The Antibiotic Bacitracin Protects Human Intestinal Epithelial Cells and Stem Cell-Derived Intestinal Organoids from Clostridium difficile Toxin TcdB

Figure 2

Bac does not inhibit the TcdB-catalyzed glucosylation of Rac1 but decreases the pH-dependent membrane translocation of TcdB in human epithelial cells. (a) Bac has no effect on the TcdB-catalyzed glucosylation of Rac1 in vitro. TcdB (300 ng) was preincubated in the presence or absence of Bac (3 mM) for 30 min at 37°C. Then, CaCo-2 lysate (40 μg protein) was added and the samples were further incubated for 2 h at 37°C. Nonglucosylated Rac1 was detected by Western blotting. Actin was detected to confirm comparable protein loading. (b) Bac decreases the pH-dependent translocation of TcdB across cell membranes into the cytosol. HeLa were preincubated in serum-free medium containing Baf A1 (200 nM) for 30 min at 37°C. Afterwards, cells were kept for 40 min on ice prior to application of TcdB (200 ng/ml) and further incubation for 40 min at 4°C to enable TcdB binding to its cell surface receptor but to prevent the “normal” uptake of the cell-bound TcdB via acidified endosomal vesicles. Subsequently, cells were exposed for 5 min at 37°C to an acidic pulse (pH 3.8) to trigger the pH-dependent TcdB translocation into the cytosol. For control (con), additional toxin-treated cells were incubated at neutral conditions (pH 7.5). Following the acidic pulse, all samples were further incubated at 37°C in pH-neutral medium containing serum and Baf A1 (200 nM). Pictures from the cells were taken and the percentages of round cells calculated from the pictures. Values are given as (). Statistical significance between toxins’ groups and the bacitracin effect groups was tested using Student’s -test (, ). To analyze the effect of Bac (1 mM) on the binding of TcdB to the cells, Bac was applied to the cells either 30 min prior and during TcdB binding and after the acidic step. To test the effect of Bac on the pH-dependent translocation of TcdB across the cytoplasmic membrane, Bac (1 mM) was only applied during the acidic step.

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