Inactivation of Plasmepsins 2 and 3 Sensitizes Plasmodium falciparum to the Antimalarial Drug Piperaquine

Dihydroartemisinin-piperaquine (DHA-PPQ), the current frontline artemisinin combination therapy used to treat Plasmodium falciparum malaria in multiple Southeast Asian countries, is now increasingly failing in Cambodia, where artemisinin resistance is nearly fixed, which suggests that PPQ resistance has emerged and is spreading rapidly in the Greater Mekong Subregion. Recent reports have shown that amplification of the genes encoding plasmepsins 2 and 3 is a molecular marker of PPQ resistance; however, whether these enzymes play a role in the mechanism of resistance is currently unknown. We show here that inactivating the genes encoding plasmepsin 2 or 3 individually in P. falciparum reference strain 3D7 results in hypersusceptibility to PPQ. Interestingly, no significant differences in the susceptibility to other antimalarials were observed, which suggests specific roles of plasmepsins 2 and 3 in PPQ susceptibility. The piperaquine hyper-sensitivity of the plasmepsin-2-and-3-inactivated lines provides direct evidence that these enzymes modulate parasite susceptibility to PPQ in the context of a single copy of PfMDR1 and independent of Kelch13 mutations conferring artemisinin resistance.