Won Hwang

Role of capsular polysaccharide (CPS) in biofilm formation and regulation of CPS production by quorum-sensing in Vibrio vulnificus.

Extracellular 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 quorum‐sensing (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.

Functional Characterization of EpsC, a Component of the Type II Secretion System, in the Pathogenicity of Vibrio vulnificus

ABSTRACT EpsC, one of the components comprising the type II secretion system (T2SS), was isolated from a human-pathogenic 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 ΔepsC 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.