FIG. 7.

Ionophore-induced membrane permeabilization by intracellular Iie⁺ parasites. Infected cells were pretreated with cytochalasin D to block parasite motility, and A23187 was added before fixing the monolayer. Immunofluorescence was used to examine the membrane integrity of host cells infected with Iie⁺ and Iie⁻ strains. A rabbit anti-SAG1 antibody was used before permeabilizing the monolayer with detergent so as to stain all the parasites that are extracellular or within permeabilized host cells. A mouse anti-SAG1 MAb was used in the presence of Triton X-100 to permeabilize all membranes and allow all the parasites to be stained regardless of their location or the state of the host cell. The two antibodies were detected with different fluorescent dyes, rabbit antibodies with Texas red and mouse antibodies with FITC, such that all parasites stained with FITC but only those that were within permeabilized host cells also stained with Texas red and thus appear yellow. (a) The effect of A23187 on the membrane integrity of host cells infected with Iie⁺ (RHΔ and MBD2.1) and Iie⁻ (MBE1.1 and MBE3.3) strains. Cells infected with RHΔ and MBD2.1 are permeabilized despite the absence of parasite motility. This permeabilization is not observed in cells infected with MBE1.1 and MBE3.3. (b) The A23187-induced permeabilization by the Iie⁺ RHΔ strain is inhibited by pretreating the infected monolayer with BAPTA-AM.