Assessment of Biological Role and Insight into Druggability of the Plasmodium falciparum Protease Plasmepsin V

Abstract

Upon infecting a red blood cell (RBC), the malaria parasite Plasmodium falciparum drastically remodels its host by exporting hundreds of proteins into the RBC cytosol. This protein export program is essential for parasite survival. Hence export-related proteins could be potential drug targets. One essential enzyme in this pathway is plasmepsin V (PMV), an aspartic protease that processes export-destined proteins in the parasite endoplasmic reticulum (ER) at the Plasmodium export element (PEXEL) motif. Despite longstanding interest in this enzyme, functional studies have been hindered by the inability of previous technologies to produce a regulatable lethal depletion of PMV. To overcome this technical barrier, we designed a system for stringent post-transcriptional regulation allowing a tightly controlled, tunable knockdown of PMV. Using this system, we found that PMV must be dramatically depleted to affect parasite growth, suggesting the parasite maintains this enzyme in substantial excess. Surprisingly, depletion of PMV arrested parasite growth immediately after RBC invasion, significantly before the death from exported protein deficit that has previously been described. The data suggest that PMV inhibitors can halt parasite growth at two distinct points in the parasite life cycle. However, overcoming the functional excess of PMV in the parasite may require inhibitor concentrations far beyond the enzyme's IC 50 .

An essential ER-resident N-acetyltransferase in Plasmodium falciparum

“…Chemical inhibition or depletion of PM V can arrest parasite growth immediately after invasion, or as early trophozoites (Boonyalai et al, 2018;Polino et al, 2020;Sleebs et al, 2014). Here we show that Pf3D7_1437000 depletion arrests parasite growth substantially later than we observe when blocking export via Hsp101 disruption.…”

“…We targeted Pf3D7_1437000 using CRISPR/Cas9 editing and the previously described pSN054 vector to replace the gene with a recodonized version that is C-terminally HA-tagged and flanked with loxP sites for gene excision, as well as TetR-binding aptamers for post-transcriptional depletion (Fig. 2A) (Polino et al, 2020). Proper genome editing was confirmed by Southern blot (Supp.…”

“…The construct for regulating Pf3D7_1437000 levels was made using the previously described pSN054 vector (Nasamu et al, 2021;Polino et al, 2020). Primer sequences are listed in Supplementary Table 1; shorthand names will be used here.…”

Redesigned TetR-Aptamer System To Control Gene Expression in Plasmodium falciparum

“…The use of a 10ϫ aptamer array in the 3= UTR alone has been sufficient to knock down the expression of several genes, but it may not be enough to achieve complete conditional control of genes that are expressed at high levels. Recent publications have overcome this issue by utilizing a linear vector that carries a recodonized copy of the target gene and homology arms corresponding to the 5= and 3= UTRs, thereby allowing simultaneous insertion of aptamers into the upstream and downstream sequences (33,43,44). This study addresses a second drawback of the TetR-DOZI system, namely, the instability of the 3= UTR aptamer array due to its repetitive nature.…”

“…Since it can be challenging to simultaneously modify the 5= and 3= UTRs of endogenous genes, several groups have effectively utilized TetR-DOZI with an aptamer array inserted only into the 3= UTR (5,26,(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44). The 3= UTR array consists of 10 aptamer copies, separated by spacers that are identical in sequence and length.…”