A
Plasmodium falciparum
genetic cross reveals the contributions of
pfcrt
and
plasmepsin II/III
to piperaquine drug resistance
Authors: Kane John , Li Xue , Kumar Sudhir , Button-Simons Katrina A. , Vendrely Brenneman Katelyn M. , Dahlhoff Haley , Sievert Mackenzie A. C. , Checkley Lisa A. , Shoue Douglas A. , Singh Puspendra P. , Abatiyow Biley A. , Haile Meseret T. , Nair Shalini , Reyes Ann , Tripura Rupam , Peto Thomas J. , Lek Dysoley , Mukherjee Angana , Kappe Stefan H. I. , Dhorda Mehul , Nkhoma Standwell C. , Cheeseman Ian H. , Vaughan Ashley M. , Anderson Timothy J. C. , Ferdig Michael T. 
Published: June 2024

Piperaquine (PPQ) is widely used in combination with dihydroartemisinin as a first-line treatment against malaria. 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 multidrug-resistant KEL1/PLA1 lineage isolate (KH004) and a drug-susceptible Malawian parasite (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 multiple copies of
pm2/3
. We recovered 104 unique recombinant parasites and examined a targeted set of progeny representing all possible combinations of variants at
pfcrt
and
pm2/3
. We performed a detailed analysis of competitive fitness and a range of PPQ susceptibility phenotypes with these progenies, including PPQ survival assay, area under the dose response curve, and a limited point IC
50
. We find that inheritance of the KH004
pfcrt
allele is required for reduced PPQ sensitivity, whereas copy number variation in
pm2/3
further decreases susceptibility but does not confer resistance in the absence of additional mutations in
pfcrt
. A deep 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. Additionally, we find that the resistance phenotype associated with parasites inheriting the G367C substitution in pfcrt is consistent with previously validated PPQ resistance mutations in this transporter.

IMPORTANCE

Resistance to piperaquine, used in combination with dihydroartemisinin, has emerged in Cambodia and threatens to spread to other malaria-endemic regions. Understanding the causal mutations of drug resistance and their impact on parasite fitness is critical for surveillance and intervention and can also reveal new avenues to limiting the evolution and spread of drug resistance. An experimental genetic cross is a powerful tool for pinpointing the genetic determinants of key drug resistance and fitness phenotypes and has the distinct advantage of quantifying the effects of naturally evolved genetic variation. Our study was strengthened since the full range of copies of KH004
pm2/3
was inherited among the progeny clones, allowing us to directly test the role of the
pm2/3
copy number on resistance-related phenotypes in the context of a unique
pfcrt
allele. Our multigene model suggests an important role for both loci in the evolution of this multidrug-resistant parasite lineage.