Population genomics and transcriptomics of Plasmodium falciparum uncover components of the artemisinin resistance genetic background.

The emergence and spread of Plasmodium falciparum lineages resistant to artemisinins is threatening the efficacy of Artemisinin Combination Therapies (ACTs), the global first-line treatment for falciparum malaria. Artemisinin resistance is a complex genetic trait in which nonsynonymous SNPs in PfK13 cooperate with other, currently unknown, genetic variations to mediate the resistance phenotype. To identify these new molecular factors, we carried out population genomic/transcriptomic analyses of P. falciparum collected from patients with uncomplicated malaria in Cambodia and Vietnam between 2018 and 2020. GWAS confirmed the strong association of mutations in PfK13, in particular C580Y, with artemisinin resistance, but also uncovered significant associations with several additional polymorphisms, including PfRad5 in which two nonsynonymous mutations (N1131I and N821K) were independently associated with artemisinin resistance since its emergence. In addition, an intronic SNP in the gene encoding a WD40 repeat-containing protein on chromosome 11 ( PfWD11 ) was associated. Population transcriptome analyses revealed a defined set of genes whose steady-state levels of mRNA and/or alternatively spliced or antisense transcripts correlated with artemisinin resistance. Using transcriptomics of sequential parasite samples after the start of ACT treatment, we also characterized in vivo transcriptional responses to artemisinins and demonstrated the capacity of the resistant parasites to decelerate their intraerythrocytic developmental cycle, which can contribute to the resistant phenotype. During this process, both PfRAD5 and PfWD11 appear to upregulate an alternatively spliced isoform suggesting a contribution to the protective response to artemisinins. Comparison with historical samples indicated that PfRad5 and PfWD11 have been under selective pressure since the emergence of artemisinin resistance in the Greater Mekong Subregion as covariates of PfK13. Hence both genes now represent new high-confidence markers presumably as the key elements of the artemisinin resistance genetic background.