Diarrheal diseases contribute to serious morbidity and mortality in humans and domestic animals world-wide. Enterotoxigenic strains of Escherichia coli (ETEC) are significant agents of watery diarrheal disease and induce illness by the elaboration of polypeptide toxins which alter the normal fluid and electrolyte balance in the gut. The ETEC heat-stable enterotoxins (STs) are peptides which mediate fluid and electrolyte loss by inducing hormone-like secretory pathways in the gut. Two STs (STa and STb) are currently recognized. STa is an 18- or 19-mer acidic peptide which binds to and activates guanylate cyclase C (GCC), a unique intestinal form of membrane guanylate cyclase. The resulting accumulation of cGMP results in chloride secretion via activation of the CFTR chloride channel. In contrast to STa, STb is a 48-mer basic peptide which causes intestinal secretion in the absence of elevated cyclic nucleotides, a hallmark of the other E. coli secretory toxins. Work in the previous funding period demonstrated that STb causes release of intestinal 5- hydroxytryptamine (serotonin, 5-HT) and formation of prostaglandin E2 (PGE2), two mediators also known to be involved in cholera toxin (CT) mediated secretion. Unlike CT, however, the cellular action of STb causes activation of a pertussis toxin-sensitive heterotrimeric G protein of the Gi subtype. Under appropriate experimental conditions, STb-mediated Gi activation causes an elevation of cytosolic calcium ions in treated cells. STb also induces G-protein dependent exocytosis from a cultured mast-like cell line resulting in release of 5-HT in vivo. Recent structural studies indicate that STb is an alpha-helical amphipathic peptide with a positively-charged polar face formed by one helix and a non-polar face formed by a second alpha helix. The STb structure and action are consistent with a broad group of compounds which directly activate G proteins following insertion or penetration of cell membranes. In this application the PI proposes to: 1) determine the specific association of STb with target cells by employing biochemical, biophysical and ultrastructural techniques; 2) determine the cellular signaling mechanism of STb and the role of G protein activation in intestinal secretion; 3) investigate the cascade of intestinal secretagogues evoked by STb action and their specific role in secretion, and 4) determine the structural features responsible for the cellular and molecular action of STb. The elucidation of the cellular and molecular action of STb, a unique bacterial toxin, will undoubtedly increase our understanding not only of bacterial enterotoxin action, but also, the means by which the intestine maintains normal fluid and electrolyte balance. The result will be the identification of new approaches to therapeutic intervention of intestinal secretory diseases.
