Single-cell analysis of Plasmodium falciparum transcripts after drug perturbation identifies feedback regulation as well as increased transmission potential.

Gene expression analysis in malaria parasites has been used to define transcriptional regulatory networks but has been used less frequently to characterize parasite response to drug treatment or to show how parasites may evade killing. Here, we applied single-cell RNA sequencing (scRNA-seq) to hundreds of thousands of individually infected asynchronous red blood cells to evaluate the parasite's response to treatment with three chemotypes that can be used for treatment (artemisinin) or prophylaxis and treatment (atovaquone, ganaplacide). We found that each treatment gave rise to different cell populations with different transcriptional profiles. Comparing single cell transcription patterns in compound-treated cells, to transcript patterns observed previously with synchronized cells showed an enrichment of cells expressing gametocyte-associated genes after artemisinin treatment but fewer lifecycle perturbations after treatment with the two other compounds. In contrast, bulk analysis showed an enrichment of pyrimidine biosynthesis transcripts for atovaquone treatment. Our results show that scRNA-seq may be used to profile diverse drug responses across many lifecycle stages and to potentially classify drug classes.