Direct Regulation by the<i>Vibrio cholerae</i>Regulator ToxT To Modulate Colonization and Anticolonization Pilus Expression

Hsiao, Ansel; Xu, Xiao Yan; Kan, Biao; Kulkarni, Rahul; Zhu, Jun

doi:10.1128/iai.01156-08

Cited by 22 publications

(27 citation statements)

References 29 publications

(36 reference statements)

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“…Although analogous leucine residues are not present in α3, if ToxT were to dimerize in a manner similar to that observed in AraC, complementary salt bridges would be formed between helix α3 residues such as D141, E142, K157, and K158 of one monomer and the same residues on the other monomer. In fact, Hsaio et al have suggested that a D141G substitution is able to repress msh promoters as a monomer, but is unable to activate tcp (25).…”

Section: Resultsmentioning

confidence: 99%

Structure of Vibrio cholerae ToxT reveals a mechanism for fatty acid regulation of virulence genes

Lowden

Skorupski²,

Pellegrini³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

152

240

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Cholera is an acute intestinal infection caused by the bacterium Vibrio cholerae. In order for V. cholerae to cause disease, it must produce two virulence factors, the toxin-coregulated pilus (TCP) and cholera toxin (CT), whose expression is controlled by a transcriptional cascade culminating with the expression of the AraCfamily regulator, ToxT. We have solved the 1.9 Å resolution crystal structure of ToxT, which reveals folds in the N-and C-terminal domains that share a number of features in common with AraC, MarA, and Rob as well as the unexpected presence of a buried 16-carbon fatty acid, cis-palmitoleate. The finding that cis-palmitoleic acid reduces TCP and CT expression in V. cholerae and prevents ToxT from binding to DNA in vitro provides a direct link between the host environment of V. cholerae and regulation of virulence gene expression.AraC | crystal structure | pathogenesis | oleic acid | palmitoleic acid

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Section: Resultsmentioning

confidence: 99%

Structure of Vibrio cholerae ToxT reveals a mechanism for fatty acid regulation of virulence genes

Lowden

Skorupski²,

Pellegrini³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

152

240

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“…These observations suggest that ToxT must be dimerized to activate transcription. However, the isolated (monomeric) C terminus has been shown to bind to the mshA promoter region, which is repressed by ToxT, suggesting that monomeric ToxT, although not transcriptionally active, can still bind certain promoters and repress their expression (13).…”

mentioning

confidence: 99%

N-terminal Residues of the Vibrio cholerae Virulence Regulatory Protein ToxT Involved in Dimerization and Modulation by Fatty Acids

Childers

Cao

Weber

et al. 2011

Journal of Biological Chemistry

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The regulatory protein ToxT is an AraC family protein that is responsible for activating transcription of the genes encoding cholera toxin and toxin coregulated pilus, which are required for virulence by the human pathogen Vibrio cholerae. The N terminus of ToxT contains dimerization and regulatory elements, whereas the C terminus contains the DNA binding domain. Bile and long chain fatty acids negatively regulate ToxT activity. Utilizing a comprehensive alanine substitution mutant library of ToxT, 19 N-terminal residues were found to be critical for dimerization and transcriptional activation. One of these mutant proteins (F151A) was confirmed to be monomeric via centrifugation and exhibited a weakened ability to bind to the tcpA promoter in a gel mobility shift assay. Moreover, a V. cholerae toxTF151A mutant failed to colonize the infant mouse intestine, emphasizing the importance of ToxT N-terminal dimerization to cholera pathogenesis. Six N-terminal alanine substitutions allowed ToxT transcriptional activity in the presence of inhibitory concentrations of bile, palmitoleic acid, and the small molecule inhibitor virstatin. Two of these mutations (N106A and L114A) enhance N-terminal dimerization in a bacterial twohybrid system reconstituted in V. cholerae, which is otherwise disrupted by bile, palmitoleic acid, and virstatin. We demonstrate that V. cholerae toxTN106A and toxTL114A strains colonize the infant mouse intestine at significantly higher levels than the wild type strain. Our results demonstrate that ToxT N-terminal dimerization is required for transcriptional activation and cholera pathogenesis and that fatty acids modulate ToxT activity via modulation of dimerization.Vibrio cholerae causes the disease cholera, a life-threatening diarrheal illness that affects thousands of people annually (1). The bacterium is acquired through the consumption of contaminated food or water and colonizes the small intestine (2). In the intestinal environment, V. cholerae expresses two critical virulence factors that facilitate colonization and disease symptoms, cholera toxin (CT) 2 and toxin-coregulated pilus (TCP). CT is an ADP-ribosylating toxin that is translocated into host cells and modifies G s ␣, causing an ion imbalance that leads to the profuse diarrhea associated with the disease (2). TCP is a type IV bundle-forming pilus that is required for intestinal colonization (3).Expression of CT and TCP is coordinately regulated by the transcriptional activator ToxT (4). ToxT, an AraC/XylS family activator, directly binds to the promoters of the ctx and tcp genes (which encode CT and TCP) and activates their transcription (5). V. cholerae strains lacking ToxT express no CT or TCP and are unable to colonize the intestine and cause disease, emphasizing the central role this regulatory protein plays in cholera pathogenesis (6). Transcription of toxT is regulated by a virulence cascade commonly referred to as the ToxR regulon, which responds to various environmental stimuli to ensure that ToxT is only expressed within the...

