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.
Researchers at the Wellcome Sanger Institute 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.
The researchers 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, they 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, the researchers sequenced a further 15,858 cells from the intraerythrocytic developmental cycle for three different species, including two human pathogens. They aligned developmental trajectories across species during the pathogenic phase of the life cycle, establishing a cross-species comparison method. Finally, they 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.
Left: Single-cell transcriptomes from across the life cycle of Plasmodium berghei were profiled (including liver, blood, and mosquito life stages). Center: Deep exploration of blood-stage parasites captured transcriptomic diversity at single-cell resolution across three different Plasmodium species by droplet sequencing. Right: Such datasets can serve as references to understand wild parasites isolated from clinical samples.
Availability – The Malaria Cell Atlas is accessible at: www.sanger.ac.uk/science/tools/mca/mca/.