Reduced expression of an essential blood-stage Plasmodium phosphatidylserine synthase does not modulate parasite resistance to Pf ATP4 inhibitors

Plasmodium falciparum ATP4 mutations A211V and G223R allow parasites to survive the lethal effects of antimalarials PA21A092 (PA92) and cipargamin (CIP), respectively. An A211V mutant line (Dd2 ^A211V ) treated with PA92 showed enhanced levels of lipid production, which prompted the idea that components of the phospholipid biosynthesis pathway could be involved in the survival mechanism of Pf ATP4 mutant parasites. As phosphatidylserine synthase ( Pf PSS) is the only enzyme that produces phosopholipid phosphatidylserine (PS) in P. falciparum parasites, we hypothesized that Pf PSS is both essential for parasite survival and that reduced PfPSS expression would cause resistant Pf ATP4 mutant parasites to become susceptible to PA92 or CIP. We created a CIP-resistant G223R mutant line (Dd2 ^G223R ) via CRISPR-Cas9 and integrated a conditional Pf PSS knockdown construct into a Dd2 ^A211V (↓PSS-Dd2 ^A211V ) and our Dd2 ^G223R line (↓PSS-Dd2 ^G223R ). We treated these knockdown lines with PA92 or CIP to determine the half-maximal effective concentration (EC ₅₀ ) of each antimalarial with normal or reduced Pf PSS levels. While we found that Pf PSS is essential for parasite survival, we did not find any significant alterations to the EC ₅₀ values of PA92 or CIP based on the reduced levels of Pf PSS in our mutant lines. Although Pf PSS does not appear to be involved, other components of the phospholipid production pathway could still affect the resistance mechanism of Pf ATP4 mutations. Identification of novel targets to counteract the mechanism by which Pf ATP4 mutant parasites resist lethal drug effects is crucial for the successful application of antimalarials in endemic countries where resistance is on the rise.