The life cycles of many parasites involve transitions between disparate host species, requiring these parasites to go through multiple developmental stages adapted to each of these specialized niches. Transmission of malaria parasites (Plasmodium spp.) from humans to the mosquito vector requires differentiation from asexual stages replicating within red blood cells into non-dividing male and female gametocytes. Although gametocytes were first described in 1880, our understanding of the molecular mechanisms involved in commitment to gametocyte formation is extremely limited and disrupting this critical developmental transition remains a long-standing goal1. We show here that expression levels of the DNA-binding protein PfAP2-G correlate strongly with levels of gametocyte formation. Using independent forward and reverse genetics approaches, we demonstrate that PfAP2-G function is essential for parasite sexual differentiation. By combining genome-wide PfAP2-G cognate motif occurrence with global transcriptional changes resulting from PfAP2-G ablation, we identify early gametocyte genes as likely targets of PfAP2-G and show that their regulation by PfAP2-G is critical for their wild-type level expression. In the asexual blood-stage parasites pfap2-g appears to be among a set of epigenetically silenced loci2,3 prone to spontaneous activation4. Stochastic activation presents a simple mechanism for a low baseline of gametocyte production. Overall, these findings identify PfAP2-G as a master regulator of sexual-stage development in malaria parasites and mark the first identification of a transcriptional switch controlling a differentiation decision in protozoan parasites.

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.

Human malaria is a devastating disease and a major cause of poverty in resource-limited countries. To develop and adapt within hosts Plasmodium falciparum undergoes drastic switches in gene expression. To identify regulatory regions in the parasite genome, we performed genome-wide profiling of chromatin accessibility in two culture-adapted isogenic subclones at four developmental stages during the intraerythrocytic cycle by using the Assay for Transposase-Accessible Chromatin by sequencing (ATAC-seq). Tn5 transposase hypersensitivity sites (THSSs) localize preferentially at transcriptional start sites (TSSs). Chromatin accessibility by ATAC-seq is predictive of active transcription and of the levels of histone marks H3K9ac and H3K4me3. Our assay allows the identification of novel regulatory regions including TSS and enhancer-like elements. We show that the dynamics in the accessible chromatin profile matches temporal transcription during development. Motif analysis of stage-specific ATAC-seq sites predicts the in vivo binding sites and function of multiple ApiAP2 transcription factors. At last, the alternative expression states of some clonally variant genes (CVGs), including eba, phist, var and clag genes, associate with a differential ATAC-seq signal at their promoters. Altogether, this study identifies genome-wide regulatory regions likely to play an essential function in the developmental transitions and in CVG expression in P. falciparum.