FIG 1.

Introduction of orthologous K13 nucleotide substitutions in P. berghei. (A) Three-dimensional homology model of P. falciparum (PF3D7_1343700) and P. berghei (PBANKA_1356700) K13 for amino acid residues 350 to 726 and 362 to 738, respectively. P. falciparum K13 mutation sites (F446I, M476I, Y493H, R539T, I543T, and C592Y) are indicated in the structure on the left, and P. berghei orthologous mutation sites are modeled on the right. Models were created in SWISS-MODEL using PDB template 4zgc.1.A. Structures were visualized and annotated using PyMOL 2.3. (B) Parasitemia growth curves monitoring recrudescence of the G2022, G2023, G2024, and G2025 lines upon artesunate (AS) challenge. Mice were infected with 2 × 10⁷ parasites by i.p. injection on day 0. Treatment with AS was commenced ∼3 h postinfection by i.p. injection and was continued for three consecutive days as indicated by arrows. Parasitemia was monitored microscopically until recrudescence was observed. Mice were bled when the parasitemia was less than 1.5% to minimize competition from wild-type parasites in case mutants carried growth defects. (C) Sanger sequencing of bulk DNA from the G2025 R551T line showing selective enrichment of this mutation upon AS treatment at 20 or 64 mg/kg. Enrichment of this mutation was also observed in the RFLP analysis (see also Fig. S2B in the supplemental material).