Open Access Dissertation
Date of Award
Immunology and Microbiology
Jeffrey H. Withey
Vibrio cholerae is responsible for the diarrheal disease cholera by producing two major virulence factors: cholera toxin (CT) and toxin-coregulated pilus (TCP). During infection, V. cholerae downregulates its motility genes, which are actively expressed in the environment, and upregulates its virulence genes, which are inactive in the environment. Virulence gene transcription is regulated by the major transcriptional activator, ToxT. ToxT is regulated, in part, by host signals, which in turn, make ToxT active or inactive in respect to virulence gene expression. Host signals include temperature and pH as well as the chemical molecules bicarbonate and bile, which are positive and negative effectors of virulence, respectively. Bile is a heterogeneous mixture that includes unsaturated fatty acids (UFAs) that have been directly implicated in virulence gene downregulation. Another small molecule, virstatin, has also been shown to cause virulence gene downregulation. This dissertation focuses on the mechanism by which bile and its UFA components, specifically linoleic acid, downregulate virulence gene expression.
The first chapter focuses on the decreased binding affinity of ToxT for various virulence gene promoters at the ToxT binding sites, termed toxboxes. These toxboxes come in different configurations and orientations, and while most promoters have two toxboxes, aldA only has one. I showed that linoleic acid can enter the cell where it can go into the cytoplasm and then interact with ToxT. It has been hypothesized that UFAs cause decreased dimerization; however at PaldA, linoleic acid still had an effect. Virstatin, which has been shown directly to decrease dimerization of ToxT, did not have an effect on ToxT DNA binding at PaldA. This suggests a mechanism in which UFAs do not affect dimerization but can affect monomeric ToxT binding to DNA.
In chapter two, we characterize an unstructured region of the N-terminal domain of ToxT in response to bile and UFAs. By mutating each of the 10 amino acids in the region, we discovered a mutant, N106F, which had decreased response to bile and UFAs. It also showed decreased response to the positive regulator bicarbonate.
Chapter three discusses the possibility of using conjugated linoleic acid (CLA) as a therapeutic or prophylactic upon exposure to V. cholerae. The in vitro work showed a strong decrease of ToxT activity in response to CLA on both gene expression and DNA binding. In the infant mouse model, CLA did not inhibit colonization, but in the rabbit ileal loop model CT production was significantly decreased, suggesting that CLA may work to reduce cholera symptoms.
In the last chapter, we looked at the possibility of using fluorescence-activated cell screening (FACS) in addition to transposon mutagenesis as a method to collect mutants that were insensitive to bile. Use of this technique would allow us to discover genes that were directly responsible for the effects of bile. Several genes were identified, including pckA, which is involved in metabolism, but could have a role in virulence. When deleted, however, the bacteria were only slightly less sensitive to bile than wild-type. FACS genetic screening is still a useful technique that, if further explored, could elucidate other genes involved in responding to effectors of virulence.
Plecha, Sarah, "The Mechanism For Inhibition Of Vibrio Cholerae Virulence Gene Expression By Bile And Its Fatty Acid Components" (2014). Wayne State University Dissertations. 1097.