Mutations in Plasmodium falciparum actin-binding protein coronin confer reduced artemisinin susceptibility

Abstract

Drug resistance is an obstacle to global malaria control, as evidenced by the recent emergence and rapid spread of delayed artemisinin (ART) clearance by mutant forms of the PfKelch13 protein in Southeast Asia. Identifying genetic determinants of ART resistance in African-derived parasites is important for surveillance and for understanding the mechanism of resistance. In this study, we carried out long-term in vitro selection of two recently isolated West African parasites (from Pikine and Thiès, Senegal) with increasing concentrations of dihydroartemisinin (DHA), the biologically active form of ART, over a 4-y period. We isolated two parasite clones, one from each original isolate, that exhibited enhanced survival to DHA in the ring-stage survival assay. Whole-genome sequence analysis identified 10 mutations in seven different genes. We chose to focus on the gene encoding PfCoronin, a member of the WD40-propeller domain protein family, because mutations in this gene occurred in both independent selections, and the protein shares the β-propeller motif with PfKelch13 protein. For functional validation, when pfcoronin mutations were introduced into the parental parasites by CRISPR/Cas9-mediated gene editing, these mutations were sufficient to reduce ART susceptibility in the parental lines. The discovery of a second gene for ART resistance may yield insights into the molecular mechanisms of resistance. It also suggests that pfcoronin mutants could emerge as a nonkelch13 type of resistance to ART in natural settings.

Keywords: actin; artemisinin resistance; coronin; evolution; malaria.

Fig. 1.

(A) Outline of in vitro selection protocol. Parasite lines were pulsed intermittently with (DHA), cycling on and off the drug until the culture was smear-negative, which took 4–12 d. Parasites were allowed to recover to 1% parasitemia in the absence of DHA, a process that took 7–60 d. The complete process of treatment to recovery is considered one selection cycle. Following each selection cycle, cultures were pulsed again with an increased concentration of DHA. (B) Drug concentrations applied to two populations originating from two field isolates from Senegal over 13 drug selection cycles. The arrow indicates the first appearance of Pfcoronin mutations in both selected lines.

Fig. 2.

RSA survival rates for Senegalese parental lines and their parasite populations that underwent drug selection for 8 or 13 cycles. Survival >1% is considered elevated. Parasites after 8 or 13 drug selection cycles had significantly higher RSA survival rates compared with their parents (P < 0.001). Results from two to three biological replicates with two technical replicates each are included.

Fig. 3.

RSA survival rates for parental Pikine and Thiès lines and their two CRISPR/Cas9 edited clones with pfcoronin mutation(s). Survival >1% is considered elevated. Pfcoronin mutations identified in Pikine_R (R100K and E107V) and Thiès_R (G50E) were knocked into their respective parents. RSA survival rates were significantly higher for both independent clones in each background, Pikine (CRISPR F4 and CRISPR F5) (P < 0.001) and Thiès (CRISPR D4 and CRISPR E4) (P < 0.01) compared with their respective parental lines. Results from three biological replicates with two technical replicates each are included.