Revisiting the initial steps of sexual development in the malaria parasite Plasmodium falciparum

Abstract

Human to vector transmission of malaria requires that some blood stage parasites abandon asexual growth and convert into non-replicating sexual forms called gametocytes. The initial steps of gametocytogenesis remain largely uncharacterized. Here we studied this part of the malaria life cycle in Plasmodium falciparum using PfAP2-G, the master regulator of sexual conversion, as a marker of commitment. We demonstrate the existence of PfAP2-G-positive sexually-committed parasite stages preceding the previously known committed schizont stage. We also found that sexual conversion can occur by two different routes: the previously described route where PfAP2-G-expressing parasites complete a replicative cycle as committed forms before converting into gametocytes upon reinvasion, or a direct route with conversion within the same cycle as initial PfAP2-G expression. The latter route is linked to early PfAP2-G expression in ring stages. Re-analysis of published single-cell RNA-seq data confirmed the presence of both routes. Consistent with these results, using plaque assays we observed that, in contrast to the prevailing model, many schizonts produced mixed plaques containing both asexual parasites and gametocytes. Altogether, our results reveal unexpected features of the initial steps of sexual development and extend the current view of this part of the malaria life cycle.

Identification of a master regulator of differentiation inToxoplasma

“…ScRNA-Seq enables profiling of thousands of cells across asynchronous processes, and has been successfully used to examine commitment to sexual differentiation in Plasmodium spp. (Bancells et al, 2019;Poran et al, 2017). Implementing these approaches in Toxoplasma retained information about cell-cycle residency and the relative timing of gene expression that is lost in bulk analyses.…”

Regulation of sexual differentiation is linked to invasion in malaria parasites

“…Commitment to sexual development generally occurs prior to schizogony, such that every merozoite within a single schizont will either enter sexual development or continue asexual multiplication following egress and re-invasion -this is known as next cycle conversion and seems to be the canonical pathway 2 . However, it is also possible for commitment to occur in the very early ring stage resulting in parasites developing directly into gametocytes without first passing through schizogony (same cycle conversion) 3 . During the 10-12 day process of gametocytogenesis, the parasite undergoes dramatic morphological changes as it passes through five distinct stages (stages I-V) 4,5 .…”

scAlign: a tool for alignment, integration and rare cell identification from scRNA-seq data

“…Over all 661 highly variable genes we analyzed, we found differentially expressed genes are enriched in genes previously established to play a role in cycle 2-specific gametocyte maturation ( Fig. 9b) ( = 1.2 × 10 −6 , Wilcox rank sum test), including Pfg27 (PF3D7_1302100) and etramp4 (PF3D7_0423700) 46 . Furthermore, for the genes we predict to be upregulated in the +Shld condition, we observed an enrichment of ap2-g targets identified via ChIP-Seq 48 ( = 6.8 × 10 −7 , Wilcox rank sum test) , consistent with the fact that the primary difference between the +/-Shld conditions is the ability to express ap2-g. Our state variation map identifies an additional eight genes not reported by Barcells and colleagues as playing a role in cycle 2-specific gametocyte maturation, but that are predicted to differ between +/-Shld (Fig.…”

“…Briefly, the life cycle of P. falciparum begins with a single naïve parasite and culminates in the commitment of an entire set of parasites towards a single fate, either asexual or sexual commitment. Sexual commitment leads to differentiation into gametocytes either in the same cycle (cycle 1) or during the next cycle (cycle 2) 46 . While the gene ap2-g is a known master regulator of sexual commitment, and its expression is necessary for sexual commitment, the events which follow ap2-g activation and lead to full sexual commitment are unknown 47 .…”

“…(d) For a single cell sequenced under any condition, we can calculate its interpolated expression profile in all conditions, then measure the predicted variance across all input conditions to calculate a state variation map for the same cell state under different conditions to identify cells whose expression profiles vary significantly across condition. 48 which genes have been reported as playing a role in cell cycle 2 gametocyte maturation 46 and which gene represents ap2-g. (c) The same state variation map of (c), but zoomed in on Cluster 13 and the genes predicted to be most differentially expressed between +/-Shld. (d) Average per-cluster expression levels of PF3D7_0220000 reported in (c), for both the +/-Shld conditions.…”