Gain-of-function PfAAT1 mutations compensate for impaired PfCRT function in Plasmodium falciparum

Mutations in the chloroquine resistance transporter (PfCRT) confer resistance to chloroquine (CQ) in the malaria parasite P. falciparum. Mutant PfCRT variants, defined by the K76T substitution, mediate efflux of CQ from the parasite digestive vacuole (DV), the site of drug action. However, these mutations impair the native function of PfCRT, which is export of hemoglobin (Hb)-derived peptides from the DV for downstream utilization as amino acids. Mutations in amino acid transporter 1 (PfAAT1) have also been associated with CQ resistance through population genomics and in vitro genetic crosses. The S258L mutation in Africa and the F313S mutation in Southeast Asia, often accompanied by additional substitutions, are found in near-complete linkage disequilibrium with pfcrt mutations, although the mechanistic basis linking these mutations to PfCRT dysfunction and CQ resistance has remained unclear. Here, we show that PfAAT1 is essential in parasites harboring mutant PfCRT, but largely dispensable in a wild-type PfCRT background. Replacing the mutant pfcrt allele with the wild-type version in otherwise isogenic parasite lines abolished the growth defect associated with pfaat1 knockdown, indicating functional overlap between PfCRT and PfAAT1. Consistent with this, PfAAT1 knockdown results in accumulation of Hb-derived peptides. Field-derived PfAAT1 variants (S258L and F313S) confer a significant fitness advantage over the wild-type allele in the mutant PfCRT background, consistent with a gain-of-function that enhances compensation for impaired PfCRT peptide transport activity. Comparative growth analyses reveal that the F313S mutation imparts a pronounced fitness advantage over the wild-type PfAAT1 under cysteine- and methionine-limiting conditions, providing a functional basis for this gain-of-function phenotype. We find that PfAAT1 mutations do not independently alter CQ sensitivity. Our data suggest that pfaat1 allele selection in the field is driven primarily by epistatic interaction with pfcrt.