Title page for ETD etd-133814659751561

Type of Document

Dissertation

Author

Obiso, Jr., Richard J

Author's Email Address

robiso@mail.vt.edu

URN

etd-133814659751561

Title

Characterization and Molecular Analysis of Fragilysin: The Bacteroides fragilis toxin

Degree

PhD

Department

Accounting (Academic)

Advisory Committee

Advisor Name	Title
Dr. Tracy D. Wilkins	chair
Dr. Dennis R. Dean	none
Dr. Eugene M. Gregory	none
Dr. G. William Claus	none
Dr. Jiann Shin Chen	none

Keywords

enterotoxin
metalloproteinases
toxin
Bacteroides fragilis
virulence

Date of Defense

1997-05-06

Availability

unrestricted

Abstract

CHARACTERIZATION AND MOLECULAR

ANALYSIS OF FRAGILYSIN: THE

BACTEROIDES FRAGILIS TOXIN by Richard

Joseph Obiso, Jr. Dr. Tracy D. Wilkins, chariman

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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