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MOLECULAR AND CELLULAR PATHOGENESIS

Structural and Functional Lesions in Brush Border of Human Polarized Intestinal Caco-2/TC7 Cells Infected by Members of the Afa/Dr Diffusely Adhering Family of Escherichia coli

Isabelle Peiffer, Julie Guignot, Alain Barbat, Christophe Carnoy, Steve L. Moseley, Bogdan J. Nowicki, Alain L. Servin, Marie-Françoise Bernet-Camard
Isabelle Peiffer
Institut National de la Santé et de la Recherche Médicale (INSERM), Unité 510, Faculté de Pharmacie Paris XI, F-92296 Châtenay-Malabry, and
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Julie Guignot
Institut National de la Santé et de la Recherche Médicale (INSERM), Unité 510, Faculté de Pharmacie Paris XI, F-92296 Châtenay-Malabry, and
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Alain Barbat
INSERM, Unité 504, F-94407 Villejuif, France;
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Christophe Carnoy
Department of Microbiology, University of Washington, Seattle, Washington 98195-7242; and
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Steve L. Moseley
Department of Microbiology, University of Washington, Seattle, Washington 98195-7242; and
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Bogdan J. Nowicki
Division of Infectious Diseases, Department of Obstetrics and Gynecology, and Department of Microbiology, The University of Texas Medical Branch, Galveston, Texas 77550
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Alain L. Servin
Institut National de la Santé et de la Recherche Médicale (INSERM), Unité 510, Faculté de Pharmacie Paris XI, F-92296 Châtenay-Malabry, and
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Marie-Françoise Bernet-Camard
Institut National de la Santé et de la Recherche Médicale (INSERM), Unité 510, Faculté de Pharmacie Paris XI, F-92296 Châtenay-Malabry, and
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DOI: 10.1128/IAI.68.10.5979-5990.2000
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  • Fig. 1.
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    Fig. 1.

    Distribution of G- and F-actin and ADF in Afa/Dr DAEC C1845-infected cultured human polarized intestinal Caco-2/TC7 cells. Control (A, C, and E) and DAEC C1845-infected cells (B, D, and F) were stained with anti-G-actin polyclonal antibody (A and B), fluorescein-labeled phalloidin (F-actin labeling) (C and D), or anti-ADF polyclonal antibody (E and F). En face micrographs of control cells show the homogenous fine labeling of G-actin, F-actin, and ADF characteristic of brush border-associated proteins. The center regions of several cells seem to be unstained because the apical faces are convex and appear out of the focus. En face micrographs showing the immunolocalization of G-actin and ADF reveal that the homogenous immunolabeling of proteins is not modified in Afa/Dr DAEC C1845-infected cells. Immunolocalization of F-actin shows that the homogenous immunolabeling of protein is dramatically modified in Afa/Dr DAEC C1845-infected cells. Note that both the F-actin and ADF immunolabeling shows lateral concentrations at points of cell-to-cell contact. Magnifications, ×100.

  • Fig. 2.
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    Fig. 2.

    CLSM analysis of F-actin immunolabeling in Afa/Dr DAEC C1845-infected Caco-2/TC7 cells. Confluent differentiated Caco-2/TC7 cells were infected apically at 37°C in a 10% CO2–90% air atmosphere for 3 h with C1845 bacteria (108CFU/well). Cells were fixed with 3.5% paraformaldehyde, washed, permeabilized with Triton X-100, and processed for immunofluorescence labeling as described in Materials and Methods. Fixed and permeabilized cells were processed for direct immunofluorescence labeling of F-actin with fluorescein-labeled phalloidin as described in Materials and Methods. Cells were examined using a confocal laser scanning microscope (model PCM 2000; Diaphot 300 microscope using a 100× Pan Fluor ELSW DM CF160 objective; Nikon). The samples were analyzed by serial optical horizontal sectioning. The section starts at the basal domain of the cells, and the following analysis was conducted until the apical domain was reached. (A and a) Control uninfected cells; (B and b) DAEC C1845-infected cells. (A and B) En face micrographs of the immunolocalization of F-actin obtained in CLSM analysis (horizontalx-y optical sections). In control cells, the majority of F-actin labeling starts on section 3 and afterwards is distributed in six sections (one section every 0.60 μm). In C1845-infected cells, the majority of the F-actin labeling starts on section 5 and afterwards is distributed in 12 sections (one section every 0.50 μm). (a and b) Lateral views of the immunolocalization of F-actin obtained in CLSM analysis (vertical x-z optical section). In control cells, F-actin labeling is localized at the apical domain in a homogenous band. In C1845-infected cells, apical F-actin labeling is dramatically modified, showing a disruption in apical labeling and delocalization of the protein in a nonhomogenous band.

  • Fig. 3.
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    Fig. 3.

