Gene disruptions demonstrate independent roles for the four falcipain cysteine proteases of Plasmodium falciparum

Erythrocytic stages of the malaria parasite Plasmodium falciparum express four related papain-family cysteine proteases, termed falcipains. Falcipain-2 and falcipain-3 are food vacuole hemoglobinases, but determination of the specific roles of these and other falcipains has been incomplete. To better characterize biological roles, we attempted disruption of each falcipain gene in the same strain (3D7) of P. falciparum. Disruption of falcipain-1, falcipain-2, and falcipain-2′ was achieved. In each case knockouts multiplied at the same rate as wild-type parasites. The morphologies of erythrocytic falcipain-1 and falcipain-2′ knockout parasites were indistinguishable from those of wild-type parasites. In contrast, consistent with previous results, falcipain-2 knockout trophozoites developed swollen, hemoglobin-filled food vacuoles, indicative of a block in hemoglobin hydrolysis and were, compared to wild-type parasites, twice as sensitive to cysteine protease inhibitors and over 1000 times more sensitive to an aspartic protease inhibitor. The falcipain-3 gene could not be disrupted, but replacement with a tagged functional copy was readily achieved, strongly suggesting that falcipain-3 is essential to erythrocytic parasites. Our data suggest key roles for falcipain-2 and falcipain-3 in the development of erythrocytic malaria parasites and a complex interplay between P. falciparum cysteine and aspartic proteases.