To obtain free amino acids for protein synthesis, trophozoite stage malaria parasites feed on the cytoplasm of host erythrocytes and degrade hemoglobin within an acid food vacuole. The food vacuole appears to be analogous to the secondary lysosomes of mammalian cells. To determine the enzymatic mechanism of hemoglobin degradation, we incubated trophozoite-infected erythrocytes with peptide inhibitors of different classes of proteinases. Leupeptin and L-transepoxy-succinyl-leucylamido-(4-guanidino)-butane (E-64) sufficient for the needs of the parasite (1). Second, the hemoglobin content of infected erythrocytes decreases 25-75% during the life cycle of erythrocytic parasites (2,3), and the concentration of free amino acids is greater in infected erythrocytes than in uninfected erythrocytes (4). Third, the composition of the amino acid pool of infected erythrocytes is similar to the amino acid composition of hemoglobin (5-7). Fourth, the infection of erythrocytes containing radiolabeled hemoglobin is followed by the appearance of labeled amino acids in parasite proteins (8-10).Hemoglobin degradation occurs predominantly during the trophozoite stage ofthe erythrocytic life cycle ofP. falciparum. Trophozoites ingest erythrocyte cytoplasm and transport it within vesicles to a large central food vacuole (1 1, 12), where the hemoglobin-rich cytoplasm is degraded. In the food vacuole the heme moiety precipitates and is a major component of malarial pigment (13), and globin is hydrolyzed to its constituent free amino acids. The food vacuole of P. falciparum is an acidic (14, 15), membrane-bound (1 1) compartment where proteins are degraded, and therefore it appears to be analogous to the secondary lysosomes of mammalian cells, where multiple proteinases hydrolyze proteins at acid pH (16,17).The enzymatic mechanism of globin degradation within the malarial food vacuole is unknown. In previous studies, aspartic proteinases that degraded denatured hemoglobin were isolated from malaria parasites (18-20). However, denatured hemoglobin is a nonspecific substrate that can be hydrolyzed by many proteinases, and the biological role of the aspartic proteinases cannot be determined from these studies. To determine the enzymatic mechanism of globin degradation in the trophozoite food vacuole we first studied the effects of class-specific proteinase inhibitors on intact parasites. We found that two peptide inhibitors of cysteine proteinases blocked globin degradation in the food vacuole of P. falciparum trophozoites. We also identified a cysteine proteinase of trophozoites that was inhibited by the same two proteinase inhibitors and had biochemical properties that were similar to those of the lysosomal cysteine proteinase cathepsin L. Our results suggest that the cysteine proteinase we identified has a critical role in hemoglobin degradation in the food vacuole of P. falciparum trophozoites.

The process of human erythrocyte invasion by Plasmodium falciparum parasites involves a calciumdependent serine protease with properties consistent with a subtilisin-like activity. This enzyme achieves the last crucial maturation step of merozoite surface protein 1 (MSP1) necessary for parasite entry into the host erythrocyte. In eukaryotic cells, such processing steps are performed by subtilisinlike maturases, known as proprotein convertases. In an attempt to characterize the MSP1 maturase, we have identified a gene that encodes a P. falciparum subtilisin-like protease (PfSUB2) whose deduced active site sequence resembles more bacterial subtilisins. Therefore, we propose that PfSUB2 belongs to a subclass of eukaryotic subtilisins different from proprotein convertases. Pfsub2 is expressed during merozoite differentiation and encodes an integral membrane protein localized in the merozoite dense granules, a secretory organelle whose contents are believed to participate in a late step of the erythrocyte invasion. PfSUB2's subcellular localization, together with its predicted enzymatic properties, leads us to propose that PfSUB2 could be responsible for the late MSP1 maturation step and thus is an attractive target for the development of new antimalarial drugs.

The pathogenesis of human cerebral malaria is suspected to be caused by blockage of cerebral microvessels by the sequestration of parasitized human red blood cells (PRBC). Examination of infected tissues indicate PRBC sequestration in microvessels is the result of PRBC knob attachment to endothelial cell surface cytoadherence receptors such as CD36, thrombospondin (TSP), and intercellular adhesion molecule-1 (ICAM-1). In lieu of fresh human tissue, several animal models for human cerebral malaria have been developed, the Plasmodium coatneyi-infected rhesus monkey model being the most versatile. To further the understanding of noncerebral malarial complications during disease, we examined noncerebral tissues of infected rhesus monkeys. Our study demonstrated similar microvessel PRBC sequestration and the presence of cytoadherence ligands in noncerebral tissues. Immunohistochemical analysis showed CD36, TSP, and ICAM-1 cytoadherence proteins in several major organs.