Escherichia coli DraE adhesin-associated bacterial internalization by epithelial cells is promoted independently by decay-accelerating factor and carcinoembryonic antigen-related cell adhesion molecule binding and does not require the DraD invasin

Abstract

The Dr family of Escherichia coli adhesins are virulence factors associated with diarrhea and urinary tract infections. Dr fimbriae are comprised of two subunits. DraE/AfaE represents the major structural, antigenic, and adhesive subunit, which recognizes decay-accelerating factor (DAF) and carcinoembryonic antigen (CEA)-related cell adhesion molecules (CEACAMs) CEA, CEACAM1, CEACAM3, and CEACAM6 as binding receptors. The DraD/AfaD subunit caps fimbriae and has been implicated in the entry of Dr-fimbriated E. coli into host cells. In this study, we demonstrate that DAF or CEACAM receptors independently promote DraE-mediated internalization of E. coli by CHO cell transfectants expressing these receptors. We also found that DraE-positive recombinant bacteria adhere to and are internalized by primary human bladder epithelial cells which express DAF and CEACAMs. DraE-mediated bacterial internalization by bladder cells was inhibited by agents which disrupt lipid rafts, microtubules, and phosphatidylinositol 3-kinase (PI3K) activity. Immunofluorescence confocal microscopic examination of epithelial cells detected considerable recruitment of caveolin, beta(1) integrin, phosphorylated ezrin, phosphorylated PI3K, and tubulin, but not F-actin, by cell-associated bacteria. Finally, we demonstrate that the DraD subunit, previously implicated as an "invasin," is not required for beta(1) integrin recruitment or bacterial internalization.

FIG. 1.

Adherence and invasion of CHO cell transfectants expressing DAF, CEA, CEACAM1, CEACAM3, or CEACAM6 from a DraE⁺ recombinant strain [AAEC191A(pCC90)]. (A) Adherence of CHO cell transfectants by DraE⁺ E. coli. The results are expressed as CFU of cell-associated bacteria per well (n = 6) (data are means ± SD). (B) Invasion of CHO cell transfectants by DraE⁺ E. coli. Cells were preincubated with 20 μM PP2, 10 μg/ml nocodazole, and 100 μM wortmannin for 1 h (PP2 and nocodazole) or 15 min (wortmannin) prior to infection. The results are expressed as percentages of cell-associated bacteria internalized (means ± SD). All assays were conducted in triplicate. *, differences are statistically significant (P < 0.001).

FIG. 2.

Differentiated primary epithelial bladder cells express DAF and CEACAM receptors and mediate adherence and internalization of DraE⁺ E. coli. (A and C) Expression of DAF by bladder cells. The cells were stained with anti-DAF antibodies and visualized by fluorescence microscopy. (B and C) Expression of CEACAM receptors by bladder cells. The cells were stained with anti-CEACAM antibodies and visualized by fluorescence microscopy. (D and E) Cells were infected with red fluorescent bacteria expressing DraE [AAEC191A(pCC90-DraEstop, pUC-R-RFP)] for 2 h. After infection, the samples were fixed and immunostained for actin expression (green fluorescence). (D) Nondifferentiated cells; (E) differentiated cells. (F) Invasion of primary epithelial bladder cells by DraE⁺ DraD⁺ E. coli [AAEC191A(pCC90)], DraE⁺ DraD⁻ E. coli [AAEC191A(pCC90-DraDstop)], and DraE⁻ DraD⁺ E. coli [AAEC191A(pCC90-DraEstop)]. The results are expressed as CFU of intracellular bacteria per well (n = 6) (data are means ± SD). All assays were conducted in triplicate. (G) Effects of inhibitors on invasion of primary epithelial bladder cells by DraE⁺ E. coli [AAEC191A(pCC90)]. Cells were preincubated with 20 μM PP2 (inhibitor of SRC kinase), 10 mM MβCD (cholesterol-depleting agent), or 10 μg/ml nocodazole (microtubule-depolymerizing agent) for 1 h prior to infection or with 100 μM wortmannin (inhibitor of PI3K) for 15 min prior to infection. The results are expressed as percentages of cell-associated bacteria internalized (means ± SD). All assays were conducted in triplicate. *, differences are statistically significant (P < 0.001).

