Clostridium perfringens enterotoxin (CPE) is a pore-forming toxin that is responsible for causing the symptoms of type A food poisoning, a leading cause of bacterial foodborne illness in the US. CPE-induced pore formation on intestinal epithelial cells results in ion permeability alterations leading to Ca2+ influx and activation of cell death pathways. Upon binding to its receptor, certain claudins, the interactions between CPE and the target membrane result in the formation of a series of toxin complexes (CH-1 and CH-2) that represent the formation of the functional CPE pore. Many bacterial toxins, particularly, pore-forming toxins, hijack cholesterol-rich lipid raft domains in the target cell membrane to aid in their virulence. Lipid rafts serve as platforms to cluster receptor proteins to allow for more efficient binding and oligomerization. Due to the pore-forming activity of CPE, we wished to determine if membrane rafts play a role in the mechanism of action of CPE. Interestingly, CPE was found to be a novel pore-forming toxin that does not require raft domains for its action in that CPE complexes do not form within lipid rafts and cholesterol depletion had no effect on CPE-induced cytotoxicity. These findings illustrate the unique interactions between CPE and target cells. Despite recent research findings indicating the presence of claudins in the various CPE complexes, these intricate interactions have not been fully elucidated, and the exact composition of the toxin complexes is unknown. Therefore, the research presented here describes the development of a two-step method of electroelution/immunoprecipitation that allows for the isolation and purification of the CPE complexes for compositional analysis by mass spectrometry. Finally, a mouse model has been developed and characterized to show that the molecular interactions that occur in cell culture models, such as complex formation and inflammatory cell death, also occur in vivo. Furthermore, the mouse model mimics the lethality that is occasionally seen in humans that suffer from type A food poisoning-related deaths.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-07272010-114552 |
| Date | 28 July 2010 |
| Creators | Caserta, Justin Angelo |
| Contributors | Russell Salter, Billy Day, Neal DeLuca, Mike Parniak, Bruce A. McClane |
| 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-07272010-114552/ |
| 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. |
Page generated in 0.0016 seconds