Critical roles for the digestive vacuole plasmepsins of Plasmodium falciparum in vacuolar function

Abstract

SummaryKnockout mutants of Plasmodium falciparum lacking pfpm1, pfpm2 and pfhap (triple-PM KO), and mutants lacking all four digestive vacuole (DV) plasmepsins (pfpm4, pfpm1, pfpm2 and pfhap; quadruple-PM KO), were prepared by double cross-over integration effecting chromosomal deletions of up to 14.6 kb. The triple-PM KO was similar to the parental line (3D7) in growth rate, morphology and sensitivity to proteinase inhibitors. The quadruple-PM KO showed a significantly slower rate of growth in standard medium, which manifested as delayed schizont maturation accompanied by reduced formation of haemozoin. In amino acid-limited medium, the reduction in growth rate of the quadruple-PM KO was pronounced. The sensitivity of both the triple-and quadruple-PM KOs to six different HIV aspartic proteinase inhibitors was comparable to that of 3D7, thus establishing that the DV plasmepsins were not the primary targets of the antimalarial activity of these clinically important compounds. Electron microscopic analysis revealed the presence of multilamellar bodies resembling ceroid in the DV of the quadruple-PM KO, and intermediates of the autophagic pathway accumulated as determined by Western blot analysis. Thus, the DV plasmepsins, although not essential, contribute significantly to the fitness of the parasite and are required for efficient degradation of endosomal vesicles delivered to the DV.

Stronger Activity of Human Immunodeficiency Virus Type 1 Protease Inhibitors against Clinical Isolates of Plasmodium vivax than against Those of P. falciparum

“…The P. falciparum food vacuole-associated plasmepsins (PMs) I, II, and IV and histoaspartic protease are thought to be involved in the initial steps of hemoglobin degradation (4). However, more-recent data from isobologram and transgenic-parasite studies suggest that these food vacuole PMs are unlikely to be the primary targets of these drugs (5,15,18,21,31). Interestingly, PMIV (GenBank accession no.…”

Toxoplasma gondii aspartic protease 1 is not essential in tachyzoites

“…The genome of P. falciparum encodes 10 ASPs termed plasmepsins (PMs), four of which (PfPMI, II, IV and HAP) are involved in haemoglobin degradation within the food vacuole, and hence critically provide amino acids for parasite growth (Bonilla et al, 2007). In addition to haemoglobinase activity, PfPMII might be involved in erythrocyte cytoskeleton remodelling and in egress by cleaving spectrin (Le Bonniec et al, 1999).…”

“…In Apicomplexans, most data on aspartic proteases concern the causative agent of malaria, P. falciparum. Successful use of aspartic protease inhibitors against Plasmodium parasites in vitro validates Plasmodium aspartic proteases as potential drug targets (Bonilla et al, 2007).…”

Identification of plasmepsin inhibitors as selective anti-malarial agents using ligand based drug design

“…4 An analysis of the binding site region of PM II reveals that the binding site regions of PM I, IV, and HAP show 84%, 68% and 39% identity, respectively, while there is a lower degree of identity between the other six PMs. 8 PM V, IX and X have been shown to be produced in intraerythrocytic parasites 1,[10][11][12][13] but to date, function has been elucidated only for PMV.…”

“…They also represent the greatest amount of available structural information, from high resolution co-crystals to large amounts of both whole cell parasite activity data and on-target DV PM inhibition data. Although these DV enzymes have shown some functional redundancy, 13 the discovery and description of novel inhibitors or chemical probes for these proteins is desirable and the design of inhibitors targeting them continues as the familial active site similarities make the prospect of a cross family inhibitor, targeting multiple PMs, possible. [16][17][18][19] The goal of this research programme was to apply computational tools to guide the discovery of novel small…”