DNA-binding protein PfAP2-P regulates parasite pathogenesis during malaria parasite blood stages

Abstract

Malaria-associated pathogenesis such as parasite invasion, egress, host cell remodelling and antigenic variation requires concerted action by many proteins, but the molecular regulation is poorly understood. Here we have characterized an essential Plasmodium-specific Apicomplexan AP2 transcription factor in Plasmodium falciparum (PfAP2-P; pathogenesis) during the blood-stage development with two peaks of expression. An inducible knockout of gene function showed that PfAP2-P is essential for trophozoite development, and critical for var gene regulation, merozoite development and parasite egress. Chromatin immunoprecipitation sequencing data collected at timepoints matching the two peaks of pfap2-p expression demonstrate PfAP2-P binding to promoters of genes controlling trophozoite development, host cell remodelling, antigenic variation and pathogenicity. Single-cell RNA sequencing and fluorescence-activated cell sorting revealed de-repression of most var genes in Δpfap2-p parasites. Δpfap2-p parasites also overexpress early gametocyte marker genes, indicating a regulatory role in sexual stage conversion. We conclude that PfAP2-P is an essential upstream transcriptional regulator at two distinct stages of the intra-erythrocytic development cycle.

Fig. 1. PfAP2-P is essential for parasite growth and development.

a, Schematic of strategy showing conditional truncation by excision of the loxP-flanked second exon. b, Confirmation of exon 2 excision in RAPA-treated parasites. c, RNA-seq reads coverage from 40 h.p.i. parasites of pfap2-p locus. d, Western blot of control (RAPA−) and rapamycin (RAPA+)-treated PfAP2-P-3HA:loxP schizont extracts probed with anti-HA and anti-histone H3 antibodies. e, Images of Giemsa-stained (control, RAPA− and treated, RAPA+; treatment at 16 h.p.i.) schizonts at end of cycle 0 (representative of four independent experiments). Scale bar, 2 μm. f, Nuclei in control (RAPA−) and treated (RAPA+) schizonts (n = 50). P value of 0.178, one-way ANOVA using Šidák’s multiple comparisons test, assumed Gaussian distribution; error bar is mean with ± standard deviation (s.d.). n.s., not significant. g, Replication of control (RAPA−) and treated (RAPA+) parasites over two growth cycles (three biological replicates, average parasitaemia ± s.d.). h, Erythrocyte invasion by control (RAPA−) and treated (RAPA+) PfAP2-P-3HA:loxP parasites under static and shaking conditions. Statistical significance: two-tailed t-test; RAPA− versus RAPA+ parasites in static conditions (n = 3, t = 14.17, degrees of freedom 4, P = 0.000013, 95% confidence interval 1.606–2.389) and RAPA– versus RAPA+ parasites in shaking conditions (n = 3, t = 12.41, degrees of freedom 4, P = 0.000021, 95% confidence interval 1.578–2.487). Error bars are means ± s.d. i,j, Immunofluorescence microscopy of control (RAPA−) and treated (RAPA+) parasites showing distribution of GAP45 (i) and MSP7 (j). Scale bar, 2 µm. DIC, differential interference contrast. k,l, Electron micrographs of iRBCs treated with compound 2 (k) or rapamycin (RAPA+) (l); R, rhoptries; FD, food vacuole; A, apical polar ring; N, nucleus; P, pellicle; scale bar, 1 µm or 100 nm (inserts). m, The different developmental stages of compound 2-treated and RAPA-treated parasites (at 49 h.p.i.). n, Confirmation of exon 2 excision in RAPA-treated parasites at 16 h.p.i. o, RNA-seq reads coverage from 16 h.p.i. parasites (cycle 1) of pfap2-p locus. p, Images of Giemsa-stained parasites in cycle 1 following parasite treatment with DMSO (RAPA−) or rapamycin (RAPA+) at 35 h.p.i. in cycle 0. Scale bar, 2 μm. Results in b–d are representative of three independent experiments and results in i–l and n–p are representative of two independent experiments. DAPI, 4′,6-diamidino-2-phenylindole. Source data

Fig. 2. PfAP2-P regulates most of the malaria pathogenesis-associated genes.

a,b, Volcano plots showing differentially expressed var genes in 16 h.p.i (cycle 1) (a) and 40 h.p.i. (cycle 0) (b) ∆pfap2-p parasites. c,d, Differential expression of var genes (n = 3 biological replicates) measured by qRT–PCR at 16 h.p.i. (c) and 40 h.p.i. (d); error bar is standard error of the mean. e, Lollipop plot of expression level of top 20 significantly down-regulated genes in ∆pfap2-p compared with control parasites at 40 h.p.i. f, Heatmaps of down-regulated known egress and invasion genes in treated (RAPA+) or control (RAPA−) parasites, grouped on the basis of the subcellular location of their products. g, Western blots of schizont extract from parental II3 and PfAP2-P-3HA parasites in the absence (−) or presence (+) of rapamycin, probed with antibodies specific for invasion proteins. BiP was detected as a loading control. Molecular mass (kDa) of standards on left side of each panel. A non-specific cross-reacting protein on the SUB1 blot is marked with an asterisk. Representative of two independent experiments. h, Left: uniform manifold approximation and projection (UMAP) of scRNA-seq data from MCA, with annotated developmental stages. Right: UMAP projections of scRNA-seq in-house data; each dot represents gene expression data from a single parasite (colours corresponding to h.p.i. and pfap2-p truncation status) plotted over MCA data. i, Distribution of developmental stages of treated (RAPA+) or control (RAPA−) parasites at 16 and 40 h.p.i. j, Violin plots of average var gene expression in treated (RAPA+) or control (RAPA−) parasites at both 16 h.p.i. and 40 h.p.i. Violin plot shows the median, Q1–Q3 range (box) and distribution of values (violin). n = 3,992; 2,747; 3,629 and 3,591 for 16 and 40 h.p.i. control and RAPA-treated cells, respectively. P values were calculated using unpaired two-sample Mann–Whitney Wilcoxon test (two-sided) with continuity correction used. k, Proportion of cells expressing one or more var genes from treated (RAPA+) or control (RAPA−) cultures (P = 6.23 × 10⁻¹³, two-sided Fisher’s exact test, odds ratio 1.53). l, Percentage of iRBCs containing control or ∆pfap2-p parasites bound by IgG from serum of malaria-infected (HS+) patients or untreated samples (HS−). Significance was determined using a two-tailed t-test (t = 6.687, degrees of freedom 4, P < 0.0001, 95% confidence interval 0.9844–2.382; n = 3); error bar is ± standard deviation. Source data

