Calpain regulates enterocyte brush border actin assembly and pathogenic Escherichia coli-mediated effacement

Abstract

This study identifies calpain as being instrumental for brush border (BB) microvillus assembly during differentiation and effacement during bacterial pathogenesis. Calpain activity is decreased by 25-80% in Caco 2 lines stably overexpressing calpastatin, the physiological inhibitor of calpain, and the effect is proportional to the calpastatin/calpain ratio. These lines exhibit a 2.5-fold reduction in the rate of microvillus extension. Apical microvillus assembly is reduced by up to 50%, as measured by quantitative fluorometric microscopy (QFM) of ezrin, indicating that calpain recruits ezrin to BB microvilli. Calpain inhibitors ZLLYCHN2, MDL 28170, and PD 150606 block BB assembly and ezrin recruitment to the BB. The HIV protease inhibitor ritonavir, which inhibits calpain at clinically relevant concentrations, also blocks BB assembly, whereas cathepsin and proteasome inhibitors do not. Microvillus effacement is inhibited after exposure of calpastatin-overexpressing cells to enteropathogenic Escherichia coli. These results suggest that calpain regulates BB assembly as well as pathological effacement, and indicate that it is an important regulator involved in HIV protease inhibitor toxicity and host-microbial pathogen interactions.

FIG. 1. Calpain activity is decreased in calpastatin-overexpressing Caco 2 Cells

Calpain activity was measured in intact cells by measuring hydrolysis of the cell permeant calpain substrate suc- LLVY-AMC by fluorimetry. Calpain activities of the calpastatin-overexpressing 2–3 and 0.5–11 lines are normalized to mean calpain activities of the control Caco 2 line, C9. Error bars shown for 2–3 and 0.5–11 indicate the S.D. of the measurements. The results shown are duplicate measurements of a representative experiment.

FIG. 2. Apical microvillus density and length are decreased in calpastatin-overexpressing Caco 2 lines

A, SEM images of the apical surface and microvilli of low and high calpastatin over-expressor Caco 2 lines (lines 2–3 and 0.5–11) are compared with the control line (C9). The cells were plated at 4-fold over confluence, fixed, and coated for SEM at 54 h of culture. The size bar shown is applicable for all images (1 µm). B, microvillus length (nm) of low and high calpastatin over-expressor Caco 2 lines (lines 2–3 and 0.5–11) is compared with the control line (C9), as a function of time following plating. Microvillus length was measured by morphometric analysis. Between 150 and 225 microvilli were measured for each time point, except for the 0.5–11 line at 6 h, where 25 microvilli were measured because of the low density of microvilli. The error bars indicate the S.D. of the mean.

FIG. 3. Ezrin content in apical microvilli of calpastatin-overexpressing Caco 2 cell lines

A, slides of confluent calpastatin-overexpressing Caco 2 cell lines were fixed, permeabilized, and immunostained with the rabbit anti-human ezrin antibody 4–170 (see “Experimental Procedures”) and a fluorescein-goat antimouse antibody. The images were photographed with a Spot CCD camera on a Zeiss Axioskop fluorescence microscope for 4 s. Fading was minimal under these conditions. Three representative fields are shown as a montage for each line. The size bar is 20 µm, for all images. B, QFM measurement of BB Ezrin. Mean pixel intensity was measured for apical microvilli, quantitatively analyzing the IF staining of calpastatin-overexpressing cells shown in A, using the QFM method. The reduction of apical ezrin staining was significant for both calpastatin-overexpressing cell lines (2–3, p < 0.0023; 0.5–11, p < 0.00010).

FIG. 4. Ezrin content in apical microvilli of calpain inhibitor-treated Caco 2 cells

A–I, Caco 2 cells were treated with Me₂SO (A), ZLLYCHN₂ (25 µm) (B, C), NH₄Cl (1 mm) (D), MDL 28,170 (50 µm) (E, F), lactacystin (1 µm) (G), PD150606 (50 µm) (H), or ritonavir (40 µm) (I). Slides were immunostained with rabbit anti-human ezrin antibody 4–170 and a fluorescein-goat anti-mouse antibody. Images were photographed with a Spot CCD camera on a Zeiss Axioskop fluorescence microscope for 4 s. Fading was minimal under these conditions. Three representative fields are shown as a montage for each cell line. The size bar is 20 µm. J, QFM measurement of BB ezrin. Mean pixel intensity was measured for apical microvilli, quantitatively analyzing the IF staining of cells shown in A–I, using the QFM method. The reduction of apical ezrin staining was significant for all calpain inhibitor conditions shown.

FIG. 5. Apical F-actin content is markedly decreased in calpastatin-overexpressing Caco 2 cells

The large rectangles contain summation of Oregon Green-phalloidin (Molecular Probes) staining along the z-axis, viewing from the basolateral surface to apical. Shown are stainings of monolayers of the control line, C9, and the calpastatin-overexpressing line, 2-1. The smaller rectangles contain vertical sections through the monolayer, located at the positions indicated by the black arrows. The apical domains of the monolayers are indicated with white arrows. The round, unspread cell located on the apical surface of the 2-1 monolayer indicates the position of the apical domain, which is poorly visualized due to decreased F-actin. Size bars are 20 µm for the C9 cells and 25 µm for the 2-1 line.

FIG. 6. Calpastatin overexpression blocks EPEC-mediated effacement of the BB

Calpastatin-overexpressing Caco 2 cell lines plated at confluence and cultured for 2 weeks on Permanox microscope slides were infected with log phase EPEC in complete medium (CM) for 30 min, without antibiotics. Infected monolayers were fixed and stained for TEM. A, control Caco 2 cell line C9 stably transfected with pRC/CMV vector is shown. The upper panel shows cells infected with 1.5 × 10⁸ EPEC/ml. The lower panel shows mock-infected cells. B, calpastatin-overexpressing Caco 2 line 0.5–11 is shown. The upper panel shows cells infected with 1.5 × 10⁸ EPEC/ml. The lower panel shows mock-infected cells. C, calpastatin-overexpressing Caco 2 line 2-1 is shown. The upper panel shows cells infected with 1.5 × 10⁸ EPEC/ml. The lower panel shows mock-infected cells. The size bar is 0.8 µm.