Phosphorylation of the flagellar regulatory protein FlrC is necessary for Vibrio cholerae motility and enhanced colonization

Abstract

SummaryThe 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 s 54 -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 s 54 -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 s 54 -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; M114I) confers constitutive transcriptional activity in the absence of phosphorylation, but a V. cholerae flrCM114I mutant strain, although flagellated and motile, was also defective in its ability to colonize. The strains carrying D54A or M114I 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 (M114I) state results in colonization defects, thereby demonstrating a requirement for modulation of FlrC activity during V. cholerae pathogenesis. Thus, the s 54 -dependent transcriptional activity of the flagellar regulatory protein FlrC contributes not only to motility, but also to colonization of V. cholerae.

Polar Flagellar Motility of the Vibrionaceae

“…The homologous gene clusters are shown in Table 4. Much is known about the role of the gene products in flagellar regulation in V. cholerae and P. aeruginosa (4,35,89,153). Based on these studies, the following model has been proposed.…”

“…V. cholerae FlrB (the FlaL homolog) is an autokinase that can transfer phosphate to FlrC (the FlaM homolog), specifically to the aspartate that is equivalent to Asp57 of CheY. FlrC must be phosphorylated to activate flagellar gene transcription (35).…”

Characterization of Enhancer Binding by the Vibrio cholerae Flagellar Regulatory Protein FlrC

“…Phosphorylation of the conserved aspartate residue (D54) in the response regulatory N-terminal domain by the histidine kinase FlrB is required for FlrC-dependent transcriptional activation, as well as enhanced intestinal colonization (5,21). FlrC shares homology over its entire length with the well-characterized 54 -dependent response regulator NtrC, and thus its mechanism of transcription activation can be inferred from studies on NtrC (29).…”

“…studied (26), as well as in Pseudomonas aeruginosa (7) and Campylobacter jejuni (14,17), suggesting that similar mechanisms underlie the regulation of class III genes in polar flagellates. Our previous results showed that strains containing FlrC locked into an inactive (D54A) or constitutively active (M114I) state demonstrated more severe defects in intestinal colonization than strains lacking FlrC entirely (5). We reasoned that this observation could be due to a repressive effect of FlrC binding upon some gene(s) necessary for V. cholerae virulence, and undertook the characterization of FlrC DNA binding.…”

Integration of Global Regulatory Mechanisms Controlling Vibrio Cholerae Behavior

“…The V. cholerae genome encodes multiple flagellin genes (fllaABCDE), but only flagellin mutants lacking FlaA are nonflagellated Klose & Mekalanos, 1998a). The expression of motility requires a hierarchical regulatory cascade involving the alternative RNA polymerase (RNAP) subunits,  54 and  28 and the  54 -dependent transcriptional activators, FlrA and FlrC (Correa et al, 2004;Correa et al, 2000;Prouty et al, 2001;Syed et al, 2009). …”