    Distribution of villin, fimbrin, and tubulin in Afa/Dr DAEC C1845-infected human cultured polarized intestinal Caco-2/TC7 cells. Experimental conditions were as in Fig. 1. Cells were stained with MAbs for antivillin (A and B), antifimbrin (C and D), and antitubulin (E and F). (A, C, and E) Control uninfected cells; (B, D, and F) DAEC C1845-infected cells. En face micrographs in control cells show the fine and homogenous labeling of villin and fimbrin characteristic of MV-associated proteins. The center regions of several cells seem to be unstained because the apical faces are convex and appear out of focus. In DAEC C1845-infected cells, disappearance of the fine and homogenous pattern and appearance of clusters of aggregated villin and fimbrin are observed. No change in tubulin distribution is observed DAEC C1845-infected cells. Magnifications, ×100.

  • Fig. 4.
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    Fig. 4.

    Distribution of apical F-actin in Afa/Dr DAEC C1845-infected Caco-2/TC7 cells treated with the chelator of intracellular Ca2+ BAPTA/AM. Experimental conditions were as in Fig. 1. En face micrographs of the immunolocalization of apical F-actin in (A) uninfected control cells, (B) uninfected cells treated with BAPTA/AM (25 μM), (C) DAEC C1845-infected cells, and (D) DAEC C1845-infected cells treated with BAPTA/AM (25 μM). In DAEC C1845-infected cells treated with BAPTA/AM, the fine, flocculated F-actin labeling centrally in the cells remains present. Note that the adhering bacteria appeared at the cell surface and that F-actin organizes around the adhering bacteria. Magnifications, ×100.

  • Fig. 5.
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    Fig. 5.

    Transmission electron microscopy of Afa/Dr DAEC C1845-infected Caco-2/TC7 cells shows the disappearance of the well-developed brush border. Experimental conditions were as in Fig. 1. (A and B) Transverse sections of the apical domain of uninfected Caco-2/TC7 cells at low and high magnifications, respectively. The apical domain of the polarized cells is uniformly organized, showing a continuous brush border with well-ordered and dense MV. (C and D) Transverse sections of the apical domain of Afa/Dr DAEC C1845-infected Caco-2/TC7 cells at low and high magnifications, respectively. The apical domain is rounded, with randomly distributed sparse and shortened MV. Note that the polarized organization of the infected cells is the same as that of uninfected cells. Low magnification, ×2,000; high magnification, ×5,000.

  • Fig. 6.
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    Fig. 6.

    CLSM analysis of SI immunolabeling in Afa/Dr DAEC C1845-infected Caco-2/TC7 cells shows a dramatic rearrangement of the brush border-associated hydrolase. Experimental conditions were as in Fig. 1. Cells were fixed with 3.5% paraformaldehyde, washed, and processed for indirect immunofluorescence labeling of SI as described in Materials and Methods. Cells were examined using a confocal laser scanning microscope (model PCM 2000; Nikon). The samples were analyzed by serial optical horizontal sectioning starting at the basal domain of the cells and following up to the apical domain. (A and a) Control uninfected cells; (B and b) DAEC C1845-infected cells. (A and B) En face micrographs of the immunolocalization of SI obtained in CLSM analysis (horizontal x-y optical sections). In control cells, the majority of the SI labeling starts on section 1 and afterwards is distributed in six successive sections (one section every 0.30 μm). In C1845-infected cells, the majority of the SI labeling starts on section 1 and afterwards is distributed in 11 sections (one section every 0.30 μm). (a and b) Lateral views of the immunolocalization of SI obtained in CLSM analysis (verticalx-z optical section). In control cells, SI labeling is restricted at the apical domain in a homogenous band. In C1845-infected cells, a profound rearrangement of the cell distribution of the brush border-associated hydrolase is observed. SI immunolabeling is dramatically modified, showing a disruption in its apical localization and the appearance of a nonhomogenous band, suggesting cytoplasmic redistribution.

  • Fig. 7.
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    Fig. 7.

    Distribution of carbohydrate transporters GLUT5 and SGLT1 on Caco-2/TC7 cells infected by the wild-type Afa/Dr DAEC strain C1845. Experimental conditions were as in Fig. 1. The cells were stained with the appropriate MAbs directed against GLUT5 (A and B) or SGLT1 (C and D). (A and C) Control uninfected cells; (B and D) DAEC C1845-infected cells. En face micrographs of the immunolocalization of GLUT5 and SGLT1 in control cells (A and C) show the typical mosaic pattern distribution characteristic of MV-associated functional proteins. In C1845-infected cells (B and D), the mosaic pattern of distribution disappears. Note that clusters of aggregated proteins were observed centrally in the cells and that lateral concentrated immunolabeling at points of cell-to-cell contact underlined the honeycomb organization of the cells. Magnifications, ×100.

Tables

  • Figures
  • Table 1.

