Fig. 4. RtxA loses its cytotoxic activity under native conditions.

a, b RtxA purified under denaturing conditions (50 mM Tri-HCl and 8 M urea; RtxA, TU) and RtxA purified under native conditions (50 mM Tris-HCl and 150 mM NaCl; RtxA, TN) were diluted 100-fold into HLaC-78 cells (1 × 10⁶/ml) (a) and erythrocytes (5 × 10⁸/ml) (b) to a final concentration of 2 µg/ml. TU and TN buffers without RtxA were used as negative controls. Cells were incubated for different times at 37 °C and analyzed as described in the Fig. 3 legend. Each point represents the mean value ± SD of four independent experiments. c, d RtxA purified under denaturing conditions was diluted 100-fold with TN buffer to concentrations of 2 and 0.5 µg/ml, respectively. The toxin samples were preincubated for different times (0, 0.5, 1, 5, and 10 min) at 37 °C and were subsequently added to pelleted HLaC-78 cells (1 × 10⁶/ml; from 2 µg/ml RtxA samples) (c) and erythrocytes (5 × 10⁸/ml; from 0.5 µg/ml RtxA samples) (d). The cells were incubated for different times at 37 °C and analyzed as described in the Fig. 3 legend. Each point represents the mean value ± SD of four independent experiments (c) or three independent determinations performed in duplicate (d)