Clinical malaria is associated with proliferation of blood-stage parasites. During the blood stage, Plasmodium parasites invade host red blood cells, multiply, egress and reinvade uninfected red blood cells to continue the life cycle. Here we demonstrate that calcium-dependent permeabilization of host red blood cells is critical for egress of Plasmodium falciparum merozoites. Although perforin-like proteins have been predicted to mediate membrane perforation during egress, the expression, activity and mechanism of action of these proteins have not been demonstrated. Here, we show that two perforin-like proteins, perforin-like protein 1 and perforin-like protein 2, are expressed in the blood stage. Perforin-like protein 1 localizes to the red blood cell membrane and parasitophorous vacuolar membrane in mature schizonts following its Ca 2 þ -dependent discharge from micronemes. Furthermore, perforin-like protein 1 shows Ca 2 þ -dependent permeabilization and membranolytic activities suggesting that it may be one of the effector proteins that mediate Ca 2 þ -dependent membrane perforation during egress.

SummaryEgress of Plasmodium falciparum merozoites from host erythrocytes is a critical step in multiplication of blood-stage parasites. A cascade of proteolytic events plays a major role in degradation of membranes leading to egress of merozoites. However, the signals that regulate the temporal activation and/or secretion of proteases upon maturation of merozoites in intra-erythrocytic schizonts remain unclear. Here, we have tested the role of intracellular Ca 2+ in regulation of egress of P. falciparum merozoites from schizonts. A sharp rise in intracellular Ca 2+ just before egress, observed by timelapse video microscopy, suggested a role for intracellular Ca 2+ in this process. Chelation of intracellular Ca 2+ with chelators such as BAPTA-AM or inhibition of Ca 2+ release from intracellular stores with a phospholipase C (PLC) inhibitor blocks merozoite egress. Interestingly, chelation of intracellular Ca 2+ in schizonts was also found to block the discharge of a key protease PfSUB1 (subtilisin-like protease 1) from exonemes of P. falciparum merozoites to parasitophorous vacuole (PV). This leads to inhibition of processing of PfSERA5 (serine repeat antigen 5) and a block in parasitophorous vacuolar membrane (PVM) rupture and merozoite egress. A complete understanding of the steps regulating egress of P. falciparum merozoites may provide novel targets for development of drugs that block egress and limit parasite growth.

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