A Subtilisin-like Protein in Secretory Organelles of Plasmodium falciparum Merozoites

Abstract

In the vertebrate host, the malaria parasite invades and replicates asexually within circulating erythrocytes. Parasite proteolytic enzymes play an essential but poorly understood role in erythrocyte invasion. We have identified a Plasmodium falciparum gene, denoted pfsub-1, encoding a member of the subtilisin-like serine protease family (subtilases). The pfsub-1 gene is expressed in asexual blood stages of P. falciparum, and the primary gene product (PfSUB-1) undergoes post-translational processing during secretory transport in a manner consistent with its being converted to a mature, enzymatically active form, as documented for other subtilases. In the invasive merozoite, the putative mature protease (p47) is concentrated in dense granules, which are secretory organelles located toward the apical end of the merozoite. At some point following merozoite release and completion of erythrocyte invasion, p47 is secreted from the parasite in a truncated, soluble form. The subcellular location and timing of secretion of p47 suggest that it is likely to play a role in erythrocyte invasion. PfSUB-1 is a new potential target for antimalarial drug development.Plasmodium falciparum, the causative agent of the most severe form of human malaria, is an obligate intracellular apicomplexan parasite. The life cycle of the organism includes a number of specialized invasive (zoite) stages. In the vertebrate host, replication of the parasite in circulating erythrocytes is initiated when the cells are invaded by merozoites. The parasite replicates asexually within the infected erythrocyte to produce a number of progeny merozoites. Upon rupture of the host cell, these are released to invade fresh erythrocytes and perpetuate the blood stage cycle. Erythrocyte invasion by the malaria merozoite has been the subject of intensive study, since intervention strategies that prevent invasion would effectively block both replication of the parasite and the associated clinical disease.Electron microscopic studies have shown that erythrocyte invasion by the malaria merozoite takes place in a number of discrete stages. Initial reversible attachment of the parasite to the red cell surface is rapidly followed by reorientation, the formation of an irreversible junction between the apical prominence of the merozoite and the host cell surface, and finally entry of the parasite into the cell by a mechanism resembling a form of induced endocytosis (1-4). The process is facilitated by the controlled release of the contents of three types of secretory organelles, called rhoptries, micronemes, and dense granules, situated at or toward the apical domain of the merozoite (2, 5, 6). There is extensive evidence indicating an essential role for parasite-derived proteases in invasion. Invasion by P. falciparum merozoites is blocked in the presence of the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) 1 (7), and invasion by merozoites of a number of Plasmodium species is prevented by chymostatin (8 -13). The inhibitory effect of chymostatin on inv...

A novel Plasmodium-specific prodomain fold regulates the malaria drug target SUB1 subtilase

“…In the case of Plasmodium, another gradient of [Ca 2 þ ] is maintained between the parasite cytoplasm (BnM), the PV (B40 mM) and the infected erythrocyte cytoplasm (B100 nM) 46,47 . In P. falciparum, the first PfSUB1 maturation event, which produces a largely enzymatically inactive 54/50 kDa doublet corresponding to the 47-45 kDa forms of PvSUB1 forms, occurs in the ER 9 , and these products remain stable through the Golgi and in the exoneme, a parasite secretory vesicle 11 . SUB1 activation needs a shift in [Ca 2 þ ] to dissociate the stable non-covalent complex formed between the pro-region and the catalytic domain.…”

“…Future anti-malarials needed to match the agenda of malaria eradication 18 should be equally efficient against both P. falciparum and P. vivax, and overcome resistance to existing drugs 19,20 by targeting yet untouched parasite biological functions of the hepatic and erythrocytic stages. Plasmodium SUB1 properties fulfill these criteria, as SUB1 is indeed highly conserved across Plasmodium species 9,21 , including P. falciparum and P. vivax, and SUB1 inhibitors have been shown to equally impair P. vivax and P. falciparum SUB1 enzymes 21 . In addition, SUB1 plays a dual essential role at both the hepatic and erythrocytic phases of the Plasmodium life cycle 12 .…”

“…falciparum SUB1 (PfSUB1) is produced as a precursor of B80 kDa reported to autoprocess in the endoplasmic reticulum (ER), giving rise to 54/47 kDa forms that accumulate in particular organelles of the Mz called exonemes 9,11 . Egress of Plasmodium Mz from infected erythrocytes is a precisely regulated proteasedependent multi-step process that requires rupture of the parasitophorous vacuole (PV) membrane followed by rupture of the red cell membrane [22][23][24] .…”

“…encodes three proteases, namely SUB1, 2 and 3, which belong to the SO8.001 subfamily of subtilases 1,8,9 . Plasmodium SUBs similarity is restricted to their catalytic domains, which display 430% sequence identity 10 , and all are expressed during the parasite blood stages [8][9][10] .…”

“…encodes three proteases, namely SUB1, 2 and 3, which belong to the SO8.001 subfamily of subtilases 1,8,9 . Plasmodium SUBs similarity is restricted to their catalytic domains, which display 430% sequence identity 10 , and all are expressed during the parasite blood stages [8][9][10] . However, although genetic approaches have shown that SUB3 is dispensable for the development of Plasmodium blood stages 11 , its precise biological role remains to be established 12 ; SUB1 and SUB2 are essential enzymes 11,13 .…”