Hierarchical transcriptional control regulates Plasmodium falciparum sexual differentiation

Abstract

Background: Malaria pathogenesis relies on sexual gametocyte forms of the malaria parasite to be transmitted between the infected human and the mosquito host but the molecular mechanisms controlling gametocytogenesis remains poorly understood. Here we provide a high-resolution transcriptome of Plasmodium falciparum as it commits to and develops through gametocytogenesis.

Results: The gametocyte-associated transcriptome is significantly different from that of the asexual parasites, with dynamic gene expression shifts characterizing early, intermediate and late-stage gametocyte development and results in differential timing for sex-specific transcripts. The transcriptional dynamics suggest strict transcriptional control during gametocytogenesis in P. falciparum, which we propose is mediated by putative regulators including epigenetic mechanisms (driving active repression of proliferation-associated processes) and a cascade-like expression of ApiAP2 transcription factors.

Conclusions: The gametocyte transcriptome serves as the blueprint for sexual differentiation and will be a rich resource for future functional studies on this critical stage of Plasmodium development, as the intraerythrocytic transcriptome has been for our understanding of the asexual cycle.

Keywords: Differentiation; Gametocyte; Gametocytogenesis; Gene expression regulation; Malaria; Plasmodium; Sexual development; Transcriptome.

Fig. 1

The developmental and associated transcriptomic profile of P. falciparum NF54 gametocytes from commitment to maturity. a Sampling and culturing strategy and stage distribution of parasites on each day of the time course. Colored lines indicate the presence of specific stages at different time points. Abbreviations indicate parasitemia (P) and hematocrit (HC) at induction, * indicate the addition of N-acetyl glucosamine (NAG) or 5% D-sorbitol. Parasite drawings were modified from freely available images (https://smart.servier.com/), under a Creative Commons Attribution 3.0 Unported Licence. b Morphological development was monitored from induction (day − 2) over 16 days of development using Giemsa-stained thin-smear microscopy. The stage distribution for each day was calculated by counting ≥100 parasites on each day of monitoring. Legend: I-V indicates different stages of gametocyte development, R = ring and T = trophozoite stage asexual parasites. c Pearson correlation coefficients of the total transcriptomes obtained for each day of development. Red boxes indicate localized phases of increased correlation. d Expression of “gold standard” asexual and gametocyte genes [43] are shown for the gametocyte time course in heatmaps. a-d Area plot designates the timing of appearance and abundance of specific stages throughout the time course

Fig. 2

Distinct clusters of expression link to biological development of the P. falciparum gametocyte. Clusters of genes expressed during gametocyte development following K10 clustering of the total transcriptome. a The 10 clusters were grouped into phases of decreased, maintained, increased or developmentally regulated transcript abundance with number of transcripts per cluster indicated in brackets and genes of interest from specific clusters highlighted next to heatmaps. Area plot designates the timing of appearance and abundance of specific stages throughout the time course. b Biological processes of interest were selected from GO enrichment (Additional File 1) of each of the clusters (P < 0.05) with the number of genes related to these functions shown for the groups of clusters in bar graphs with generic descriptions of the gene sets used to describe their function

Fig. 3

Commitment and development are distinctly regulated processes. a The genes increased in expression during commitment [18, 20, 25] were compared to transcripts increased in abundance during gametocytogenesis (Clusters 6–10, 2425 transcripts) with overlapping genes of interest: ap2-g (pf3d7_1222600), sap18 (pf3d7_0711400), sir2a (pf3d7_1328800), lsd2 (pf3d7_ 0801900), lsd1 (pf3d7_ 1,211,600), set3 (pf3d7_ 0827800) and genes only increased during commitment hp1 (pf3d7_1220900), hda2 (pf3d7_1008000), gdv1 (pf3d7_0935400), iswi (pf3d7_0624600), sn2fl (pf3d7_1104200) highlighted in heatmaps. b The increased and developmentally regulated gene clusters also contained significantly enriched regulatory 5′ and 3′ UTR motifs identified using the FIRE algorithm [54]

Fig. 4

Stage-specific increases in gene expression contribute to the extended differentiation of P. falciparum gametocytes. a During stage I-III of development genes in cluster 8 sharply increased in expression (indicated with dotted line) with the abundance of these transcripts indicated by ribbon plot with mean ± SD. GO enrichment of genes involved in regulation of transcription (GO:0010468, 11 transcripts, P = 0.029) is present in this cluster, with presence of protein for these genes in stage I/II and V indicated in black [, –41] and the corresponding Interpro domains (https://www.ebi.ac.uk/interpro/) of proteins with unknown function indicated on the right. b The timing of sexually dimorphic transcript profiles [35] are shown in line graphs while the association of male-and female-enriched transcripts with specific clusters [–10] are shown as standardized residuals and significance of these associations indicated (P < 0.05*,0.001**,0.0001***, Fisher’s exact test). Genes of interest for each sex are highlighted in heatmaps next to male and female symbols. c The genes expressed during maturation (cluster 10) showed a significant association (P < 0.0001, two-tailed Fisher’s exact test) with genes stabilized post-transcriptionally during commitment [18] and H3K36me3-associated genes in asexual development [16, 61] before a sharp increase at stage IV-V of development (dashed line). Blocks indicate the timing of stabilization of the transcripts [18] or abundance of the H3K36me3 mark [57] and the overlap between the 3 datasets are indicated in the Venn diagram. Genes of interest within the three functional datasets are highlighted in heatmap. a-c Area plot designates the timing of appearance and abundance of specific stages throughout the time course

Fig. 5

ApiAP2 transcription factors act as regulatory elements during gametocytogenesis. a ApiAP2 transcription factors increased in transcript abundance during gametocytogenesis were evaluated for their expression throughout gametocyte development with blocks indicating periods of increased abundance. Area plot designates the timing of appearance and abundance of specific stages throughout the time course. b The transcription factors were also probed for regulatory activity using coexpression analysis by GRENITS. Transcription factors with known binding sites [13], were probed against genes containing the transcription factor binding sites indicated or the total transcriptome if their binding site was unknown. The targets of each transcription factor are shown by shaded ribbons, with correlated transcripts indicated in red and anticorrelated transcripts indicated in blue. Newly identified motifs were associated with genes coexpressed with pf3d7_0611200 using the FIRE algorithm [54] and are indicated on the graph. Generic functional terms describing enriched gene ontology terms or individual gene products are indicated in red (increased transcripts) or blue (decreased transcripts)

Fig. 6

Molecular model of regulatory modules that shape cellular differentiation during gametocytogenesis. Specific regulatory events are mapped out over the extended gametocyte development of P. falciparum parasites. Molecular regulators are highlighted in red while specific events or epigenetic marks are shown in black. Colored blocks indicate the span of specific phases of transcript abundance, with dotted lines indicating transition points in gametocyte development and grey triangles indicate the timing of repressive mechanisms in gametocyte development. Parasite drawings were modified from freely available images (https://smart.servier.com/), under a Creative Commons Attribution 3.0 Unported Licence