Mutation in the Plasmodium falciparum BTB/POZ Domain of K13 Protein Confers Artemisinin Resistance

Abstract

Partial artemisinin resistance, defined in patients as a delayed parasite clearance following artemisinin-based treatment, is conferred by non-synonymous mutations in the Kelch beta-propeller domain of the Plasmodium falciparum k13 (pfk13) gene. Here, we carried out in vitro selection over a 1-year period on a West African P. falciparum strain isolated from Kolle (Mali) under a dose-escalating artemisinin regimen. After 18 cycles of sequential drug pressure, the selected parasites exhibited enhanced survival to dihydroartemisinin in the ring-stage survival assay (RSA^0-3h = 9.2%). Sanger and whole-genome sequence analyses identified the PfK13 P413A mutation, localized in the BTB/POZ domain, upstream of the propeller domain. This mutation was sufficient to confer in vitro artemisinin resistance when introduced into the PfK13 coding sequence of the parasite strain Dd2 by CRISPR/Cas9 gene editing. These results together with structural studies of the protein demonstrate that the propeller domain is not the sole in vitro mediator of PfK13-mediated artemisinin resistance, and highlight the importance of monitoring for mutations throughout PfK13.

Keywords: BTB/POZ domain; K13 mutation; artemisinin resistance; malaria.

FIG 1

(A) In vitro selection of ART resistance by exposure of the parental field isolate SMT010 to escalating drug pressure cycles. Each cycle of drug pressure exposed ring-stage parasites at 3% parasitemia to sequential and increasing doses of ART for 24 h. After drug removal, parasites were placed back in drug-free culture conditions until they reached 3% parasitemia. Selected lineages are annotated SMT010pX (“p” for pressure and “X” for the number of pressure cycles). (B) ART concentration applied for each pressure cycle across the year of selection process (blue line) and the corresponding time to recovery of parasites (time necessary to go back to 3% parasitemia) after the drug pressure (black bars). The star indicates the appearance at p18 of the P413A mutation in the pfk13 gene. (C) RSA^0-3h survival rates 72 h after exposure to a 6 h pulse of 700 nM dihydroartemisinin (DHA) for the parental strain SMT010 and the selected lineages (all the selected lineages after 18 pressure cycles of drug pressure are K13-mutated). Mean survival rates and numbers of independent experiments for each strain are presented in supplemental Table S1. P values were derived by using the Mann-Whitney U test, comparing selected strains to the parental strain. *** P < 0.001.

FIG 2

(A) pfk13 editing strategy. (B) RSA^0-3h survival rate of parental and edited Dd2 P. falciparum strains 72 h after exposure to a 6 h pulse of 700 nM DHA. Dd2-P413A groups the three clones Dd2-C8^P413A/D8^P413A/F3^P413A. Mean survival rates and numbers of independent experiments for each strain are presented in supplemental Table S1. P values were derived by using the Mann-Whitney U test, comparing edited strains to the parental strain. ** P < 0.01.

FIG 3

(A) Location of residue P413 on the PfK13 BTB/POZ-propeller structure. The structure is shown as surface, and residue P413 is colored in red. (B) Location of residue P413 on a dimeric structure of PfK13 BTB/POZ-Propeller structure. The structure is shown as surface; monomer A is colored in green, monomer B is colored in blue, and P413 is highlighted in red. (C) Torsion angles of residue P413. (D) Location of P413 on the PfK13 BTB/POZ structure. The structure is colored according to secondary structures. Two other prolines, P417 and P419, are localized on the same structural segment. (E) Evolution of secondary structures for each residue of PfK13 BTB/POZ-propeller structures during 100 ns simulation. The arrow indicates the location of position 413. T, Turn; E, Extended configuration (β-sheet); B, Isolated bridge; H, ɑ-helix; G, 3-10 helix; C, Coil. (F) PfK13 amino acids in contact with P413. Distances are expressed in Angströms. The structure is shown as a cartoon, and amino acids close to P413 are sticks. P413 is colored red and labeled. (G) Cɑ-based root mean square fluctuations (RMSFs) at each residue of the PfK13 BTB/POZ-propeller structure. RMSF values were calculated from the 100 ns molecular dynamics simulations. The arrow indicates the location of the residue 413. (H) Electrostatic surface potential of the homodimeric PfK13 BTB/POZ-Propeller structure, estimated with the APBS method. Electrostatic potential values are in units of kT/e at 298 K, on a scale of −5 kT/e (red) to +5 kT/e (blue). White color indicates a neutral potential.