Exposure of macrophages to bacteria or LPS mediates activation of signaling pathways that induce expression of self defense-related genes. Pathogenic Yersinia species impair activation of transcription factor NF-κB and trigger apoptosis in macrophages. In this study, we dissected the mechanism of apoptosis induction by Yersinia. Selectively, Yersinia enterocolitica strains producing the effector protein Yersinia outer protein P (YopP) hampered NF-κB activation and subsequently conferred apoptosis to J774A.1 macrophages. Thereby, YopP bound and inhibited the macrophage NF-κB-activating kinase IKKβ. YopP- and Yersinia-, but not Salmonella-induced apoptosis was specifically prevented by transient overexpression of NF-κB p65, giving evidence that YopP mediates cell death by disrupting the NF-κB signaling pathway. Transfection of J774A.1 macrophages with YopP induced a moderate, but significant degree of apoptosis (40–50% of transfected cells). This effect was strongly enhanced by additional initiation of LPS signaling (80–90%), indicating a synergism between LPS-induced signal transduction and inhibition of NF-κB by YopP. This reflects a strategy of a bacterial pathogen that takes advantage of LPS, serving as cofactor, to impair the macrophage.
Pathogenic Yersinia spp. neutralize host defense mechanisms by engaging a type III protein secretion system that translocates several Yersinia outer proteins (Yops) into the host cell. Although the modulation of the cellular responses by individual Yops has been intensively studied, little is known about the fate of the translocated Yops inside the cell. In this study, we investigated involvement of the proteasome, the major nonlysosomal proteolytic system in eukaryotic cells, in Yop destabilization and repression. Our data show that inhibition of the proteasome in Yersinia enterocolitica-infected cells selectively stabilized the level of YopE, but not of YopH or YopP. In addition, YopE was found to be modified by ubiquitination. This suggests that the cytotoxin YopE is physiologically subjected to degradation via the ubiquitin-proteasome pathway inside the host cell. Importantly, the increased levels of YopE upon proteasome inhibition were associated with decreased activity of its cellular target Rac. Thus, the GTPase-down-regulating function of YopE is enhanced when the proteasome is inhibited. The stabilization of YopE by proteasome inhibitor treatment furthermore led to aggravation of the cytotoxic YopE effects on the actin cytoskeleton and on host cell morphology. Together, these data show that the host cell proteasome functions to destabilize and inactivate the Yersinia effector protein YopE. This implies the proteasome as integral part of the cellular host immune response against the immunomodulatory activities of a translocated bacterial virulence protein.
Pathogenic Yersinia spp. use a panel of virulence proteins that antagonize signal transduction processes in infected cells to undermine host defense mechanisms. One of these proteins, Yersinia enterocolitica outer protein P (YopP), down-regulates the NF-κB and MAPK signaling pathways, which suppresses the proinflammatory host immune response. In this study, we explored the mechanism by which YopP succeeds to simultaneously disrupt several of these key signaling pathways of innate immunity. Our data show that YopP operates upstream of its characterized eukaryotic binding partner IκB kinase-β to shut down the NF-κB signaling cascade. Accordingly, YopP efficiently impaired the activities of TGF-β-activated kinase-1 (TAK1) in infected cells. TAK1 is an important activator of the IκB kinase complex in the TLR signaling cascade. The repression of TAK1 activities correlated with reduced activation of NF-κB- as well as AP-1-dependent reporter gene expression in Yersinia-infected murine macrophages. This suggests that the impairment of the TAK1 enzymatic activities by Yersinia critically contributes to down-regulate activation of NF-κB and of MAPK members in infected host cells. The inhibition of TAK1 potentially results from the blockade of signaling events that control TAK1 induction. This process could involve the attenuation of ubiquitination of the upstream signal transmitter TNFR-associated factor-6. Together, these results indicate that, by silencing the TAK1 signaling complex, Yersinia counteracts the induction of several conserved signaling pathways of innate immunity, which aids the bacterium in subverting the host immune response.
Pathogenic Yersinia spp. counteract host defense mechanisms by modulating the cellular signal relay in response to infection. Subversion of the antiapoptotic NF-B 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 yopPknockout 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-B-activating kinase IKK in macrophages. However, selectively, YopPO8 mediated efficient inhibition of IKK activities, which led to substantial suppression of NF-B 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 IKK activity and survival of macrophages.Pathogenic Yersinia spp. have evolved a series of strategies for evasion and neutralization of host defense mechanisms. The ability to escape the host immune response largely depends on the presence of the 70-kb virulence plasmid pYV, which is common to the three Yersinia species that are pathogenic for rodents and humans. Y. pestis is the causative agent of plague, and Y. enterocolitica and Y. pseudotuberculosis cause gastrointestinal syndromes, lymphadenitis, and septicemia (6, 9). The Yersinia virulence plasmid pYV encodes a sophisticated bacterial virulence system for subverting eukaryotic cells (9). It encompasses the genes for a type III protein secretion machinery and for a set of at least six Yersinia effector proteins (Yersinia outer proteins [Yops] YopE, YopH, YopM, YopT, YopO/YpkA, and YopP/YopJ). The type III protein secretion system is activated upon host-cell contact and specifically mediates the delivery of the Yersinia effector proteins inside eukaryotic cell, there perturbing key cellular functions. By interference with the actin cytoskeleton dynamics, Yersinia blocks its phagocytosis by macrophages and polymorphonuclear neutrophils (4, 9).
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