Open Access Dissertation
Date of Award
Immunology and Microbiology
Vibrio cholerae is the bacterium responsible for the severe diarrheal disease cholera. The disease is directly caused by cholera toxin, which is secreted by the bacterium in the upper small intestinal lumen during the course of infection. Expression of cholera toxin, along with other virulence genes, is activated by the positive transcriptional regulator, ToxT. ToxT binds to DNA sequences known as toxboxes that are found within promoters of virulence genes and subsequently ToxT activates transcription. However, the toxboxes have not been previously characterized in arguably the most important virulence promoter in V. cholerae, the cholera toxin promoter (PctxAB). Additionally, H-NS, a global transcriptional repressor found in Gram-negative bacteria including V. cholerae, also binds to PctxAB. To activate transcription of PctxAB, ToxT needs to overcome the repression mediated by H-NS. The mechanism for this, along with characterization of ToxT and H-NS binding to PctxAB, is investigated in this dissertation.
Chapter one characterizes ToxT binding to PctxAB and the experimental results identified two functional toxboxes in the promoter. Mutagenesis to either of the toxboxes resulted in a significant defect in ToxT-dependent transcriptional activation and ToxT binding to DNA. ToxT was also unable to bind the DNA when both toxboxes contained mutations and this led to a complete loss of ToxT activation of PctxAB. Although there are other potential ToxT binding sites within the PctxAB promoter, ToxT requires only these specific regions of DNA for activation.
Chapter two investigates the interplay between ToxT and H-NS at PctxAB. Different V. cholerae biotypes contain a varying number of heptad repeats that are also H-NS binding regions and it was previously thought that H-NS needs to be completely displaced from the DNA to relieve its repressive role. However, the binding sites of ToxT and H-NS overlap in a small region of DNA and complete disassociation of H-NS from the promoter may not be necessary for PctxAB activation by ToxT. Further experiments also revealed that ToxT and H-NS bind to the DNA simultaneously in promoters containing six heptad repeat sequences. This does not occur in the promoter that contains only three heptad repeat sequences. These results suggest there is a direct correlation between the number of these heptad repeat sequences and the overall repression exerted by H-NS.
Dittmer, Jennifer, "Functional Characterization Of The Cholera Toxin Promoter Of vibrio Cholerae" (2013). Wayne State University Dissertations. 650.