Single-cell RNA-seq reveals novel heterogeneity in the malaria parasite Plasmodium falciparum

The complex life cycle of Plasmodium falciparum includes multiple stages in the human host and Anopheles mosquito. Most of these unicellular parasites in the human host follow an asexual cycle characterized by a tightly synchronized continuous cascade of gene expression, a phenomenon described using bulk-cell analyses. While individual parasites are known to exhibit transcriptional variation to evade the host immune system or commit to a sexual fate, these expression changes are largely undetectable in bulk-cell analyses. In an effort to understand the transcriptional variations among individual P. falciparum, we used single-cell RNA sequencing (scRNA-seq) to uncover the heterogeneity of P. falciparum populations during the intraerythrocytic developmental cycle (IDC). We utilized a Smart-Seq protocol optimized for the Fluidigm C1 and prepared sequencing libraries with the Nextera XT kit. These libraries were sequenced in one lane on an Illumina HiSeq 2500 with 50 bp single-end reads. In total, we sequenced the transcriptomes of 49 late stage asexual parasites approximately 34 hours post-invasion and 5 late stage gametocytes 9 days after gametocyte induction. Three late stage asexual parasites clustered with gametocytes and are likely early stage gametocytes due to their expression of sex-specific genes, including Pfs16. Our analyses of the remaining parasites identified a number of novel gametocyte-specific genes as well as three distinct clusters of late stage asexual parasites. Two clusters were characteristic of cell cycle progression (expression of SERA versus rhoptry genes, respectively), while the third cluster exhibited enhanced expression of genes not known to be stage specific. Furthermore, individual parasites showed wide variations in their expression of cluster-specific genes at the single-cell level, representing an additional layer of transcriptional regulation and variability not seen in previous analyses. This heterogeneity seen through scRNA-seq will aid in our understanding of the parasite's adaptive responses to the host immune system, drug treatment, and competition from other parasites.