Single-cell transcriptomics reveals transcriptional programs underlying male and female cell fate during Plasmodium falciparum gametocytogenesis
The Apicomplexa constitute a large phylum of parasitic protozoans with complex life cycles that typically include meiotic sex. The life cycle of the malaria parasite, Plasmodium falciparum, includes obligate transition and stage development between a human and mosquito host. Asexual parasite replication in the human erythrocytes is followed by differentiation which leads to the formation of a precursor gamete stage, referred to as gametocytes. The gametocyte stage is solely responsible for malaria transmission into the mosquito vector where gamete fusion followed by meiosis occurs. How the parasite differentiates into male and female gametocytes in the absence of sex chromosomes largely remains an open question. Here, we combine FACS-based cell enrichment of a gametocyte reporter line followed by single-cell RNA-seq, to enable targeted characterization of the entire gametocyte developmental stage. Our data defines differential transcription programs during male and female gametocyte development and highlights a bifurcation point for sexual cell fate. We perform prediction analyses of novel candidate driver genes underlying P. falciparum sexual cell fate. Additionally, we delineate the timing of expression of members of the ApiAP2 family of transcription factors and predict their specificity in male or female P. falciparum gametocyte development. Overall design: Using P. falciparum NF54-tdTomato, we established five consecutive time points (Day 1, 3, 5, 7, and 9) for time courses scRNA-seq to cover a broad range of gametocytes throughout their developmental period. The culture samples were then purified using Magnetic cell separation (MACs) sorter MidiMACS™ from day 3 to day 9 for contaminating asexual forms removal. The gametocytes were then counted using a hemocytometer and the gametocytaemia was set to 400.000 cells/ml. On day one, 2?µg/ml of Hoechst 33342 was added to the gametocytes sample, incubated for 15 mins, and FACs-sorted, single-cell sorting was performed with a MoFlo Astrios EQ (Beckman Coulter, USA) according to tdTomato fluorescence into a total volume of 4ul of sterile-filtered 1 x PBS. Hoechst 33342 was detected using the 488?nm laser with the 450/50?nm filter and tdTomato (RFP) was detected at the 561?nm laser with a 586/16?nm filter. Using a targeted population strategy, we sorted single cells from each time point into a defined bulk population to ensure enough targeted cells per time point. We then pooled all of the defined bulk population into a single container before single-cell RNA-sequencing was performed using a chromium single-cell barcoding system instructions to recover 5000 to 8000 cells/ul for each time point.