APlasmodium falciparumgenetic cross reveals the contributions ofpfcrtandplasmepsin II/IIIto piperaquine drug resistance

Abstract

Piperaquine (PPQ) is widely used in combination with dihydroartemisinin (DHA) as a first-line treatment against malaria parasites. Resistance to this artemisinin combination therapy (ACT) has emerged in Cambodia and threatens to spread to other malaria-endemic regions. Multiple drivers of PPQ resistance have been reported, including mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and increased copies of plasmepsin II/III (pm). Experimental genetic crosses allow for the precise quantification of the relative contributions of specific loci to a phenotype and have the distinct advantage of assaying the effects of naturally evolved genetic variation. We generated a cross between a Cambodia-derived multi-drug resistant KEL1/PLA1 lineage parasite 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 pm, while KH004 has a chloroquine-resistant (Dd2-like) pfcrt allele with an additional G367C substitution and four copies of pm. We cloned 101 unique recombinant progeny from this cross, and from this pool, we identified a targeted set of progeny representing all possible combinations of variants at pfcrt and pm for detailed analysis of a range of PPQ response and competitive fitness phenotypes. Fortuitously, progeny clones that inherited the KH004 pm allele displayed a range of different copies, allowing us to directly test the role of pm copy number on a range of resistance phenotypes, including IC50, area under the curve (AUC), and PSA as well as competitive fitness. We find that all progeny inheriting both pfcrt and pm alleles from KH004 are PPQ resistant by (PSA and AUC) and that inheritance of KH004 pfcrt is required for resistance (including progeny that inherited the single copy pm from Mal31). Our findings clearly display how progeny clones from experimental crosses can be used to parse apart the contributions of pfcrt, pm, and parasite genetic background, to a range of PPQ-related traits and confirm the critical role of the PfCRT G367C substitution on PPQ resistance.

Temporal and spatial dynamics ofPlasmodium falciparumclonal lineages in Guyana

“…At least two mutations that are segregating in Guyana confer resistance to piperaquine: a point C350R mutation in the chloroquine resistance transporter ( pfcrt ) gene that is endemic to the Guiana Shield region and has been increasing in frequency over the last 20 years [28,30,31], and copy number amplification of the plasmepsin 2 ( Pfpm2 - PF3D7_1408000) and/or plasmepsin 3 ( Pfpm3 - PF3D7_1408100) genes [30]. The pfcrt C350R mutation and plasmepsin 2/3 amplifications interact epistatically to yield piperaquine resistance [32], adding credibility to the hypothesis that clonal transmission may be adaptive under DHA-piperaquine pressure.…”

Incomplete Plasmodium falciparum growth inhibition following piperaquine treatment translates into increased parasite viability in the in vitro parasite reduction ratio assay

“…> 10%) was not only associated with abnormal dose-response curves, but also with treatment failure in the patients from whom the parasite isolate originated ( Duru et al., 2015 ). Finally, studies aiming at identifying genetic markers of PPQ resistance pointed to an association with amplifications in plasmepsin 2 and 3 ( Agrawal et al., 2017 ; Amato et al., 2017 ; Witkowski et al., 2017 ; Bopp et al., 2018 ; Kane et al., 2023 ), or single nucleotide polymorphisms in exonuclease ( Amato et al., 2017 ) and chloroquine-resistance transporter ( crt ) ( Duru et al., 2015 ; Agrawal et al., 2017 ; Dhingra et al., 2017 ; Ross et al., 2018 ; Dhingra et al., 2019 ; Gomez et al., 2023 ; Kane et al., 2023 ).…”