Genetic Disruption of the Plasmodium falciparum Digestive Vacuole Plasmepsins Demonstrates Their Functional Redundancy

Abstract

The digestive vacuole plasmepsins PfPM1, PfPM2, PfPM4, and PfHAP (a histoaspartic proteinase) are 4 aspartic proteinases among 10 encoded in the Plasmodium falciparum malarial genome. These have been hypothesized to initiate and contribute significantly to hemoglobin degradation, a catabolic function essential to the survival of this intraerythrocytic parasite. Because of their perceived significance, these plasmepsins have been proposed as potential targets for antimalarial drug development. To test their essentiality, knockout constructs were prepared for each corresponding gene such that homologous recombination would result in two partial, nonfunctional gene copies. Disruption of each gene was achieved, as confirmed by PCR, Southern, and Northern blot analyses. Western and two-dimensional gel analyses revealed the absence of mature or even truncated plasmepsins corresponding to the disrupted gene. Reduced growth rates were observed with PfPM1 and PfPM4 knockouts, indicating that although these plasmepsins are not essential, they are important for parasite development. Abnormal mitochondrial morphology also appeared to accompany loss of PfPM2, and an abundant accumulation of electron-dense vesicles in the digestive vacuole was observed upon disruption of PfPM4; however, those phenotypes only manifested in about a third of the disrupted cells. The ability to compensate for loss of individual plasmepsin function may be explained by close similarity in the structure and active site of these four vacuolar enzymes. Our data imply that drug discovery efforts focused on vacuolar plasmepsins must incorporate measures to develop compounds that can inhibit two or more of this enzyme family.

Plasmodium falciparum ensures its amino acid supply with multiple acquisition pathways and redundant proteolytic enzyme systems

“…Previous studies have shown that individual knockouts of each of the four food vacuole plasmepsins and falcipain-2 have nearly normal growth phenotypes in RPMI medium 1640 (5,16,17). A plasmepsin IV͞I double knockout has a doubling time Ϸ10% longer than the parental strain; this phenotype is accentuated slightly in 5AA (5).…”

“…Inhibitors of aspartic and cysteine proteases kill parasites in culture and animal models, although their specificity is not strict (13)(14)(15). Knockouts of plasmepsins individually or in combination (plasmepsin IV͞I double knockout) give parasites with only slightly impaired growth (5,16), and a falcipain-2 knockout shows normal growth (17). The effect of aspartic protease inhibitors is potentiated when combined with cysteine protease inhibitors or used in falcipain-2 knockout parasites (15,17).…”

Anti-malaria drug development targeting the M1 alanyl and M17 leucyl aminopeptidases

“…13,18,19 However, knockout experiments have shown that the DV plasmepsins and falcipains 2 and 2′ are individually not essential to parasite survival, and because of this redundancy between the function of these enzyme classes it has proved difficult to develop drugs that effectively block the digestive process within the DV. [21][22][23][24] Peptides produced in the DV may be further processed to dipeptides by a dipeptidyl aminopeptidase I, an orthologue of mammalian cathepsin C. 25,26 Small peptides and dipeptides may also be reduced to free aminoacids within the DV and/or transported to the parasite cytosol for processing by aminopeptidases. 20,[27][28][29][30][31][32][33] Aminopeptidases are essential in releasing aminoacids from haemoglobin-derived peptides and are thus central to the growth and development of malaria parasites in the erythrocyte.…”

Novel diaryl ureas with efficacy in a mouse model of malaria

“…The diaryl ureas presented herein, however, do not appear to significantly target the plasmepsins as they equally inhibit the growth of both wild type and knockouts of PfPM1 through 4 (See Supplementary Data Table S1), attained via a genetic disruption methodology in the Dd2 background. 20 GlaxoSmithKline disclosed the whole-cell efficacy of screening hit TCMDC-139010 (Supplementary Data Figure S1; XC 50 = 930 nM vs. 3D7 strain), but without information concerning the biochemical target. 4 We also reported in 2005 the preparation of triclosan-based 4'-ureas (Supplementary Data Figure S1) that were less potent against cultured parasite (EC 50 values of ~100 μM) than 12 -25, but exhibited IC 50 values of ~100 nM against purified PfFabI assayed in vitro.…”