Cation Regulation And Proteomic Analysis Of Phase Variation And Biofilm Formation In The Human Pathogen Vibrio Vulnificus

Vibrio vulnificus, a Gram-negative halophile which is ubiquitously present in the marine environments, can cause diseases like gastroenteritis, septicemia and wound infections in susceptible human beings. Production of capsular polysaccharide results in an opaque phenotype, which is pathogenic to humans. Loss or reduction of capsule results in translucent colonies composed of cells that are nonpathogenic or have diminished virulence, respectively. The opaque and the translucent phenotypes, upon production of an exopolysaccharide, give rise to a dry, wrinkled rugose form that can form copious biofilms. These three phenotypes can spontaneously switch from one form to another in a process known as phase variation. In this study, we have sought to identify environmental factors and genetic mechanisms that affect phase variation and biofilm formation in V. vulnificus. Elements such as Ba2+, Mn2+, and Sr2+, were tested to determine their effect on V. vulnificus phase variation and we found that manganese at micromolar to millimolar concentrations, induces V.vulnificus polysaccharide phase variation from opaque to translucent or rugose forms in this species. Since calcium was previously shown to induce significant phase variation in different V.vulnificus strains, we have also attempted to compare the effects of equimolar addition of manganese and calcium on phase variation of different V.vulnificus strains. Here, we have also attempted to better understand the process of V. vulnificus rugose colony formation. Our study has identified 28 proteins that include translational elongation factors, various metabolic enzymes, proteases, sugar binding proteins, amino acid transporters, polar flagellins and an uncharacterized protein that are differentially expressed in the biofilm proficient rugose phase variant compared to the parent isogenic opaque phase variant. One protein of particular interest, MalE (VVA0397 gene product), a maltose binding periplasmic protein, was identified as being expressed at higher amounts, and concomitantly showed higher transcript levels in the rugose variant compared to the opaque variant. Interestingly, generation of a targeted mutant of the malE gene did not knock out rugosity, indicating that the malE (VVA0397) gene is not required for rugosity in V.vulnificus. However, its up-regulation at both the transcriptional and translational levels suggests that MalE protein is involved in rugose colony formation in a way that remains to be determined