Bile Salts Promote ToxR Regulon Activation during Growth under Virulence-Inducing Conditions

Abstract

Cholera is an epidemic disease caused by the Gram-negative bacterium Vibrio cholerae . V. cholerae is found in aquatic ecosystems and infects people through the consumption of V. cholerae contaminated food or water. Following ingestion, V. cholerae responds to host cues to activate the expression of critical virulence genes that are under control of a hierarchical regulatory system called the ToxR regulon. The ToxR regulon is tightly regulated and is only expressed in vitro under special growth conditions referred to as AKI conditions. AKI conditions have been instrumental in elucidating V. cholerae virulence regulation, but the chemical cues within AKI medium that activate virulence gene expression are unknown. In this study, we fractionated AKI medium on a reverse phase chromatography column (RPCC) and showed that the virulence activating molecules were retained on the RPCC column, and recovered in the eluate. LC-HRMS analysis of the eluate revealed the presence of a known ToxR regulon activator, taurocholate, and other bile salts. The RPCC eluate activated the ToxR regulon when added to non-inducing media and promoted TcpP dimerization in a two-hybrid system, consistent with taurocholate being responsible for the virulence-inducing activity of AKI medium. Additional experiments using purified bile salts showed that ToxR regulon was preferentially activated in response to primary bile acids. The results of this study shed light on the chemical cues involved in V. cholerae virulence activation and suggested that V. cholerae virulence genes are modulated in response to regional-specific bile acid species in the intestine.

Adaptation to bile and anaerobicity limitsVibrio choleraephage adsorption

“…Also indicative of ToxR regulon activity and bile resistance, ompU expression was increased in overnight comparisons, although the corresponding downregulation of ompT 30 was only observed in one of the overnight comparisons (Supplementary Figure S4B). Bile and anaerobicity are both known activators of the ToxR regulon 42,43 , which is consistent with the observation of its transcriptional upregulation in these conditions. ToxR induces a regulatory cascade for V. cholerae virulence gene expression induced in the human gut in the context of infection 12 , and has been shown to be highly transcriptionally upregulated in both rabbit and mouse animal models of cholera 44 .…”

Vibrio cholerae RND efflux systems: mediators of stress responses, colonization and pathogenesis

“…Interestingly, ToxR’s periplasmic sensing domain is required for activation of leuO transcription suggestive of an efflux-dependent small molecule feedback mechanism tying ToxR to leuO expression. Consistent with these observations, ToxR induced leuO expression in the presence of multiple RND efflux substrates including bile acids, indole, and diketopiperazines, the former two being RND efflux substrates that are abundant in the human gastrointestinal tract ( Bina et al., 2009 ; Bina and Bina, 2010 ; Bina et al., 2013 ; Ante et al., 2015b ; Howard et al., 2019 ; Bina et al., 2021 ). Furthermore, the RND efflux inhibitor phenylalanine-arginine β-naphthylamide induced leuO expression by a mechanism dependent on the presence of the ToxR periplasmic sensing domain suggestive of a regulatory feedback circuit that inhibits efflux of the transporter’s native substrates to modulate the ToxR regulon.…”

Holistic analysis of lysine acetylation in aquaculture pathogenic bacteria Vibrio alginolyticus under bile salt stress

“…In this study, we also found that ToxR protein, a key transcription activator, is acetylated. Bile salt can promote the activation of toxR regulator and participate in the regulation of bacterial virulence genes ( 48 , 49 ). Bacterial toxin factor ( AOG25_14550 ) is a Hcp1 family type VI secretion system effector, which also acetylated under bile salt stress in V. alginolyticus .…”