Single cell transcriptional changes across the blood stages of artemisinin resistant K13C580Y mutant Plasmodium falciparum upon dihydroartemisinin exposure

Artemisinins have been a cornerstone of malaria control, but resistance in Plasmodium falciparum, due to mutations in the Kelch 13 gene, threaten these advances. Artemisinin exposure results in a dynamic transcriptional response across multiple pathways, but most work has focused on ring stages and ex vivo transcriptional analysis, limiting evaluation of all life cycle stages. We applied single cell RNAseq to two unsynchronized isogenic parasite lines (K13C580 and K13580Y) over 6 hrs after a pulse exposure to dihydroartemisinin (DHA). Transcription was altered across all stages, with the greatest occurring at the early trophozoite and mid ring stage in both lines. This response involved the arrest of metabolic processes and the enhancement of protein trafficking and the unfolded protein response. While similar, the response was enhanced in the K13580Y mutant, which may lead to the dormancy phenomenon upon treatment. Increased surface protein expression was seen in mutant parasites at baseline and upon drug exposure, highlighted by the increased expression of PfEMP1 and GARP, a potential therapeutic target. Antibody targeting GARP maintained anti-parasitic efficacy in mutant parasites. This work provides single cell insight of gene transcription across all life cycle stages revealing transcriptional changes that could initiate dormancy state and mediate survival.