FIG 6.

Binding of mutant and wild-type CFA/I pili to glycosphingolipids and Caco-2 intestinal cells. ELISA measurements of pilus binding to asialo-GM1 (A), GM1 (B), and Caco-2 cells (C) are shown. Wild-type CFA/I pili (black squares) bound strongly to asialo-GM1 and Caco-2 cells, while pili containing CfaE1 (R181A substitution, black diamonds) and CfaE2 (R67A, black triangles) showed significantly reduced binding (P < 0.05 at all pilus concentrations). Binding of pili containing CfaE3 (R67A R181A; black circles) was no greater than that of heat-inactivated wild-type pili (gray squares) or heat-inactivated CfaE3-containing pili (gray circles). Active and heat-inactivated wild-type pili and pili containing CfaE3 failed to bind to GM1 (B), which served as a negative control, confirming the specificity of glycosphingolipid binding by CFA/I.