The human pathogen Vibrio cholerae specifically expresses virulence factors within the host, including cholera toxin (CT) and the toxin co-regulated pilus (TCP), which allow it to colonize the intestine and cause disease. V. cholerae is a highly motile organism by virtue of a polar flagellum, and motility has been inferred to be an important aspect of virulence, yet the exact role of motility in pathogenesis has remained undefined. The two-component regulatory system FlrB/FlrC is required for polar flagellar synthesis; FlrC is a σ54-dependent transcriptional activator. We demonstrate that the transcriptional activity of FlrC affects both motility and colonization of V. cholerae. In a purified in vitro reaction, FlrB transfers phosphate to the wild-type FlrC protein, but not to a mutant form in which the aspartate residue at amino acid position 54 has been changed to alanine (D54A), consistent with this being the site of phosphorylation of FlrC. The wildtype FlrC protein, but not the D54A protein, activates σ54-dependent transcription in a heterologous system, demonstrating that phospho-FlrC is the transcriptionally active form. A V. cholerae strain containing a chromosomal flrCD54A allele did not synthesize a flagellum and had no detectable levels of transcription of the critical σ54-dependent flagellin gene flaA. The V. cholerae flrCD54A mutant strain was also defective in its ability to colonize the infant mouse small intestine, approximately 50-fold worse than an isogenic wildtype strain. Another mutation of FlrC (methionine 114 to isoleucine; M1141) confers constitutive transcriptional activity in the absence of phosphorylation, but a V. cholerae flrCM114l mutant strain, although flagellated and motile, was also defective in its ability to colonize. The strains carrying D54A or M1141 mutant FlrC proteins expressed normal levels of CT and TCP under in vitro inducing conditions. Our results show that FlrC 'locked' into either an inactive (D54A) or an active (M114l) state results in colonization defects, thereby demonstrating a requirement for modulation of FlrC activity during V. cholerae pathogenesis. Thus, the σ54-dependent transcriptional activity of the flagellar regulatory protein FlrC contributes not only to motility, but also to colonization of V. cholerae.
ASJC Scopus subject areas
- Molecular Biology