The overall goals of this proposal are to increase our understanding of the pathogenicity of enterotoxigenic E. coli and Y. enterocolitica through the study of heat stable toxins produced by these organisms. These toxins are important factors in mediating fluid and electrolyte secretion and diarrhea. We have purified and characterized a heat stable toxin (ST) from a human isolate of E. coli; the purified ST has been sequenced and contains 18 amino acids. The sequence closely corresponds to the carboxy terminus of two 72 amino acid gene analysis sequences obtained from two unrelated ST producing strains of E. coli. Our data show that these E. coli STs bind to receptors located on the brush borders of rat small bowel enterocytes. Furthermore, our data also suggest that another heat stable enterotoxin Yersinia enterocolitica ST, binds to the same receptors. The specific objectives of this proposal are to: a) purify the rat enterocyte receptor for E. coli ST using affinity chromatographic techniques; b) characterize the ST receptor with respect to composition, molecular size, subunit structure and binding characteristics for E. coli and Y. enterocolitica STs; c) purify by traditional and/or affinity chromatographic procedures, then sequence and characterize biochemically and immunologically STs produced by 2 strains of E. coli for which predicted amino acid sequences have been determined by gene analysis; and d) purify by traditional and/or chromatographic procedures an ST from Y. enterocolitica, then sequence and characterize the purified toxin to determine its structural, biochemical and immunological relationship to E. coli STs. These studies will yield considerable insight into the nature of small peptide toxin induced diarrheas, and into the use of these peptides as molecular probes for receptor characterization. Ultimately we will be able to design specific modes of therapy to interrupt or prevent ST induced diarrheas. Reagents and procedures developed during this study will aid in the development of rapid diagnostic tests for in vivo ST production of E. coli and Y. enterocolitica. Finally, protein sequence data coupled with gene analysis predicted amino acid sequences of these toxins will yield considerable formation on toxin processing before elaboration by both E. coli and Y. enterocolitica.
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