Evaluation of the usefulness of selected virulence markers for identification of virulent Yersinia enterocolitica strains. II. Genotypic markers associated with the pYV plasmid

Pathogenic strains of Yersinia enterocolitica bear virulence associated plasmid pYV. Unfortunately plasmid pYV is easily lost by these bacteria incubated at elevated temperatures (37 degrees C) or long stored at room temperatures. This sometimes makes difficult the detection of the virulence plasmid, especially by its isolation or biochemical tests. On the other hand, observations done by some authors suggest that polymerase chain reaction (PCR) could be useful for demonstration of the pYV plasmid of Yersinia strains. Accordingly to this observation the aim of the presented study was to check the usefulness of plasmid-localised genes virF and yadA, detected by PCR, for the identification of the virulent strains of Y. enterocolitica. In the presented study one hundred and fifty two clinical strains of Y. enterocolitica belonging to serogroup O3 were investigated by the PCR for the presence of genes virF and yadA. Bacterial strains were first tested for the presence of pYV plasmid. In addition the phenotypic features: calcium dependence, Congo red binding and autoagglutination were determined. In this way the virulence plasmid was found in 130 of 152 examined strains. For PCR studies also forty plasmid-cured strains of Y. enterocolitica and 32 non-Y. enterocolitica, Enterobacteriaceae strains were included. The obtained results show that the tested genes were present only in Yersinia strains possessing the pYV plasmid and no one non-specific PCR product was observed. The detection level of these genes in nested PCR permits to detect pathogenic Y. enterocolitica in suspension composed of 1 x 10(3) CFU/ml of pYV+ bacilli and 3 x 10(9) CFU/ml plasmid-cured, isogenic bacteria. In the study it was shown that genes virF and yadA were useful virulence markers, which could be helpful in clinical studies for the detection of the virulence plasmid in Y. enterocolitica strains long stored or incubated at elevated temperatures.     

LcrF is the temperature-regulated activator of the yadA gene of Yersinia enterocolitica and Yersinia pseudotuberculosis.

The virulence plasmid of human pathogenic Yersinia species, pYV, encodes secreted proteins, Yop proteins, and an outer membrane protein, YadA. YadA has been associated with binding to a variety of substrates and with interference with host defense. YadA is regulated by temperature and is expressed only at 37 degrees C. Unlike the yop regulon, the yadA gene is not under Ca2+ regulation. Here, we show that LcrF (VirF), the temperature-regulated activator of the yop regulon, also acts as an activator for yadA.     

The pYV plasmid of Yersinia encodes a lipoprotein, YlpA, related to TraT

A series of lipoproteins was detected in the membrane fraction of Yersinia enterocolitica W227, a typical strain from serotype O:9. At least two of them, YlpA and YlpB, are encoded by the pYV plasmid. The sequence of ylpA reveals the presence of a typical lipoprotein signal peptide. The mature YlpA protein would be 223 residues long with a calculated molecular weight of 23798 for the proteic moiety of the molecule. YlpA shares 88% identical residues with the TraT protein encoded by plasmid pED208, 80% identity with TraT proteins encoded by plasmids R100 and F, and 77% identity with the TraT protein encoded by the virulence plasmid of Salmonella typhimurium. The ylpA gene hybridized with the pYV plasmid of Yersinia pseudotuberculosis, suggesting that this gene is conserved among Yersinia spp. The production of YlpA is controlled by virF and only occurs at 37 degrees C in the absence of Ca2+ ions. This co-regulation with the yop genes suggests that ylpA is a virulence determinant. However, mutations in ylpA clearly affect neither the resistance to human serum nor the virulence for intravenously inoculated mice.     

The Yersinia enterocolitica pYV Virulence Plasmid Contains Multiple Intrinsic DNA Bends Which Melt at 37°C

Temperature has a pleiotropic effect on Yersinia enterocolitica gene expression. Temperature-dependent phenotypes include the switching between two type III protein secretion systems, flagellum biosynthesis (相似文献   

Involvement of the virulence gene products of Yersinia enterocolitica in the immune response of infected mice

The virulence of Yersinia enterocolitica is known to be highly dependent on its virulence plasmid. However, it remains unclear whether the virulence plasmid is engaged also in the induction of cell-mediated immunity that is essential for protective immunity in the host. In this study, we have compared the induction of type 1 helper T cell immunity against Y. enterocolitica using a virulent strain (P+) harboring the pYV plasmid and an avirulent strain (P-) harboring no pYV. Spleen cells from both groups of mice immunized with 1/10 LD50 of P+ strain and those with 1/10 LD50 of P- strain produced a high level of gamma interferon (IFN-gamma) upon stimulation with heat-killed bacteria, and CD4+ T cells were exclusively responsible for IFN-gamma production. When crude Yersinia outer proteins (Yops) were used for antigenic stimulation, IFN-gamma response of immune spleen cells against crude Yops was observed only in mice immunized with P+ strain. Flowcytometric analysis revealed a significant level of increase in IFN-gamma-producing CD8+ T cells as well as the increase in IFN-gamma-producing CD4+ T cells against crude Yops. These results suggest that the virulence plasmid of Y. enterocolitica is involved in the induction of Th1-type of possibly protective T cells in infected mice.     

Yop fusions to tightly folded protein domains and their effects on Yersinia enterocolitica type III secretion

Yersinia enterocolitica organisms secrete Yop proteins via the type III pathway. Translational fusion of yop genes to ubiquitin or dihydrofolate reductase results in hybrid proteins that cannot be secreted. The folding of hybrids prevents their own transport, but it does not hinder the type III secretion of other Yops.     

Translocation of YopE and YopN into eukaryotic cells by Yersinia pestis yopN,tyeA, sycN,yscB and lcrG deletion mutants measured using a phosphorylatable peptide tag and phosphospecific antibodies

Yersinia pestis, the causative agent of plague, exports a set of virulence proteins called Yops upon contact with eukaryotic cells. A subset of these Yops is translocated directly into the cytosol of host cells. In this study, a novel protein tag-based reporter system is used to measure the translocation of Yops into cultured eukaryotic cells. The reporter system uses a small bipartite phosphorylatable peptide tag, termed the Elk tag. Translocation of an Elk-tagged protein into eukaryotic cells results in host cell protein kinase-dependent phosphorylation of the tag at a specific serine residue, which can subsequently be detected with phosphospecific antibodies. The YopN, TyeA, SycN, YscB and LcrG proteins function to prevent Yop secretion before host cell contact. The role of these proteins was investigated in the translocation of Elk-tagged YopE (YopE129-Elk) and YopN (YopN293-Elk) into HeLa cells. Y. pestis yopN, tyeA, sycN and yscB deletion mutants showed reduced levels of YopE129-Elk phosphorylation compared with the parent strain, indicating that these mutants translocate reduced amounts of YopE. We also demonstrate that YopN293-Elk is translocated into HeLa cells and that this process is more efficient in a Yersinia yop polymutant strain lacking the six translocated effector Yops. Y. pestis sycN and yscB mutants translocated reduced amounts of YopN293-Elk; however, tyeA and lcrG mutants translocated higher amounts of YopN293-Elk compared with the parent strain. These data suggest that TyeA and LcrG function to suppress the secretion of YopN before host cell contact, whereas SycN and YscB facilitate YopN secretion and subsequent translocation.