Michie, K.; Bishop, E.; Corredor, V.; Echeverry, D. F.; Deane, J. E.; Rayner, J. C.

Antimalarial drug resistance remains one of the most significant challenges to global malaria control. The emergence of resistance to chloroquine, the first truly globally distributed antimalarial, has been extensively studied but is still not fully understood. While mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) primarily drive resistance, the phenotype is complex and multigenic, with mutations in the putative amino acid transporter PfAAT1 recently confirmed to play a modulatory role. To date studies have focused on PfAAT1 mutations found in African and Southeast Asian P. falciparum lineages, but chloroquine resistance emerged independently in South America, where there may be novel PfAAT1 polymorphisms that are functionally important. We used AlphaFold modelling to reveal high homology between PfAAT1 and the human lysosomal arginine transporter SLC38A9, which allows prediction of membrane orientation and identifies a partially open channel accessible from the cytoplasm. Several PfAAT1 mutations found only in South American isolates sit near the entrance of this pore, most notably V231 where mutation to aspartate is predicted to alter channel conformation and influence transport, while nearby P446A (which is always found in combination with V231D) and I248T are predicted to impact pore flexibility and local structural stability. To functionally validate these insights, we employed CRISPR/Cas9 gene editing across parasite strains with diverse geographic origins. Reverting the regional V231D mutation in the South American 7G8 strain significantly reduced CQ resistance, providing the first functional evidence that this residue modulates drug susceptibility. Furthermore, introducing the apparently Colombia-specific I248T mutation significantly enhanced parasite multiplication rates in 7G8, demonstrating complex fitness and sensitivity trade-offs. Our findings reinforce the distinct evolutionary trajectory for South American CQ resistance and highlight the necessity of including additional pfaat1 mutations in global molecular surveillance strategies.