Erythrocytic
malaria parasites utilize
proteases for a number of cellular processes, including hydrolysis of
hemoglobin,
rupture of erythrocytes by mature schizonts, and subsequent invasion of erythrocytes by free merozoites. However, mechanisms used by
malaria parasites to control
protease activity have not been established. We report here the identification of an endogenous
cysteine protease inhibitor of
Plasmodium falciparum, falstatin, based on modest homology with the Trypanosoma cruzi
cysteine protease inhibitor chagasin. Falstatin, expressed in Escherichia coli, was a potent reversible inhibitor of the P. falciparum
cysteine proteases falcipain-2 and
falcipain-3, as well as other parasite- and nonparasite-derived
cysteine proteases, but it was a relatively weak inhibitor of the P. falciparum
cysteine proteases falcipain-1 and dipeptidyl
aminopeptidase 1. Falstatin is present in schizonts, merozoites, and rings, but not in trophozoites, the stage at which the
cysteine protease activity of P. falciparum is maximal. Falstatin localizes to the periphery of rings and early schizonts, is diffusely expressed in late schizonts and merozoites, and is released upon the
rupture of mature schizonts. Treatment of late schizionts with
antibodies that blocked the inhibitory activity of falstatin against native and recombinant
falcipain-2 and
falcipain-3 dose-dependently decreased the subsequent invasion of erythrocytes by merozoites. These results suggest that P. falciparum requires expression of falstatin to limit proteolysis by certain host or parasite
cysteine proteases during erythrocyte invasion. This mechanism of regulation of proteolysis suggests new strategies for the development of
antimalarial agents that specifically disrupt erythrocyte invasion.