作者: Brenneman, Katelyn M Vendrely ; Shoue, Douglas A ; Peto, Tom ; Checkley, Lisa A ; Haile, Meseret T ; Tripura, Rupam ; Nair, Shalini ; Dhorda, Mehul ; Abatiyow, Biley A ; Cheeseman, Ian H ; Anderson, Timothy J C ; Button-Simons, Katrina A ; Vaughan, Ashley M ; Kappe, Stefan H I ; Reyes, Ann ; Lek, Dysoley ; Kane, John ; Li, Xue ; Ferdig, Michael T ; Kumar, Sudhir ; Dahlhoff, Haley ; Sievert, Mackenzie A C ; Nkhoma, Standwell C ; Singh, Puspendra P
Piperaquine (PPQ) is widely used in combination with dihydroartemisinin (DHA) as a first-line treatment against malaria parasites. Multiple genetic drivers of PPQ resistance have been reported, including mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and increased copies of plasmepsin II/III (pm2/3). We generated a cross between a Cambodia-derived multi-drug resistant KEL1/PLA1 lineage isolate (KH004) and a drug susceptible parasite isolated in Malawi (Mal31). Mal31 harbors a wild-type (3D7-like) pfcrt allele and a single copy of pm2/3, while KH004 has a chloroquine-resistant (Dd2-like) pfcrt allele with an additional G367C substitution and four copies of pm2/3. We recovered 104 unique recombinant progeny and examined a targeted set of progeny representing all possible combinations of variants at pfcrt and pm2/3 for detailed analysis of competitive fitness and a range of PPQ susceptibility phenotypes, including PPQ survival assay (PSA), area under the dose-response curve (AUC), and a limited point IC50 (LP-IC50). We find that inheritance of the KH004 pfcrt allele is required for PPQ resistance, whereas copy number variation in pm2/3 further enhances resistance but does not confer resistance in the absence of PPQ-R-associated mutations in pfcrt. Deeper investigation of genotype-phenotype relationships demonstrates that progeny clones from experimental crosses can be used to understand the relative contributions of pfcrt, pm2/3, and parasite genetic background, to a range of PPQ-related traits and confirm the critical role of the PfCRT G367C substitution in PPQ resistance.
