The type IV leader peptidase/N-methyltransferase of Vibrio vulnificus controls factors required for adherence to HEp-2 cells and virulence in iron-overloaded mice.

Vibrio vulnificus expresses a number of potential virulence determinants that may contribute to its ability to cause a severe and rapidly disseminating septicemia in susceptible hosts. We have cloned and characterized two genes encoding products related to components of the type IV pilus biogenesis and general secretory (type II) pathways by complementation of a type IV peptidase/N-methyltransferase (PilD) mutant of Pseudomonas aeruginosa with a V. vulnificus genomic library. One of the genes (vvpD) encodes a protein homologous to PilD and other members of the type IV peptidase family that completely restores this activity in a P. aeruginosa mutant deficient in the expression of PilD. The other gene (vvpC) encodes a homolog of PilC from P. aeruginosa, where it is essential for assembly of type IV pili. Phenotypic characterization of a V. vulnificus vvpD mutant, constructed by allelic exchange, showed that VvpD is required for the expression of surface pili, suggesting that the pili observed on V. vulnificus are of the type IV class. This mutant was also unable to secrete at least three extracellular degradative enzymes, and the localization of one of these (the cytolysin/hemolysin) to the periplasmic space indicates that these proteins are normally exported via the type II secretion pathway. Loss of VvpD resulted in significant decreases in CHO cell cytotoxicity, adherence to HEp-2 cells, and virulence in a mouse model. Capsule formation and serum resistance were not affected in the vvpD mutant, indicating that in addition to capsule, virulence of V. vulnificus requires type IV pili and/or extracellular secretion of several exoenzymes.

Amino acid substitutions in PilD, a bifunctional enzyme of Pseudomonas aeruginosa. Effect on leader peptidase and N-methyltransferase activities in vitro and in vivo.

Subunits of type IV pili and a subset of proteins of the type II extracellular protein secretion apparatus undergo two consecutive post-translational modifications: leader peptide cleavage, followed by methylation of the newly created N-terminal amino acid. These two reactions are carried out by a single bifunctional enzyme encoded in Pseudomonas aeruginosa by the pilD gene. Properties of PilD mutants at positions Gly95 and/or Lys96 which were differentially affected in leader peptidase and N-methyltransferase function were characterized. None of the single amino acid substitutions showed a significant alteration in their ability to cleave the prepilin leader peptide; however, two double mutants did exhibit a modest reduction in the efficiency of cleavage. In contrast, a significant decrease of N-methyltransferase activity was detected in PilD having substitutions at Gly95. Mutants with substitutions at position Lys96 showed a variable effect on N-methyltransferase activity with an apparent requirement for any charged amino acid at this position. Absence of N-methyltransferase activity did not appear to interfere with the ability of P. aeruginosa to assemble functional pili. Moreover, pilin monomers isolated from P. aeruginosa expressing PilD with Gly95 substitutions were not methylated. Although complete methylation does not appear to be absolutely required for pilus assembly in P. aeruginosa, this modification may be important for pilus function in its natural habitat.

Temperature-dependent regulation of Yersinia enterocolitica Class III flagellar genes.

Temperature is a key environmental cue for Yersinia enterocolitica as well as for the two other closely related pathogens, Yersinia pestis and Yersinia pseudotuberculosis. Between the range of 30 degrees C and 37 degrees C, Y. enterocolitica phase-varies between motility and plasmid-encoded virulence gene expression. To determine how temperature regulates Y. enterocolitica motility, we have been dissecting the flagellar regulatory hierarchy to determine at which level motility is blocked by elevated temperature (37 degrees C). Here we report the cloning, DNA sequences, and regulation of the two main regulators of Class III flagellar genes, fliA (sigma F) and flgM (anti-sigma F), and a third gene, flgN, which we show is required for filament assembly. Identification of the Y. enterocolitica fliA and flgM genes was accomplished by functional complementation of both S. typhimurium and Y. enterocolitica mutations and by DNA sequence analysis. The Y. enterocolitica fliA gene, encoding the flagellar-specific sigma-factor, sigma F, maps immediately downstream of the three flagellin structural genes. The flgM and flgN genes, encoding anti-sigma F and a gene product required for filament assembly, respectively, map downstream of the invasin (inv) gene but are transcribed in the opposite (convergent) direction. By using Northern blot analyses we show that transcription of both fliA and flgM is immediately arrested when cells are exposed to 37 degrees C, coincident with the timing of virulence gene induction. Unlike S. typhimurium flgM mutants, Y. enterocolitica flgM mutants are fully virulent.

