Genetic Variation in the Vibrio vulnificus Group 1 Capsular Polysaccharide Operon

Abstract

Vibrio vulnificus produces human disease associated with raw-oyster consumption or wound infections, but fatalities are limited to persons with chronic underlying illness. Capsular polysaccharide (CPS) is required for virulence, and CPS expression correlates with opaque (Op) colonies that show "phase variation" to avirulent translucent (Tr) phenotypes with reduced CPS. The results discussed here confirmed homology of a V. vulnificus CPS locus to the group 1 CPS operon in Escherichia coli. However, two distinct V. vulnificus genotypes or alleles were associated with the operon, and they diverged at sequences encoding hypothetical proteins and also at unique, intergenic repetitive DNA elements. Phase variation was examined under conditions that promoted high-frequency transition of Op to Tr forms. Recovery of Tr isolates in these experiments showed multiple genotypes, which were designated TR1, TR2, and TR3: CPS operons of TR1 isolates were identical to the Op parent, and cells remained phase variable but expressed reduced CPS. TR2 and TR3 showed deletion mutations in one (wzb) or multiple genes, respectively, and deletion mutants were acapsular and locked in the Tr phase. Complementation in trans restored the Op phenotype in strains with the wzb deletion mutation. Allelic variation in repetitive elements determined the locations, rates, and extents of deletion mutations. Thus, different mechanisms are responsible for reversible phase variation in CPS expression versus genetic deletions in the CPS operon of V. vulnificus. Repetitive-element-mediated deletion mutations were highly conserved within the species and are likely to promote survival in estuarine environments.Vibrio vulnificus causes systemic human infections with high mortality (Ͼ50%), and death can occur within hours or days following exposure (3). Illness is generally a consequence of raw oyster consumption or exposure of wounds to seawater. Underlying liver disease, diabetes, hemochromatosis (iron overload), and immune system dysfunction are prerequisites for life-threatening infections. The pathogenesis of V. vulnificus has been reviewed (14, 54), and disease symptoms resemble endotoxic shock. Although the contribution of lipopolysaccharide (LPS) to virulence remains unclear (31, 38), lethality in animal models is clearly related to capsular polysaccharide (CPS) expression (38,49,62,63,64,69). Both CPS expression and virulence are associated with opaque (Op) colony morphology, but colonies can spontaneously revert to the translucent (Tr) phenotype in a process termed phase variation. Tr colonies have reduced CPS expression and diminished virulence in mouse models (64). These phenotypes are reversible for both Op3Tr and Tr3Op transitions at rates of about 10 Ϫ3 to 10 Ϫ4 , and colony morphology is currently the most reliable virulence marker for V. vulnificus (51).Bacterial phase variation typically is related to reversible mutations that alter the expression of cell surface structures (CPS, LPS, pili, flagella, or outer membrane proteins) and increase...

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

“…4A). The pattern of expression of the wcrABCDF genes was markedly different from the pattern of expression of many of the other exopolysaccharide genes studied, such as wza, a CPS transport gene of V. vulnificus, which has been reported to be missing in some TrS strains (7,58) and which, in contrast, was expressed in this study by all phase variants at all time points tested (Table 3 and Fig. 4B).…”

“…The group 1-like operon is present on the larger chromosome of V. vulnificus YJ016 and CMCP6 and of Vibrio species other than V. cholerae, which does not possess homologs of this operon (7,8). The wcr gene cluster is present on the smaller chromosome in both of the sequenced V. vulnificus strains, YJ016 and CMCP6 (8,25).…”

“…A portion of the wcrABCDFHIJK locus was originally identified as a low-identity (Ͻ25% amino acid identity; no DNA identity) homolog of the V. vulnificus group 1-like CPS operon containing wza (7,8). The group 1-like operon is present on the larger chromosome of V. vulnificus YJ016 and CMCP6 and of Vibrio species other than V. cholerae, which does not possess homologs of this operon (7,8).…”

“…It was reported recently that there are at least three different genotypes that are associated with the spontaneous appearance of the TrS phenotype and that two of the three genotypes appear to be the result of accumulated deletions in the operon, which result in strains that are locked in the TrS phase (7). Although the genetic basis of the OpS-to-TrS transition in the spontaneous TrS strain AZ(T) used in our study is unknown, AZ(T) (TrS) can convert back to the OpS phenotype, although it does so infrequently, and it was the only TrS isolate tested previously that never produced an R derivative during phenotypic switching assays (14).…”

Sticky Situations: Key Components That Control Bacterial Surface Attachment

“…appear to have negative roles. For example, loss of the group 1 capsular polysaccharide (CPS) in V. vulnificus is associated with increased attachment and, subsequently, reduced biofilm formation (23,73). V. cholerae O139 contains a locus with genes for CPS and lipopolysaccharide (LPS) O antigen biosynthesis that also plays a negative role in biofilm formation (77).…”