mammalian immune system and a human pathogen.

Cell membrane is impaired, accompanied by enhanced type III secretion system expression in Yersinia pestis deficient in RovA regulator.

BackgroundIn the enteropathogenic Yersinia species, RovA regulates the expression of invasin, which is important for enteropathogenic pathogenesis but is inactivated in Yersinia pestis. Investigation of the RovA regulon in Y. pestis at 26°C has revealed that RovA is a global regulator that contributes to virulence in part by the direct regulation of psaEFABC. However, the regulatory roles of RovA in Y. pestis at 37°C, which allows most virulence factors in mammalian hosts to be expressed, are still poorly understood.Methodology/Principal FindingsThe transcriptional profile of an in-frame rovA mutant of Y. pestis biovar Microtus strain 201 was analyzed under type III secretion system (T3SS) induction conditions using microarray techniques, and it was revealed that many cell-envelope and transport/binding proteins were differentially expressed in the ΔrovA mutant. Most noticeably, many of the T3SS genes, including operons encoding the translocon, needle and Yop (Yersinia outer protein) effectors, were significantly up-regulated. Analysis of Yop proteins confirmed that YopE and YopJ were also expressed in greater amounts in the mutant. However, electrophoresis mobility shift assay results demonstrated that the His-RovA protein could not bind to the promoter sequences of the T3SS genes, suggesting that an indirect regulatory mechanism is involved. Transmission electron microscopy analysis indicated that there are small loose electron dense particle-like structures that surround the outer membrane of the mutant cells. The bacterial membrane permeability to CFSE (carboxyfluorescein diacetate succinimidyl ester) was significantly decreased in the ΔrovA mutant compared to the wild-type strain. Taken together, these results revealed the improper construction and dysfunction of the membrane in the ΔrovA mutant.Conclusions/SignificanceWe demonstrated that the RovA regulator plays critical roles in the construction and functioning of the bacterial membrane, which sheds considerable light on the regulatory functions of RovA in antibiotic resistance and environmental adaptation. The expression of T3SS was upregulated in the ΔrovA mutant through an indirect regulatory mechanism, which is possibly related to the altered membrane construction in the mutant.

The many faces of the YopM effector from plague causative bacterium Yersinia pestis and its implications for host immune modulation

The Yersinia outer protein (Yop) M effector from the Yersinia pestis bacterium is well-known for being a critical virulence determinant; however, structural insight vis-à-vis its role in Y. pestis pathogenesis has been elusive. Here, we investigate the intact sequence of the YopM protein through our recently developed fold identification and homology modeling tools, and analyze the immune modulatory potential of its constituent domains. We identify a putative novel E3 ligase (NEL) domain towards the C-terminal tail of YopM and characterize its active site, to show that YopM could function as an autoregulated bacterial type E3 ubiquitin ligase. We further identify unreported NEL domains in several other bacteria and note remarkable similarity in sequence, structure, surface, and electrostatics for the family of NEL-containing bacterial effectors that suggests conserved function and potentially similar host targets for these proteins. Based on these observations and recent empirical evidence for degradation of the human proteins HLA-DR, thioredoxin, and NEMO/IKKγ by other members of the NEL-containing bacterial family, we discuss the potential for YopM to modulate a wide spectrum of immune signal transduction pathways. The key immune modulatory effects highlighted are suppression of MHC class II antigen presentation, dampening of nuclear factor (NF)-κB mediated inflammatory response, and intonation of mitogen-activated protein kinase (MAPK) signaling. Additionally, our analysis of the modeled YopM LRR domain reveals structural features akin to the Toll-like receptor 4 (TLR4) LRR motif. We propose that YopM LRR could be a 'molecular mimic' of TLR4 LRR, permitting reduced immunogenicity and potentially mitigating bacterial lipopolysaccharide surveillance of the innate immune system. Our identification and characterization of the YopM NEL domain, taken together with our analysis of the YopM LRR domain, provides plausible insight into subversion of host immunity by Y. pestis YopM and perhaps could set the stage for design of new therapeutic opportunities.

