Generation of a mutator parasite to drive resistome discovery in Plasmodium falciparum

In vitro evolution of drug resistance is a powerful approach for identifying antimalarial targets, however, key obstacles to eliciting resistance are the parasite inoculum size and mutation rate. Here we sought to increase parasite genetic diversity to potentiate resistance selections by editing catalytic residues of Plasmodium falciparum DNA polymerase delta. Mutation accumulation assays reveal a similar to 5-8 fold elevation in the mutation rate, with an increase of 13-28 fold in drug-pressured lines. Upon challenge with the spiroindolone PfATP4-inhibitor KAE609, high-level resistance is obtained more rapidly and at lower inocula than wild-type parasites. Selections also yield mutants with resistance to an irresistible compound, MMV665794 that failed to yield resistance with other strains. We validate mutations in a previously uncharacterised gene, PF3D7_1359900, which we term quinoxaline resistance protein (QRP1), as causal for resistance to MMV665794 and a panel of quinoxaline analogues. The increased genetic repertoire available to this mutator parasite can be leveraged to drive P. falciparum resistome discovery.

Automated summary

By editing the catalytic residues of Plasmodium falciparum DNA polymerase delta, parasite genetic diversity and resistance to antimalarial drugs can be enhanced. Mutation accumulation assays show a significant increase in mutation rate, particularly under drug pressure. Through selections, high-level resistance to both known and previously resistant compounds can be obtained, and the causal mutations can be identified. This mutator parasite provides a valuable genetic repertoire for discovering the P. falciparum resistome.