Plasmodium berghei K13 Mutations Mediate Artemisinin Resistance That Is Reversed by Proteasome Inhibition.

The recent emergence of parasite resistance to the first line antimalarial drug artemisinin is of particular concern. Artemisinin resistance is primarily driven by mutations in the K13 protein, which enhance survival of early ring-stage parasites treated with the artemisinin active metabolite dihydroartemisinin and associate with delayed parasite clearance However, association of K13 mutations with artemisinin resistance has been problematic due to the absence of a tractable model. Herein, we have employed CRISPR/Cas9 genome editing to engineer selected orthologous K13 mutations into the gene of an artemisinin-sensitive rodent model of malaria. Introduction of the orthologous K13 F446I, M476I, Y493H, and R539T mutations into K13 yielded gene-edited parasites with reduced susceptibility to dihydroartemisinin in the standard 24-h assay and increased survival in an adapted ring-stage survival assay. Mutant K13 parasites also displayed delayed clearance upon treatment with artesunate and achieved faster recrudescence upon treatment with artemisinin. Orthologous C580Y and I543T mutations could not be introduced into , while the equivalents of the M476I and R539T mutations resulted in significant growth defects. Furthermore, a -selective proteasome inhibitor strongly synergized dihydroartemisinin action in these K13 mutant lines, providing further evidence that the proteasome can be targeted to overcome artemisinin resistance. Taken together, our findings provide clear experimental evidence for the involvement of K13 polymorphisms in mediating susceptibility to artemisinins and, most importantly, under conditions. Recent successes in malaria control have been seriously threatened by the emergence of parasite resistance to the frontline artemisinin drugs in Southeast Asia. artemisinin resistance is associated with mutations in the parasite K13 protein, which associates with a delay in the time required to clear the parasites upon drug treatment. Gene editing technologies have been used to validate the role of several candidate K13 mutations in mediating artemisinin resistance under laboratory conditions. Nonetheless, the causal role of these mutations under conditions 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, Phenotyping of these K13 mutant parasites provides evidence of their role in mediating artemisinin resistance , which supports artemisinin resistance observations. However, we were unable to introduce some of the K13 mutations (C580Y and I543T) into the corresponding amino acid residues, while other introduced mutations (M476I and R539T equivalents) carried pronounced fitness costs. Our study provides evidence of a clear causal role of K13 mutations in modulating susceptibility to artemisinins and using the well-characterized model. We also show that inhibition of the proteasome offsets parasite resistance to artemisinins in these mutant lines.