The Acetyltransferase Activity of the Bacterial Toxin YopJ of Yersinia Is Activated by Eukaryotic Host Cell Inositol Hexakisphosphate

Plague, one of the most devastating diseases in human history, is caused by the bacterium Yersinia pestis. The bacteria use a syringe-like macromolecular assembly to secrete various toxins directly into the host cells they infect. One such Yersinia outer protein, YopJ, performs the task of dampening innate immune responses in the host by simultaneously inhibiting the MAPK and NFκB signaling pathways. YopJ catalyzes the transfer of acetyl groups to serine, threonine, and lysine residues on target proteins. Acetylation of serine and threonine residues prevents them from being phosphorylated thereby preventing the activation of signaling molecules on which they are located. In this study, we describe the requirement of a host-cell factor for full activation of the acetyltransferase activity of YopJ and identify this activating factor to be inositol hexakisphosphate (IP6). We extend the applicability of our results to show that IP6 also stimulates the acetyltransferase activity of AvrA, the YopJ homologue from Salmonella typhimurium. Furthermore, an IP6-induced conformational change in AvrA suggests that IP6 acts as an allosteric activator of enzyme activity. Our results suggest that YopJ-family enzymes are quiescent in the bacterium where they are synthesized, because bacteria lack IP6; once injected into mammalian cells by the pathogen these toxins bind host cell IP6, are activated, and deregulate the MAPK and NFκB signaling pathways thereby subverting innate immunity.

The bacterial effector protein YopM is a selfdelivering immune therapeutic agent that reduces inflammation in rheumatoid arthritis

Synovial fibroblasts (RASF) are active drivers of joint destruction in rheumatoid arthritis (RA) through the production of cytokines that maintain the inflammatory process. The Yersinia outer protein M (YopM) is...

Neutrophils Are Resistant to Yersinia YopJ/P-Induced Apoptosis and Are Protected from ROS-Mediated Cell Death by the Type III Secretion System

BackgroundThe human innate immune system relies on the coordinated activity of macrophages and polymorphonuclear leukocytes (neutrophils or PMNs) for defense against bacterial pathogens. Yersinia spp. subvert the innate immune response to cause disease in humans. In particular, the Yersinia outer protein YopJ (Y. pestis and Y. pseudotuberculosis) and YopP (Y. enterocolitica) rapidly induce apoptosis in murine macrophages and dendritic cells. However, the effects of Yersinia Yop J/P on neutrophil fate are not clearly defined.Methodology/Principal FindingsIn this study, we utilized wild-type and mutant strains of Yersinia to test the contribution of YopJ and YopP on induction of apoptosis in human monocyte-derived macrophages (HMDM) and neutrophils. Whereas YopJ and YopP similarly induced apoptosis in HMDMs, interaction of human neutrophils with virulence plasmid-containing Yersinia did not result in PMN caspase activation, release of LDH, or loss of membrane integrity greater than PMN controls. In contrast, interaction of human PMNs with the virulence plasmid-deficient Y. pestis strain KIM6 resulted in increased surface exposure of phosphatidylserine (PS) and cell death. PMN reactive oxygen species (ROS) production was inhibited in a virulence plasmid-dependent but YopJ/YopP-independent manner. Following phagocytic interaction with Y. pestis strain KIM6, inhibition of PMN ROS production with diphenyleneiodonium chloride resulted in a reduction of PMN cell death similar to that induced by the virulence plasmid-containing strain Y. pestis KIM5.ConclusionsOur findings showed that Yersinia YopJ and/or YopP did not induce pronounced apoptosis in human neutrophils. Furthermore, robust PMN ROS production in response to virulence plasmid-deficient Yersinia was associated with increased PMN cell death, suggesting that Yersinia inhibition of PMN ROS production plays a role in evasion of the human innate immune response in part by limiting PMN apoptosis.

