Figure 4.

Cytolytic activities of purified HlyA, ApxIA, and RtxA toxins. (a,b) Washed sheep erythrocytes (5 × 10⁸/mL) were incubated at 37 °C with 20 ng/mL of HlyA (a) or 1 µg/mL of ApxIA (b) and erythrocyte lysis was measured in time as the amount of released hemoglobin by photometric determination at 541 nm (A₅₄₁). Average values ± standard deviations from four independent determinations are shown. (c) HLaC-78 cells (1 × 10⁶/mL) were incubated with 1 µg/mL of RtxA for indicated time at 37 °C. Cell viability was determined by a vital dye staining using 1 µg/mL of Hoechst 33258 followed by flow cytometry. The initial viability of cells incubated without RtxA was taken as 100%. Each point represents the mean value ± standard deviation of three independent experiments. (d) Sheep erythrocytes (5 × 10⁸/mL) were incubated at 37 °C in the presence of 200 ng/mL of RtxA and erythrocyte lysis was measured as above.