Recruitment of CD55 and CD66e brush border-associated glycosylphosphatidylinositol-anchored proteins by members of the Afa/Dr diffusely adhering family of Escherichia coli that 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 border-associated 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.

FIG. 1

Change in CD55 distribution in Afa/Dr DAEC C1845-infected human fully differentiated intestinal Caco-2/TC7 cells. Cells were infected with E. coli C1845 or recombinant strain HB101(pSSS1) (F1845⁺) for 3 h at 37°C before washing and fixing. The paraformaldehyde-fixed cells were stained with MAb CY-CD55 and examined at the apical domain. (A) CD55 in control cells showing punctate labeling and mosaic pattern distribution. (B) Immunolabeling of CD55 in C1845-infected cells showing disappearance of CD55 mosaic pattern distribution and appearance of positive CD55 immunolabeling around adhering bacteria indicating CD55 clustering. (C) CD55 in E. coli recombinant strain HB101(pSSS1) (F1845⁺)-infected cells showing disappearance of CD55 mosaic pattern distribution and appearance of positive CD55 immunolabeling around adhering bacteria indicating CD55 clustering. (D and E) As a control, C1845 plated on glass slide and immunolabeled with anti-Dr or anti-CD55 antibodies, respectively, shows positive Dr labeling (D) and negative CD55 labeling (E). Magnifications, ×100.

FIG. 2

CD55 clustering around adhering Afa/Dr DAEC C1845 infecting CHO cell transfectant clones that stably express human CD55 cDNA, cDNA deletion constructs, or a construct for a TM version of CD55. Cells were infected with strain C1845 for 3 h at 37°C before washing and fixing. The paraformaldehyde-fixed cells were stained with MAb CY-CD55. (A and B) Expression of CD55 in uninfected DAF/A9 clone (A) and CD55 clustering around adhering bacteria (B) in infected DAF/A9 clone. (C and D) No CD55 clustering (C) around adhering bacteria observed by phase-contrast microscopy (D) in infected DAFΔSCR1/029-6B clone. (E and F) CD55 clustering around adhering bacteria in infected DAFΔSCR4/054-5×4 and DAF-TM/2H clones, respectively. magnifications, ×100.

FIG. 3

Change in apical distribution of the GPI-anchored CD66e and AP in Afa/Dr DAEC C1845-infected human fully differentiated intestinal Caco-2/TC7 cells. Cells infected with E. coli C1845 or recombinant strains for 3 h at 37°C before washing and fixing. Paraformaldehyde-fixed cells were stained with MAb anti-CD66E or anti-AP and examined at the apical domain. (A) CD66e in control cells showing punctate labeling and mosaic pattern distribution. (B) Disappearance of CD66e mosaic pattern distribution in C1845-infected cells and appearance of positive CD66e immunolabeling around adhering bacteria. (C) Disappearance of CD66e mosaic pattern distribution in E. coli recombinant strain HB101(pSSS1) (F1845⁺)-infected cells and appearance of positive CD66e immunolabeling around adhering bacteria. (D) AP in control cells showing punctate labeling and mosaic pattern distribution. (E) Disorganization of the AP mosaic pattern distribution in C1845-infected cells and absence of AP labeling around infecting bacteria. We noticed that as for CD55 (Fig. 1), the C1845 bacteria plated on glass slide and immunolabeled with appropriate antibodies presented no immunolabeling of CD66e or AP around adhering bacteria (not shown). Magnifications, ×100.

FIG. 4

CD66e clustering around adhering Afa/Dr DAEC bacteria infecting stably transfected HeLa cells expressing CD66e. HeLa cells were infected with strains C1845 (B, F, and H), HB101(pSSS1) (F1845⁺) (C), and EC901(pBN406) (Dr⁺) (D). The paraformaldehyde-fixed cells were stained with MAb anti-CEA D14HD11 recognizing CD66e (A, B, E to H), anti-CD55 (C and D) or with polyclonal anti-Dr antibody (A). (A to C) HeLa cells containing the expression vector (HeLa-SFFV.neo) alone. (D to H) HeLa cells stably transfected with CD66e cDNA (HeLa-CD66e). (A) Immunolabeling with anti-Dr antibody in C1845-infected HeLa cells-SFFV.neo reveals adhering bacteria. (B) No clustering of CD66e around C1845 bacteria infecting the HeLa-SFFV.neo cells. (C) Clustering of CD55 around C1845 bacteria infecting HeLa-SFFV.neo cells. (D) Clustering of CD55 around C1845 bacteria infecting HeLa-CD66e cells. (E) Positive CD66e immunofluorescence in HeLa-CD66e cells. (F to H) Clustering of CD66e around adhering C1845 (F), HB101(pSSS1) (F1845⁺) (G), and EC901(pBN406) (Dr⁺) (H) bacteria infecting the HeLa-CD66e cells. Magnifications, ×100.

FIG. 5

CD55 and CD66e immunolabeling in human fully differentiated Caco-2/TC7 cells infected by recombinant E. coli(pCC90) and a Dr mutant carrying pCC90-D54C. Experimental conditions were as for Fig. 1. (A) CD55 clustering around adhering pCC90 (Dr⁺-carrying bacteria. (B and C) No CD55 (B) and CD66e (C) clustering around pCC90-D54C-carrying adhering bacteria. Arrows indicate adhering bacteria. Magnifications, ×100.