Seroepidemiological Survey of Pathogenic Yersinia in Breeding Squirrel Monkeys in Japan

To investigate the prevalence of antibodies to pathogenic Yersinia in breeding squirrel monkeys, the serum samples of 252 squirrel monkeys from 9 zoological gardens in Japan were tested by ELISA using plasmid-encoded Yersinia outer membrane protein (Yops) as the antigen. The cutoff value was calculated by using the serum samples of the squirrel monkeys from Suriname, where no prevalence of pathogenic Yersinia have been reported. According to the cutoff value, 164 of 252 (65.1%) squirrel monkeys were considered positive against pathogenic Yersinia. These positive monkeys belonged to 8 of the 9 zoological gardens, and the percentage of the seropositive monkeys ranged from 22.2 to 89.4%. Furthermore, in one zoological garden, the positive rate of the squirrel monkeys which were over 1 year old (95.7%) was significantly higher than those which were under 1 year old (23.3%). These results suggested that pathogenic Yersinia is highly prevalent among breeding monkeys in Japan.

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.

Recombinant YopJ induces apoptosis in murine peritoneal macrophages in vitro: involvement of mitochondrial death pathway

Yersinia species during infection adhere to host immune cells primarily to macrophages and employ its secretary proteins known as Yersinia outer proteins to trigger death in infected cells. In the present study, it is shown that recombinant Yersinia outer protein J (rYopJ) could induce apoptosis in murine peritoneal macrophages in vitro as assessed by morphological features, internucleosomal DNA fragmentation, change in mitochondrial membrane potential (MMP) (Deltapsim), activation of caspases and Annexin V binding. rYopJ-induced cell death was dose and time dependent. Pre-treatment with broad-spectrum caspase inhibitor Z-VAD-FMK, caspase-3 inhibitor Ac-DEVD-CHO and caspase-8 inhibitor Z-IETD-FMK prevented the change in MMP and DNA fragmentation, suggesting caspase-dependent apoptosis of rYopJ-treated macrophages. Blocking the endocytosis by pre-treatment of cells with cytochalasin B did not prevent the rYopJ-induced macrophages apoptosis. The data further suggest that rYopJ-induced apoptosis is mediated by molecules upstream of caspase-8 and relay through mitochondrial pathway involving Bax, Bcl-2, activation of caspase-8 and caspase-3, Bid and polyadenosine diphosphate-ribose polymerase cleavage, cytochrome c release and DNA fragmentation.

Yersinia pestis and host macrophages: immunodeficiency of mouse macrophages induced by YscW

The virulence of the pathogenic Yersinia species depends on a plasmid-encoded type III secretion system (T3SS) that transfers six Yersinia outer protein (Yop) effector proteins into the cytoplasm of eukaryotic cells, leading to disruption of host defence mechanisms. It is shown in this study that Yersinia pestis YscW, a protein of the T3SS injectisome, contributes to the induction of a deficiency in phagocytosis in host macrophages and a reduction in their antigen-presenting capacity. A Y. pestis strain lacking yscW had no effect on uptake by host macrophages. In mice infected with wild-type Y. pestis, the yscW mutant or a complement strain, immunodeficiency was observed in host macrophages compared with those from uninfected mice. However, the phagocytosis and antigen presenting capacities of macrophages infected by yscW mutant strain both in vivo and in vitro were significantly higher than those by wild type strain. Consistent with this finding, when YscW was expressed in the RAW264.7 macrophage cell line, phagocytosis and antigen-presenting capacities were significantly lower than those of the control groups. These results indicate that Y. pestis YscW may directly induce immunodeficiency in murine macrophages by crippling their phagocytosis and antigen-presenting capacities. These data provide evidences to Y. pestis pathogenesis that some proteins in T3SS injectisome, such as YscW protein, might play independent roles in disrupting host defense apart from their known functions.

In Vitro Intracellular Trafficking of Virulence Antigen during Infection by Yersinia pestis

