Fig. 1.

Host calpain-1 is associated with P. falciparum egress. (A) Human RBCs were infected with 3D7 strain parasites, synchronized in sorbitol, treated at 42 hours postinfection (hpi) with either dimethyl sulfoxide (control) or 3 μM DCG04, and fixed and stained 6 to 18 hours later. Normal schizonts were evident in both samples at 48 hours, but whereas controls emerged to establish new ring stage infections by 60 hours, parasites treated with DCG04 appeared unable to egress, remaining arrested as schizonts. Scale bar, 5 μm. (B) Synchronized infected cultures were treated with 3 μM DCG04 for 2 hours beginning at various times throughout the intraerythrocytic life cycle, followed by incubation with 0.02% saponin to permeabilize the RBC and parasitophorous vacuole membrane (but not parasites). Parasites were removed by centrifugation, and the remaining material fractionated to yield a host + parasitophorous vacuole membrane pellet and a soluble fraction. Blotting identified proteins biotinylated by DCG04 (labeling any active cysteine protease), as well as the RBC membrane marker band 4.9, and calpain-1 (a cytoplasmic RBC protein when inactive). The single prominent ~80-kD band observed in membranes isolated from RBCs harboring schizont-stage parasites was identified as host cell calpain-1 (table S1).