<i>E</i>. <i>coli</i> AfaE-III suppresses epithelial exfoliation in CEAtg mice.

<p><i>(A)</i> Whole mount urogenital tracts of uninfected wild-type or CEAtg female mice were fixed and processed for scanning electron microscopy (SEM). Pictures show the luminal surface of the upper vaginal and cervical regions and are representative for five animals each treatment group. <i>(B)</i> Wild-type or CEAtg female mice were infected with <i>E</i>. <i>coli</i> AfaE-III or <i>E</i>. <i>coli</i> ΔAfaE-III for 24 h and the genital tracts processed as in (A). SEM pictures (at two different magnifications, as indicated by the scale bars) show the luminal surface of the upper vaginal and cervical regions. Whereas massive epithelial exfoliation is evident in infected wildtype mice and in CEAtg mice infected with <i>E</i>. <i>coli</i> ΔAfaE-III, a strongly reduced detachment of epithelial cells is observed in CEAtg mice infected with CEACAM-binding <i>E</i>. <i>coli</i> AfaE-III. Adherent bacteria can be observed at higher magnifications (arrows). Pictures are representative for five animals each treatment group. <i>(C)</i> Quantification of exfoliating epithelial cells from samples in (B). Bars represent mean ± S.D. of exfoliating cells from at least n = 26 areas (~0.075 mm²) derived from at least five animals each treatment group. Results were compared by Mann-Whitney U-test and highly significant differences (p<0.001) are indicated by ***.</p

The interaction of <i>E</i>. <i>coli</i> Opa_CEA with CEA facilitates mucosal colonization and leads to CD105 expression in epithelial cells.

<p><i>(A)</i> Wild-type (●) or CEAtg (▼) female mice were infected with the indicated bacterial strains and 24 h later, bacteria were re-isolated. Each data point in the graph reflects the number of bacteria re-isolated from an individual animal (n = 10; except for CEAtg animals infected with <i>E</i>. <i>coli</i> Opa_CEA, where n = 15). Data were compiled from four independent experiments. The median for each experimental group of animals is indicated by a line; groups were compared by Mann-Whitney U-test and highly significant differences (p<0.001) are indicated by ***. <i>(B)</i> Individual re-isolated bacterial colonies from the genital tract of CEAtg mice infected with either <i>E</i>. <i>coli</i> or <i>E</i>. <i>coli</i> Opa_CEA were plated on LB-ampicillin agar plates (post infection). Five isolates for each strain were analysed by Western blotting with an antibody against Opa protein. As a control, lysates of the <i>E</i>. <i>coli</i> or <i>E</i>. <i>coli</i> Opa_CEA used for infection (input) were also analysed. <i>(C)</i> Genital tracts from CEAtg mice infected for 24 hours with <i>E</i>. <i>coli</i> or <i>E</i>. <i>coli</i> Opa_CEA were excised, and cryosections were co-stained with antibodies against <i>E</i>. <i>coli</i> (green) and against CEA (red). Cell nuclei were stained with Hoechst dye (blue). Arrowheads indicate host-associated <i>E</i>. <i>coli</i>. Pictures are representative for three independent biological replicates. <i>(D)</i> Cryosections as in (C) were co-stained with antibodies against <i>E</i>. <i>coli</i> (green) and a rat monoclonal antibody against murine CD105 (red). Cell nuclei were visualized by Hoechst (blue). CD105 expression on the mucosal surface of CEAtg mice infected with <i>E</i>. <i>coli</i> Opa_CEA is highlighted by small arrows. Pictures are representative for three independent biological replicates.</p

<i>E</i>. <i>coli</i> AfaE-III shows improved mucosal colonization and induces CD105 expression.

<p><i>(A)</i> Wild-type or CEAtg female mice were infected with <i>E</i>. <i>coli</i> AfaE-III or <i>E</i>. <i>coli</i> ΔAfaE-III. 24 h later, bacteria were re-isolated. Each data point in the graph reflects the number of bacteria re-isolated from an individual animal (n = 10). Data were compiled from two independent experiments. The median for each experimental group of animals is indicated by a line; numbers of recovered bacteria were compared by Mann-Whitney U-test and highly significant differences (p<0.001) are indicated by ***. <i>(B</i>, <i>C)</i> Animals were infected as in (A) and genital tracts were excised, fixed and frozen. <i>(B)</i> Cryosections of genital tracts were co-stained with rabbit antibodies against <i>E</i>. <i>coli</i> (green) and a mouse monoclonal antibody against CEA. Cell nuclei were visualized by Hoechst (blue). Numerous <i>E</i>. <i>coli</i> AfaE-III can be detected in close association with the CEA-positive epithelium (arrowhead), whereas non-CEACAM binding <i>E</i>. <i>coli</i> are rarely observed. (C) Cryosections were co-stained with rabbit antibodies against <i>E</i>. <i>coli</i> (green) and a rat monoclonal antibody against murine CD105 (red). Cell nuclei were visualized by Hoechst (blue). Strong local expression of CD105 can be observed on the mucosal surface of CEAtg mice upon association with <i>E</i>. <i>coli</i> AfaE-III (arrowhead). Pictures in B) and C) are representative for three independent biological replicates.</p

CEA engagement by <i>E</i>. <i>coli</i> AfaE-III triggers enhanced cell-matrix adhesion via integrin activation.

