Recruitment of CD55 and CD66e Brush Border-Associated Glycosylphosphatidylinositol-Anchored Proteins by Members of the Afa/Dr Diffusely Adhering Family ofEscherichia coliThat Infect the Human Polarized Intestinal Caco-2/TC7 Cells

Abstract

The Afa/Dr family of diffusely adhering Escherichia coli (Afa/Dr DAEC) includes bacteria expressing afimbrial adhesins (AFA), Dr hemagglutinin, and fimbrial F1845 adhesin. We show that infection of human intestinal Caco-2/TC7 cells by the Afa/Dr DAEC strains C1845 and IH11128 is followed by clustering of CD55 around adhering bacteria. Mapping of CD55 epitopes involved in CD55 clustering by Afa/Dr DAEC was conducted using CD55 deletion mutants expressed by stable transfection in CHO cells. Deletion in the short consensus repeat 1 (SCR1) domain abolished Afa/Dr DAEC-induced CD55 clustering. In contrast, deletion in the SCR4 domain does not modify Afa/Dr DAEC-induced CD55 clustering. We show that the brush borderassociated glycosylphosphatidylinositol (GPI)-anchored protein CD66e (carcinoembryonic antigen) is recruited by the Afa/Dr DAEC strains C1845 and IH11128. This conclusion is based on the observations that (i) infection of Caco-2/TC7 cells by Afa/Dr DAEC strains is followed by clustering of CD66e around adhering bacteria and (ii) Afa/Dr DAEC strains bound efficiently to stably transfected HeLa cells expressing CD66e, accompanied by CD66e clustering around adhering bacteria. Inhibition assay using monoclonal antibodies directed against CD55 SCR domains, and polyclonal anti-CD55 and anti-CD66e antibodies demonstrate that CD55 and CD66e function as a receptors for the C1845 and IH11128 bacteria. Moreover, using structural draE gene mutants, we found that a mutant in which cysteine replaced aspartic acid at position 54 displayed conserved binding capacity but failed to induce CD55 and CD66e clustering. Taken together, these data give new insights into the mechanisms by which Afa/Dr DAEC induces adhesin-dependent cross talk in the human polarized intestinal epithelial cells by mobilizing brush border-associated GPI-anchored proteins known to function as transducing molecules.

Characterization of AfaE Adhesins Produced by Extraintestinal and Intestinal Human Escherichia coli Isolates: PCR Assays for Detection of Afa Adhesins That Do or Do Not Recognize Dr Blood Group Antigens

“…The Afa/Dr ϩ adhesins bind to human DAF, and this attachment results in a dense local accumulation of DAF molecules readily detectable on HeLa and Caco-2 cells by immunofluorescence (22,24; M. Jouve, M.-I. Garcia, P. Courcoux, S. Bouzari, A. Labigne, P. Gounon, and C. Le Bouguénec, Abstr.…”

Pathogenesis of Human Enterovirulent Bacteria: Lessons from Cultured, Fully Differentiated Human Colon Cancer Cell Lines

“…The Dr or F1845 adhesin promotes hDAF and hCEA receptor clustering around bacteria adhering to the brush border of fully differentiated Caco-2 cells (Fig. 8C) (526).…”

“…These adhesins are assembled via the chaperone-usher pathway (260,520). The DraE adhesin subunit expresses two separate adhesion domains; the first recognizes the human decay-accelerating factor (hDAF) at the complement control protein (CCP) 2 and 3 domains (521)(522)(523)(524), and the second recognizes the N domains of several hCEACAMs (525)(526)(527)(528)(529). The physiological role of hDAF is to inhibit the complement cascade at the level of the critical C3 convertase step for the protection of normal cells from complement-mediated attack during innate activation, and in addition hDAF serves as a receptor for certain strains of pathogenic E. coli, Helicobacter pylori, and certain types of enteroviruses (530).…”

Infection strategies of enteric pathogenic Escherichia coli

“…4). All Afa/Dr adhesins bind the decay-accelerating factor (DAF), while a subfamily of Afa/Dr adhesins (AfaE-III, Dr and F1845 adhesins) can also bind carcinoembryonic antigen-related molecules (CEACAMs) 69,70 and the Dr adhesin can also bind type IV collagen.…”