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

Abstract

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.

The role of clathrin-dependent endocytosis in bacterial internalization

“…Data coming from studies using drugs inhibiting endocytosis and microscopy studies also suggest a possible role of clathrin-dependent endocytosis in the entry of E. coli [38][39][40][41], Staphylococcus aureus [42,43], Streptococcus pneumoniae [44,45], Streptococcus dysgalactiae [46], Ehrlichia risticii [17,47], Brucella abortus [48], Klebsiella pneumoniae [49], Legionella pneumophila [50], Campylobacter jejuni [51] and Citrobacter freundii [51]. More controversial is the role of clathrin-dependent endocytosis in Neisseria and Chlamydia invasion.…”

Pathogenesis of Human Diffusely Adhering Escherichia coli Expressing Afa/Dr Adhesins (Afa/Dr DAEC): Current Insights and Future Challenges

“…Moreover, DA adhesion onto Hep-2 or HeLa cells has been also observed for UPEC strains expressing Afa-I (37), Afa-III (28), and Dr (38). Goluszko et al (39) have proposed a HeLa cell receptor assay designated the diffuse clustering assay (DCA), which associates the cell diffuse adhesion of Afa/Dr DAEC with the property of Afa/Dr adhesins to promote hDAF receptor clustering around adhering bacteria (40)(41)(42). However, the DCA did not detect all the E. coli strains bearing Afa/Dr adhesins, considering that AfaE-VII and AfaE-VIII adhesins did not recognize hDAF (26).…”

“…Nuclear magnetic resolution and biophysical studies have revealed that the structural organization of Afa-III adhesin develops by assembly onto the bacterial membrane usher of AfaE-III adhesins subunits (158,159) capped by the AfaD-III subunit (166)(167)(168). The AfaD-III subunit also has the ability to separate from the Dr fimbriae (42). A diffuse and not well-ordered cell surface localization of the AfaD-III subunit has been observed by immunoelectron microscopy (42,168,169).…”

“…The AfaD-III subunit also has the ability to separate from the Dr fimbriae (42). A diffuse and not well-ordered cell surface localization of the AfaD-III subunit has been observed by immunoelectron microscopy (42,168,169). Using chimeras constructed from the afa-3 and daa operons, a study has revealed that the afimbrial or fimbrial morphologies of the adhesins were influenced by the order in the genes coding for the afimbrial or fimbrial adhesin subunits (28).…”

“…As shown in the analysis of epidemiological studies below, Afa-possessing E. coli strains have been found to be ex- (26,44) (Table 1). Like the Afa-III adhesin, the Afa-VII and Afa-VIII adhesins can aggregate to form amorphous masses (26,42). Among the four sRNA genes (171) present in the PAI AL862 strain and expressed by afa-8-positive E. coli (44), the AfaR small RNA, the transcription of which is temperature controlled, regulates the expression of the AfaD-VIII subunit (172).…”