Fatty Acid Regulation of Virulence in Vibrio cholerae: A Structural Analysis of Unbound, Ligand‐Bound, and DNA‐Bound ToxT

Abstract

Vibrio cholerae is an infectious bacterium that causes activation of transmembrane ion channels responsible for mediating the tonicity of cells in the small intestine. Activation of these ion channels results in osmotic movement into the small intestine, thus dehydrating cells and the infected individual. The transcription factor, ToxT, upregulates production of cholera toxin (CT) and the toxin coregulated pilus (TCP), key virulence factors in the progression of cholera. ToxT, a member of the AraC family of transcriptional regulators, has been crystallized in the presence of an unsaturated fatty acid (UFA) revealing a repressed form of the transcription factor in a conformation that prevents DNA binding. Two □‐helices in the DNA binding domain (DBD), □6 and □9, must be parallel to bind consecutive major grooves in the DNA. In the UFA‐bound structure, these helices are non‐parallel, supporting the model that ligand binding to ToxT prevents DNA binding. ToxT has been shown biochemically to be homodimeric when bound to DNA. However, crystallization of the DNA bound form of ToxT has been challenging due to instability of the complex. Recently, a natural variant of ToxT with increased solubility has been crystallized in the presence and absence of UFA. Crystallization of the apo form of ToxT required a lysine to alanine substitution at position 231 in the ligand‐binding pocket of the protein to prevent UFA binding. Although the structure of apo‐ToxT is highly similar to the UFA‐bound structure, the apo version was shown to be more flexible than ligand‐bound ToxT, supporting a model in which □6 and □9 in each subunit of dimeric ToxT are parallel and capable of binding to DNA. Using the structures described in this study, The Pingry School SMART (Students Modeling A Research Topic) Team used a 3D‐printer from the Milwaukee School of Engineering (MSOE) to model the apo structure and the dimeric DNA‐bound model of ToxT. These models support an in‐depth analysis of ToxT structural conformations in support of the proposed mechanism of regulation. A detailed structural understanding of ToxT may support future development of vaccines and therapeutics for cholera and other pathogens whose toxicity is regulated by AraC family members.