Abstract
Class 5 fimbriae of enterotoxigenic Escherichia coli (ETEC) comprise eight serologically discrete colonization factors that mediate small intestinal adhesion. Their differentiation has been attributed to the pressure imposed by host adaptive immunity. We sequenced the major pilin and minor adhesin subunit genes of a geographically diverse population of ETEC elaborating CFA/I (n = 31), CS17 (n = 20), and CS2 (n = 18) and elucidated the functional effect of microevolutionary processes. Between the fimbrial types, the pairwise nucleotide diversity for the pilin or adhesin genes ranged from 35-43%. Within each fimbrial type, there were 17 non-synonymous and 1 synonymous point mutations among all pilin or adhesin gene copies, implying that each fimbrial type was acquired by ETEC strains very recently, consistent with a recent origin of this E. coli pathotype. The 17 non-synonymous allelic differences occurred in the CFA/I pilin gene cfaB (two changes) and adhesin gene cfaE (three changes), and CS17 adhesin gene csbD (12 changes). All but one amino acid change in the adhesins clustered around the predicted ligand-binding pocket. Functionally, these changes conferred an increase in cell adhesion in a flow chamber assay. In contrast, the two mutations in the non-adhesive CfaB subunit localized to the intersubunit interface and significantly reduced fimbrial adhesion in this assay. In conclusion, naturally occurring mutations in the ETEC adhesive and non-adhesive subunits altered function, were acquired under positive selection, and are predicted to impact bacteria-host interactions.