Probing Plasmodium falciparum sexual commitment at the single-cell level

Abstract

Background: Malaria parasites go through major transitions during their complex life cycle, yet the underlying differentiation pathways remain obscure. Here we apply single cell transcriptomics to unravel the program inducing sexual differentiation in Plasmodium falciparum. Parasites have to make this essential life-cycle decision in preparation for human-to-mosquito transmission. Methods: By combining transcriptional profiling with quantitative imaging and genetics, we defined a transcriptional signature in sexually committed cells. Results: We found this transcriptional signature to be distinct from general changes in parasite metabolism that can be observed in response to commitment-inducing conditions. Conclusions: This proof-of-concept study provides a template to capture transcriptional diversity in parasite populations containing complex mixtures of different life-cycle stages and developmental programs, with important implications for our understanding of parasite biology and the ongoing malaria elimination campaign.

Application of single-cell transcriptomics to kinetoplastid research

“…The former methods encapsulate each cell with an adaptor-coated bead using microfluidics, whereas the latter sort cells into individual wells of plates along with the unique barcoded adaptors. Alternative plate-based assays include SCRB-seq (Soumillon et al, 2014) and the NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® (New England Biolabs Inc. n.d.), both of which have been used to study P. falciparum (Brancucci et al, 2018;Real et al, 2020). Detailed summaries of the available methods can be found in reviews elsewhere (Haque et al, 2017;Hwang et al, 2018).…”

“…Parasitology 3 ∼$30 per cell (Brancucci et al, 2018). In the case of high-throughput droplet-based methods, the cost can be reduced by mixing species with distinct transcriptomes that can be easily separated bioinformatically (Tung et al, 2017;Kang et al, 2018), as has been done with P. berghei and P. knowlesi (Reid et al, 2018;Howick et al, 2019), and T. brucei and L. mexicana (Briggs et al, 2020;Warren et al, unpublished).…”

“…In the protozoan parasite studies discussed here, SMART-seq2 frequently recovered the most genes per cell (Table 1). The plate-based method SCRB-seq (Soumillon et al, 2014) has also been used to study protozoan parasites (P. falciparum) (Brancucci et al, 2018), and although the number of genes captured per cell was lower compared to the SMART-seq2 analysis of extracted P. falciparum (Reid et al, 2018), insight into sexual commitment was still gained. NEB also offers a single cell/low input RNA Illumina library preparation kit (New England Biolabs Inc.), which Real et al, used to perform scRNA-seq of P. falciparum and documented the parasite's journey through the mosquito in detail (Real et al, 2020).…”

“…The cost of scRNA-seq experiments is largely determined by the high price of library preparation consumables, reviewed here (Ziegenhain et al, 2017)). Notably, SCRB-seq (Soumillon et al ., 2014) employed by Brancucci et al was highly cost affective, at ~$30 per cell (Brancucci et al ., 2018). In the case of high-throughput droplet-based methods, the cost can be reduced by mixing species with distinct transcriptomes that can be easily separated bioinformatically (Tung et al ., 2017; Kang et al ., 2018), as has been done with P. berghei and P. knowlesi (Reid et al ., 2018; Howick et al ., 2019), and T. brucei and L. mexicana (Briggs et al ., 2020; Warren et al ., unpublished).…”

Single-cell analyses of polyclonal Plasmodium vivax infections and their consequences on parasite transmission

“…The advent of single cell genomics and the ability to generate gene expression profiles from individual parasite by single cell RNA sequencing (scRNA-seq) provides a unique opportunity to disentangle this complexity. scRNA-seq has been successfully applied to different Plasmodium species, including P. vivax , to extensively characterize variations in gene expression throughout the life cycle of the parasites 26 , 37 – 45 . However, since scRNA-seq relies on sequencing the mRNA molecules, the data generated can also be leveraged to analyze DNA polymorphisms in these regions and genotype individual parasites 46 .…”