SummaryExtracellular polysaccharides, such as lipopolysaccharide and loosely associated exopolysaccharides, are essential for Vibrio vulnificus to form biofilms. The role of another major component of the V. vulnificus extracellular matrix, capsular polysaccharide (CPS), which contributes to colony opacity, has been characterized in biofilm formation. A CPS-deficient mutant, whose wbpP gene encoding UDP-GlcNAc C4-epimerase was knocked out, formed significantly more biofilm than wild type, due to increased hydrophobicity of the cell surface, adherence to abiotic surfaces and cell aggregation. To elucidate the direct effect of CPS on biofilm structure, extracted CPS and a CPS-degrading enzyme, α-N-acetylgalactosaminidase, were added in biofilm assays, resulting in reduction and increment of biofilm sizes respectively. Therefore, it is suggested that CPS play a critical role in determining biofilm size by restricting continual growth of mature biofilms. Since CPS is required after maturation, CPS biosynthesis should be controlled in a cell density-dependent manner, e.g. by quorumsensing (QS) regulation. Analysing transcription of the CPS gene cluster revealed that it was activated by SmcR, a QS master regulator, via binding to the upstream region of the cluster. Therefore, CPS was produced when biofilm cell density reached high enough to turn on QS regulation and limited biofilms to appropriate sizes.

Results of our previous studies have shown that the chemiluminescence response of human neutrophils (polymorphonuclear leukocytes [PMNs]) is inhibited by plasmid-mediated cell surface components from Yersinia enterocolitica. In this study we examined the susceptibility to phagocytosis of Y. enterocolitica cells with or without plasmid-mediated surface structure and the effect of isolated outer membrane fragments on phagocytosis of Escherichia coli by PMNs in vitro. Y. enterocolitica cells with expressed plasmid-mediated surface structure were much less sensitive to ingestion by PMNs than those without it, and the resistance to phagocytosis was readily eliminated in a dose-dependent fashion by pronase treatment of whole cells, which was shown to remove plasmid-encoded outer membrane proteins. Ingestion and intracellular killing of E. coli were inhibited significantly in the presence of isolated outer membrane fragments derived from plasmid-bearing Y. enterocolitica cells. To assess the interaction of Y. enterocolitica with phagocytic cells in vivo, two isogenic strains of Y. enterocolitica, differing only in the presence or absence of the virulence plasmid, were inoculated intradermally into the backs of rabbits; and tissue sections obtained at 12 h postinoculation were examined by light and electron microscopy. The plasmidless strain was found almost entirely in PMNs or mononuclear cells. In contrast, the plasmid-bearing strain was found to be surrounded by, or interspersed with, PMNs and mononuclear cells; but most bacteria were extracellular, with little evidence of phagocytosis. These results suggest that plasmid-mediated cell surface components of Y. enterocolitica act as antiphagocytic factors, thus facilitating the survival and proliferation of the organism in the host tissue.

Summary. Rabbits were given, by the intra-gastric route, two isogenic strains of Yersinia enterocolitica that differed only in the presence or absence of the virulence plasmid. Clinical illness and characteristic morphological lesions of Y . enterocolitica infection were seen only in rabbits infected with the plasmid-bearing strain (MCH700S). Although rabbits infected with a strain lacking the plasmid (MCH700L) remained healthy, mild histological changes in the small intestine, consisting of epithelial-cell damage, dilatation of lymphatics and a slight increase in neutrophil polymorphonuclear leukocytes in lamina propria, were seen in the first 12 h after inoculation. Bacteria, which were identified as Y . enterocolitica by indirect fluorescent anti body staining, were seen in dilated lymphatics. These early lesions tended to abate quickly and were no longer detectable at 24 h. Strain MCH700L was recovered from the mesenteric lymph nodes in increasing numbers until 24 h after inoculation; the number then began to decrease rapidly. In contrast, the early lesions in rabbits givFn strain MCHIIOOS progressed to micro-abscesses, focal destruction of villi, and ulcerations beginning 24 h after inoculation; the number of bacteria recovered from the lymph nodes continued to increase beyond 24 h after inoculation. Bacteria were also recovered from the liver and spleen. These results suggest that both plasmidbearing and non-bearing strains of Y. enterocolitica are capable of penetrating the intestinal mucosa. However, the virulence plasmid is required for invading bacteria to proliferate in the host tissue and to establish infection.

EpsC, one of the components comprising the type II secretion system (T2SS), was isolated from a humanpathogenic bacterium, Vibrio vulnificus, to evaluate its role in eliciting virulence. An espC-deleted mutant of V. vulnificus displayed a reduced cytotoxicity to the human cell line HEp-2 and an attenuated virulence in a mouse model. This mutant exhibited dramatic defects in the secretion of diverse extracellular proteins, such as outer membrane proteins, transporters, and the known secreted factors, notably, a hemolysin (VvhA) and an elastase (VvpE). A defect in its secretion of proteins was restored by in trans complementation of the intact epsC gene. Analyses of cellular fractions revealed that VvhA and VvpE of the ⌬epsC mutant were not excreted outside the cell but were present mainly in the periplasmic space. Examination of a V. vulnificus mutant deficient in TolC, a component of the T1SS, showed that it is not involved in the secretion of VvhA and VvpE but that it is necessary for the secretion of another major toxin of V. vulnificus, RtxA. Therefore, the T2SS is required for V. vulnificus pathogenicity, which is mediated by at least two secreted factors, VvhA and VvpE, via facilitating the secretion and exposure of these factors to host cells.