Abstract
Pathogens have to balance transmission with persistence. For Plasmodium falciparum, the most widespread and virulent malaria parasite, persistence within its human host requires continuous asexual replication within red blood cells, while its mosquito-borne transmission depends on intra-erythrocytic differentiation into non-replicating sexual stages called gametocytes1. Commitment to either fate is determined during the preceding cell cycle that begins with invasion by a single, asexually committed merozoite and ends, 48 hours later, with a schizont releasing newly formed merozoites, all committed to either continued asexual replication or differentiation into gametocytes2,3. Sexual commitment requires the transcriptional activation of ap2-g (PF3D7_1222600)4,5, the master regulator of sexual development, from an epigenetically silenced state during asexual replication6,7. AP2-G expression during this ‘commitment cycle’ prepares gene expression in nascent merozoites to initiate sexual development through a hitherto unknown mechanism2,4. To maintain a persistent infection, the expression of ap2-g is limited to a sub-population of parasites (1–30%, depending on genetic background and growth conditions). As sexually committed schizonts comprise only a sub-population and are morphologically indistinguishable from their asexually committed counterparts, defining their characteristic gene expression has been difficult using traditional, bulk transcriptome profiling8. Here we use highly parallel, single-cell RNA sequencing9 of malaria cultures undergoing sexual commitment to determine the transcriptional changes induced by AP2-G within this sub-population. By analysing more than 18,000 single parasite transcriptomes from a conditional AP2-G knockdown line and NF54 wild-type parasites at multiple stages of development, we show that sexually committed, AP2-G+ mature schizonts specifically upregulate additional regulators of gene expression, including other AP2 transcription factors, histone-modifying enzymes, and regulators of nucleosome positioning. These epigenetic regulators may act to facilitate the expression and/or repression of genes that are necessary for the initiation of gametocyte development in the subsequent cell cycle.
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Acknowledgements
We would like to thank the WCM Genomics and Flow Cytometry core facilities, and G. Suppa for technical assistance. This work was supported by WCM internal startup funds (B.F.C.K.) and the NSF CAREER award (DBI-10549646, to O.E.), LLS SCOR (7006-13 and 7012016, O.E.), Hirschl Trust Award (O.E.), Starr Cancer Consortium (I6-A618, to O.E.) and NIH 1R01CA194547 (O.E.). A.P. and C.N. were supported by WCM graduate fellowships.
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B.F.C.K. conceived the study with input from O.E. B.F.C.K. performed culturing and sample preparation for a single initial Drop-seq experiment. A.P. established and optimized the Drop-seq platform in the laboratory of O.E., and carried out Drop-seq and library preparation. C.N. carried out all other parasite culturing and sample processing for Drop-seq and RNA FISH. O.A. assisted in library preparation. N.M.-T. aided in probe design, optimization of RNA FISH, and performed flow cytometry. M.L.G. aided in optimization of flow cytometry for RNA FISH, and D.C.H. suggested methods for RNA FISH and supervised N.M.-T. C.T.H. acquired RNA FISH micrographs. A.P. and B.F.C.K. developed and performed bioinformatic analyses with contributions from C.N. B.F.C.K. analysed flow cytometry data. B.F.C.K. and A.P. wrote the manuscript, designed and generated figures, with notable input by C.N. and O.E.
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Extended data figures and tables
Extended Data Figure 1 Drop-seq single-cell sequencing and analysis workflow.
a, Single, infected RBCs (iRBCs) and uniquely barcoded beads are captured in droplets of cell lysis buffer using a microfluidics device. b, Released mRNAs are captured on individually barcoded poly-dT oligonucleotides. c, Template-switch cDNA synthesis labels each captured transcript with a cell-specific barcode and a unique molecular identifier (UMI). Following library preparation and Illumina sequencing, individual transcripts are mapped and counted within each cell. d, The resulting expression matrix of single-cell transcriptomes is used for clustering and analysis using the Seurat package of scRNA-seq analysis tools.
Extended Data Figure 2 Experimental overview.
Fusion of the endogenous ap2-g coding sequence with the FKBP destabilization domain (DD) makes sexual commitment conditional on treatment with 0.5 μM Shield1 ligand. Unless ligand is added, AP2-G is targeted for proteolytic degradation. At the start of the commitment cycle, AP2-G-DD parasites were split into two cultures and treated with ligand or solvent control. Cultures were maintained under conditions inducing sexual commitment and infected RBCs were purified for Drop-seq at 30, 36 and 42 hpi during the commitment cycle, and after 42 h of gametocyte development in the subsequent cycle.
Extended Data Figure 3 Analysis of AP2-G-DD cluster composition.
a, Cluster assignment of SCTs collected at 30, 36, and 42 hpi and from stage I gametocytes. b, Cluster composition by sample time point. c, Cluster-wise enrichment for treated versus untreated cells collected during the commitment cycle. Positive and negative log-odds-ratio values indicate enrichment for treated and untreated cells, respectively. Error bars indicate the 95% confidence interval. The number of cells (n) in each sample is shown.
Extended Data Figure 4 Single-cell transcriptomes averaged by collection time match corresponding bulk RNA-seq time points and pseudo-time assignment.
a, The averaged single-cell expression profiles for each of the collected samples (n24–36 h = 1,202, n30–42 h = 1,536, n36–48 h = 6,035) were correlated with published bulk RNA-seq time points. Pearson’s correlation coefficients are shown in heatmap boxes. Maximal correlation values indicate agreement between collection time point and transcriptome mapping. b, The distributions of pseudo-time assignment of treated cluster 1–11 SCTs grouped by collection time (n30 h = 508, n36 h = 821, n42 h = 3,356). Boxes indicate the interquartile range; whiskers extend 1.5× interquartile range from the box.
