SHIGELLA TOXIN: STRUCTURE & FUNCTION RELATIONSHIPS

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Shigellosis is a world wide disease producing significant morbidity and in developing countries, a leading cause of diarrheal disease mortality. Pathogenesis involves invasion of colonic epithelial cells. A protein toxin is now believed to be a second improtant virulence factor. The same or very similar toxin is produced by non-invasive enteropathogenic E. coli and other organisms. The toxin molecule consists of one A chain and 5 B chains. A functional role of the B chains is to mediate toxin binding to the eukaryotic cell surface. A glycolipid has been shown to be a toxin receptor and a glycoprotein receptor with a different sugar specificity (GlcNAc3) is also involved. This proposal is designed to study basic structure-function relationships of the toxin B chain. Particular emphasis will be placed on the structrual location of the two binding domains and on their biological roles in mediating the biological activities of the toxin. Thorugh the use of the glycoprotein receptor analogue, chitotetraose, direct evidence for the two binding domains will first be documented. Since the B chains exist in multimer form in the individual monomers and/or regions of B to B interaction. A chemical cross-linker will be used to determine the inter or intra- chain location of the binding domains. By intraluminal injections of toxin with soluble receptor analogues the role of the two receptors in mediating fluid secretion in rabbits wil be analyzed. Immunization of rabbits with synthetic peptides derived from the B chain amino acid sequence will lead to antibodies that will be tested for their ability to neutralized the biological activity of toxin. Information from this study will be useful for the future design of safe and effective toxin vaccines. Finally by both specific chemical modification of the B chain and by the study of B chain mutants information on the location of amino acids involved in important B chain functions will be determined.