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 discovered that exogenous unsaturated fatty acids (UFAs) 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. In the El Tor biotype, the current pandemic strain, we have recently discovered that exogenous UFAs also influence the transcription, translation and stability of ToxT by different mechanisms through the master regulator of FA metabolism, FadR. We have also recently discovered that the virulence regulator ToxR is influenced by a signalling process referred to as regulated intramembrane proteolysis (RIP) similar to that controlling the levels of TcpP. The RIP of ToxR occurs in response to nutrient limitation at alkaline pH, a condition associated with the late stages of infection, which might signal to the bacterium to terminate virulence and prepare for entry back into the environment. This proposal will build upon the ToxT structural and functional data, as well as our recent studies, in order to elucidate several key mechanisms involved in regulating the expression of the virulence cascade.
In Aim 1, we propose to determine the mechanisms controlling the dimerization of ToxT and its inhibition by UFAs as well as the small molecule virstatin.
In Aim 2, we propose to elucidate how FadR influences both the translation of ToxT and its stability.
In Aim 3, we propose to determine the factors involved in the RIP of ToxR and its role in V. cholerae pathogenesis. 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.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
High Priority, Short Term Project Award (R56)
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Special Emphasis Panel (ZRG1-IDM-S (02))
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Hall, Robert H
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Dartmouth College
Schools of Medicine
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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:
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
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
Shi, Wei; Kovacikova, Gabriela; Lin, Wei et al. (2015) The 40-residue insertion in Vibrio cholerae FadR facilitates binding of an additional fatty acyl-CoA ligand. Nat Commun 6:6032
Almagro-Moreno, Salvador; Kim, Tae K; Skorupski, Karen et al. (2015) Proteolysis of virulence regulator ToxR is associated with entry of Vibrio cholerae into a dormant state. PLoS Genet 11:e1005145
Almagro-Moreno, Salvador; Pruss, Kali; Taylor, Ronald K (2015) Intestinal Colonization Dynamics of Vibrio cholerae. PLoS Pathog 11:e1004787
Almagro-Moreno, Salvador; Root, Michael Z; Taylor, Ronald K (2015) Role of ToxS in the proteolytic cascade of virulence regulator ToxR in Vibrio cholerae. Mol Microbiol 98:963-76
Taylor, Jennifer L; De Silva, Rukman S; Kovacikova, Gabriela et al. (2012) The crystal structure of AphB, a virulence gene activator from Vibrio cholerae, reveals residues that influence its response to oxygen and pH. Mol Microbiol 83:457-70
Son, Mike S; Taylor, Ronald K (2012) Growth and maintenance of Escherichia coli laboratory strains. Curr Protoc Microbiol Chapter 5:Unit 5A.4.
Son, Mike S; Megli, Christina J; Kovacikova, Gabriela et al. (2011) Characterization of Vibrio cholerae O1 El Tor biotype variant clinical isolates from Bangladesh and Haiti, including a molecular genetic analysis of virulence genes. J Clin Microbiol 49:3739-49

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