Single-cell RNA sequencing reveals a signature of sexual commitment in malaria parasites

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Abstract
Highly parallel single-cell transcriptome profiling of Plasmodium falciparum blood stages provides insight into the role AP2-G plays in early sexual development of this eukaryotic pathogen. Mosquito-borne transmission of the malaria parasite requires a sub-population of the asexual form of the parasite to differentiate into sexual stages called gametocytes. This process is controlled by the transcriptional regulator AP2-G, but the mechanism involved is poorly understood. In this first application of single-cell RNA sequencing to a eukaryotic pathogen, Björn Kafsack and colleagues determine the transcriptional changes induced by AP2-G, identifying other AP2 transcription factors, histone-modifying enzymes and regulators of nucleosome positioning. These findings form the basis for further work aimed at elucidating the mechanistic underpinnings of gametocytogenesis in this important pathogen. Pathogens have to balance transmission with persistence. For Plasmodium falciparum, the most widespread and virulent malaria parasite, persistence within its human host requires continuous asexual replication within red blood cells, while its mosquito-borne transmission depends on intra-erythrocytic differentiation into non-replicating sexual stages called gametocytes1. Commitment to either fate is determined during the preceding cell cycle that begins with invasion by a single, asexually committed merozoite and ends, 48 hours later, with a schizont releasing newly formed merozoites, all committed to either continued asexual replication or differentiation into gametocytes2,3. Sexual commitment requires the transcriptional activation of ap2-g (PF3D7_1222600)4,5, the master regulator of sexual development, from an epigenetically silenced state during asexual replication6,7. AP2-G expression during this ‘commitment cycle’ prepares gene expression in nascent merozoites to initiate sexual development through a hitherto unknown mechanism2,4. To maintain a persistent infection, the expression of ap2-g is limited to a sub-population of parasites (1–30%, depending on genetic background and growth conditions). As sexually committed schizonts comprise only a sub-population and are morphologically indistinguishable from their asexually committed counterparts, defining their characteristic gene expression has been difficult using traditional, bulk transcriptome profiling8. Here we use highly parallel, single-cell RNA sequencing9 of malaria cultures undergoing sexual commitment to determine the transcriptional changes induced by AP2-G within this sub-population. By analysing more than 18,000 single parasite transcriptomes from a conditional AP2-G knockdown line and NF54 wild-type parasites at multiple stages of development, we show that sexually committed, AP2-G+ mature schizonts specifically upregulate additional regulators of gene expression, including other AP2 transcription factors, histone-modifying enzymes, and regulators of nucleosome positioning. These epigenetic regulators may act to facilitate the expression and/or repression of genes that are necessary for the initiation of gametocyte development in the subsequent cell cycle.

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