Activation of Rho GTPases by Escherichia coli cytotoxic necrotizing factor 1 increases intestinal permeability in Caco-2 cells

Abstract

The cytotoxic necrotizing factor 1 (CNF1) activates Rho GTPases by deamidation of glutamine-63 and thereby induces redistribution of the actin cytoskeleton and formation of stress fibers. Here, we have studied the effects of CNF1 on the transepithelial resistance of Caco-2 cells, a human intestinal epithelial cell line, in comparison with the Rho-inactivating toxin B of Clostridium difficile. Whereas toxin B decreased the transepithelial resistance of Caco-2 cells by about 80% after 4 h, CNF1 reduced it by about 40%. Significant changes of the transepithelial resistance induced by CNF1 were detected after 3 h of incubation. Half-maximal effects were observed with 10 and 41 ng of CNF1 and toxin B per ml, respectively. Flux measurement revealed no CNF1-induced increase of fluorescein isothiocyanate-dextran permeation within the first 4 h of incubation and a 2.9-fold increase after 24 h of incubation. In contrast, toxin B induced a 28-fold increase of permeation after 24 h. As detected by rhodamine-phalloidin staining, CNF1 increased polymerization of F actin at focal contacts of adjacent cells and induced formation of stress fibers. The data indicate that not only depolymerization but also polymerization of actin and subsequent reorganization of the actin cytoskeleton alter the barrier function of intestinal tight junctions.

FIG. 1

Effect of GST-CNF1 on the transepithelial resistance of Caco-2 cells. (A) Caco-2 cells incubated for the indicated times with increasing concentrations of GST-CNF1. Thereafter, transepithelial resistance (TER) was determined as described in Materials and Methods. (B) EC₅₀ of GST-CNF1 (9.85 ng/ml) calculated from changes in transepithelial resistance after 4 h of incubation with the toxin. The effect of GST-CNF showed a lag period of 0.5 to 1 h, in which no significant changes in transepithelial resistance were detectable. Data are means ± standard deviations (n = 4).

FIG. 2

Effects of GST-CNF1 and GST-CNF1-C866S on transepithelial resistance. Caco-2 cells were incubated with GST-CNF1 (100 ng/ml), CNF1 liberated from GST (100 ng/ml), heat-inactivated (10 min, 95°C) GST-CNF1 (200 ng/ml) and the enzymatically inactive mutant GST-CNF1-C866S (200 ng/ml), and purified GST (200 ng/ml) for 4 h. Thereafter, transepithelial resistance (TER) was determined. Data are means ± standard deviations (n = 4, *P < 0.05).

FIG. 3

Effect of C. difficile toxin B on transepithelial resistance (TER). (A) Caco-2 cells treated with increasing concentrations of toxin B for the indicated times. Thereafter, transepithelial resistance was determined. (B) EC₅₀ (41 ng/ml) of toxin B calculated for changes in the transepithelial resistance occurring after 4 h of incubation with the toxin. Data are means ± standard deviations. (n = 5 to 10).

FIG. 4

Flux measurement with FITC-dextran. Caco-2 cell monolayers were treated with GST-CNF1 (100 ng ml⁻¹) or toxin B (100 ng ml⁻¹) for 4 and 24 h, respectively. At the indicated times, the permeability of the monolayer was determined by measurement of FITC-dextran fluxes as described in Materials and Methods. As a positive control, the cells were treated with 0.05% Triton X-100. Data are means ± standard deviations (n = 4 to 5; ∗, P < 0.05).

FIG. 5

Effects of CNF1 on toxin B-induced decrease in transepithelial resistance (TER). Caco-2 cell monolayers were incubated for 1 h with CNF1 (100 ng/ml [upper panel]) and toxin B (ToxB, [100 ng/ml [lower panel]), respectively. Thereafter, toxin B (100 ng/ml [upper panel]) or CNF1 (100 ng/ml [lower panel]) was added, and the incubation was continued for the indicated times. At the indicated time points, transepithelial resistance was determined. Data are means ± standard deviations (n = 5).

FIG. 6

[³²P]ADP ribosylation of Rho in lysates of CNF1- and toxin B-treated Caco-2 cells. Caco-2 cells were treated for 4 h without (control) and with GST-CNF1 (100 ng/ml) and toxin B (100 ng/ml), respectively. Thereafter, cells were lysed and Rho proteins were [³²P]ADP ribosylated in the presence of [³²P]NAD and C3 exoenzyme. The labeled proteins were analyzed by SDS-PAGE and phosphorimaging (shown). C3 labeled Rho with an apparent molecular mass of 23 kDa. Treatment of cells with GST-CNF1, which deamidates Rho, causes a gel shift to higher apparent molecular mass. Toxin B-induced glucosylation of Rho prevents subsequent ADP ribosylation by C3.

FIG. 7

Phase-contrast microscopy of CNF1- and toxin B-treated Caco2 cells. Caco-2 cells were untreated (A) or treated with GST-CNF1 (100 ng ml⁻¹) (B), with GST-CNF1C866S (100 ng ml⁻¹) (C), or with toxin B (100 ng ml⁻¹) (D) for 6 h. (E) Cells were also treated with CNF1 (100 ng ml⁻¹) for 1 h, and then toxin B was added for a further 5 h. After treatment, the cells were fixed and applied for phase-contrast microscopy.

FIG. 8

Rhodamine-phalloidin staining of CNF1- and toxin B-treated Caco-2 cells. Caco-2 cells were untreated (control) (A) or were treated with GST-CNF1 (100 ng ml⁻¹) (B), GST-CNF1C866S (100 ng ml⁻¹) (C), toxin B (100 ng ml⁻¹) (D), and toxin B after 1 h of preincubation with GST-CNF1 (each 100 ng ml⁻¹) (E) for 6 h. Thereafter, the cells were washed, and F actin was stained with rhodamine-phalloidin. Arrows indicate prominent formation of stress fibers and actin filaments located at the adherens junctions of adjacent cells.