Yersinia pestis, the causative agent of plague, encodes several essential virulence factors on a 70 kb plasmid, including the Yersinia outer proteins (Yops) and a multifunctional virulence antigen (V). V is uniquely able to inhibit the host immune response; aid in the expression, secretion, and injection of the cytotoxic Yops via a type III secretion system (T3SS)-dependent mechanism; be secreted extracellularly; and enter the host cell by a T3SS-independent mechanism, where its activity is unknown. To elucidate the intracellular trafficking and target(s) of V, time-course experiments were performed with macrophages (MΦs) infected with Y. pestis or Y. pseudotuberculosis at intervals from 5 min to 6 h. The trafficking pattern was discerned from results of parallel microscopy, immunoblotting, and flow cytometry experiments. The MΦs were incubated with fluorescent or gold conjugated primary or secondary anti-V (antibodies [Abs]) in conjunction with organelle-associated Abs or dyes. The samples were observed for co-localization by immuno-fluorescence and electron microscopy. For fractionation studies, uninfected and infected MΦs were lysed and subjected to density gradient centrifugation coupled with immunoblotting with Abs to V or to organelles. Samples were also analyzed by flow cytometry after lysis and dual-staining with anti-V and anti-organelle Abs. Our findings indicate a co-localization of V with (1) endosomal proteins between 10–45 min of infection, (2) lysosomal protein(s) between 1–2 h of infection, (3) mitochondrial proteins between 2.5–3 h infection, and (4) Golgi protein(s) between 4–6 h of infection. Further studies are being performed to determine the specific intracellular interactions and role in pathogenesis of intracellularly localized V.

Yersinia outer protein YopE affects the actin cytoskeleton in Dictyostelium discoideumthrough targeting of multiple Rho family GTPases

BackgroundAll human pathogenic Yersinia species share a virulence-associated type III secretion system that translocates Yersinia effector proteins into host cells to counteract infection-induced signaling responses and prevent phagocytosis. Dictyostelium discoideum has been recently used to study the effects of bacterial virulence factors produced by internalized pathogens. In this study we explored the potential of Dictyostelium as model organism for analyzing the effects of ectopically expressed Yersinia outer proteins (Yops).ResultsThe Yersinia pseudotuberculosis virulence factors YopE, YopH, YopM and YopJ were expressed de novo within Dictyostelium and their effects on growth in axenic medium and on bacterial lawns were analyzed. No severe effect was observed for YopH, YopJ and YopM, but expression of YopE, which is a GTPase activating protein for Rho GTPases, was found to be highly detrimental. GFP-tagged YopE expressing cells had less conspicuous cortical actin accumulation and decreased amounts of F-actin. The actin polymerization response upon cAMP stimulation was impaired, although chemotaxis was unaffected. YopE also caused reduced uptake of yeast particles. These alterations are probably due to impaired Rac1 activation. We also found that YopE predominantly associates with intracellular membranes including the Golgi apparatus and inhibits the function of moderately overexpressed RacH.ConclusionThe phenotype elicited by YopE in Dictyostelium can be explained, at least in part, by inactivation of one or more Rho family GTPases. It further demonstrates that the social amoeba Dictyostelium discoideum can be used as an efficient and easy-to-handle model organism in order to analyze the function of a translocated GAP protein of a human pathogen.

A Family of Bacterial Cysteine Protease Type III Effectors Utilizes Acylation-dependent and -independent Strategies to Localize to Plasma Membranes

Bacterial phytopathogens employ a type III secretion system to deliver effector proteins into the plant cell to suppress defense pathways; however, the molecular mechanisms and subcellular localization strategies that drive effector function largely remain a mystery. Here, we demonstrate that the plant plasma membrane is the primary site for subcellular localization of the Pseudomonas syringae effector AvrPphB and five additional cysteine protease family members. AvrPphB and two AvrPphB-like effectors, ORF4 and NopT, autoproteolytically process following delivery into the plant cell to expose embedded sites for fatty acylation. Host-dependent lipidation of these three effectors directs plasma membrane localization and is required for the avirulence activity of AvrPphB. Surprisingly, the AvrPphB-like effectors RipT, HopC1, and HopN1 utilize an acylation-independent mechanism to localize to the cellular plasma membrane. Although some AvrPphB-like effectors employ acylation-independent localization strategies, others hijack the eukaryotic lipidation machinery to ensure plasma membrane localization, illustrating the diverse tactics employed by type III effectors to target specific subcellular compartments.

