Single-cell transcriptional changes across the blood stages of artemisinin-resistant K13580Y Plasmodium falciparum upon dihydroartemisinin exposure.

Artemisinin-based therapies have been central to malaria control, but partial resistance in Plasmodium falciparum, driven by mutations in the Kelch13 (K13) gene, threatens these gains. To investigate the molecular basis of this resistance, we applied single-cell RNA sequencing to coisogenic parasite lines, K13 wild-type (K13-C580) and the artemisinin-resistant mutant (K13-580Y), following a 6 hour pulse of dihydroartemisinin (DHA). This approach enabled high-resolution profiling across all intraerythrocytic stages. Both lines exhibited stage-specific transcriptional responses, with the most pronounced changes in ring and trophozoite stages. These included downregulation of metabolic genes and upregulation of stress response pathways, including the unfolded protein response and protein trafficking. Transcriptional signatures consistent with a dormancy-like state were observed, particularly in the downregulation of SERA family genes. Notably, K13-580Y parasites showed a more consistent and robust transcriptional adaptation across stages, suggesting enhanced survival under drug-induced stress. Using MELD, a computational framework for quantifying transcriptional perturbation, we found that K13-580Y parasites exhibited higher DHA likelihood scores across the asexual cycle, indicating broad and sustained transcriptional shifts in response to treatment. Additionally, increased expression of surface proteins such as PfEMP1 and GARP was observed in K13-580Y parasites at baseline and following DHA exposure. Functional assays confirmed that anti-GARP antibodies retained efficacy against both lines, supporting its potential as a therapeutic target. These findings provide a comprehensive view of the cellular responses that may facilitate artemisinin resistance, identify molecular features of dormancy and pathogenesis, and highlight surface proteins like GARP and PfEMP1 as promising intervention targets. This work underscores the power of single-cell approaches to dissect drug responses and guide strategies to overcome resistance in malaria parasites.