Date of Award

Spring 1990

Project Type

Dissertation

Program or Major

Microbiology

Degree Name

Doctor of Philosophy

First Advisor

Frank G Rodgers

Abstract

Campylobacter jejuni is a major cause worldwide of human gastroenteritis. In this study, the pathogenicity of the organism was examined in fertile hens' eggs and the mortality data and histopathological findings induced by both the organism and bacteria-free filtered broth were similar. The absence in chick embryo tissues of either bacteria or an inflammatory infiltrate suggested a toxic etiology. A protein was obtained from the filtrate by gel electroelution and this was sensitive to heat, pH changes and trypsin treatment and was lethal for the fertile hen's egg. It showed cytotoxic effects in primary chick embryo fibroblast (PCEF), Chinese hamster ovary (CHO) and intestinal 407 (Int 407) cells. Subsequently, a 68 kilodalton protein was isolated by polyacrylamide gel electrophoresis from the eluted toxic protein (ETP). A monoclonal antibody (CETPMAb$\sb4$) raised to the ETP abolished toxicity and bound only to the 68 kilodalton protein. No homology between C. jejuni ETP and Vibrio cholerae toxin and their respective antisera was observed. In enzyme-linked immunosorbent assay (ELISA) tests, the ETP did not bind to GM$\sb1$ ganglioside. Binding of ETP to PCEF and Int 407 host cell membranes was maximal after 2 h and this adherence was significantly reduced by prior treatment of the cells with proteolytic enzymes, neuraminidase or glutaraldehyde but not with $\beta$-galactosidase, lipase, Nonidet P-40 or sodium metaperiodate. In competitive binding assays, sugars (except N-acetyl neuraminic acid), lectins or GM$\sb1$ ganglioside did not adversely influence uptake of the ETP by these cells. Electron microscopic examination of the Int 407 cells treated with either C. jejuni or ETP showed similar ultrastructural damage. Western blot and ELISA tests of bacterial sub-components probed with CETPMAb$\sb4$ indicated that the toxin was associated with the outer membranes of the organism. Immunoassays on ETP treated eukaryotic host cells demonstrated that the toxin was attached only to the cell membranes and was not internalized. The present study indicates that the production of a membrane adhering cytotoxin that binds to protein or glycoprotein receptors on host cell membranes may be an important virulence factor responsible for the inflammatory diarrhea caused by many C. jejuni strains.

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