Molecular determinants of enterotoxigenic Escherichia coli heat-stable toxin secretion and delivery

Abstract

Enterotoxigenic Escherichia coli (ETEC), a heterogeneous diarrheal pathovar defined by production of heat-labile (LT) and/or heat-stable (ST) toxins, remain major causes of mortality among children in developing regions, and cause substantial morbidity in individuals living in or traveling to endemic areas. Studies demonstrating a major burden of ST-producing ETEC have focused interest on ST toxoids for ETEC vaccines. We therefore examined fundamental aspects of ETEC ST biology using ETEC H10407, which carries estH and estP genes encoding ST-H and ST-P, respectively, in addition to eltAB genes responsible for LT. In this background, we found that deletion of estH significantly diminished cGMP activation in target epithelia, while deletion of estP had a surprisingly modest impact, and a dual estH/estP mutant was not appreciably different than the estH mutant. Nevertheless, either ST-H or ST-P recombinant peptides stimulated cGMP production. We also found that the TolC efflux protein was essential for both toxin secretion and delivery, providing a potential avenue for efflux inhibitors in treatment of acute diarrheal illness. In addition, we demonstrated that the EtpA adhesin is required for optimal delivery of ST and that antibodies against either the adhesin or ST-H significantly impaired toxin delivery and cGMP activation in target T84 cells. Finally, we used FLAG epitope fusions to demonstrate that the ST-H pro-peptide sequence is secreted by the bacteria, potentially providing additional targets for antibody neutralization. These studies collectively extend our understanding of ETEC pathogenesis and potentially inform additional avenues to mitigate disease by these common diarrheal pathogens.