Inhibition of Plasmodium falciparum cysteine proteases by the sugarcane cystatin CaneCPI-4

Malaria is a disease caused by Plasmodium parasites that affects hundreds of millions of people. Plasmodium proteases are involved in invasion, erythrocyte egress and degradation of host proteins. Falcipains are well-studied cysteine peptidases located in P. falciparum food vacuoles that participate in hemoglobin degradation. Cystatins are natural cysteine protease inhibitors that are implicated in a wide range of regulatory processes. Here, we report that a cystatin from sugarcane, CaneCPI-4, is selectively internalized into P. falciparum infected erythrocytes and is not processed by the parasite proteolytic machinery. Furthermore, we demonstrated the inhibition of P. falciparum cysteine proteases by CaneCPI-4, suggesting that it can exert inhibitory functions inside the parasites. The inhibition of the proteolytic activity of parasite cells is specific to this cystatin, as the addition of an anti-CaneCPI-4 antibody completely abolished the inhibition. We extended the studies to recombinant falcipain-2 and falcipain-3 and demonstrated that CaneCPI-4 strongly inhibits these enzymes, with IC50 values of 12nM and 42nM, respectively. We also demonstrated that CaneCPI-4 decreased the hemozoin formation in the parasites, affecting the parasitemia. Taken together, this study identified a natural molecule as a potential antimalarial that specifically targets falcipains and also contributes to a better understanding of macromolecule acquisition by Plasmodium falciparum infected RBCs. Schematic model of recombinant sugarcane cystatin (CaneCPI-4) action in Plasmodium infected erythrocytes. N – nucleus; DV – digestive vacuole; Hb – Hemoglobin; Hz – Hemozoin; PVM – Parasitophorous Vacuole Membrane. Black arrow – hemoglobin internalization by cystotome vesicle formation. Red dashed arrow – CaneCPI-4 entry through infected cell membranes. [Display omitted] •CaneCPI-4 inhibited the P. falciparum cysteine proteases falcipains 2 and 3.•CaneCPI-4 is internalized to the P. falciparum cytosol, but is not hydrolyzed.•CaneCPI-4 decreased the hemozoin formation in vitro, affecting the parasite growth.