An assay to probe Plasmodium falciparum growth, transmission stage formation and early gametocyte development

Abstract

Conversion from asexual proliferation to sexual differentiation initiates the production of the gametocyte, which is the malaria parasite stage required for human-to-mosquito transmission. This protocol describes an assay designed to probe the effect of drugs or other perturbations on asexual replication, sexual conversion and early gametocyte development in the major human malaria parasite Plasmodium falciparum. Synchronized asexually replicating parasites are induced for gametocyte production by the addition of conditioned medium, and they are then exposed to the treatment of interest during sexual commitment or at any subsequent stage of early gametocyte development. Flow cytometry is used to measure asexual proliferation and gametocyte production via DNA dye staining and the gametocyte-specific expression of a fluorescent protein, respectively. This screening approach may be used to identify and evaluate potential transmission-blocking compounds and to further investigate the mechanism of sexual conversion in malaria parasites. The full protocol can be completed in 11 d.

Probing Plasmodium falciparum sexual commitment at the single-cell level

“…The increase of cells with detectable ap2-g transcripts upon LysoPC depletion ( Figure 2b) with the proportion of sexual progeny in the same experiment, confirming that ap2-g activation in a cell is predictive of its differentiation state. Considering the mixed commitment of parasites to either the sexual or the asexual pathway under inducing conditions in vitro 17 , the transcriptional profile from our population-level RNA-seq experiment likely represented a combination of different transcriptional signatures. Specifically, we hypothesized that the changes induced by LysoPC depletion represent both a general response in all cells as well as a sexual commitment-specific activation of ap2-g and other factors required to initiate sexual differentiation in a subset of cells.…”

“…( a ) Analysis of progeny from 96 single cells in the Pf2004/164tdTom reporter line (expressing tdTomato reporter under the gametocyte-specific PF10_0164 promoter 17 ). Each parasite produces only asexual or gametocyte progeny (left panel).…”

“…P. falciparum cell culture was performed as described previously 17, 41 . Culture media consisted of RPMI-1640 supplemented with 25 mM HEPES, 100 μM hypoxanthine, 24 mM sodium bicarbonate, and gentamicin (all from Sigma-Aldrich).…”

“…All cell cultures were maintained in a 5% CO 2 , 1% O 2 and 94 % N 2 gas mixture (Med-Tech Gases). All experiments used a clone of the Pf2004/164TdTomato P. falciparum line 17 , which requires addition of 4 nM WR99210 (Jacobus Pharmaceuticals) to select for stable maintenance of episomes.…”

“…We have shown that LysoPC is the major building block for phosphatidylcholine biosynthesis in the parasite, and its depletion results in the induction of a compensatory metabolic response and triggers AP2-G activation and sexual commitment in a large part of the parasite population. This observation was facilitated through development of a highly controlled and quantitative in vitro assay to induce gametocyte formation 16, 17 . Here we apply this assay to define the transcriptional signature of individual cells at different stages during sexual commitment and validate key findings experimentally.…”