Single-cell transcriptomics reveals transcriptional programs underlying male and female cell fate during Plasmodium falciparum gametocytogenesis

Single-cell transcriptomics reveals transcriptional programs underlying male and female cell fate during Plasmodium falciparum gametocytogenesis

The Apicomplexa constitute a large phylum of parasitic protozoa 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 transcriptional 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 male and female lineage development. Our data indicate that a large panel of genes linked to the inner membrane complex, known to be involved in morphological life cycle changes, appears to be uniquely expressed in the female gametocyte lineage. Additionally, we delineate the timing of expression of members of the ApiAP2 family of transcription factors and predict their specificity in male and female P. falciparum gametocyte development. A motif-driven gene regulatory network analysis indicates a major role for AP2-G5 in downstream gene regulation along the male lineage developmental trajectory. In total, we anticipate that this study provides the malaria community with an important resource for the development of transmission-blocking intervention strategies.

data_resource directory contains the P. falciparum cis-target motif database used in this study to generate motif-driven gene regulatory networks in scRNA-seq data.

• cis-target database in .feather formats prepared using create cis-target motif database Cis-target-motif using position weight matrices generated by Campbell et al 2010
• Additional sub-folders contain predicted isoforms from smart-seq2 and 10Xchromium single-cell data using StringTie
• motif collections in the cluster buster format folder contain converted position weight matrices (PWM) motifs ready to reconstruct motif database for P.falciparum
• Annotation file with transcription factor linked to motif name
• motifs from Campbell et al 2010 position weight matrices in jasper and .meme formats 
• P.falciparum reference gtf file ready to upload together with StringTie predicted isoforms in integrative genomics viewer IGV 
• cells metadata contains annotation and clusters for single-cell data 
• Raw expression matrix file used for the analyses of single-cell data
• Scanpy object in h5ad format output of single-cell data analyses collectively
• Single-cell data can be interactively visualized via Mohammed et al
• Scope viewer Mohammed et al