Further Characterization of Vibrio vulnificus Rugose Variants and Identification of a Capsular and Rugose Exopolysaccharide Gene Cluster

Abstract

Capsular polysaccharide (CPS) is a major virulence factor in Vibrio vulnificus, and encapsulated strains have an opaque, smooth (OpS) colony morphology, while nonencapsulated strains have a translucent, smooth (TrS) colony morphology. Previously, we showed that OpS and TrS parental strains can yield a third colony type, rugose (R), and that the resulting strains, with the OpR and TrR phenotypes, respectively, form copious biofilms. Here we show that while OpR and TrR strains both produce three-dimensional biofilm structures that are indicative of rugose extracellular polysaccharide (rEPS) production, OpR strains also retain expression of CPS and are virulent in an iron-supplemented mouse model, while TrR strains lack CPS and are avirulent. Chlorine resistance assays further distinguished OpR and TrR isolates as exposure to 3 g/ml NaOCl eradicated both OpS and OpR strains, while both TrS and TrR strains survived, but at rates which were significantly different from one another. Taken together, these results further emphasize the importance of CPS for virulence of V. vulnificus and establish a correlation between CPS expression and chlorine sensitivity in this organism. Using reverse transcriptase PCR, we also identified a nine-gene cluster associated with both CPS and rEPS expression in V. vulnificus, designated the wcr (capsular and rugose polysaccharide) locus, with expression occurring primarily in R variants. The latter results set the stage for characterization of functional determinants which individually or collectively contribute to expression of multiple EPS forms in this pathogen.Vibrio vulnificus is a gram-negative marine and estuarine bacterium capable of causing severe disease in susceptible individuals. This bacterium is normally found in fish and shellfish, including oysters. Several medical conditions can predispose a person to being susceptible to infection by this organism, including diabetes, liver disease, hemachromatosis, and a compromised immune system. When people with such conditions consume raw oysters or other seafood containing V. vulnificus, they are at risk of developing a rapidly progressing primary septicemia that can be fatal within 24 to 48 h. In an otherwise healthy individual, if a break in the skin is exposed to seawater containing this organism, a severe wound infection may develop that could necessitate amputation if treatment is not begun soon after the onset of symptoms. V. vulnificus does not infect as many people as other members of the genus Vibrio, but it is the leading reported cause of death from the consumption of seafood in the United States (28, 48).V. vulnificus produces several virulence factors, including multiple enzymes, siderophores, RtxA toxin, and a polysaccharide capsule (for a review, see reference 15). While the other factors may assist in virulence, the capsular polysaccharide (CPS) is considered a major virulence factor and has been reported to protect the bacteria from phagocytosis and complement-mediated killing by the host immune system. When grown ...

Identification of a c-di-GMP-Regulated Polysaccharide Locus Governing Stress Resistance and Biofilm and Rugose Colony Formation in Vibrio vulnificus

“…Two of these loci coded for known CPS and putative LPS biosynthesis genes, respectively. We focused our attention on one of the four remaining loci (vv21574 to vv21582), as its expression was recently reported to be associated with the R variant (24). However, its regulation and impact, if any, on biofilm or rugose colony development were not known.…”

“…The "c" designation stemmed from the observation that a translucent (CPS Ϫ ) mutant with a Tn insertion in vv21580 failed to generate opaque colonies in a reversion assay that would be indicative of restored CPS production (24). However, the mutant was never complemented to verify that vv21580 was required for CPS production; hence the role of the wcr locus in CPS production was not definitively demonstrated.…”

“…This locus was previously designated wcr, so named because polysaccharide nomenclature traditionally designates "w" for any putative polysaccharide gene (64) and because it was preferentially expressed in the rugose ("r") variant (24). The "c" designation stemmed from the observation that a translucent (CPS Ϫ ) mutant with a Tn insertion in vv21580 failed to generate opaque colonies in a reversion assay that would be indicative of restored CPS production (24).…”

“…The rugose phenotype was shown to enhance biofilm formation, and the R variant was more resistant to serum killing than the parental strain. However, the phenotype did not correlate with resistance to any other environmental stresses (24). The CPS of V. vulnificus is essential for virulence (25,26,37,45,53,76) but is not required for biofilm or rugose colony formation (25,26,37,45,53,76).…”

“…Comparative proteomics of ntrC mutant and wild-type strains grown under planktonic and biofilm conditions suggested that NtrC exerted its effect, in part, via regulation of an ADP-glycero-manno-heptose-6-epimerase that is normally involved in biosynthesis of the inner core region of lipopolysaccharides (LPS) (14,75). Recently, reverse transcription-PCR (RT-PCR) was used to identify a polysaccharide locus whose transcription was increased in the R variant of V. vulnificus (24). However, it was not determined if this locus played any role in biofilm formation or the development of the rugose phenotype.…”