The Vibrio cholerae Flagellar Regulatory Hierarchy Controls Expression of Virulence Factors

Abstract

Vibrio cholerae is a motile bacterium responsible for the disease cholera, and motility has been hypothesized to be inversely regulated with virulence. We examined the transcription profiles of V. cholerae strains containing mutations in flagellar regulatory genes (rpoN, flrA, flrC, and fliA) by utilizing whole-genome microarrays. Results revealed that flagellar transcription is organized into a four-tiered hierarchy. Additionally, genes with proven or putative roles in virulence (e.g., ctx, tcp, hemolysin, and type VI secretion genes) were upregulated in flagellar regulatory mutants, which was confirmed by quantitative reverse transcription-PCR. Flagellar regulatory mutants exhibit increased hemolysis of human erythrocytes, which was due to increased transcription of the thermolabile hemolysin (tlh). The flagellar regulatory system positively regulates transcription of a diguanylate cyclase, CdgD, which in turn regulates transcription of a novel hemagglutinin (frhA) that mediates adherence to chitin and epithelial cells and enhances biofilm formation and intestinal colonization in infant mice. Our results demonstrate that the flagellar regulatory system modulates the expression of nonflagellar genes, with induction of an adhesin that facilitates colonization within the intestine and repression of virulence factors maximally induced following colonization. These results suggest that the flagellar regulatory hierarchy facilitates correct spatiotemporal expression patterns for optimal V. cholerae colonization and disease progression.Vibrio cholerae causes the human diarrheal disease cholera. The bacteria are natural inhabitants of aquatic environments and are introduced into the human population through the ingestion of contaminated food or water. Within the human host, V. cholerae expresses virulence factors that facilitate colonization of the intestine (e.g., toxin-coregulated pilus [TCP]) and that stimulate dramatic fluid loss from host tissues (cholera toxin [CT]) (5, 61). A regulatory cascade consisting of a number of different proteins, including ToxR, TcpP, and ToxT, induces the coordinated expression of CT and TCP maximally within the intestine and under specific in vitro growth conditions (for a review, see reference 7).V. cholerae is a highly motile organism by virtue of its single polar flagellum. Flagellar genes are transcribed in a four-tiered transcriptional hierarchy (51). The single class I gene product FlrA activates 54 -dependent transcription of class II genes, which encode components of the MS ring-switch-export apparatus as well as the two-component system FlrBC (31). Phosphorylated FlrC activates 54 -dependent transcription of class III genes, which encode the basal body-hook and the flagellin FlaA (10, 11). Finally, the antisigma factor FlgM is secreted through the basal body-hook to allow 28 -dependent transcription of class IV genes, which encode four additional flagellins and some of the motor components (9, 30). Motility has been linked to the virulence of V. cholerae. Spontaneous nonmot...

Neutrophils Are Essential for Containment of Vibrio cholerae to the Intestine during the Proinflammatory Phase of Infection

“…It has been proposed that optimal next generation live attenuated vaccines would have defined mutations to remove flagellins to prevent reactogenic diarrhea in vaccine recipients (2). However, due to the transcriptional hierarchy of the flagellin system, class II nonmotile mutants such as the ⌬flaA mutant have a consequence of increased transcription of numerous adherence and virulence factors, including tcp genes, gbpA, hlx, frhA, and hlyA (52). These factors may be responsible for the residual reactogenicity of ⌬fla mutants resulting in 12% of animals developing mild to severe diarrhea (47).…”

Vibrio cholerae Flagellar Synthesis and Virulence

“…cdGMP is an important signaling molecule that modulates complex behaviors in bacteria, most notably biofilm formation (discussed below). The results demonstrate that the flagellar hierarchy controls the transcription of non-flagellar genes that contribute to other aspects of the V. cholerae lifecycle besides motility (Syed, Beyhan et al 2009). …”

“…Spontaneous non-motile V. cholerae strains were characterized as less virulent than motile strains in several in vivo and in vitro rabbit models of cholera. Mutations that adversely affect flagellar synthesis and motility generally lead to decreased intestinal colonization in infant mice (Guentzel and Berry 1975;Montie, Doyle-Huntzinger et al 1982;Carsiotis, Weinstein et al 1984;Weinstein, Carsiotis et al 1984;Schmitt, Darnell et al 1994;Kennedy, Rosey et al 1997;Watnick, Lauriano et al 2001;Syed, Beyhan et al 2009). Non-motile live attenuated V. cholerae vaccine strains exhibit reduced reactogenicity (disease symptoms) in human volunteers, when compared to motile isogenic strains.…”

“…Utilizing whole genome transcription profiling of V. cholerae strains with mutations in the key flagellar regulatory genes (rpoN, flrA, flrC, and fliA), it was observed that non-flagellated strains exhibit increased transcription of known (CT, TCP) and putative virulence factors (T6SS, hemolysins, etc) (Syed, Beyhan et al 2009). The results suggest coordinate regulation by the flagellar regulatory hierarchy over a variety of virulence factors whose regulation was previously thought to be unlinked (Syed, Beyhan et al 2009). …”

“…The transcriptional profiling of the flagellar regulatory mutants identified the flagellar-regulated hemolysin as TLH, which is encoded adjacent to HlyA, the "El Tor" hemolysin (Syed, Beyhan et al 2009). Also identified was the flagellar-regulated hemagglutinin, FrhA, which is a large cadherin-containing protein that enhances binding to epithelial cells in vitro and intestinal colonization in both infant and adult mice.…”