Molecular Determinants of Enterotoxigenic Escherichia coli Heat-Stable Toxin Secretion and Delivery

Abstract

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.

Phosphodiesterase 5 (PDE5) restricts intracellular cGMP accumulation during enterotoxigenic Escherichia coli infection

“…Both ST subtypes contribute to disease symptoms, 25 although the relative amounts produced by a particular ETEC strain may vary. 26 In the next assessment of the infection model, we evaluated total ST secretion (ST h and ST p ) and subsequent induction of cGMP production in HEMs following exposure to WT H10407 or mutant strains deficient in ST h , ST p , LT, EatA, EtpA, or CfaE expression ( Figure 2a ). Each of the mutants (other than ST-targeted mutations) secreted approximately equivalent amounts of ST compared to WT, suggesting no impairment in ST synthesis or release mechanisms.…”

“…Measurement of intracellular cAMP and cGMP in HEMs colonized by H10407 was recently reported. 24,26 However, the characterization of infection-associated cyclic nucleotide efflux has not been explored in the HEM model. Additionally, evaluation of an approximate pathophysiological ST concentration for experimental reference has not been described.…”

Research in a time of enteroids and organoids: how the human gut model has transformed the study of enteric bacterial pathogens

“…Interestingly, intracellular cGMP accumulation was markedly restricted by phosphodiesterase PDE5-mediated degradation in enteroids and colonoids, whereas Caco-2 and T84 polarized monolayers incubated with ST yielded high amounts of both intracellular and extracellular cGMP. Toxin delivery by ETEC is known to be enhanced by several virulence factors, 96 and this was reinforced in the observed dependence on expression of adhesins CFA/I and EtpA and the mucinase EatA by ETEC H10407 to elicit ST-induced cGMP production in enteroids.…”

Heat-Stable Enterotoxins of Enterotoxigenic Escherichia coli and Their Impact on Host Immunity

“…These intramolecular disulfide bridges ensure correct folding of the mature STa peptide, which closely resembles that of two mammalian peptides, guanylin and uroguanylin, and are important for its function [41]. Secretion of mature STh and STp into the extracellular environment requires the efflux protein TolC (Figure 1) [42]. Interestingly, these authors also showed that the propeptide (pro-STh) is secreted, reaffirming earlier reports on propeptide secretion by human ETEC strains [43].…”