Piperaquine-resistant PfCRT mutations differentially impact drug transport, hemoglobin catabolism and parasite physiology in Plasmodium falciparum asexual blood stages

The emergence of Plasmodium falciparum parasite resistance to dihydroartemisinin + piperaquine (PPQ) in Southeast Asia threatens plans to increase the global use of this first-line antimalarial combination. High-level PPQ resistance appears to be mediated primarily by novel mutations in the P. falciparum chloroquine resistance transporter (PfCRT), which enhance parasite survival at high PPQ concentrations in vitro and increase the risk of dihydroartemisinin + PPQ treatment failure in patients. Using isogenic Dd2 parasites expressing contemporary pfcrt alleles with differential in vitro PPQ susceptibilities, we herein characterize the molecular and physiological adaptations that define PPQ resistance in vitro. Using drug uptake and cellular heme fractionation assays we report that the F145I, M343L, and G353V PfCRT mutations differentially impact PPQ and chloroquine efflux. These mutations also modulate proteolytic degradation of host hemoglobin and the chemical inactivation of reactive heme species. Peptidomic analyses reveal significantly higher accumulation of putative hemoglobin-derived peptides in the PPQ-resistant mutant PfCRT isoforms compared to parental PPQ-sensitive Dd2. Joint transcriptomic and metabolomic profiling of late trophozoites from PPQ-resistant or -sensitive isogenic lines reveals differential expression of genes involved in protein translation and cellular metabolism. PPQ-resistant parasites also show increased susceptibility to an inhibitor of the P. falciparum M17 aminopeptidase that operates on short globin-derived peptides. These results reveal unique physiological changes caused by the gain of PPQ resistance and highlight the potential therapeutic value of targeting peptide metabolism in P. falciparum. Author summary The emergence of multidrug resistance in Plasmodium falciparum parasites presents a significant obstacle to the malaria elimination agenda. Resistance to piperaquine (PPQ), an important first-line partner drug, has spread across Southeast Asia where it has contributed to widespread treatment failures. The genetic basis of high-level resistance to PPQ is primarily attributable to a novel set of amino acid substitutions in the P. falciparum chloroquine resistance transporter (PfCRT). Using genetically-modified parasite lines, we show that PPQ-resistant asexual blood stage parasites only modestly reduce the intracellular concentration of PPQ, in contrast with the related drug chloroquine where resistant forms of PfCRT lead to minimal accumulation. We also report that PPQ-resistant, PfCRT mutant parasites accumulate higher levels of putative hemoglobin-derived peptides than do their PPQ-sensitive counterparts. These two groups of parasites exhibit differential expression of genes involved in multiple processes including protein translation and phosphorylation, cellular metabolism, and RNA processing. We also observed increased susceptibility of the PPQ-resistant mutant PfCRT parasites to inhibition of an enzyme involved in cleaving globin-derived peptides, supporting the hypothesis that PfCRT mutations interfere with the vital process of hemoglobin catabolism.