Clostridium perfringens is a Gram-positive, anaerobic, spore-forming, rod-shaped bacterium that causes histotoxic infections and enterotoxaemias in humans and domestic animals. This bacterium owes its pathogenicity to the production of a large arsenal of toxins, including the C. perfringens enterotoxin (CPE) and beta2 toxin (CPB2). Type A, cpe-chromosomal isolates cause human food-poisoning (FP), whereas type A, cpe-plasmid isolates cause human antibiotic associated diarrhea (AAD). Symptoms of AAD are more severe and longer in duration than symptoms of FP. We hypothesized that AAD isolates may produce an accessory toxin, which would contribute to pathogenesis, as an explanation for these symptomatic differences. Consequently, the goal of this dissertation was to determine; 1) whether the recently discovered, plasmid-encoded, CPB2 toxin was produced by AAD isolates, 2) how this toxin functions in vitro, and 3) its role in vivo (in the context of human and animal disease). PCR analysis, pulsed-field gel electrophoresis/Southern blotting, and DNA sequencing determined that the cpb2 gene is preferentially associated with AAD isolates (but not FP isolates), in part, due to its presence on some cpe-encoding plasmids. Sequencing of cpe and cpe/cpb2 plasmids found a large region of plasmid DNA, encoding a conjugal-gene cluster, which appears conserved amongst most C. perfringens virulence plasmids. Subsequent sequencing of a number of cpb2 genes identified two CPB2 variants, both of which are produced (detected using Western blot). Neutral red cytotoxicity assays using purified CPB2 demonstrated that CPB2h1 is more cytotoxic for Caco-2 cells than CPB2h2 and this activity is heat labile. Further in vitro work (using 86Rubidium release assays and osmotic stabilizers) suggested that CPB2 acts by disrupting membrane permeability. In vivo experiments confirmed that our CPB2 preparations also were lethal using a mouse intravenous injection model. However, efforts to develop a more realistic model of pathogenesis (which initiates in the intestines) should be continued to further
analyze the role of CPB2 in pathogenesis. Ultimately, the production of CPB2 by most AAD isolates, as opposed to FP isolates, could provide an explanation for the more severe symptoms associated with AAD cases and suggests that CPB2 may be involved in human gastrointestinal disease.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-10232006-145618 |
| Date | 24 October 2006 |
| Creators | Fisher, Derek James |
| Contributors | Dr. Timothy Mietzner, Dr. Bruce McClane, Dr. Lee Harrison, Dr. Saleem Khan, Dr. James Carroll |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-10232006-145618/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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