Additional PfCRT mutations driven by selective pressure for improved fitness can result in the loss of piperaquine resistance and altered Plasmodium falciparum physiology

Malaria elimination efforts in Southeast Asia have been hindered by multidrug-resistant Plasmodium falciparum. High-grade resistance to piperaquine (PPQ, used in combination with dihydroartemisinin) is associated with PfCRT mutations that arose in strains expressing the PfCRT Dd2 isoform, which mediates resistance to the related 4-aminoquinoline chloroquine (CQ). The PPQ-resistant PfCRT haplotype Dd2 + F145I mediates the highest level resistance but causes a significant growth defect in intra-erythrocytic parasites. Recently, three separate mutations (F131C, I347T and C258W) have been observed on Dd2 + F145I PfCRT either during extended parasite culture or in Southeast Asian isolates no longer subject to PPQ pressure. Competitive growth assays with pfcrt-edited parasites reveal that these compensatory mutations reduce the fitness defect caused by F145I. PPQ survival assays on edited lines show a loss of PPQ resistance in two of the three variants, including the field mutant (C258W). The latter restores CQ resistance. None of these variants alter parasite susceptibility to the first-line partner drug, mefloquine. Utilizing drug transport assays with purified PfCRT isoforms reconstituted into proteoliposomes, we identify differences in mutant PfCRT-mediated transport of PPQ and CQ. Molecular dynamics energy minimization calculations predict that these same mutations cause small but significant conformational changes in PfCRT regions implicated in drug interactions. Metabolomic analyses of isogenic parasite lines reveal differences in hemoglobin-derived peptide accumulation as a hallmark of PfCRT variation. These studies highlight the transient nature of PPQ resistance upon removal of drug pressure and suggest a strategy for employing this drug as part of multiple first-line therapies. IMPORTANCE Our study leverages gene editing techniques in Plasmodium falciparum asexual blood stage parasites to profile novel mutations in mutant PfCRT, an important mediator of piperaquine resistance, which developed in Southeast Asian field isolates or in parasites cultured for long periods of time. We provide evidence that increased parasite fitness of these lines is the primary driver for the emergence of these PfCRT variants. These mutations differentially impact parasite susceptibility to piperaquine and chloroquine, highlighting the multifaceted effects of single point mutations in this transporter. Molecular features of drug resistance and parasite physiology were examined in depth using proteoliposome-based drug uptake studies and peptidomics, respectively. Energy minimization calculations, showing how these novel mutations might impact the PfCRT structure, suggested a small but significant effect on drug interactions. This study reveals the subtle interplay between antimalarial resistance, parasite fitness, PfCRT structure, and intracellular peptide availability in PfCRT-mediated parasite responses to changing drug selective pressures.