Pathogenesis of defined invasion mutants of Yersinia enterocolitica in a BALB/c mouse model of infection.

It has been hypothesized for many years that the ability of Yersinia spp. to invade tissue culture cells is reflective of their ability to penetrate the intestinal epithelium and that this capacity is an important aspect of the disease process. Three different genes from Yersinia spp. that are involved in the tissue culture invasion phenotype have been identified: inv, ail, and yadA. It was previously shown that inv is necessary for efficient penetration of the intestinal epithelium by Yersinia enterocolitica. The present study was initiated to determine whether other known Yersinia invasion factors could promote uptake of the bacteria by mice in the absence of invasion. In addition, the roles of these three invasion factors in the survival of the bacteria, lethality for mice, and development of pathology were compared. We found that YadA is necessary for persistence of Y. enterocolitica in Peyer's patches, and consistent with this observation, the yadA mutant was avirulent for mice infected either orally or intraperitoneally. In addition, the inv yadA double mutant was avirulent. Histological and immunohistological examination of the Peyer's patches of infected mice indicated that despite the presence of large numbers of CFU at 24 h the yadA and ail yadA mutants cause only minimal pathology and recruitment of macrophages. At 42 h postinfection, Peyer's patches from mice infected with the inv mutant showed no pathology, despite the prediction that some of the mice by this time would be colonized. However, at 72 h, inflammation and necrosis were evident in some Peyer's patches. Together, these observations suggest that for visible pathology to develop, a threshold number of bacteria (> 10(5)) is needed and the bacteria need to persist for more than 24 h. Lastly, YadA but not Ail may play a role in the less efficient, delayed invasion of the intestinal epithelium observed for the inv mutant.

Growth phase and low pH affect the thermal regulation of the Yersinia enterocolitica inv gene.

The inv gene encodes the protein invasin, which is the primary invasion factor for Yersinia enterocolitica in vitro and in vivo. Previous studies of Yersinia species have shown that inv expression and entry into mammalian cells are temperature regulated. Invasin production is reduced at the host temperature of 37 degrees C as compared to production at ambient temperature; consequently, this study was initiated to determine whether other host environmental signals might induce inv expression at 37 degrees C. An inv::phoA translational fusion was recombined on to the Y. enterocolitica chromosome by allelic exchange to monitor inv expression. Molecular characterization of expression of the wild-type inv gene and the inv::phoA fusion showed that invasin is not produced until early stationary phase in bacteria grown at 23 degrees C. Y. enterocolitica grown at 37 degrees C and pH 5.5 showed levels of inv expression comparable to those observed in bacteria grown at 23 degrees C. An increase in Na+ ions caused a slight increase in expression at 37 degrees C. However, expression at 37 degrees C was unaffected by anaerobiosis, growth medium, calcium levels, or iron levels. Additionally, Y. enterocolitica expressed invasin in Peyer's patches two days after being introduced intragastrically into BALB/c mice. These results suggest that invasin expression in Y. enterocolitica may remain elevated early during interaction with the intestinal epithelium, a site at which invasin was shown to be necessary.

Cloning of the YenI restriction endonuclease and methyltransferase from Yersinia enterocolitica serotype O8 and construction of a transformable R-M+ mutant.

Two different clonal groups of pathogenic Yersinia enterocolitica strains, American and non-American, have been recognized. These are distinguished by a number of criteria, including their virulence in a murine model of infection. However, genetic analysis of virulence in American strains has been hampered due to the severe restriction of transformed or electroporated DNA. Thus, we cloned the yenIMR locus from the American serotype strain 8081c, which encodes YenI, an isoschizomer of PstI. This clone encodes both the restriction endonuclease and methyltransferase. The location of the genes on the clone was determined and this information was used to construct a small deletion (400 bp) that results in an R-M+ phenotype. This mutation was recombined onto the Y. enterocolitica chromosome to give an R-M+ mutant which showed at least a 1000-fold increase in electroporation frequency compared to the wild-type strain. Southern analysis using a probe derived from yenIMR indicated that American serotype strains have this locus whereas non-American serotype strains do not.

