A protease cascade regulates egress of Plasmodium falciparum from the human erythrocyte

Malaria parasites invade erythrocytes and replicate inside a parasitophorous vacuole (PV). Invasive merozoites eventually egress in a process that involves sequential rupture of first the PV membrane (PVM) then the erythrocyte membrane. Egress is protease-dependent, with both cysteine and serine proteases implicated. The parasite serine protease SUB1 is stored in merozoite secretory organelles that are discharged into the PV 10 minutes before erythrocyte membrane rupture. Pharmacological inhibition of SUB1 activity or discharge blocks egress, but the mechanism by which SUB1 regulates egress is unclear. In the PV, SUB1 cleaves multiple substrates including SERA6, a putative cysteine protease. In asexual blood stages of Plasmodium falciparum, the agent of the most dangerous form of malaria, SERA6 is believed to be essential but its function and whether this depends on SUB1 is unknown. Here it is shown that conditional disruption of the P. falciparum SUB1 or SERA6 genes produces two distinct, lethal phenotypes. SUB1-null parasites undergo none of the morphological changes that precede egress and fail to rupture the PVM. In contrast, PVM rupture and the typical erythrocyte membrane poration occur normally in SERA6-null parasites but erythrocyte membrane rupture does not occur. Complementation studies demonstrate that SERA6 is an enzyme and that processing by SUB1 is required for its function. This study concludes that SUB1 and SERA6 play distinct, essential roles in a coordinated proteolytic cascade that enables sequential rupture of the two bounding membranes leading to egress