B-153. Heat-Labile Enterotoxin Secretion, Lipopolysaccharide Association, and Outer Membrane Vesicle Release
Background: Enterotoxigenic E. coli (ETEC) is a leading cause of traveler’s diarrhea. ETEC expresses a major virulence factor, heat-labile enterotoxin (LT), which is highly homologous to cholera toxin. Research from our lab has shown that LT associates with lipopolysaccharide (LPS) on the outer surface of ETEC after secretion. Moreover, LT associates with outer membrane vesicles (OMVs) released by ETEC. We hypothesized that OMV release would be stimulated in vivo and that we could generate a mutation in LT that impaired its binding to LPS without disrupting toxic host effects. Recent work by Holmner, et al. uncovered a sugar binding pocket on LT’s periphery, which we reasoned may also play a role in LPS binding. Methods: We compared broth-grown ETEC and ETEC recovered after intragastric inoculation in mice by scanning electron microscopy. LT binding pocket mutants were assayed for their ability to bind the surface of E. coli. In order to verify that other properties of LT were not altered, each mutant was introduced into an LT-deficient ETEC strain, and levels of toxin in the supernatant and periplasm were measured by ELISA. Toxicity was verified with a cell culture assay. Results: We found that ETEC recovered from intestinal lumen produced significantly more OMVs than ETEC in broth. One mutant expressed at wildtype levels in ETEC, but was strongly impaired for surface binding without secretion or toxicity defects. Isogenic strains carrying mutant and wildtype LT were further compared for toxicity, adherence to host cells, and induction of other host responses. We also isolated two mutants which reduced secretion efficiency through the type II secretion system (TTSS). These mutants were not unstable or hypersensitive to degradation, and each assembled into a holotoxin. One mutation is of particular interest, since it resides in the sugar binding pocket. Conclusion: This work shows ETEC OMV production is stimulated in vivo and that LT’s secretion and binding to LPS can be uncoupled from its other properties by mutation. Further characterization of the mutants and of OMVs shed by ETEC will reveal the importance of the LT-LPS interaction in pathogenesis.