Three Asparagine insertions in the K13-propeller led to Plasmodium falciparum becoming resistant to multiple antimalarial drugs

Drug resistance in Plasmodium falciparum represents a significant challenge in malaria treatment. Identifying the molecular markers associated with P. falciparum resistance will effectively detect resistance and enhance treatment efficiency. In this study, we utilized the advanced CRISPR/Cas9 technology to precisely insert one, two, or three asparagine residues into the Kelch 13(K13) gene of the 3D7 strain, positioned after the 142nd amino acid residue, resulting in 1N-3D7, 2N-3D7, and 3N-3D7. Using ring-stage survival assays (RSA), drug sensitivity evaluations, and in vitro developmental assessments, our findings revealed a trend: 1) the insertion of asparagine residues into the parasite genome increased RSA, with more asparagine insertions leading to higher RSA. 2) According to the IC50 values, 1N-3D7 and 2N-3D7 exhibited similar sensitivity profiles across all ten tested drugs, with both demonstrating resistance to Naphthoquine, indicating that the insertions of one or two asparagines played an equivalent role in conferring resistance. However, the insertion of three asparagine residues resulted in significantly higher IC50 values compared to the first two forms when tested with Artesunate, Artemether, Dihydroartemisinin, Pyronaridine Phosphate, and Naphthoquine, showing resistance to all five drugs. Furthermore, 3N-3D7 exhibited a prolonged ring phase and a shortened trophozoite phase within red blood cells; the schizont phase appeared synchronous with the others, yet its mature schizonts contained fewer merozoites. Additionally, 3N-3D7 exhibited a fitness defect, with the proportion decreasing gradually during co-culture with 3D7, its fitness cost calculated as 14.88 ± 2.87. All these results support the opinion that the insertion of three asparagines was a molecular marker of resistance to artemisinin derivatives, Pyronaridine Phosphate, and Naphthoquine in P. falciparum.