Elucidation of the molecular mechanisms of Vibrio cholerae virulence and biofilm gene induction following host cell contact.

Vibrio cholerae, etiological agent of the disease cholera, is known to form biofilms for persistence in the environment. Previously it has been shown that host cell contact can induce virulence in adhered bacteria. The effect of host cell contact on biofilm formation was however not known. It is demonstrated here that about 7 fold upregulation of the biofilm regulatory gene vpsT was observed within 30 minutes of adherence of V. cholerae to the intestinal cell line INT 407 and a massive induction of about 700 fold was observed in rabbit ileal loops. The upregulation was observed in both the classical and El Tor biotype strains of serogroup O1 that is most frequently associated with epidemic cholera. Though a number of regulators are known to regulate vpsT in vitro, vpsT upregulation was primarily dependent on the virulence master regulator AphA in adhered bacteria with VpsR playing a secondary role. Quorum sensing regulator LuxO, secondary molecules c-di- GMP or negative regulator HNS were found to have no role in vpsT upregulation. Though AphA is a virulence master regulator, the induction of biofilms upon host contact appears to be independent of virulence induction and a distinct temporal speciation exists between the two events. It was observed that during infection, biofilm genes are upregulated almost immediately after host cell contact and microscopic observation indicated that biofilm formation is initiated within 30 mins after adherence of V. cholerae to intestinal cells. Of possible clinical relevance was the observation that V. cholerae in the INT 407 associated biofilms was significantly more resistant to antibiotics than unadhered planktonic cells which could lead to a number of complications during treatment of cholera. The induction of vpsT was observed in vivo in rabbit ileal loops as well and it was found to be independant of the strain or biotype of V. cholerae. Overall this work is the first direct evidence that V. cholerae forms antibiotic resistant biofilm within the host in an entirely host contact dependent manner