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“…The P toxT -lacZ plasmid reporter was constructed by cloning toxT promoter DNA into LacZ transcriptional reporter plasmid pAH6 (30). Overnight cultures of the V. cholerae ⌬tcpPH ⌬toxRS mutant containing P toxT -lacZ reporters and various P BAD -tcpPH and P tac -toxRS plasmids were diluted 1:50 (for microaerophilic growth) or 1:2,000 (for aerobic growth) into fresh AKI medium (1.5% Bacto peptone, 0.4% yeast extract, 0.5% sodium chloride, pH 7.5) supplemented with the appropriate antibiotics, 0.01% arabinose, and 1 M IPTG and incubated at 37°C (stationary or shaking) until the OD 600 reached 0.3.…”

Section: Methodsmentioning

confidence: 99%

Enhanced Interaction of Vibrio cholerae Virulence Regulators TcpP and ToxR under Oxygen-Limiting Conditions

Fan

Jabeen

et al. 2014

Infect Immun

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dVibrio cholerae is the causative agent of the diarrheal disease cholera. The ability of V. cholerae to colonize and cause disease requires the intricately regulated expression of a number of virulence factors during infection. One of the signals sensed by V. cholerae is the presence of oxygen-limiting conditions in the gut. It has been shown that the virulence activator AphB plays a key role in sensing low oxygen concentrations and inducing the transcription of another key virulence activator, TcpP. In this study, we used a bacterial two-hybrid system to further examine the effect of oxygen on different virulence regulators. We found that anoxic conditions enhanced the interaction between TcpP and ToxR, identified as the first positive regulator of V. cholerae virulence genes. We further demonstrated that the TcpP-ToxR interaction was dependent on the primary periplasmic protein disulfide formation enzyme DsbA and cysteine residues in the periplasmic domains of both ToxR and TcpP. Furthermore, we showed that in V. cholerae, an interaction between TcpP and ToxR is important for virulence gene induction. Under anaerobic growth conditions, we detected ToxR-TcpP heterodimers, which were abolished in the presence of the reducing agent dithiothreitol. Our results suggest that V. cholerae may sense intestinal anoxic signals by multiple components to activate virulence.

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Direct Regulation by theVibrio choleraeRegulator ToxT To Modulate Colonization and Anticolonization Pilus Expression

Cited by 22 publications

References 29 publications

Structure of Vibrio cholerae ToxT reveals a mechanism for fatty acid regulation of virulence genes

Structure of Vibrio cholerae ToxT reveals a mechanism for fatty acid regulation of virulence genes

N-terminal Residues of the Vibrio cholerae Virulence Regulatory Protein ToxT Involved in Dimerization and Modulation by Fatty Acids

Enhanced Interaction of Vibrio cholerae Virulence Regulators TcpP and ToxR under Oxygen-Limiting Conditions

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