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

Abstract

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 first two leucines of the AraC, UreR, and XylS triads align with F151 and F152 of Vibrio cholerae ToxT. An F151 alanine substitution results in a monomeric form of ToxT that is unable to bind the tcpA promoter to wild-type levels, resulting in loss of murine intestinal colonization (31). For ExsA, pairwise combinations of the L140A, K141A, and L148A substitutions were sufficient to generate mutants with significant defects in self-association and promoter activation (Fig.…”

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“…V. cholerae strain KKV2426 (C6706 ΔtoxT::Cm) was created via chitin transformation (27) using genomic DNA from strain SY1002 (28). V. cholerae strains KKV2333 (O395 toxT 5′ UTR U5C), KKV2368 (O395 toxT 5′ UTR U7C), KKV2425 (O395 toxT 5′ UTR U5C, U7C), KKV2382 (C6706 toxT 5′ UTR U5C, U7C), as well as KKV598 (O395 native toxT 5′ UTR) and KKV2372 (C6706 native toxT 5′ UTR), were constructed by using plasmids pKEK1445, pKEK1468, pKEK1469, and pKEK1808 to introduce the mutations into V. cholerae strains KKV2288 (29) and KKV2426 by allelic exchange. A complete list of primers and plasmids used is provided in Tables S1 and S2.…”

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