The role of CFA/I in adherence and toxin delivery by ETEC expressing multiple colonization factors in the human enteroid model.

Enterotoxigenic Escherichia coli (ETEC) is a primary causative agent of diarrhea in travelers and young children in low-to-middle-income countries (LMICs). ETEC adhere to intestinal epithelia via colonization factors (CFs) and secrete heat-stable toxin (ST) and/or heat-labile toxin (LT), causing dysregulated cellular ion transport and water secretion. ETEC isolates often harbor genes encoding more than one CF that are targets as vaccine antigens. CFA/I is a major CF that is associated with ETEC that causes moderate-to-severe diarrhea and plays an important role in pathogenesis. The Global Enteric Multicenter Study finding that 78% of CFA/I-expressing ETEC also encode the minor CF CS21 prompted investigation of the combined role of these two CFs. Western blots and electron microscopy demonstrated growth media-dependent and strain-dependent differences in CFA/I and CS21 expression. The critical role of CFA/I in adherence by ETEC strains expressing CFA/I and CS21 was demonstrated using the human enteroid model and a series of CFA/I- and CS21-specific mutants. Furthermore, only anti-CFA/I antibodies inhibited adherence by global ETEC isolates expressing CFA/I and CS21. Delivery of ST and resulting cGMP secretion was measured in supernatants from infected enteroid monolayers, and strain-specific ST delivery and time-dependent cGMP production was observed. Interestingly, cGMP levels were similar across wildtype and CF-deficient strains, reflecting a limitation of this static aerobic infection model. Despite adherence by ETEC and delivery of ST, the enteroid monolayer integrity was not disrupted, as shown by the lack of decrease in transepithelial electrical resistance and the lack of IL-8 cytokines produced during infection. Taken together, these data demonstrate that targeting CFA/I in global clinical CFA/I-CS21 strains is sufficient for adherence inhibition, supporting a vaccine strategy that focuses on blocking major CFs. In addition, the human enteroid model has significant utility for the study of ETEC pathogenesis and evaluation of vaccine-induced functional antibody responses. Author summary: Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in travelers and in young children in low-to-middle-income countries (LMIC). ETEC infects the small intestine using surface located adhesins called colonization factors (CFs) and delivers toxins to the intestine. These CFs are optimal targets for ETEC vaccine development for the induction of colonization-blocking responses. In the current study we investigated the role of 2 CFs, namely CFA/I and CS21 when they are simultaneously expressed in geographically diverse ETEC clinical isolates, in infection using the human enteroid model. This system is the most human relevant and sensitive model to study enteric pathogens. We determined that CFA/I was important for adherence to the human enteroid by ETEC strains expressing CFA/I and CS21. Strain-specific levels of toxin delivery were quantified but there was no difference between wildtype and CF-deficient mutant strains. This result highlights the opportunity to consider the effects of intestinal movement and oxygen levels in the model. Infected enteroids did not have disrupted cell monolayers or produce inflammatory signals. This study supports the strategy to target CFA/I in ETEC vaccine development and underscores the enteroid as a significant model system. [ABSTRACT FROM AUTHOR]