A transcriptional switch underlies commitment to sexual development in malaria parasites

Abstract

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 goal. Here we show 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 probable 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 loci prone to spontaneous activation. 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 discovery of a transcriptional switch controlling a differentiation decision in protozoan parasites.

Figure 1. PfAP2-G transcript levels mirror gametocyte production

A) PfAP2-G relative transcript abundance in synchronized (early schizont stage) cultures as measured by qPCR varies significantly between 3D7-A and 3D7-B populations as well as the 3D7-B subclones E5, A7, and B11. Values are normalized against seryl tRNA synthetase (PF07_0073) (n=3, standard deviation shown). B) Percent commitment to gametocyte differentiation in these lines mirrors relative PfAP2-G transcript levels (mean of n=2).

Figure 2. Disrupting PfAP2-G function results in loss of gametocyte production

A) Positions of pfap2-g mutations in the gametocyte non-producer lines F12 and GNP-A4 and the targeted deletion of Δpfap2-g. B) Southern blot showing successful disruption of the pfap2-g locus by homologous recombination (also see Supplementary Fig. 4). Single replicate. C) pfap2-g mutants fail to produce gametocytes (n=3, standard error shown, scalebar = 5 μm). D) Ligand-regulatable gametocyte formation in PfAP2-G-ddFKBP (bottom row images) but not in the E5 parent (top row images) Representive of n=4. E) Quantification of ligand-regulatable gametocyte formation (n=4, standard error shown).

Figure 3. Identification of PfAP2-G targets

A) PfAP2-G-HAx3 localizes to the nuclei of schizonts in asexually growing parasites (see Fig S7 for additional stages, scale bar = 1μm). Representative of n=4. B) Relative abundance of transcripts with greater than 2-fold average difference in both Δpfap2-g and F12 with respect to 3D7 clone E5 across the intra-erythrocytic developmental cycle at 6h intervals. Columns on the right indicate whether genes are known gametocyte markers (blue), detected in 2+ gametocyte proteomes (orange), enriched in early gametocyte proteome (purple), and the number of PfAP2-G cognate motifs within 2kB upstream of the start codon. C) Binding of recombinant PfAP2-G AP2 domain to three gametocyte promoters occurs only in the presence of the wild-type cognate motif (+). Representative of n=3. D) Relative luciferase activity under the control of wild-type gametocyte promoters in 3D7 E5 (blue) and Δpfap2-g (red), or in 3D7 E5 under control of promoters lacking the PfAP2-G motif (green). (18-30h post-invasion, n=3, standard error is shown, two-sided t-test used, NA: Not tested.)

Figure 4. Activation of PfAP2-G

A) Only a small fraction (1-6%) of asexually growing subclone 9A schizonts (see Fig. S7 for details) express detectable levels of PfAP2-G-HAx3 (first row). H3K4me3 staining was performed in parallel to confirm full permeabilization (second row), scale bar = 10μm. Representative of n=3. B) The percentage of PfAP2-G-HAx3 positive cells is highly predictive (R² = 0.94) of subsequent gametocyte formation levels. C) Model of PfAP2-G activation and function.