Thesis (Ph.D.(Microbiology))--University of Pretoria, 2006. / Includes bibliographical references. Available on the Internet via the World Wide Web.

No description available.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 135-141).

Cholera toxin (CT) is a classic A-B type protein toxin that has an A subunit (A1 + A2) and a pentameric B subunit. The catalytic A1 domain is linked to the A2 domain via a disulfide linkage. CTA1 must be dissociated from the rest of the toxin to cause a cytopathic effect. Protein disulfide isomerase (PDI) can reduce the CTA1/CTA2 disulfide bond, but disassembly of the reduced toxin requires the partial unfolding of PDI that occurs when it binds to CTA1. This unfolding event allows PDI to push CTA1 away from the rest of the toxin. My research question is whether the efficiency of PDI in disassembling CT would be affected by molecular crowding, where a dense internal cell environment is recreated in vitro by the use of chemical agents such as Ficoll. This will give insight on how CT behaves inside a cell. Our hypothesis was that molecular crowding would make CTA1 disassembly more efficient by recreating the tight packing of macromolecules in cells, which provides an extra nudge to enhance toxin disassembly. We then used enzyme-linked immunosorbent assays (ELISAs), a pull-down assay and a biochemical assay to determine how molecular crowders affect the binding, reduction, and disassembly of CT by PDI. Our results will bring about a deeper understanding of the cellular events that may affect the course of a cholera infection. From the preliminary results, molecular crowders increased PDI's ability to bind to CTA1 and did not prevent PDI from cleaving the CTA1/CTA2 disulfide bond. Based off the disassembly results, molecular crowders reduced PDI's ability to displace CTA1 from the rest of the toxin. This contradicts our original hypothesis. Our new hypothesis is that crowders block PDI unfolding, which is required for CT disassembly. Biophysical experiments using Fourier Transform Infrared Spectroscopy will test this prediction in future work.

Cholera, caused by the secretion of cholera toxin (CT) by Vibrio cholerae within the intestinal lumen, triggers massive secretory diarrhea which may lead to life-threatening dehydration. CT is an AB5-type protein toxin that is comprised of an enzymatically active A1 chain, an A2 linker, and a cell-binding B pentamer. Once secreted, the CT holotoxin moves from the cell surface to the endoplasmic reticulum (ER) of a host target cell. To cause intoxication, CTA1 must be displaced from CTA2/CTB5 in the ER and is then transferred to the cytosol where it induces a diarrheal response by stimulating the efflux of chloride ions into the intestinal lumen. Protein disulfide isomerase (PDI), a resident ER oxidoreductase and chaperone, is involved in detaching CTA1 from the holotoxin. The PDI domain(s) that binds to CTA1 and precisely how this interaction is involved in CTA1 dissociation from the holotoxin are unknown. The goal of this project is to identify which domain(s) of PDI is responsible for binding to and dislodging CTA1 from the CT holotoxin. Through incorporation of ELISA, surface plasmon resonance (SPR), and Fourier transform infrared (FTIR) spectroscopy techniques in conjunction with a panel of purified PDI deletion constructs, this project aims to provide important molecular insight into a crucial interaction of the CT intoxication process.

Vibrio cholerae, the causative agent of cholera, has been a lethal enteric pathogen to humans for most of recorded history. Even though it is well studied, it still kills many people every year due to rapid and severe dehydrations from diarrhea. Part of what makes V. cholerae such an effective pathogen is its ability to control virulence factors depending on its environment. ToxR is a major virulence protein that has upstream control of most of the virulence genes that are turned on when in a human host. Two of the most critical virulence factors, toxin coregulated pilus and cholera toxin are controlled by ToxR. CsrA is a protein that regulates many cellular functions in V. cholerae, including glycogen synthesis, motility, and biofilm production. Preliminary data suggests a link between CsrA and the regulation of ToxR. In order to study CsrA as it relates to ToxR regulation, a csrA mutant must be generated in V. cholerae. CsrA plays such an important role in glycogen metabolism that a csrA mutant is not viable due to excessive glycogen levels. In order to make a viable csrA mutant, glycogen synthesis has to be turned off. In this research, I attempt to make a viable V. cholerae csrA mutant by deleting csrA in a strain that is deficient for glycogen synthesis (glg). Normally without CsrA, glycogen in the cell would increase to a detrimental level. Since a glg⁻ csrA⁻ mutant lacks the ability to make glycogen, the levels never reach a lethal level, allowing the mutant to survive without functional CsrA. Such a glg- csrA- double mutant's ToxR regulation can be studied by growth in various media by measuring OmpU and OmpT expression. Using PCR, restriction enzymes, and DNA ligase, a suicide plasmid was created containing sequences that flank the csrA gene but instead of the csrA gene, a chloramphenicol resistance cassette was inserted. Through bacterial conjugation this plasmid was introduced into three V. cholerae glg- strains. Allelic exchange was carried out utilizing the homology between the DNA flanking wild type csrA and the csrA deletion with chloramphenicol cassette. This first crossover event was initiated with the requirement of the [pi] protein for the plasmid to replicate. Without the pir gene to create [pi] protein, selection for antibiotic resistance required that the plasmid integrate into the genome. This was selected based on the plasmid encoded ampicillin resistance. After the second crossover event, there were two possible outcomes of excision: reverting to wild type csrA or retention of the csrA mutation. The csrA mutant was selected based on its sucrose and chloramphenicol resistance and ampicillin sensitivity. / text

No description available.

Thesis (M.S.)--University of Texas at Dallas, 2007. / Includes vita. Includes bibliographical references (leaves 37-39)

Thesis (Ph.D. in Microbiology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 183-207). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

The Cholera outbreak at Tiberias on the Sea of Galilee in Oct., 1918, that I am about to describe was of great importance from a military point of view. Consequently, the author and a bacteriologist, (Capt. A. Compton, R.A.M.C., O.C. 32 Mobile Laboratory) were dispatched to the scene of the outbreak in order to investigate the conditions and, in short, to adopt any measures possible to suppress the epidemic. The matter was of considerable urgency. Tiberias lay on the direct line of communication for troops operating in the Damascus area, and it was essential that all reinforcements and food supplies pass through the town. The hilly nature of the country, the arid soil, the daily extremes of temperature at that season of the year - for the days were hot and the nights bitterly cold - the deficient and defective water supply - all these factors had a bearing upon the health of the troops and the nature of the problem. Had Cholera spread at that critical period to any great extent it might have brought, what proved to be, one of the most brilliant feats in military history, to an untimely close.