The long term objective of this proposal is determine the role of a cytolytic enterotoxin (Act) of A. hydrophila in the pathogenesis of disease. The investigators have isolated a 52 kDa polypeptide from a diarrheal isolate of A. hydrophila (SSU) that is associated with hemolytic, cytotoxic and enterotoxic activities and is lethal for mice. The toxin gene has been cloned, expressed and sequenced by the investigators. Site directed mutagenesis studies recently completed suggest different loci coding for the three biological activities associated with the toxin. The suggestion that Act plays a significant role in the infectious process is supported by the lack of virulence of an Act minus isolate of A. hydrophila and transposon mutants with reduced biological activity. More recent studies with an isogenic mutant of strain SSU that is without in vitro hemolytic and cytotoxic activity also supports the belief that this toxin is an important virulence factor. Characterization of the role of the toxin will proceed using the toxin negative mutant recently produced. Probing the antigenic and biologically active domains of the toxin will be accomplished following hyperexpression of the Act gene with a multi-host range vector. The functional domains of the toxin molecule will be mapped by generating anti-peptide antibodies and monoclonal antibodies. Site directed mutagenesis will be used to refine the role of amino acid residues in the biological activities of the toxin. The investigators also propose to study the regulation of the Act gene either by fusing it with a reporter gene or by mutagenesis of the chromosome of Aeromonas using a transposon which imparts antibiotic resistance to screen for toxin negative mutants. Finally, the investigators propose to study the mechanism of action of the toxin by probing the mechanism of cell damage and by examining the toxin receptor on the cell surface.
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