In this proposal, the role and significance of the Shiga family of toxins in diseases due to Shigella (dysenteriae Type 1 and Escherichia coli producing the related """"""""Shiga-like"""""""" toxins (or SLT's) will be studied. The health relatedness of this research is clear: Shiga family toxins play an important role in the intestinal manifestations of S. dysenteriae Type l and probably SLT-producing E. coli. They are the only common explanation of the strong association of these pathogens with severe and fatal hemolytic-uremic syndrome (HUS). In the U.S., HUS is primarily associated with SLT-producing E. coli acquired from hamburgers at fast-food restaurants, and is the most common cause of acute renal failure in children. There are three specific aims: 1) to study how toxin is internalized by susceptible cells and the relationship of this process to the glycolipid toxin receptor, globotriaosylceramide, using a intestinal cell lines in culture, 2) to study the impact of toxin, with or without lipopolysaccharide endotoxin, on endothelial cells in culture, or with in vitro reconstituted blood vessels designed to resemble the target organ, the renal glomerulus, on the production of biological mediators relevant to initiation and propagation of HUS; and 3) to use the techniques of molecular genetics to engineer a vaccine candidate based on development of neutralizing antibody to the non-toxic binding (B) subunit of the toxin. The last aim includes development of vaccines to deliver a protective antigen, and the study of an experimental oral cholera vaccine strain and a totally new approach to utilize the spore of Bacillus subtilis (a nonpathogenic heat and environmentally resistant microbial form) as vehicles to deliver antigen. The research utilizes cell biology to study toxin-receptor interactions and uptake mechanisms; cell physiology to study responses of endothelial cells and vascular smooth muscle cells to toxin and endotoxin challenge; and molecular genetics to clone and express toxin B-subunit in selected oral delivery systems for use as a vaccine. We will correlate in vitro and in vivo data to guide the research. At the end of the project we will know more about the mechanisms of toxin interactions with intestinal epithelial cells and vascular tissue and the pathophysiology of HUS, and we will develop strategies to block impending HUS and a vaccine candidate to prevent this problem.
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