Bile acids induce cholera toxin expression in Vibrio cholerae in a ToxT-independent manner

Abstract

The production of cholera toxin (CT) during Vibrio cholerae infection results in the hallmark diarrhea that characterizes the disease cholera. The transmembrane protein ToxR was originally identified as a functional transcriptional activator of ctxAB in a heterologous Escherichia coli system. However, direct ToxR activation of the ctxAB promoter in V. cholerae has not been previously demonstrated. Instead, a regulatory cascade has been defined in which the activators ToxRS and TcpPH modulate ctxAB expression by acting in concert to transcriptionally activate another regulator, ToxT. ToxT, in turn, directly activates ctxAB expression as well as expression of the tcp genes and other virulence-associated genes. In this study, we show that ToxRS directly activates ctxAB in a ToxT-independent manner in a classical biotype V. cholerae, and that this activation requires the presence of bile acids. Although the levels of CT induced by this mechanism are lower than levels induced under other in vitro conditions, the bile-dependent conditions described here are more physiologic, being independent of pH and temperature. We further show that the inability of bile acids to stimulate ToxRS-dependent expression of CT in El Tor biotype strains is related to the differences between classical and El Tor ctxAB promoters, which differ in the number of heptad TTTTGAT repeats in their respective upstream regions. The ability of bile acids to stimulate direct activation of ctxAB by ToxRS depends upon the transmembrane domain of ToxR, which may interact with bile acids in the inner membrane of V. cholerae. The Gram-negative bacterium Vibrio cholerae is the infectious agent that causes the severe human diarrheal disease cholera (1). Two major virulence factors, cholera toxin (CT) and the toxin-coregulated pilus (TCP), play major roles in the pathogenesis of this infection. Secretion of CT, an ADP-ribosylating toxin encoded in the genome of the filamentous, lysogenic CTX⌽ phage, results in elevated cAMP levels in intestinal epithelial cells and subsequent secretory diarrhea (2). The regulation of CT and TCP expression has been studied intensely and has been reported to respond to temperature, pH, osmolarity, bile salts, and certain amino acids in vitro (3, 4); nevertheless, a clear understanding of the in vivo environmental stimuli that trigger CT and TCP production remains elusive.The first transcriptional regulator of CT production identified was ToxR, a 32-kDa inner-membrane protein that activates the ctx promoter in Escherichia coli and whose deletion in V. cholerae results in the inability to produce CT (5). Deletion analysis of the ctxAB promoter localized the binding site of ToxR to multiple heptad repeats, TTTTGAT, upstream of ctxAB (6). However, ToxR activation of ctxAB transcription in V. cholerae has not been demonstrated (7). Instead, ToxR in V. cholerae binds with its downstream enhancer ToxS to the upstream region of the toxT gene, which encodes another transcriptional activator (8). ToxT belongs to the AraC family of trans...

Bile salt–induced intermolecular disulfide bond formation activates Vibrio cholerae virulence

“…Bile salts have previously been implicated in virulence in Vibrio parahaemolyticus (19), but the mechanism of induction is unclear. Interestingly, it has been shown that in a classical strain of V. cholerae (20), bile salts also may affect ToxR activity to enhance ctx production independent of ToxT. Although the induction in this case was rather minimal compared with the maximal ctx expression, it does suggest that bile salts may play additional roles in regulating virulence.…”

Modulation ofVibrio choleraePorin Function by Acidic pH

“…2 These two proteins play a major role in controlling the production of virulence factors, such as cholera toxin; in addition, ToxR controls the expression of the two general diffusion porin genes ompU and ompT. [3][4][5][6][7] Once inside the host, the activation of ToxR is believed to turn on ompU expression while repressing ompT expression. Although OmpU and OmpT are not required for virulence, 8 the type of porin present in the outer membrane may have an impact on the ability of V. cholerae to colonize the duodenum.…”

Molecular Analysis of BcrR, a Membrane-bound Bacitracin Sensor and DNA-binding Protein from Enterococcus faecalis

“…ToxR contains a single transmembrane ␣-helix separating a cytoplasmic DNA-binding domain from a short C-terminal periplasmic domain (10). A role in signal transduction by ToxR may be likely in the presence of bile salts, where it regulates cholera toxin production (12) and expression of the outer membrane porins OmpU and OmpT (13).…”