The biological role of invasin during a Yersinia enterocolitica infection.

Epidemiological data suggest that the ability to invade the intestinal epithelium of mammals is an essential virulence determinant of Yersinia enterocolitica. The chromosomally encoded Y. enterocolitica 8081v invasion gene, inv, was disrupted to assess its role in pathogenesis. The inv mutant was unable to invade cultured epithelial cells as efficiently as wild type. Furthermore, when mice were infected intragastrically, the inv mutant was extremely deficient at penetrating the murine intestinal epithelium. Analysis of the course of infection showed that the inv mutant had distinct differences relative to wild type in the distribution of visible infectious foci and in tissue colonization; however, the mutant and wild-type strains had similar median lethal doses for both orally and intraperitoneally infected mice. The invasion defect of the inv mutant was fully complemented in vitro and in vivo by introduction of the wild-type inv gene in trans. The inv gene product, invasin, appears to play a vital role in promoting entry during the initial stage of infection. During the subsequent establishment of a systemic infection, invasin may be of secondary importance, since the Y. enterocolitica inv mutant was as proficient as wild-type at causing a fatal infection in mice. The possible role of invasin in a naturally occurring Y. enterocolitica infection is discussed.

Yersinia enterocolitica invasin: a primary role in the initiation of infection.

The ability to invade the intestinal epithelium of mammals is an essential virulence determinant of Yersinia enterocolitica. The chromosomally encoded Y. enterocolitica 8081v invasion gene, inv, was disrupted to assess its role in pathogenesis. The inv mutant (JP273v) was approximately 80-fold less invasive than wild type for cultured epithelial cells. When mice were infected intragastrically, up to 10(7) fewer JP273v were recovered from Peyer's patches early (6-18 hr) after infection compared with wild type. Analysis of the course of infection revealed that the inv mutant had distinct differences relative to wild type in the distribution of visible infectious foci and in tissue colonization; however, the mutant and wild-type strains had similar LD50 values for both orally and intraperitoneally infected mice. The invasion defect of the inv mutant was fully complemented in vitro and in vivo by introduction of the wild-type inv gene in trans. The inv gene product, invasin, appears to play a vital role in promoting entry during the initial stage of infection. During the subsequent establishment of a systemic infection, invasin may be of secondary importance, since the Y. enterocolitica inv mutant was as proficient as wild type at causing a fatal infection in mice. Based on these data, we discuss the role of invasin in a naturally occurring Y. enterocolitica infection.

The Yersinia enterocolitica inv gene product is an outer membrane protein that shares epitopes with Yersinia pseudotuberculosis invasin.

An essential virulence attribute for Yersinia enterocolitica and Yersinia pseudotuberculosis is the ability to invade the intestinal epithelium of mammals. The chromosomal invasin gene (inv) has been cloned from both of these Yersinia species, and the Y. pseudotuberculosis invasin has been well characterized (R. R. Isberg, D. L. Voorhis, and S. Falkow, Cell 50:769-778, 1987). Here we constructed TnphoA translational fusions to the Y. enterocolitica inv gene to identify, characterize, and localize the inv protein product in Escherichia coli. The cloned Y. enterocolitica inv locus encoded a unique protein of ca. 92 kilodaltons when expressed in minicells. A protein of comparable size was detected in immunoblots by using monoclonal antibodies directed against the Y. pseudotuberculosis invasin. This protein, which we also refer to as invasin, promoted both attachment to and invasion of cultured epithelial cells. These two functions were not genetically separable by insertional mutagenesis. We determined that the Y. enterocolitica invasin was localized on the outer membrane and that it was exposed on the bacterial cell surface, which may have implications for how invasin functions to mediate invasion.