Characterization of immune responses to Yersinia pestis infection: resistant versus susceptible mice(129.29)

Abstract We have previously reported that inbred B10.T(6R) mice are resistant to Yersinia pestis infection, compared to susceptible strains, e.g. Swiss Webster mice; the mean LD50's for B10.T(6R) females is 14000 CFU's, compared to Swiss Webster females, at 22 CFU's. Interestingly, B10.T(6R) mice demonstrated a more robust pro-inflammatory response early in infection, and a lesser anti-inflammatory response later on, when compared to susceptible Swiss Webster mice. Significantly higher levels of TNF-alpha were detected in B10.T(6R) on day 1 while TNF-alpha responses were delayed in Swiss Webster mice. Conversely, IL-10 levels were not changed in B10.T(6R) mice while IL-10 levels increased significantly in Swiss Webster mice. Yersinia outer membrane proteins (YOP) are translocated into the host cell's cytoplasm via a type three secretion system. After contact with a eukaryotic cell, YOPs then interact with cellular components to disrupt the host's immune response and induce the production of suppressive cytokines, e.g. IL-10. We postulate that the resistant B10.T(6R) strain of mice are able to overcome initial infection with Y. pestis through higher levels of pro-inflammatory cytokines and decreased levels of immune suppressive cytokines later in infection.

Targeting Type III Secretion in Yersinia pestis

Yersinia pestis, the causative agent of plague, utilizes a plasmid-encoded type III secretion system (T3SS) to aid it with its resistance to host defenses. This system injects a set of effector proteins known as Yops (Yersinia outer proteins) into the cytosol of host cells that come into contact with the bacteria. T3SS is absolutely required for the virulence of Y. pestis, making it a potential target for new therapeutics. Using a novel and simple high-throughput screening method, we examined a diverse collection of chemical libraries for small molecules that inhibit type III secretion in Y. pestis. The primary screening of 70,966 compounds and mixtures yielded 421 presumptive inhibitors. We selected eight of these for further analysis in secondary assays. Four of the eight compounds effectively inhibited Yop secretion at micromolar concentrations. Interestingly, we observed differential inhibition among Yop species with some compounds. The compounds did not inhibit bacterial growth at the concentrations used in the inhibition assays. Three compounds protected HeLa cells from type III secretion-dependent cytotoxicity. Of the eight compounds examined in secondary assays, four show good promise as leads for structure-activity relationship studies. They are a diverse group, with each having a chemical scaffold not only distinct from each other but also distinct from previously described candidate type III secretion inhibitors.

Cross-reaction between Yersinia outer membrane proteins and anti-Borreli antibodies in sera of patients with Lyme disease

Diagnosis of Yersinia infections accompanied by reactive arthritis could be complicated by cross-reaction with other arthritogenic bacteria. The possible cross-reaction between Yersinia antigens and anti-Borrelia antibodies in blood sera of patients with Lyme disease was studied. The occurrence of specific IgA, IgG and IgM antibodies was analyzed in serum samples from 30 patients with Yersinia-triggered reactive arthritis, 30 patients with Lyme disease and five samples from healthy blood donors. For anti-Borrelia IgG antibodies, cross-reaction was detected with YopH, YopB, V-ag, YopD, YopN, YopP and YopE, and for IgA with YopD. For IgM, no cross-reaction was detected. Owing to cross-reactivity with Borrelia, the diagnosis of Yersinia-triggered reactive arthritis should be based on a combination of serological and clinical findings.

Too early to dismiss Yersinia enterocolitica infection in the aetiology of Graves’ disease: evidence from a twin case–control study

Yersinia enterocolitica (YE) infection has long been implicated in the pathogenesis of Graves' disease (GD). The association between YE and GD could, however, also be due to common genetic or environmental factors affecting the development of both YE infection and GD. This potential confounding can be minimized by investigation of twin pairs discordant for GD. To examine whether YE infection is associated with GD. We first conducted a classical case-control study of individuals with (61) and without (122) GD, and then a case-control study of twin pairs (36) discordant for GD. Immunoglobulin (Ig)A and IgG antibodies to virulence-associated Yersinia outer membrane proteins (YOPs) were measured. The prevalence of YOP IgA and IgG antibodies. Subjects with GD had a higher prevalence of YOP IgA (49%vs. 34%, P = 0.054) and YPO IgG (51%vs. 35%, P = 0.043) than the external controls. The frequency of chronic YE infection, reflected by the presence of both IgA and IgG YOP antibodies, was also higher among cases than controls (49%vs. 33%, P = 0.042). Similar results were found in twin pairs discordant for GD. In the case-control analysis, individuals with GD had an increased odds ratio (OR) of YE infection: IgA 1.84 (95% CI 0.99-3.45) and IgG 1.90 (95% CI 1.02-3.55). In the co-twin analysis, the twin with GD also had an increased OR of YE infection: IgA 5.5 (95% CI 1.21-24.81) and IgG 5.0 (95% CI 1.10-22.81). The finding of an association between GD and YE in the case-control study and within twin pairs discordant for GD supports the notion that YE infection plays an aetiological role in the occurrence of GD, or vice versa. Future studies should examine the temporal relationship of this association in more depth.

