Flagellar regulation in enterotoxigenic E. coli modulates innate immune activation.

Enterotoxigenic Escherichia coli (ETEC) remains a leading cause of diarrheal disease in low- and middle-income countries, yet vaccine efforts have struggled to achieve broad and durable protection. To better understand early steps in host-pathogen interactions, we utilized human stem cell-derived jejunal organoid monolayers to compare epithelial responses to ETEC H10407 and E24377A, prototypical strains frequently used in controlled human infection challenges. Transcriptomic analysis revealed that H10407 uniquely stimulated a robust innate immune response, characterized by upregulation of TLR5-dependent genes, including IL-17C and CXCL6. This response was found to be driven by sustained expression of flagella, the result of a naturally occurring fliZ gene disruption in H10407, which allows flagellin production despite ample nutrient availability. Complementation with fliZ restored repression of flagellar genes, confirming its regulatory role. In epithelial-macrophage co-cultures, H10407-expressed flagella enhanced macrophage sampling of bacteria as well as IL-6 production, effects that were not replicated by recombinant flagellin alone. However, exogenous flagellin did induce epithelial IL-17C release, a cytokine implicated in adaptive immune priming. These findings identify constitutive flagellar expression as a key factor in the heightened innate immune response and virulence of H10407, and highlight its potential utility in optimizing mucosal vaccine design against ETEC.