Enterotoxigenic (ETEC), a heterogeneous diarrheal pathovar defined by production of heat-labile (LT) and/or heat-stable (ST) toxins, causes substantial morbidity among young children in the developing world. Studies demonstrating a major burden of ST-producing ETEC have focused interest on ST toxoids for ETEC vaccines. We examined fundamental aspects of ST biology using ETEC strain H10407, which carries and genes encoding STh and STp, respectively, in addition to genes responsible for LT. Here, we found that deletion of significantly diminished cyclic GMP (cGMP) activation in target epithelia, while deletion of had a surprisingly modest impact, and a dual mutant was not appreciably different from the mutant. However, we noted that either STh or STp recombinant peptides stimulated cGMP production and that the loss of was compensated by enhanced transcription. We also found that the TolC efflux protein was essential for 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 STh significantly impaired toxin delivery and cGMP activation in target T84 cells. Finally, we used FLAG epitope fusions to demonstrate that the STh propeptide sequence is secreted by ETEC, potentially providing additional epitopes 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.

32Enterotoxigenic Escherichia coli (ETEC), a heterogeneous diarrheal pathovar defined by 33 production of heat-labile (LT) and/or heat-stable (ST) toxins, remain major causes of mortality 34 among children in developing regions, and cause substantial morbidity in individuals living in or 35 traveling to endemic areas. Studies demonstrating a major burden of ST-producing ETEC have 36 focused interest on ST toxoids for ETEC vaccines. We therefore examined fundamental aspects 37 of ETEC ST biology using ETEC H10407, which carries estH and estP genes encoding ST-H and 38 ST-P, respectively, in addition to eltAB genes responsible for LT. In this background, we found 39 that deletion of estH significantly diminished cGMP activation in target epithelia, while deletion 40 of estP had a surprisingly modest impact, and a dual estH/estP mutant was not appreciably 41 different than the estH mutant. Nevertheless, either ST-H or ST-P recombinant peptides 42 stimulated cGMP production. We also found that the TolC efflux protein was essential for both 43 toxin secretion and delivery, providing a potential avenue for efflux inhibitors in treatment of 44 acute diarrheal illness. In addition, we demonstrated that the EtpA adhesin is required for 45 optimal delivery of ST and that antibodies against either the adhesin or ST-H significantly 46 impaired toxin delivery and cGMP activation in target T84 cells. Finally, we used FLAG epitope 47 fusions to demonstrate that the ST-H pro-peptide sequence is secreted by the bacteria, 48 potentially providing additional targets for antibody neutralization. These studies collectively 49 extend our understanding of ETEC pathogenesis and potentially inform additional avenues to 50 mitigate disease by these common diarrheal pathogens. 51 52 475

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