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

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.

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.

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.

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.

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.