Single Cell RNA Sequencing Reveals Transcriptional Heterogeneity and Stochastic Gene Expression in Plasmodium falciparum Schizonts

Genetically identical cells are known to exhibit differential phenotypes in the same environmental conditions. These phenotypic variants are linked to transcriptional stochasticity and have been shown to contribute towards adaptive flexibility of a wide range of unicellular organisms such as yeast and bacteria. Here, we investigated transcriptional heterogeneity and stochastic gene expression in P. falciparum by performing single cell RNA sequencing on blood stage schizonts. Our data reveals significant transcriptional variations in the schizonts stage that appear to reflect several diversification processes including putative developmental “checkpoint”; transcriptomically distinct parasite cells sub-populations; transcriptional switches in variable gene families and a distinct group of highly variable transcripts. Most of these features of transcriptional variability were preserved in isogenic parasite cell populations (albeit with a lesser amplitude) suggesting a role of epigenetic factors in cell-to-cell transcriptional variations in human malaria parasites. Finally, to validate our findings, we applied quantitative qRT-PCR and RNA-FISH approach and confirmed stochastic expression of several genes such as msp1, msp3, msp7, rhopH2 and eba181, some of which represent key candidates for invasion blocking vaccines Overall design: Examination of stochastic mRNA variations between single parasites in non-isogenic and isogenic lines.