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 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 ⌬yopT deletion mutant colonizes mouse tissues to a greater extent than the parental strain. The ⌬yopO, ⌬yopP, and ⌬yopE mutants were only slightly attenuated after oral infection since they were still able to colonize the spleen and liver and cause systemic infection. The ⌬yopO mutant was lethal for mice, whereas ⌬yopP and ⌬yopE mutants were successfully eliminated from the spleen and liver 2 weeks after infection. In contrast the ⌬yopH, ⌬yopM, and ⌬yopQ mutants were highly attenuated and not able to colonize the spleen and liver on any of the days tested. The ⌬yopH, ⌬yopO, ⌬yopP, ⌬yopE, ⌬yopM, and ⌬yopQ mutants had only modest defects in the colonization of the small intestine and Peyer's patches. The ⌬yopE mutant was eliminated from the small intestine 3 weeks after infection, whereas the ⌬yopH, ⌬yopP, ⌬yopM, and ⌬yopQ mutants continued to colonize the small intestine at this time.

BackgroundE-cadherin is a major component of adherens junctions. Impaired expression of E-cadherin in the small intestine and colon has been linked to a disturbed intestinal homeostasis and barrier function. Down-regulation of E-cadherin is associated with the pathogenesis of infections with enteropathogenic bacteria and Crohn's disease.Methods and FindingsTo genetically clarify the function of E-cadherin in intestinal homeostasis and maintenance of the epithelial defense line, the Cdh1 gene was conditionally inactivated in the mouse intestinal epithelium. Inactivation of the Cdh1 gene in the small intestine and colon resulted in bloody diarrhea associated with enhanced apoptosis and cell shedding, causing life-threatening disease within 6 days. Loss of E-cadherin led cells migrate faster along the crypt-villus axis and perturbed cellular differentiation. Maturation and positioning of goblet cells and Paneth cells, the main cell lineage of the intestinal innate immune system, was severely disturbed. The expression of anti-bacterial cryptidins was reduced and mice showed a deficiency in clearing enteropathogenic bacteria from the intestinal lumen.ConclusionThese results highlight the central function of E-cadherin in the maintenance of two components of the intestinal epithelial defense: E-cadherin is required for the proper function of the intestinal epithelial lining by providing mechanical integrity and is a prerequisite for the proper maturation of Paneth and goblet cells.

Abstract. We have identified the yeast homologue of Neurospora crassa MOM72, the mitochondrial import receptor for the ADP/ATP carrier (AAC), by functional studies and by eDNA sequencing. Mitochondria of a yeast mutant in which the gene for MOM72 was disrupted were impaired in specific binding and import of AAC. Unexpectedly, we found a residual, yet significant import of AAC into mitochondria lacking MOM72 that occurred via the receptor MOM19. We conclude that both MOM72 and MOM19 can direct AAC into mitochondria, albeit with different efficiency. Moreover, the precursor of MOM72 apparently does not require a positively charged sequence at the extreme amino terminus for targeting to mitochondria.

The flagellum is believed to be the common ancestor of all type III secretion systems (TTSSs). In Yersinia enterocolitica, expression of the flagellar TTSS and the Ysc (Yop secretion) TTSS are inversely regulated. We therefore hypothesized that the Ysc TTSS may adopt flagellar motor components in order to use the pathogenicity-related translocon in a drill-like manner. As a prerequisite for this hypothesis, we first tested a requirement for the proton motive force by both systems using the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Motility as well as type III-dependent secretion of Yop proteins was inhibited by CCCP. We deleted motAB, which resulted in an immotile phenotype. This mutant, however, secreted amounts of Yops to the supernatant comparable to those of the wild type. Translocation of Yops into host cells was also not affected by the motAB deletion. Virulence of the mutant was comparable to that of the wild type in the mouse oral infection model. Thus, the hypothesis that the Ysc TTSS might adopt flagellar motor components was not confirmed. The finding that, in addition to consumption of ATP, Ysc TTSS requires the proton motive force is discussed.

SummaryYersinia spp. inject effector proteins (Yersinia outer proteins, Yops) into target cells via a type III secretion apparatus. The effector YopE was recently shown to possess GAP activity towards the Rho GTPases RhoA, Rac and CDC42 in vitro. To investigate the intracellular,`in vivo' targets of YopE we generated a Yersinia enterocolitica strain [WA(pYLCR1E)] that injects`life-like' amounts of YopE as only effector. Primary human umbilical vein endothelial cells (HUVEC) were infected with WA(pYLCR1E) and were then stimulated with: (i) bradykinin to induce actin microspikes followed by ruffles as an assay for CDC42 activity followed by CDC42 stimulated Rac activity; (ii) sphingosine-1-phosphate to form ruffles by direct Rac activation; or (iii) thrombin to generate actin stress fibres through Rho activation. In WA(pYLCR1E)-infected HUVEC microspike formation stimulated with bradykinin remained intact but the subsequent development of ruffles was abolished. Furthermore, ruffle formation after stimulation with sphingosine-1-phosphate or thrombin induced production of stress fibres was unaltered in the infected cells. These data suggest that YopE is able to inhibit Rac-but not Rho-or CDC42-regulated actin structures and, more specifically, that YopE is capable of blocking CDC42Hs dependent Rac activation but not direct Rac activation in HUVEC. This provides evidence for a considerable specificity of YopE towards selective Rac-mediated signalling pathways in primary target cells of Yersinia.

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Pathogenic bacteria of the genus Yersinia counteract host defense by interfering with eukaryotic signal transduction pathways. YpkA of Yersinia pseudotuberculosis shares significant homology with eukaryotic Ser/Thr protein kinases, is translocated into the host cell and has been shown to be an essential virulence factor in a mouse infection model. In this study, we identify the small GTPases RhoA and Rac-1 as eukaryotic binding partners of YpkA and its homolog YopO of Yersinia enterocolitica. We demonstrate that the interaction is independent of phosphorylation of YpkA and nucleotide loading state of the GTPases. The interaction with RhoA and Rac-1 might provide an important clue to how YpkA interferes with eukaryotic signaling on a molecular level. ß