Adhesion to and invasion of HeLa cells by pathogenic Escherichia coli carrying the afa-3 gene cluster are mediated by the AfaE and AfaD proteins, respectively

The afa-3 gene cluster, expressed by uropathogenic and diarrhea-associated Escherichia coli strains, determines the formation of an afimbrial adhesive sheath composed of the AfaD and AfaE-III adhesins. The adherence to HeLa cells by recombinant HB101 strains producing both or only one of these two adhesins was investigated, Ultrastructural analyses of the interaction and gentamicin protection assays showed adherence to HeLa cells by HB101 producing both the AfaD and AfaE-III proteins and internalization of a subpopulation of the bacteria into the cells, The interactions of HeLa cells either with HB101 mutants producing AfaD or AfaE-III or with polystyrene beads coated with purified His(6)-tagged AfaD or His(6)-tagged AfaE-III proteins were studied, These experiments demonstrated that AfaE-III allows binding to HeLa cells and that AfaD mediates the internalization of the adherent bacteria, Ultrastructural analyses of the interaction of His(6)-AfaD-gold complexes with HeLa cells confirmed that AfaD is able to bind to the HeLa cell surface and indicated that it penetrates the cells via clathrin vesicles. These data demonstrate that the afa gene cluster is unique among bacteria, as alone it encodes both adhesion to and invasion of epithelial cells.