Single-Cell RNA Sequencing Reveals Cellular Heterogeneity and Stage Transition under Temperature Stress in Synchronized Plasmodium falciparum Cells

SUMMARY The malaria parasite has a complex life cycle exhibiting phenotypic and morphogenic variations in two different hosts by existing in heterogeneous developmental states. To investigate this cellular heterogeneity of the parasite within the human host, we performed single-cell RNA sequencing of synchronized Plasmodium cells under control and temperature treatment conditions. Using the Malaria Cell Atlas ( https://www.sanger.ac.uk/science/tools/mca ) as a guide, we identified 9 subtypes of the parasite distributed across known intraerythrocytic stages. Interestingly, temperature treatment results in the upregulation of the AP2-G gene, the master regulator of sexual development in a small subpopulation of the parasites. Moreover, we identified a heterogeneous stress-responsive subpopulation (clusters 5, 6, and 7 [∼10% of the total population]) that exhibits upregulation of stress response pathways under normal growth conditions. We also developed an online exploratory tool that will provide new insights into gene function under normal and temperature stress conditions. Thus, our study reveals important insights into cell-to-cell heterogeneity in the parasite population under temperature treatment that will be instrumental toward a mechanistic understanding of cellular adaptation and population dynamics in Plasmodium falciparum . IMPORTANCE The malaria parasite has a complex life cycle exhibiting phenotypic variations in two different hosts accompanied by cell-to-cell variability that is important for stress tolerance, immune evasion, and drug resistance. To investigate cellular heterogeneity determined by gene expression, we performed single-cell RNA sequencing (scRNA-seq) of about 12,000 synchronized Plasmodium cells under physiologically relevant normal (37°C) and temperature stress (40°C) conditions phenocopying the cyclic bouts of fever experienced during malarial infection. In this study, we found that parasites exhibit transcriptional heterogeneity in an otherwise morphologically synchronized culture. Also, a subset of parasites is continually committed to gametocytogenesis and stress-responsive pathways. These observations have important implications for understanding the mechanisms of drug resistance generation and vaccine development against the malaria parasite.