Human Decay-Accelerating Factor and CEACAM Receptor-Mediated Internalization and Intracellular Lifestyle of Afa/Dr Diffusely Adhering Escherichia coli in Epithelial Cells

We used transfected epithelial CHO-B2 cells as a model to identify the mechanism mediating internalization of Afa/Dr diffusely adhering Escherichia coli. We provide evidence that neither the α5 or β1 integrin subunits nor α5β1 integrin functioned as a receptor mediating the adhesion and/or internalization of Dr or Afa-III fimbria-positive bacteria. We also demonstrated that (i) whether or not the AfaD or DraD invasin subunits were present, there was no difference in the cell association and entry of bacteria and that (ii) DraE or AfaE-III adhesin subunits are necessary and sufficient to promote the receptor-mediated bacterial internalization into epithelial cells expressing human decay-accelerating factor (DAF), CEACAM1, CEA, or CEACAM6. Internalization of Dr fimbria-positive E. coli within CHO-DAF, CHO-CEACAM1, CHO-CEA, or CHO-CEACAM6 cells occurs through a microfilament-independent, microtubule-dependent, and lipid raft-dependent mechanism. Wild-type Dr fimbria-positive bacteria survived better within cells expressing DAF than bacteria internalized within CHO-CEACAM1, CHO-CEA, or CHO-CEACAM6 cells. In DAF-positive cells, internalized Dr fimbria-positive bacteria were located in vacuoles that contained more than one bacterium, displaying some of the features of late endosomes, including the presence of Lamp-1 and Lamp-2, and some of the features of CD63 proteins, but not of cathepsin D, and were acidic. No interaction between Dr fimbria-positive-bacterium-containing vacuoles and the autophagic pathway was observed.Diffusely adhering Escherichia coli (DAEC) organisms comprise two classes of strains, the typical DAEC and the atypical DAEC strains, each subdivided into two subclasses of strains (67). These pathogenic E. coli strains belong to group six of enterovirulent E. coli (38). Typical Afa/Dr DAEC strains have been shown to be involved in age-dependent diarrhea in infants (48, 63). Typical Afa/Dr DAEC strains have been shown to belong to the recently reported type IV pathotype of uropathogenic E. coli (UPEC) (46). Typical Afa/Dr DAEC strains are involved in urinary tract infections (UTIs), since 25 to 50% of children with cystitis and 30% of pregnant women with pyelonephritis are infected with E. coli bearing Afa/Dr fimbriae (30, 57). Moreover, typical Afa/Dr DAEC strains are involved in recurrent UTIs, and the vast majority of the typical Afa/Dr DAEC isolates (90%) are multiantibiotic resistant (29). The typical Afa/Dr DAEC strain expresses a family of genes that is organized to form a family of afa-, dra-, daa-, and/or nfa-related operons encoding Afa-I, Afa-II, Afa-III, Afa-V, Dr, Dr-II, F1845, and Nfa-I fimbriae (67). The genes are organized in similar ways, with at least five genes (A to E), of which the last, the E gene, encodes a major structural adhesin subunit (70). The D gene encodes the invasin subunit (21, 35, 72). Importantly, DraE and AfaE-III proteins display 98% sequence identity, whereas DraD and AfaD-III share 100% sequence identity. Atomic resolution models of Dr and Afa-III fibrils have revealed that the structural basis for assembly occurs by donor strand complementation and that the architecture of capped surface fibers results from the assembly of several DraE or AfaE subunits, with one invasin subunit, DraD or AfaD-III, at their distal ends (1, 14).Typical Afa/Dr fimbriae govern the adhesion of the bacteria to host epithelial cells and the cells'' responses to their presence. All these fimbriae (Afa/Dr_DAF) are able to bind specifically to the complement control protein repeats 2 and 3 (CCP2 and CCP3), domains of human decay-accelerating factor (DAF; CD55) (56). The DAF binding domain into the Afa/Dr adhesin subunit is located in its central part, localized on strands B and E of DraE (1, 31). Moreover, our group has recently reported that a subclass of Afa/Dr fimbriae, including Afa-III, Dr, and F1845 (Afa/Dr_CEACAM), recognized members of the human CEACAM family (3), which includes CEACAM1 (biliary glycoprotein; CD66a), CEA (carcinoembryonic antigen; CD66e), and CEACAM6 (nonspecific cross-reacting antigen; CD66c) (4, 27). AfaE-I, AfaE-III, AfaE-V, DraE, and DaaE adhesin subunits of Afa/Dr DAEC targeted the N-terminal domains of CEACAMs (40). The CEACAM binding site is located primarily in the A, B, E, and D strands of the Dr adhesin opposite the beta-sheet encompassing the previously determined binding site for DAF (40). Typical Afa/Dr DAEC strains have been described as invasive in unpolarized epithelial cells expressing several Afa/Dr fimbriae receptors but with a low level of efficiency (24, 26, 35). In cultured human polarized intestinal cells forming a cell monolayer that mimics an epithelium, these bacteria are apically uninvasive and enter the cells via the basolateral domain (26). The process of internalization into unpolarized epithelial cells involves lipid rafts (26, 37, 66) and dynamic, unstable microtubules (24, 26). Previous studies conducted with Dr and Afa-III fimbriae have not elucidated the processes by which typical Afa/Dr DAEC strains enter epithelial cells, which remain controversial. Two different working hypotheses have been proposed. According to one possible mechanism, after the Dr fimbriae have recognized the membrane-bound human DAF, the entire dra operon is necessary to trigger a receptor-mediated internalization of the bacteria (24). Selvarangan et al. (66) have shown that a transposon mutant with a mutation in the DraE adhesin subunit lacks adhesiveness and consequently fails to enter the cells. More convincingly, Das et al. (15) have demonstrated that the DraE subunit is both an adhesin and an invasin, since by mutagenesis to replace selected amino acids in hydrophilic domain II of the DraE protein, bacterial internalization was reduced or abolished without modifying the bacterial cell association. The second possible mechanism would imply that DraD and AfaD-III invasin subunits recognize the membrane-bound α5β1 integrin, and this is sufficient to trigger the entry of bacteria (26, 61) via a zipper-like mechanism (37). For this second mechanism, it has not been clearly established whether there is an initial step of recognition of the membrane-bound receptor DAF or CEACAM members by the AfaE/DraE adhesin subunits.We decided to conduct a series of experiments to analyze the role of DAF and/or α5β1 integrin in the receptor-mediated internalization of Afa/Dr DAEC. We extended our investigation by analyzing the role of the CEACAMs that act as receptors for Afa/Dr_CEACAM adhesins during the receptor-mediated internalization of Afa/Dr DAEC. We also investigated the role of AfaE/DraE adhesins and/or AfaD/DraD invasin subunits in bacterial internalization. We also examined the intracellular lifestyle of Dr fimbria-positive bacteria by comparing the survival rates of the intracellular bacteria in a series of cell lines, each of which expresses one of the membrane-bound epithelial receptors of Afa/Dr fimbriae, and by characterizing the late vacuole-containing internalized bacteria in a DAF-positive epithelial cell line.