Novel Genetic Mechanism of Artemisinin Resistance for Malaria

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Recent progress in malaria control has reduced the incidence and saved the lives of hundreds of thousands of children, but effective strategies depend on a combination of measures that includes special emphasis on artemisinin-based combination therapies (ACTs). The predicted dire consequences of the evolution and spread of artemisinin (ART) resistance have been borne out tragically in Southeast Asia, where ART resistance evolved quickly and spread rapidly. The ART-resistance determinants reside in the propeller domain of the Pfkelch13 locus (K13), which is thought to facilitate protein quality control and modulate stress responses. Importantly, the K13 determinant was first identified through five-year in vitro selection and sequencing of a resistant strain from Tanzania; K13 was only later confirmed in the field in Southeast Asia. Were ART resistance to take hold and spread in Africa it would be truly catastrophic. Why it has not is open to speculation, however one possibility is because K13 effects are strongly dependent on genetic background, and there are significant genetic differences between African parasite and SE Asian lineages. But persistent, strong selection pressure from ART treatment in Africa will—as every evolutionary biologist knows—result almost inevitably in the evolution of resistance determinants in Africa. Based on the hypothesis that ART resistance might depend on genetic background, four years ago (prior to the K13 report) we began selecting independent replicate lines of parasites from Senegal. We are now able to report that high-level resistance has evolved in three independent lines. Our ART resistance lines show all of the known in vitro phenotypic hallmarks of clinical ART resistance, but they are not K13 mutants! Remarkably, three independent selected lines each contain a different mutation in the gene PF3D7_1251200, which encodes Coronin, one of a family of WD-repeat proteins containing a beta propeller structure. The importance of Pf1251200 has already been demonstrated experimentally in our laboratory using CRISPR/Cas9 replacements. These findings demonstrate the existence of at least two distinct genetic mechanisms of ART resistance. Investigating the Pf1251200 mutants and their interactions with K13 and other genes will help elucidate the mechanism of action of artemisinin, which is still unknown, and perhaps more important will provide markers for early detection of non-K13 ART resistance in clinical settings in Africa as well as SE Asia where a significant proportion of ART-resistant isolates have no mutations in K13.