Figure 3.

CyaA-S606A and CyaA-D1079A toxins exhibit an almost identical specific cytotoxic activity against sheep erythrocytes and mouse macrophages J774A.1 as intact CyaA. Proteins were expressed in E. coli BL-21 cells and purified from urea extracts on DEAE Sepharose. (a) Sheep erythrocytes (5 × 10⁸ cells/mL) were incubated at 37 °C in the presence of 2 mM calcium (+Ca²⁺) or 5 mM EDTA (+EDTA) with 1 µg/mL (~0.4 U/mL) of the purified CyaA proteins and after 30 min. Aliquots were used for determination of the cell-associated AC activity (binding) and of the AC activity internalized into erythrocytes and protected against digestion by externally added trypsin (invasive AC). Activities are expressed as percentages of intact CyaA activity and represent average values ± standard deviations from at least three independent determinations performed in duplicate with two different toxin preparations (n = 6–8). (b) Sheep erythrocytes (5 × 10⁸ cells/mL) in Tris 50 mM, NaCl 150 mM, CaCl₂ 2 mM, pH 7.4 buffer (TNC) were incubated at 37 °C with intact CyaA or with its mutant variants (10 µg/mL). Hemolytic activity was measured as the amount of released hemoglobin by photometric determination (A₅₄₁), (n = 3). (c) Binding of intact CyaA or its mutant variants to J774A.1 cells (1 × 10⁶) was determined as the amount of total cell-associated AC enzyme activity upon incubation of cells with 1 µg/mL (~0.4 U/mL) of the protein for 30 min at 4 °C. cAMP intoxication was assessed by determining the intracellular concentration of cAMP generated in cells after 30 min of incubation of J774A.1 cells (2 × 10⁵) with four different toxin concentrations from within the linear range of the dose-response curve (100, 50, 25, and 12.5 ng/mL, i.e., ~40, 20, 10, 5 mU/mL, respectively). Activities are expressed as percentages of intact CyaA activity and represent average values ± standard deviations from at least three independent determinations performed in duplicate with two different toxin preparations (n = 6–8).