Vibrio cholerae O1 causes the fatal epidemic disease cholera. The ability of V. cholerae to cause disease in humans is dependent upon two primary virulence factors, the toxin-coregulated pilus (TCP), a critical colonization factor, and cholera toxin (CT). The expression of these factors is controlled by a highly regulated transcriptional cascade that serves as a paradigm for the regulation of bacterial virulence. Expression of the cascade is initiated at the tcpPH promoter by a cooperative interaction between the regulators AphA and AphB. TcpPH and ToxRS are homologous pairs of transmembrane regulators that then cooperate to activate expression from the toxT promoter. ToxT, an AraC-type regulator, directly activates the expression of TCP and CT. Transcriptional activation of the virulence cascade is strongly dependent upon a variety of stimuli from the external environment. The long-term goals of this proposal are to understand the molecular basis of virulence gene regulation in V. cholerae so as to facilitate the development of new strategies to control its infectivity. Through a collaborative effort involving laboratories with expertise in structural biology, virulence gene regulation and pathogenesis, we have found that exogenous unsaturated fatty acids (UFAs), which are components of human bile, are capable of binding to ToxT and impairing its ability to activate virulence gene expression. UFAs bind into a ligand pocket in the N-terminal domain of ToxT and inhibit its dimerization as well as its ability to bind to DNA. Bicarbonate, which neutralizes the acid that comes from the stomach, has been shown to function as a second in vivo signal that, in contrast to UFAs, stimulates ToxT and enhances its DNA binding through an unknown mechanism. We have recently identified a new link between fatty acids (FAs) and virulence gene expression in the current pandemic strain of V. cholerae with the discovery that the master regulator of FA metabolism, FadR, influences the translation of ToxT by an unknown mechanism. This proposal will build upon the ToxT structural and functional data, as well as our recent studies involving FadR, in order to elucidate several key mechanisms involved in regulating the expression of the virulence cascade.
In Aim 1, we propose to elucidate the allosteric mechanisms controlling the dimerization of ToxT and to investigate the opposing effects of UFAs and bicarbonate on this process.
In Aim 2, we propose to elucidate the mechanism by which FadR influences the translation of ToxT in V. cholerae. These studies will contribute significantly toward our understanding of how virulence gene expression is regulated in V. cholerae and will likely provide new avenues for antivirulence drug discovery.

Public Health Relevance

Cholera is an extremely virulent disease. It affects both adults and children and it can kill a person within hours. There are an estimated three to five million cholera cases every year, with about 100,000 to 120,000 deaths. Despite many years of research on V. cholerae, the causative agent of cholera, there are still no effective vaccines or specific antivirulence drugs. The studies that are proposed within the current application are relevant to the prevention and control of cholera and may also be applicable to a number of other enteric diseases caused by gram-negative bacterial infections world-wide, which addresses major goals of the NIH mission.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI120068-01A1
Application #
9174511
Study Section
Special Emphasis Panel (ZRG1-IDM-R (02)M)
Program Officer
Hall, Robert H
Project Start
2016-07-01
Project End
2021-06-30
Budget Start
2016-07-01
Budget End
2017-06-30
Support Year
1
Fiscal Year
2016
Total Cost
$371,251
Indirect Cost
$142,084
Name
Dartmouth College
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
041027822
City
Hanover
State
NH
Country
United States
Zip Code
03755
Midgett, Charles R; Almagro-Moreno, Salvador; Pellegrini, Maria et al. (2017) Bile salts and alkaline pH reciprocally modulate the interaction between the periplasmic domains of Vibrio cholerae ToxR and ToxS. Mol Microbiol 105:258-272
Kovacikova, Gabriela; Lin, Wei; Taylor, Ronald K et al. (2017) The Fatty Acid Regulator FadR Influences the Expression of the Virulence Cascade in the El Tor Biotype of Vibrio cholerae by Modulating the Levels of ToxT via Two Different Mechanisms. J Bacteriol 199:
Woodbrey, Anne K; Onyango, Evans O; Pellegrini, Maria et al. (2017) A new class of inhibitors of the AraC family virulence regulator Vibrio cholerae ToxT. Sci Rep 7:45011
Privett, Britney R; Pellegrini, Maria; Kovacikova, Gabriela et al. (2017) Identification of a Small Molecule Activator for AphB, a LysR-Type Virulence Transcriptional Regulator in Vibrio cholerae. Biochemistry 56:3840-3849