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dc.contributor.authorObiso, Jr., Richard J.en_US
dc.date.accessioned2014-03-14T20:21:16Z
dc.date.available2014-03-14T20:21:16Z
dc.date.issued1997-05-06en_US
dc.identifier.otheretd-133814659751561en_US
dc.identifier.urihttp://hdl.handle.net/10919/30292
dc.description.abstractCHARACTERIZATION AND MOLECULAR ANALYSIS OF FRAGILYSIN: THE BACTEROIDES FRAGILIS TOXIN by Richard Joseph Obiso, Jr. Dr. Tracy D. Wilkins, chairman Department of Biochemistry and Anaerobic Microbiology (ABSTRACT) Bacteroides fragilis is a gram negative, anaerobic rod, that is a member of the normal colonic microflora of most mammals, and it is the anaerobe most commonly isolated from human soft tissue infections. During the past decade, strains of B. fragilis that produce an enterotoxin have been implicated as the cause of diarrhea in a number of animals, including humans. The extracellular enterotoxin has been purified and characterized as a single polypeptide (Mr~ 20,600) that causes rapid morphological changes in human colon carcinoma cell lines, particularly, HT-29. This dissertation research began in 1993 with the purpose of determining how this enterotoxin, termed fragilysin, causes diarrhea. The deduced amino acid sequence revealed a signature zinc binding consensus motif (His-Glu-Xx-Xxx-His-Xxx-Xxx-Gly-Xxx-Xxx-His/Met) characteristic of metalloproteinases. Sequence analysis showed close identity with metalloproteinases within the zinc-binding and Met-turn regions. Purified fragilysin contained 1 gram atom of zinc per molecule, and it hydrolyzed a number of proteins, including gelatin. Optimal proteolytic activity occurred at 37° C and pH 6.5. Activity was inhibited by metal chelators but not by inhibitors of other classes of proteinases. When fragilysin is injected into ligated ileal and colonic loops of animals, there is significant tissue damage and a subsequent dose dependent fluid response. Histological examination revealed mild necrosis of epithelial cells, crypt elongation, villus attenuation, and hyperplasia. There was extensive detachment and rounding of surface epithelial cells and an infiltration of neutrophils. Enterotoxic activity was inhibited by the metal chelators EDTA and 1,10-phenanthroline; and, to some degree, the enterotoxic activity could be reconstituted by the addition of zinc to chelated toxin. Fragilysin rapidly increased the permeability of the paracellular barrier of epithelial cells to ions (decrease in electrical resistance across monolayers) and to larger molecules (increase in mannitol flux across monolayers). Furthermore, there is a direct effect on the tight junction proteins. Fragilysin appears to cause diarrhea by proteolytically degrading the paracellular barrier of epithelial cells. Fragilysin is a recently discovered virulence factor that could contribute to the pathogenesis of B. fragilis in both intestinal and soft tissue infections. This research was supported by a Public Health Service grants AI 322940 and AI 32940-03 from the National Institute of Allergy and Infectious Diseases, and by the Commonwealth of Virginia project 6127250en_US
dc.publisherVirginia Techen_US
dc.relation.haspartRJObiso-etd.pdfen_US
dc.rightsI hereby grant to Virginia Tech or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University Libraries in all forms of media, now or hereafter known. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation.en_US
dc.subjectBacteroides fragilisen_US
dc.subjectvirulenceen_US
dc.subjecttoxinen_US
dc.subjectmetalloproteinasesen_US
dc.subjectenterotoxinen_US
dc.titleCharacterization and Molecular Analysis of Fragilysin: The Bacteroides fragilis Toxinen_US
dc.typeDissertationen_US
dc.contributor.departmentBiochemistry and Anaerobic Microbiologyen_US
dc.description.degreePh. D.en_US
thesis.degree.namePh. D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen_US
thesis.degree.disciplineBiochemistry and Anaerobic Microbiologyen_US
dc.contributor.committeechairWilkins, Tracy D.en_US
dc.contributor.committeememberDean, Dennis R.en_US
dc.contributor.committeememberChen, Jiann-Shinen_US
dc.contributor.committeememberGregory, Eugene M.en_US
dc.contributor.committeememberClaus, George Williamen_US
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-133814659751561/en_US
dc.date.sdate1998-07-26en_US
dc.date.rdate1998-06-05
dc.date.adate1997-06-05en_US


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