title: 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 two-hybrid 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.
authors.affiliation: South Texas Center for Emerging Infectious Diseases and Department of Biology, University of Texas, San Antonio, Texas 78249, USA.
authors.forename: Brandon M
authors.initials: BM
authors.lastname: Childers
authors.forename: Xiaohang
authors.initials: X
authors.lastname: Cao
authors.forename: Gregor G
authors.initials: GG
authors.lastname: Weber
authors.forename: Borries
authors.initials: B
authors.lastname: Demeler
authors.forename: P John
authors.initials: PJ
authors.lastname: Hart
authors.forename: Karl E
authors.initials: KE
authors.lastname: Klose
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001646:Bile / Q000378:metabolism / Q000382:microbiology; D002087:Butyrates / Q000378:metabolism*; D002771:Cholera / Q000235:genetics / Q000378:metabolism*; D005229:Fatty Acids, Monounsaturated / Q000378:metabolism*; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000382:microbiology; D051379:Mice; D020125:Mutation, Missense; D053644:Naphthalimides / Q000378:metabolism*; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics; D014734:Vibrio cholerae / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D014774:Virulence / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D002087:Butyrates; D005229:Fatty Acids, Monounsaturated; D053644:Naphthalimides; D014157:Transcription Factors; C505381:virstatin; C075626:tcpN protein, Vibrio cholerae; C008757:palmitoleic acid