    Effect of calcium blockers on DAEC C1845-induced apical F-actin disassembly in Caco-2/TC7 cellsa

    Cells (treatment)Concn (μM)Mean relative F-actin immunofluorescence intensity ± SEMMean % of cells presenting apical F-actin disassembly ± SEM
    Control2.80 ± 0.180
    Infected0.32 ± 0.0995 ± 5
    Infected (BAPTA/AM)7.51.12 ± 0.17*60 ± 10*
    252.30 ± 0.19*25 ± 5*
    Infected (dantrolene)1000.75 ± 0.20*75 ± 8*
    2002.65 ± 0.35*15 ± 6*
    • ↵a Apical F-actin distribution was revealed by direct immunofluorescence labeling using phalloidin-fluorescein. Relative F-actin immunofluorescence intensity was reported as the mean ± SEM of arbitrary units measured as described in Materials and Methods. Results are also presented as the mean (± SEM) percentage of cells presenting apical F-actin disorganization relative to the total number of cells observed (54). Dantrolene and BAPTA/AM were present in the incubation medium 60 min before and during infection. Statistical analysis comparing infected cells with infected-treated cells was performed with a Student t test. ∗, significant difference (P < 0.01).

  • Table 2.

    Effect of point mutations within Dr hemagglutinin on binding and apical F-actin disassembly in Caco-2/TC7 cellsa

    PlasmidBindingb (mean % ± SEM)Apical F-actin disorganization (mean relative fluorescence intensity ± SEM)
    pCC905.5 ± 0.495 ± 4
    pCC90-D54stop0.7 ± 0.2*0d
    BN17 (draE)0.5 ± 0.2*0d
    pCC90-D54V5.1 ± 0.596 ± 4
    pCC90-D54Y5.5 ± 0.295 ± 5
    pCC90-T90M5.8 ± 0.493 ± 8
    pCC90-I113T5.8 ± 0.593 ± 7
    pCC90-D54G2.0 ± 0.3*15 ± 10*
    pCC90-D54C3.6 ± 0.5*5 ± 4*
    • ↵a Statistical analysis comparing pCC90 with the mutants was performed with a Student t test. ∗, Significant difference (P < 0.01).

    • ↵b Percentage of 14C-radiolabeled bacteria bound ± SEM.

    • c Apical F-actin organization was revealed by direct immunofluorescence labeling using phalloidin-fluorescein. Results are presented as the mean (± SEM) percentage of cells presenting apical F-actin disorganization relative to the total number of cells (54). Relative immunofluorescence intensities: uninfected cells, 2.78 ± 0.25; pCC90-infected cells, 0.42 ± 0.15; pCC90-D54G, 2.03 ± 0.40*; pCC90-D54C, 2.68 ± 0.30* (arbitrary units).

    • ↵d Although E. coli(pCC90-D54stop) and BN17 (draE) did not adhere to Caco-2/TC7 cells, isolated adhering E. coli could be observed in randomly distributed cells without alteration of the apical F-actin network.

  • Table 3.

    Effect of infection by wild-type Afa/Dr DAEC strains C1845 and IH11128 on apical distribution and enzyme activity of brush border-associated hydrolasesa

    EnzymeCellsMean relative immunofluorescence intensity ± SEMMean enzyme sp act (mU/mg of protein) ± SD (% inhibition)
    SIControls2.78 ± 0.32135 ± 10
    C1845 infected0.40 ± 0.14*35 ± 15** (74)
    IH11128 infected0.55 ± 0.20*50 ± 20** (63)
    DPPIVControls1.80 ± 0.25172 ± 7
    C1845 infected1.25 ± 0.15*124 ± 6* (28)
    IH11128 infected1.30 ± 0.10*123 ± 12* (28)
    • ↵a Apical SI and DPPIV distribution was observed by indirect immunolabeling as described in Materials and Methods. Relative immunofluorescence intensity is reported as the mean ± SEM (arbitrary units). SI and DPPIV enzyme activities were determined on the total membrane fraction from control and infected (3 h postinfection) cells as described in Materials and Methods. Results are means ± standard deviation for five independent experiments conducted with successive cell passages. Percent inhibition of enzyme activity in infected cells calculated relative to enzyme activity in control cells is shown in parentheses. Statistical analysis comparing infected with control cells was performed with a Student ttest. Significant difference: ∗, P < 0.05; ∗∗, P < 0.01.

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Structural and Functional Lesions in Brush Border of Human Polarized Intestinal Caco-2/TC7 Cells Infected by Members of the Afa/Dr Diffusely Adhering Family of Escherichia coli
Isabelle Peiffer, Julie Guignot, Alain Barbat, Christophe Carnoy, Steve L. Moseley, Bogdan J. Nowicki, Alain L. Servin, Marie-Françoise Bernet-Camard
Infection and Immunity Oct 2000, 68 (10) 5979-5990; DOI: 10.1128/IAI.68.10.5979-5990.2000

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Structural and Functional Lesions in Brush Border of Human Polarized Intestinal Caco-2/TC7 Cells Infected by Members of the Afa/Dr Diffusely Adhering Family of Escherichia coli
Isabelle Peiffer, Julie Guignot, Alain Barbat, Christophe Carnoy, Steve L. Moseley, Bogdan J. Nowicki, Alain L. Servin, Marie-Françoise Bernet-Camard
Infection and Immunity Oct 2000, 68 (10) 5979-5990; DOI: 10.1128/IAI.68.10.5979-5990.2000
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KEYWORDS

Adhesins, Escherichia coli
Escherichia coli
Intestinal Mucosa
Microvilli

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