Vibrio cholerae leuO Transcription Is Positively Regulated by ToxR and Contributes to Bile Resistance

Abstract

Vibrio cholerae is an aquatic organism and facultative human pathogen that colonizes the small intestine. In the small intestine, V. cholerae is exposed to a variety of antimicrobial compounds, including bile. V. cholerae resistance to bile is multifactorial and includes alterations in the membrane permeability barrier that are mediated by ToxR, a membrane-associated transcription factor. ToxR has also been shown to be required for activation of the LysR family transcription factor leuO in response to cyclic dipeptides. LeuO has been implicated in the regulation of multiple V. cholerae phenotypes, including biofilm production and virulence. In this study, we investigated the effects of bile on leuO expression. We show that leuO transcription increased in response to bile and bile salts but not in response to other detergents. The bile-dependent increase in leuO expression was dependent on ToxR, which was found to bind directly to the leuO promoter. The periplasmic domain of ToxR was required for basal leuO expression and for the bile-dependent induction of both leuO and ompU transcription. V. cholerae mutants that did not express leuO exhibited increased bile susceptibility, suggesting that LeuO contributes to bile resistance. Our collective results demonstrate that ToxR activates leuO expression in response to bile and that LeuO is a component of the ToxR-dependent responses that contribute to bile resistance. IMPORTANCEThe success of Vibrio cholerae as a human pathogen is dependent upon its ability to rapidly adapt to changes in its growth environment. Growth in the human gastrointestinal tract requires the expression of genes that provide resistance to host antimicrobial compounds, including bile. In this work, we show for the first time that the LysR family regulator LeuO mediates responses in V. cholerae that contribute to bile resistance. Vibrio cholerae is a Gram-negative bacterial pathogen and the causal agent of the severe diarrheal disease cholera. V. cholerae exists naturally in aquatic reservoirs and is capable of colonizing the human small intestine. The transition of V. cholerae from the aquatic ecosystem to growth in the human gastrointestinal tract is mediated by transcriptional responses that are required for colonization and disease development. Many of the genes that contribute to intestinal colonization are under the control of the membrane-associated regulatory protein ToxR, which functions as one of the primary regulators in the ToxR regulon (reviewed in reference 1). The ToxR regulon is divided into two branches, a ToxTdependent branch, which controls the expression of virulence factors, and a ToxT-independent branch, which reciprocally regulates the production of the outer membrane porins OmpU and OmpT. The ToxT-dependent branch of the ToxR regulon is a hierarchical regulatory cascade that regulates the expression of genes encoding the production of cholera toxin (CT) and the toxin-coregulated pilus (TCP) in response to environmental cues in the host.ToxR is a membrane-associated ...

Vibrio cholerae LeuO Links the ToxR Regulon to Expression of Lipid A Remodeling Genes

“…ToxR also activates leuO transcription in response to bile salts and cyclic dipeptides (10, 11). LeuO has been linked to multiple phenotypes in V. cholerae, including biofilm production, virulence repression, acid tolerance, and bile resistance (10,11,19,20). Taken together, these results suggest that in addition to reg- …”

“…The observation that ToxR only represses carRS transcription in the presence of leuO is consistent with the conclusion that ToxR functions indirectly at the carRS promoter. This conclusion is further supported by the fact that the carRS promoter lacks a consensus ToxR binding site that is found in the promoter region of many ToxR-regulated genes, including leuO (10,43).…”

“…In the toxT-independent branch, ToxR regulates the expression of the ompU and ompT porins (8,9). Recent studies in our laboratory have shown that ToxR also regulates the expression of the LysR family transcription factor leuO independent of toxT (10,11).…”

“…OmpU serves as a sensor for the sigma E membrane stress response, and cells that lack ompU (i.e., ompU or toxR mutants) fail to activate the sigma E membrane stress response upon membrane perturbation, a phenotype that has been linked to increased CAMP susceptibility (17,18). ToxR also activates leuO transcription in response to bile salts and cyclic dipeptides (10,11). LeuO has been linked to multiple phenotypes in V. cholerae, including biofilm production, virulence repression, acid tolerance, and bile resistance (10,11,19,20).…”

“…The periplasmic domain is thought to function as a sensor which transduces extracellular cues to effect the activity of the cytoplasmic DNA binding domain. ToxR has been shown to directly regulate ompU, ompT, and leuO expression in response to bile salts (10,14). In the presence of bile salts, ToxR activates ompU expression while repressing ompT expression.…”