Enterotoxigenic Escherichia coli (ETEC) is one of the major causes of childhood morbidity and mortality in developing countries. In 2010, annual mortality from illness due to ETEC was estimated at 157,000 deaths (9 percent of all deaths attributed to diarrhea) and approximately 1 percent of all deaths in children 28 days to 5 years of age. Development of natural immunity to ETEC requires years of recurrent infections, which raises the question as to what about this organism prevents the development of immunity. ETEC cause disease by colonizing the proximal small intestine with colonization factor antigens and through production of heat-labile (LT) or heat-stable (ST) enterotoxins. LT is highly immunogenic and induces antibodies to itself when given to human volunteers. On the other hand ST is a small non-immunogenic peptide. We have recently begun to investigate the contribution of ST on ETEC pathogenesis. Our preliminary data indicate (1) sta1 gene (encoding STh) is up-regulated under anaerobic conditions, as could be found within the intestinal tract (2) ST binds both iron and iron-sulfur (FeS) clusters under these anaerobic conditions, (3) iron and FeS cluster bound ST elicits less cGMP from intestinal epithelial cells than native ST, and (4) ST suppresses fecal IgA and Th17 immune responses against ETEC H10407 (whole cell and O78-LPS). Based on the preliminary data, the hypotheses are that (1) sta1 is up-regulated in response to anaerobic conditions by the oxygen-sensitive transcriptional regulator FNR (2) ST can bind iron and FeS clusters, which regulate its toxicity, and (3) ST suppresses mucosal immunity to ETEC antigens thereby maintaining susceptibility of the host to recurrent infections by the same organism mediated by iron sensing. The specific aims are 1) Determine the environmental conditions that regulate transcription of genes encoding ST, 2) Characterize the iron and iron-sulfur cluster binding properties of ST and how this controls ST enterotoxicity, and 3) Determine how ST modifies mucosal immune responses to ETEC antigens. At the conclusion of these studies, growth conditions that result in enhanced ST expression and secretion will be elucidated. In addition, the role of iron and FeS cluster bound ST on ST toxicity will be expounded. Lastly, mechanisms concerning ST-mediated suppression of mucosal immune responses to ETEC antigens will provide compelling evidence for why it takes years of recurrent ETEC infections to develop natural immunity.