Vibrio cholerae is the etiologic agent of cholera, a devastating dehydrating diarrhea disease endemic in many parts of the world, imposing a significant burden of morbidity and mortality, especially on developing countries. The field of V. cholerae pathogenesis research has recently identified many of the molecular pathways by which colonization and toxin production, the key mechanisms by which these bacteria cause cholera, are tightly regulated. Missing from this equation, however, has been the key element in the virulence regulatory pathway: the host signals that V. cholerae hijack to induce bacteria's transcriptional commitments to pathoge- nesis and survival in the host. Using a novel ex vivo anaerobic intestinal tissue model we recently developed, we have identified two host-derived small molecules conserved among many mammalian species that can activate V. cholerae virulence gene transcription. We have named these compounds the Virulence-inducing Factors (VIFs). We propose in three specific aims to characterize VIFs and how V. cholerae sense these factors and integrate them into their virulence regulation. First, we will determine the virulence-inducing factor-2 (VIF-2) structure and mechanisms of action. We have found that both VIF-1 and VIF-2 activate key virulence determinants and require major virulence regulators AphB, TcpP, and ToxT to function. The VIF-2 has been partially determined as C12H17O6 and is likely to a novel class of molecules. We will solve VIF-2's structure using a number of chemistry analyses and then perform de novo synthesis to confirm the activity of VIF-2. Preliminary results suggest that AphB, a LysR-family protein with a conserved structure comprised of an N-terminal DNA-binding and a C-terminal ligand-binding domain, binds to VIF-2 and enhances tcpP expression. We thus propose that VIF-2 serves as the ligand of AphB and we will further investigate how AphB senses and responds to VIF-2 to regulate tcpP expression using in vitro purified systems and genetic analyses. Next, we will investigate how VIF-1 regulates virulence gene expression. We have successfully purified and identified VIF-1 as taurocholic acid, a bile acid. Preliminary studies suggest that VIF-1 enhances the activity of TcpP. We will apply biochemical and genetic methods to test our hypotheses that VIF-1 may directly interact with TcpP proteins to affect protein conformation and function. Alternatively, VIF-1 may affect TcpP activity indirectly, by interacting with TcpH, a TcpP stabilizing protein, or other V. cholerae components to enhance TcpP activity. Finally, we will determine the spatiotemporal and dynamic virulence gene expression in response to two host virulence-inducing factors. Our preliminary studies indicate that VIF-1 and VIF-2 feed into the virulence regulatory cascades by modulating TcpP protein levels (transcriptional) and activity (post-translational). We hypothesize that the availability of VIF- 1 and VIF-2 may vary in intestines with redundant signaling ensuring the appropriate induction of virulence genes at different intestinal locations or a gradient of virulence regulator levels controlling different target genes with varying activation affinities. Alternatively, both VIF-1 and VIF-2 may work synergistically to jump-start induction of virulence genes upon entering the host. We will quantify VIF concentration in different intestinal locations using HPLC-MS analysis. We will take advantage of our simple, accessible ex vivo anaerobic tissue model to mimic the VIF levels seen in vivo to monitor and model virulence gene expression patterns using qRT-PCR and fluorescence microscopy. By understanding the properties and role of VIFs, we hope to overcome the greatest stumbling block in the full understanding of V. cholerae colonization and pathogenesis, the identification of the signals that induce bacteria to take up residence in the host and cause disease. Our studies will hopefully lead to novel therapies that can target these most basic of V. cholerae virulence regulatory steps and shed new light on the host- pathogen interactions that underpin infections by related enteric bacterial pathogens.

Public Health Relevance

Vibrio cholerae has figured prominently in the history of infectious diseases as a cause of periodic global epidemics. Despite major improvements in medical treatment as well as a better understanding of the molecular processes involved in the virulence of V. cholerae, cholera still occurs widely in epidemic form particularly in South Asia, Africa, and Latin America. This study proposed will shed light on importance of V. cholerae's virulence gene regulations in host-pathogen interactions, with the goal of better understanding V. cholerae pathogenesis and, potentially, discovering novel treatment options for the cholera disease.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Bacterial Pathogenesis Study Section (BACP)
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Hall, Robert H
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University of Pennsylvania
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