The polybasic region of Rho GTPases defines the cleavage by Yersinia enterocolitica outer protein T (YopT)

Pathogenic Yersinia strains evade the innate immune responses of the host by producing effector proteins ( Yersinia outer proteins [Yops]), which are directly injected into mammalian cells by a type III secretion system (TTSS). One of these effector proteins (YopT) disrupts the actin cytoskeleton of the host cell resulting in cell rounding. YopT is a cysteine protease that cleaves Rho proteins directly upstream of the post-translationally modified cysteine. Thereby, it releases the GTPases from the membrane leading to inactivation. Small GTPases are modified by isoprenylation of the cysteine of the CAAX box, cleavage of the -AAX tripeptide, and methylation of the cysteine. We have shown that isoprenylation and the endoproteolytic cleavage of the tripeptide of Rho GTPases are essential for YopT-induced cleavage, whereas carboxyl methylation is not required. In the present study, we post-translationally modified RhoA, Rac, Cdc42, and several mutants in vitro and characterized the YopT-induced cleavage with recombinant YopT. We show that farnesylated RhoA is a preferred substrate of YopT compared with the geranylgeranylated GTPase. Geranylgeranylated RhoA, however, is the preferred substrate for YopT-catalyzed cleavage with a threefold faster turnover rate over Rac and Cdc42. Moreover, our data indicate that the composition of the polybasic region of the GTPases defines the specificity and efficiency of the YopT-induced cleavage, and that a space between the polybasic stretch of amino acids at the C terminus and the CAAX box enhances the turnover rate of YopT-catalyzed cleavage.

Differential roles of Yersinia outer protein P-mediated inhibition of nuclear factor-kappa B in the induction of cell death in dendritic cells and macrophages

Yersinia outer protein P (YopP) induces cell death in macrophages and dendritic cells (DC). In DC this YopP-dependent cell death coincides with the inhibition of nuclear factor-kappa B (NF-kappaB) activation. However, as shown by measurement of propidium iodide uptake via disrupted cellular membranes, the preincubation of DC with several NF-kappaB inhibitors prior to infection with Yersinia did not restore the death-inducing capacity of a YopP-deficient Yersinia mutant. These results suggest that in contrast to macrophages, in DC the YopP-dependent inhibition of NF-kappaB activation is not causative for the induction of cell death. Instead, in DC, the inhibition of mitogen-activated protein kinases (MAPKs), in particular, p38 and c-Jun N-terminal kinase, prior to infection with a YopP-deficient Yersinia mutant substituted the death-inducing capacity of the Yersinia wild-type strain, indicating that the YopP-dependent inhibition of MAPKs mediates Yersinia-induced DC death. The differences between DC and macrophages in the mechanisms of cell death induction by YopP presented herein might be crucial for the function of these antigen-presenting cells.

Yersinia as oral live carrier vaccine: Influence of Yersinia outer proteins (Yops) on the T-cell response

Attenuated enteropathogenic Yersinia strains are attractive candidates for the development of oral live carrier vaccines. Yersiniae colonize the small intestine and invade lymphoid tissue of the terminal ileum where they replicate extracellularly. Yersiniae can be engineered to secrete or translocate heterologous antigens into the cytosol of antigen-presenting cells by their type 3 secretion system (T3SS). This results in the induction of both cellular and humoral immune responses to heterologous antigens of viral, bacterial and parasitic origin. In this review, we summarize the progress in developing Yersinia-based vaccine carrier strains by mutating the T3SS effector proteins of Yersinia called Yops ( Yersinia outer proteins) to both attenuate the strains and to modulate the T-cell response.