Extended Data Figure 5 Analysis of NF54 late-stage SCTs clustered independently or co-clustered with treated AP2-G-DD SCTs.
a–d, SCTs clustered independently (left) or co-clustered with treated AP2-GG-DD (right). a, tSNE plot of cluster 1–11 SCTs. b, Gene set expression as a function of pseudo-time. Colour bar indicates cluster assignment along pseudo-time. c, tSNE plot showing AP2-G+ cells in red. d, Mean ap2-g expression per 10,000 transcripts by cluster. Error bars are s.e.m. The number of cells (n) in each sample is shown.
Extended Data Figure 6 Differential expression analysis in treated AP2-G-DD and NF54 cells.
a, b, The fold change of differentially expressed genes in treated AP2-G+ cells compared to AP2-G− cells in AP2-G-DD SCTs (a) or independently clustered NF54 SCTs (b). Grey denotes not detected.
Extended Data Figure 7 Homology-based functional annotation of PF3D7_0801900 as a putative histone lysine-specific demethylase.
a, Alignment of conserved blocks (C-blocks) for P. falciparum (Pf) LSD2 (PF3D7_0801900) and syntenic orthologues in P. vivax (Pv) (PVP01_0118300), P. ovale curtisi (Poc) (PocGH01_01025900), P. gallinaceum (Pg) (PGAL8A_00078100), and P. berghei (Pb) (PBANKA_1228300). Coloured residues are conserved. Key functional residues for the flavin-containing amine oxidoreductase (PF01593, yellow) and extended plant homeodomain (PHD) finger (cd15571, green) domains are indicated. b, NCBI Conserved Domain Database hits for P. vivax LSD2 conserved blocks. c, NCBI Conserved Domain Database hits for P. falciparum LSD2 conserved blocks. d, Single PANTHER database functional annotation hit for P. falciparum LSD2 conserved blocks.
Extended Data Figure 9 Validation of single cell findings.
a, Fold change in expression of treated versus untreated AP2-G-DD schizonts as determined by qRT–PCR. Four independent biological replicates; the asterisk indicates significantly higher than 1.0 (one-sided P < 0.05, two-sided Welch’s two-sample t-test). Bar height indicates the mean fold change across replicates, error bars are s.e.m. b, RNA FISH quantification gating schema (top) and fluorescence minus-one controls (bottom) for data shown in Fig. 4d. Results are representative of three independent experiments.
Extended Data Figure 10 Expression correlation coefficients for pairs of significantly co-expressed genes by cluster.
For each cluster, all genes across all the treated AP2-G-DD and NF54 cells in the cluster were evaluated for co-expression (ϕ > 0.3) with each of the 19 shared hits in Fig. 3d. Spearman’s correlation of expression is shown for highly co-expressed gene pairs. Solid symbols indicate gene pairs including AP2-G. The number of cells (n) used to evaluate co-expression is shown for each cluster.
Supplementary information
Supplementary data
Alignment of LSD2. This zipped data file contains the full alignment of P. falciparum LSD2 (PF3D7_0801900) with syntenic orthologs in P. vivax (PVP01_0118300), P. ovale curtisi (PocGH01_01025900), P. gallinacium (PGAL8A_00078100), and P. berghei (PBANKA_1228300) that was generated using the phylogeny-aware multiple sequence aligner webPRANK. (ZIP 21 kb)
Supplementary Table 1
This table provides information about the samples analyzed with scRNA-seq, including culture conditions, drug treatments, the life cycle stage, numbers of cells sequenced, read depth, read mapping, the mean transcripts, and genes detected per cell. Each row represents an independently carried out experiment. (XLSX 45 kb)
Supplementary Table 2
This table provides a list of modules of genes related to specific transcriptional programs activated during different stages of the cell cycle of P. falciparum. (XLSX 62 kb)
Supplementary Table 3
This table lists genes significantly differentially expressed between shield1-treated AP2-G positive cells and shield1-treated AP2-G negative cells, for clusters 1–11 in AP2-G-DD and clusters 1–9 in NF54. (XLSX 44 kb)
Supplementary Table 4
This table lists genes that are significantly co-expressed in each cluster, based on a Fisher’s exact test that detected significant co-expression between A) ap2-g and the 19 shared differentially expressed genes and B) all other genes of the genome. (XLSX 63 kb)
Supplementary Table 5
This table lists the sequences of the custom probe sets used for PrimeFlow RNA FISH that were designed and synthesized against unique regions of the transcripts of interest by the manufacturer. (XLSX 16 kb)
Supplementary Table 6
This table lists the sequences of the gene-specific primer sets used for determining relative transcript abundance by qRT-PCR. (XLSX 37 kb)
Three dimensional tSNE plot of AP2-G-DD cells
This video shows the three dimensional tSNE plot of AP2-G-DD cells that results from unsupervised clustering based on similarity in overall gene expression. The 10,509 quality-filtered AP2-G-DD single cell transcriptomes can be seen self-organized in a continuous arc comprised of eleven clusters (1-11) surrounding five central clusters (12-16). Cells are colored by cluster assignment. (MP4 1737 kb)
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Poran, A., Nötzel, C., Aly, O. et al. Single-cell RNA sequencing reveals a signature of sexual commitment in malaria parasites. Nature 551, 95–99 (2017). https://doi.org/10.1038/nature24280
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DOI: https://doi.org/10.1038/nature24280
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