Tailored ß-Cyclodextrin Blocks the Translocation Pores of Binary Exotoxins from C. Botulinum and C. Perfringens and Protects Cells from Intoxication
Figure 7
AMBnTßCD protects Vero cells from intoxication with iota toxin from C. perfringens and inhibits the pH-dependent membrane translocation of the toxin.
A. Time- and concentration-dependent inhibition of the intoxication of Vero cells with iota toxin. Vero cells grown in 24-well dishes to subconfluency were treated for 30 min at 37°C with 2, 5, 10 and 20 µM final concentrations of AMBnTßCD or without AMBnTßCD for control. Iota toxin (200 ng/ml Ib+100 ng/ml Ia) was added and cells were further incubated at 37°C with the toxin in the absence or presence of AMBnTßCD. Pictures were taken after the indicated incubation periods, the number of total cells and round cells were counted and the percentages of round cells calculated. Values are given as mean ± S.D. (n = 3) and significance was tested for each time point between iota toxin-treated samples without and with the respective concentration of AMBnTßCD by using the student's t-test (***p<0.0005; ***<0.005; *<0.05). B. The time point of AMBnTßCD application determines the protective effect against iota toxin. AMBnTßCD (10 µM) was applied to Vero cells at 30, 15 or 5 min before iota toxin (200 ng/ml Ib+100 ng/ml Ia), together with iota toxin or 5, 15 or 30 min after the toxin. As a control, cells were treated with medium or with iota toxin alone. The cells were incubated for 3 h at 37°C and pictures were taken to determine the percentages of round cells. Values are given as mean ± S.D. (n = 3) and significance was tested between cells treated with iota toxin alone and cells treated with toxin and AMBnTßCD by using the student's t-test (***p<0.0005; **p<0.005). C. AMBnTßCD inhibits the pH-dependent membrane translocation of iota toxin across the cytoplasmic membranes of intact Vero cells. Cells were incubated for 30 min at 37°C with 100 nM Baf A1 and subsequently for 30 min at 4°C in serum-free medium with iota toxin (1000 ng/mL Ib+500 ng/ml Ia) or without toxin for control. Then, 10 µM of AMBnTßCD were added (for control no AMBnTßCD) and the pH of the medium was adjusted to 4.5 with HCl (for control pH 7.5) and cells were exposed for 15 min to 37°C to trigger pore formation by Ib membrane translocation of Ia. Subsequently, cells were further incubated at 37°C in neutral medium containing Baf A1 and pictures were taken after 45 min and 3 h of incubation. The percentages of round (i. e. intoxicated) cells were determined, values are given as mean ± S.D. (n = 3). Significance was tested for each time point between samples treated with iota toxin under acidic condition in the absence or presence of AMBnTßCD by using the student's t-test (***p<0.0005).