The Malaria Cell Atlas: Single parasite transcriptomes across the complete Plasmodium life cycle

INTRODUCTION Plasmodium parasites, the causative agent of malaria, are single-celled organisms with distinct morphological developmental stages each specialized to inhabit vastly different environments and host cell types. Underlying this morphological diversity is tight regulation of a compact genome, where the functions of ~40% of genes remain unknown, hampering the rate of effective drug and vaccine development. Single-cell RNA sequencing (scRNA-seq) has allowed high-resolution mapping of developmental processes, cellular diversity, and cell-to-cell variation, and its application to unicellular organisms reveals individual-level variation between parasites across their full life cycle. RATIONALE We have assembled a Malaria Cell Atlas that presents the transcriptomic profiles of individual Plasmodium parasites across all morphological life cycle stages. The ambition of such an atlas is to (i) inform gene function and usage throughout the life cycle, (ii) understand the gene regulatory mechanisms underlying developmental transitions, (iii) discover parasite bet-hedging patterns, and (iv) provide a reference dataset that can be used to understand parasite biology by the malaria community in both lab and natural infections for multiple Plasmodium species. RESULTS We isolated 1787 parasites using cell sorting and profiled full-length transcriptomes at 10 time points covering all life cycle stages across both the vector mosquito and the mammalian host. From these data, we could understand fine-scale transcriptional patterns of development and identify marker genes associated with parasite stage, cellular strategy (replicative, growth, and sexual phases), and host environment. Comparing single-cell gene expression patterns across the life cycle revealed groups of genes expressed in similar patterns during development. The resulting clusters of genes that behave similarly enables inference of possible function for the ~40% of genes that remain uncharacterized. Using droplet sequencing, we sequenced a further 15,858 cells from the intraerythrocytic developmental cycle for three different species, including two human pathogens. We aligned developmental trajectories across species during the pathogenic phase of the life cycle, establishing a cross-species comparison method. Finally, we developed a protocol for preserving wild parasites collected from naturally infected carriers and used scRNA-seq, together with the Malaria Cell Atlas as a reference, to identify wild parasite developmental stages and characterize a natural mixed-species infection at single-cell resolution. CONCLUSION We generated transcriptomes for all life cycle stages of Plasmodium and released these via the interactive Malaria Cell Atlas website, www.sanger.ac.uk/science/tools/mca/mca/. The Malaria Cell Atlas provides new insights into gene function and parasite developmental progression. We have demonstrated that it can serve as a transcriptomic reference, facilitating the interpretation of data from multiple species and multiple technologies. The characterization of wild Plasmodium parasites with immense genetic diversity will advance the study of the pathology and transmission of malaria directly from infected carriers. We envision that the Malaria Cell Atlas will support the development of much-needed new drugs, vaccines, and transmission-blocking strategies.