Type of Document Dissertation Author Obiso, Jr., Richard J Author's Email Address firstname.lastname@example.org URN etd-133814659751561 Title Characterization and Molecular Analysis of Fragilysin: The Bacteroides fragilis toxin Degree PhD Department Biochemistry and Anaerobic Microbiology Advisory Committee
Advisor Name Title Chen, Jiann-Shin Claus, G. William Dean, Dennis R. Gregory, Eugene M. Wilkins, Tracy D. Committee Chair Keywords
- Bacteroides fragilis
Date of Defense 1997-05-06 Availability unrestricted 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 6127250
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