FIG. 3.

Binding of DraE⁺ E. coli [AAEC191A(pCC90)] to bladder cells elicits aggregation of tubulin around bacteria. After infection, the samples were fixed and processed for double immunofluorescence labeling with anti-Dr adhesin antibodies (red fluorescence) (A and C) and anti-α-tubulin antibody (green) (B and C). Arrows indicate examples of tubulin colocalized with bacteria.

FIG. 4.

Binding of DraE⁺ E. coli to bladder cells triggers recruitment and activation of ERM around bacteria. The cells were infected with red fluorescent bacteria (red fluorescence in panels A, C, D, and E) expressing DraE [AAEC191A(pCC90-DraEstop, pUC-R-RFP)] for 2 h. After infection, the samples were fixed and immunofluorescently labeled with anti-ERM antibodies (green fluorescence in panels B and C) or anti-p-ezrin antibodies (green fluorescence in panels D and E). (E) Confocal microscopy x-z section showing E. coli fluorescence (red) and p-ezrin (green) immunofluorescence in bladder cells. Arrows indicate examples of colocalization of ERM or p-ezrin with cell-associated bacteria.

FIG. 5.

DraE⁺ E. coli is associated with caveolin and phosphorylated PI3K in bladder cells. The cells were infected with red fluorescent bacteria (red fluorescence in panels A, C, D, and F) expressing DraE [AAEC191A(pCC90-DraEstop, pUC-R-RFP)] for 2 h. After infection, the samples were fixed and immunofluorescently labeled with anti-caveolin antibodies (green fluorescence in panels B and C) or anti-p-PI3K antibodies (green fluorescence in panels E and F). Arrows indicate examples of bacteria colocalized with caveolin or p-PI3K.

FIG. 6.

Independent recruitment of DAF and CEACAM to Dr-fimbriated E. coli adhered to bladder cells. Bladder cells were infected for 2 h with E. coli expressing Dr fimbriae comprised of DraE (A, B, and C), the DraE D61A mutant (D, E, and F), or NfaE (G, H, and I) [AAEC191A(pCC90-DraEstop, pUC-R), AAEC191A(pCC90-DraEstop, pUC-R-D61A), or AAEC191A(pCC90-DraEstop, pUC-NfaE), respectively]. After infection, the samples were fixed and processed for double immunofluorescence labeling with anti-DAF (red) (A, C, D, F, G, and I) and anti-CEACAM (green) (B, C, E, F, H, and I). Arrows denote bacteria.

FIG. 7.

DraE binding to GPI-anchored receptors is required for β₁ integrin clustering around bacteria. Caco-2 cells were infected with DraE⁺ DraD⁺ [AAEC191A(pCC90-DraEstop, pUC-R)] (A, B, and C), DraE⁺ DraD⁻ [AAEC191A(pCC90-ΔSacI, pUC-R)] (D, E, and F), or NfaE⁺ DraD⁺ [AAEC191A(pCC90-DraEstop, pUC-NfaE)] (G, H, and I) E. coli for 2 h. After infection, the samples were fixed and processed for double immunofluorescence labeling with anti-CEACAM (green) (A, C, D, and F), anti-DAF (green) (G and I), or anti-β₁ integrin (red) (B, C, E, F, H, and I). Arrows indicate colocalization of CEACAM or DAF with β₁ integrin at the site of bacterial adherence.

FIG. 8.

SPR analysis of interactions between DraD and α₅β1 integrin. (A) Sensogram depicting the binding of DraD to immobilized α₅β₁ integrin (800 response units). (B) Equilibrium measurements shown in panel A were analyzed with BIAevaluation 3.0 software to globally fit the data and to derive the dissociation constants (K_d). A fit of these data is shown. The injection time was 120 s, and the flow rate was 20 μl/ml. RU, response units; Req, response units at equilibrium; conc, M, molar concentration of DraD.