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

ABSTRACT 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.


Figure S6
A B pH at 5.5 pH at 7.4   S6.

Fig. S7 .
Fig. S7.Number of differentially accumulated peptides shown as a function of peptide charge of accumulated peptides at pH 5.5 or 7.4.Plots show differences in peptide levels between lines with a given mutation and Dd2 Dd2+F145I .Peptides are classified by (A) charge at pH 5.5 (representing the DV lumen) or (B) charge at pH 7.4 (representing the cytosol).Details are provided in TableS6.

Fig. S8 .
Fig. S8.Peptides HVDDM and VDPVNF inhibit 3 H-PPQ and 3 H-CQ transport via PfCRT.Uptake of (A) 50 nM 3 H-PPQ by 7G8+F145I PfCRT or (B) 3 H-CQ by 7G8 PfCRT was measured for 1 min in the presence or absence of the 25 µM HVDDM or VDPVNF.Values were normalized to the signal in the absence of the non-radiolabelled compound ("none").CQ, PPQ, and the non-PfCRT related drugs pyrimethamine (PYR) and atovaquone (ATQ) were also tested at 25 μM.Data are means ± s.d. of 3 independent experiments.

Table S1
Piperaquine survival assay values of pfcrt -modified parasite lines.
Piperaquine survival assay (PSA) values (nM) indicate the mean ± SEM, as determined in 2 to 8 independent assays performed in duplicate.Parasite survival is defined as the ratio of the parasitemias of the PPQ-treated to the no-drug control wells.This assay measures the survival of synchronous ring-stage parasites (0-6 hr post-invasion) exposed to PPQ for 72 hr, prior to measuring parasitemias by flow cytometry.N, number of independent assays.Statistical significance was determined via non-parametric Mann-Whitney U tests.P values are reported for comparisons with the parasite lines Dd2

Table S4
All salt bridges found for all PfCRT isoforms.
Interaction lifetimes are given as a percentage of time where the interacting species are within 4 Å of each other.Values shown are for interactions that exist for ≥10% of simulation time for at least one isoform.Green indicates a higher percentage and red indicates a low percentage.

Table S5 (
page 1) Averaged log 2 fold change of the baseline peptide levels in the variant PfCRT lines versus Dd2 Dd2+F145I .

Table S5 (page 2)
detected in either positive or negative mode.Blank spaces denote missing or undetectable values.Averaged log 2 fold change of the baseline peptide levels in the variant PfCRT lines versus Dd2 Dd2+F145I .

Table S6 (page 1)
List of peptides showing significantly different levels for Dd2 Dd2 , Dd2 Dd2+F145I+F131C , Dd2 Dd2+F145I+I347T , and Dd2 Dd2+F145I+C258W compared to Dd2 Dd2+F145I .Data were obtained from three independent experiments with technical triplicates.For each peptide, the isoelectric point (IEP), pH at 5.5, and pH 7.4 are displayed.P values were calculated from unpaired t tests.Gray indicates peptides that were detected in more than one line.

Table S6 (page 2)
List of peptides showing significantly different levels for Dd2

Table S7
List of oligonucleotides used in this study.Sequencing primer for pfcrt .Integration PCR #1.2.5 kb yes/no.integration at 3' end Sequences exons 2-3.
F145I SDM on pfcrt sequence codon optimized for S. cerevisiae.