<p><i>(A)</i> 293 cells were transfected with a control vector (GFP), or plasmids encoding CEA or CD105. Cells were left uninfected or infected for 8 h with the indicated bacteria. After infection, cells were used in adhesion assays on collagen. Bars represent means ± S.D. of 8 replicates. Two-tailed student’s t-test; *** p < 0.001, n.s.–not significant. Shown is one representative experiment out of three independent biological replicates <i>(B)</i> 293 cells were transfected with CEA and seeded on 25 μg/ml collagen. Confluent layers were left uninfected or infected for 14 h with <i>E</i>. <i>coli</i>, <i>E</i>. <i>coli</i> AfaE-III, or <i>E</i>. <i>coli</i> ΔAfaE-III. Following infection, cells were washed and remaining cells were stained with crystal violet. Representative areas with remaining cells were photographed. Pictures are representative for three independent biological replicates. <i>(C)</i> 293 cells were infected and stained as in (B). Staining intensity was determined after dye elution in a spectrophotometer at 550 nm. Bars represent mean ± S.D. of 8 replicates. Two-tailed student’s t-test; *** p < 0.001, n.s.—not significant. Shown is one representative experiment out of three independent biological replicates. <i>(D)</i> 293 cells were transfected with plasmids encoding CEA or CD105. Cells were left uninfected or infected for 8 h with the indicated bacteria. After infection, cells were used in adhesion assays on collagen in the absence or presence of 1 mM Mn²⁺. Bars represent means ± S.D. of 8 replicates. Two-tailed student’s t-test; *** p < 0.001, n.s.—not significant. Shown is one representative experiment out of three independent biological replicates. <i>(E)</i> CEA-transfected 293 cells were infected for 14 h with the indicated bacteria and analyzed by flow cytometry for CD105 expression. Gray area indicates staining of uninfected cells with an isotype matched control antibody. Shown is one representative experiment out of three independent biological replicates. <i>(F</i>, <i>G)</i> 293 cells were transfected with plasmids encoding either CEA or CD105. As indicated, transfected cells were infected with bacteria for 14 h or left uninfected. After infection, cells were replated onto collagen-coated culture dishes for 90 min and stimulated or not for 5 min with 1 mM Mn²⁺ before fixation. Fixed samples were either stained with a rat monoclonal integrin β1 antibody (clone AIIB2), which recognizes the integrin β1 extracellular domain irrespective of its conformation (total integrin) (F) or samples were stained with an activation-epitope specific rat monoclonal integrin β1 antibody (clone 9EG7), which recognizes the extended, ligand-bound conformation of integrin β1 (active integrin)(G). Bars represent the mean ± S.D. of 5 replicates. Two-tailed student’s t-test; *** p < 0.001, n.s.—not significant. Shown is one representative experiment out of three independent biological replicates.</p

Uropathogenic <i>E</i>. <i>coli</i> expressing the AfaE-III adhesin selectively bind to the amino terminal domain of human CEACAM family members.

<p><i>(A)</i> The indicated soluble CEACAM-GFP fusion proteins (human CEACAM1-NT, CEA-NT, and CEACAM8-NT) were produced in 293 cells and the resulting cell culture supernatants were incubated with <i>E</i>. <i>coli</i> AfaE-III or with the AfaE-III-deficient strain (<i>E</i>. <i>coli</i> ΔAfaE-III). After washing, bacteria-associated CEACAM-GFP fusion proteins were detected by Western blotting with a monoclonal anti-GFP antibody (Pull-down; upper two panels). To verify the presence of equal amounts of the CEACAM-GFP fusion proteins, cell culture supernatants were analyzed by Western blotting with anti-GFP antibody (Supe; lowest panel). Shown is a representative experiment out of three independent biological replicates. (<i>B</i>) Cell culture supernatants containing the soluble N-terminal domains of murine CEACAM1 (mCEA1), bovine CEACAM1a (bCEA1a), bovine CEACAM1b (bCEA1b), canine CEACAM1 (cCEA1), human CEACAM1 (hCEA1) as GFP-fusion proteins, or GFP alone (GFP) were incubated with <i>E</i>. <i>coli</i> AfaE-III. After washing, bacteria-associated CEACAM1 was detected as in (A) (Pull-down; upper panel). The presence of CEACAM-GFP fusion proteins was analyzed as in (A) (Supe; lower panel). Shown is a representative experiment out of three independent biological replicates. <i>(C)</i> ME-180 cells were infected or not with the indicated bacteria for 2 h, fixed and co-stained with antibodies against endogenous CEACAMs (clone D14HD11; red) and rabbit anti-<i>E</i>. <i>coli</i> (green). Arrowheads indicate bacteria bound to clustered CEA. Pictures are representative for four independent biological replicates.</p