Plasmodium bergheiK13 Mutations MediateIn VivoArtemisinin Resistance That is Reversed by Proteasome Inhibition
Authors: Simwela Nelson V. , Stokes Barbara H. , Aghabi Dana , Bogyo Matt , Fidock David A. , Waters Andrew P. 
Published: August 2020

ABSTRACTThe recent emergence ofPlasmodium falciparum(PF) parasite resistance to the first line antimalarial drug artemisinin is of particular concern. Artemisinin resistance is primarily driven by mutations in the PF K13 protein, which enhance survival of early ring stage parasites treated with the artemisinin active metabolite dihydroartemisininin vitroand associate with delayed parasite clearancein vivo. However, association of K13 mutations within vivoartemisinin resistance has been problematic due to the absence of a tractable model. Herein, we have employed CRISPR/Cas9 genome editing to engineer selected orthologous PF K13 mutations into theK13gene of an artemisinin-sensitive,P.berghei(PB) rodent model of malaria. Introduction of the orthologous PF K13 F446I, M476I, Y493H and R539T mutations into PB K13 produced gene-edited parasites with reduced susceptibility to dihydroartemisinin in the standard 24-hourin vitroassay and increased survival in an adaptedin vitroring-stage survival assay. Mutant PB K13 parasites also displayed delayed clearancein vivoupon treatment with artesunate and achieved faster recrudescence upon treatment with artemisinin. Orthologous C580Y and I543T mutations could not be introduced into PB while the equivalent of the M476I and R539T mutations resulted in significant growth defects. Furthermore, aPlasmodium-selective proteasome inhibitor strongly synergized dihydroartemisinin action in these PB K13 mutant lines, providing further evidence that the proteasome can be targeted to overcome ART resistance. Taken together, our work provides clear experimental evidence for the involvement of K13 polymorphisms in mediating susceptibility to artemisininsin vitro, and most importantly underin vivoconditions.IMPORTANCERecent successes in malaria control have been seriously threatened by the emergence ofPlasmodium falciparumparasite resistance to the frontline artemisinin drugs in Southeast Asia.P.falciparumartemisinin resistance is associated with mutations in the parasite K13 protein, which associates with a delay in the time required to clear the parasites upon treatment with the drug. Gene editing technologies have been used to validate the role of several candidate K13 mutations in mediatingP.falciparumartemisinin resistancein vitrounder laboratory conditions. Nonetheless, the causal role of these mutations underin vivoconditions has been a matter of debate. Here, we have used CRISPR/Cas9 gene editing to introduce K13 mutations associated with artemisinin resistance into the related rodent-infecting parasite,P.berghei. Phenotyping of theseP.bergheiK13 mutant parasites provides evidence of their role in mediating artemisinin resistancein vivo, which supportsin vitroartemisinin resistance observations. However, we were unable to introduce some of theP.falciparumK13 mutations (C580Y, I543T) into the corresponding amino acid residues, while other introduced mutations (M476I, R539T equivalents) carried a pronounced fitness cost. Our study provides evidence of a clear causal role of K13 mutations in modulating susceptibility to artemisininsin vitroandin vivousing the well-characterizedP.bergheimodel. We also show that inhibition of theP.bergheiproteasome offsets parasite resistance to artemisinins in these mutant lines.