The Role of Colonization Factors CFA/I and CS21 in Enterotoxigenic E.coli (ETEC) Pathogenesis in the Human Enteroid Model

Abstract

Enterotoxigenic Escherichia coli (ETEC) is a primary causative agent of diarrhea in travelers and in 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 and are prime targets as vaccine antigens. Many clinical isolates express CFA/I and CS21; however, a role for CS21 alone or with CFA/I has not been defined. We hypothesize that expression of both CFs confers increased adherence and toxin delivery to the human enteroid. Clinical strains expressing CFA/I and/or CS21 were evaluated, and CF-deficient mutants were engineered. After confirming CF expression using western blot and electron microscopy, assays demonstrated CFA/I was important for CFA/I-CS21 ETEC adherence, as CFA/I-deficient mutants and strains pre-incubated with anti-CFA/I antibody had significantly reduced adherence to enteroid monolayers compared to wildtype. In contrast, CS21 was not required as CS21-deficient mutants and strains pre-incubated with anti-CS21 antibody adhered at similar levels as wildtype. These data demonstrate that targeting CFA/I in CFA/I-CS21 ETEC is sufficient for significant adherence reduction. Delivery of ST by CFA/I-CS21 ETEC was evaluated. Strain-specific levels of toxin delivery were detected but CF-dependent ST delivery was not observed, which may reflect the lack of flow and stretch in the current enteroid model. Upon investigation of host responses to ETEC, the enteroid monolayer integrity was not disrupted, as shown by the increase in transepithelial electrical resistance and the lack of inflammatory cytokines produced. Infection with ETEC strains resulted in decreased mucus (MUC2) production, but this was not CF-dependent. Further studies of strain-specific CFA/I expression revealed that it may be transcriptionally or post-transcriptionally regulated, following observation of nearly identical CFA/I operon sequences and many shared CF-specific regulators at the genomic level. Overall, these data support the role of CFA/I in CFA/I-CS21 ETEC adherence and reinforces CFA/I as a main target for vaccines. These data also highlight the human enteroid model to study ETEC pathogenesis and for evaluation of preclinical therapeutics.