Diarrheal disease is a serious medical and agricultural problem of global significance. Strains of enterotoxigenic Escherichia coli which cause secretory diarrheal disease do so by the elaboration of protein toxins which disturb the normal function of the gut epithelium. One class of E. coli enterotoxins, the heat-stable enterotoxins (STa and STb) are peptides which, although share heat-stability and their genetic location on composite bacterial transposons, bear no functional or structural similarity to one another. The mechanism for STa-mediated secretion is partially understood in that, toxin-induced activation of the brush-border membrane guanylate cyclase is followed by a rapid increase in the mucosal cGMP content which is coupled to net chloride secretion probably through the intestinal epithelial cell cGMP dependent protein kinase. In contrast to our understanding of the mechanism of STa action, little is known of the mechanism by which STb induces intestinal secretion. Preliminary findings suggest that STb induces electrogenic ion transport independent of cAMP or cGMP elevation. In this application, we propose to: 1) investigate the nature and distribution of STb receptors by standard ligand-receptor interaction studies; 2) char- acterize the second messenger response to STb and determine the signal which yields electrogenic ion transport in the gut epithelium. In this part of the study we will investigate the potential role of calcium-dependent ion transport mechanisms including the inter-relationship between phospholipase C, activation and hydrolysis of phosphoinositides, the potential role of protein kinase C, or the involvement of eicosanoid metabolism in response to toxin action. Finally, we will analyze the structural features of STb which contribute receptor binding and biological action. We will accomplish this by a combination of oligonucleotide-directed mutagenesis and epitope mapping of STb. The completion of these aims will add to our understanding of secretory diarrheal disease mechanisms and provide potential rational approaches for disease intervention.