Fig. 3. PfAP2-P regulates pathogenesis-associated genes via promoter binding.

a, Genome-wide occupancy of PfAP2-P at 16 h.p.i. and 40 h.p.i., determined by ChIP–seq. Subtelomeric and internal regions of chromosome 7 (~1,450 kb) containing var genes with PfAP2-P bound to their promoter regions are shown as an example. Chromosomal positions are indicated. Results are representative of two independent replicates. b, Pie charts showing the proportion of each family of genes with PfAP2-P bound to the promoter region (in blue) at 16 h.p.i. c, PfAP2-P occupancy at 16 h.p.i. in putative promoter regions of genes implicated in iRBC remodelling and parasite development. Two biological replicate ChIP versus input tracks are shown (input-subtracted PfAP2-P-ChIP). Positions on chromosome (chr) 09, 10, 11 and 13 are indicated. x axis shows the genomic position, and numbers on the right show the enrichment score (Methods). d, Expression levels of 27 P. falciparum AP2 genes during different IDC stages (AP2-P gene ID highlighted in red) and in merozoites are depicted by the diameter of the circles. On the right, blue, brown and green circles indicate the binding of PfAP2-P to the promoter at either or both 16 h.p.i. and 40 h.p.i.; the heatmap displays the expression status of all 26 api-ap2s in ∆pfap2-p parasites compared with controls at 16 and 40 h.p.i. The heatmap for pfap2-p is black because ∆pfap2-p parasites only express RNA from the first exon and have no functional AP2-P protein. TPM, transcripts per million.

Fig. 4. Depletion of PfAP2-P increases chromatin accessibility.

a, The most significantly enriched motif bound by PfAP2-P at 16 h.p.i. b,c, The top most (b) and the second most (c) significantly enriched motifs bound by PfAP2-P at 40 h.p.i. d, The most enriched motif (c) at 40 h.p.i. is similar to the PfAP2-I binding motif. e, The numbers of genes with promoters bound by PfAP2-P at either 16 h.p.i., 40 h.p.i. or both. f,g, Genomic regions uniquely bound by PfAP2-P at 16 h.p.i. (f) or at both 16 and 40 h.p.i. (g). x axis shows the genomic position and numbers on the right show the enrichment score. h,i, Label-free quantitative proteomic analysis of P. falciparum proteins enriched in PfAP2-P immunoprecipitates at 16 h.p.i. (h) and 40 h.p.i. (i). j, Chromatin contact count heatmap of chromosome 7 at 16 h.p.i. (first three) and 40 h.p.i. (last three) for the control and Δpfap2-p parasites, as well as the ${\log }_2$ fold change in interactions (third and sixth panels from left) between control and Δpfap2-p parasites. Blue indicates a loss of interactions and red indicates an increase of interactions of Δpfap2-p over control (WT). Chr, chromosome.

Fig. 5. PfAP2-P master regulator of malaria pathogenesis.

Deletion of pfap2-p before the first peak of expression at 16 h.p.i. blocks parasite development beyond late trophozoite/early schizont stages. Deletion of pfap2-p well before 40 h.p.i., but after the first peak of expression at 16 h.p.i., affects merozoite development and blocks parasite egress from iRBCs. At this late stage of intra-erythrocytic development, the second peak of pfap2-p expression activates many genes associated with invasion, egress, antigenic variation, host cell remodelling and protein phosphorylation, either directly by binding to their promoter or indirectly through other downstream ApiAP2 transcription factors and regulators. PfAP2-P is a direct repressor of var genes and an indirect repressor of many gametocytogenesis-associated marker genes. Deletion of pfap2-p derepresses expression of most of var genes leading to the display of the corresponding PfEMP1 on the iRBC surface. PfAP2-P acts as a direct activator of many other genes, such as rifins, stevors and pfmc-2tms, coding for antigenically variant proteins, and it binds to the promoter of 14 other ApiAP2s (50% of all P. falciparum ApiAP2 genes), suggesting that it is an upstream regulator of gene expression cascades during the IDC. PfAP2-P associates with many known and putative histone modifiers and chromatin remodellers that probably participate in PfAP2-P associated gene regulation. Altogether, PfAP2-P regulates the expression of most known pathogenic factors (associated with antigenic variation and parasite growth) in P. falciparum, suggesting it is a master regulator of malaria pathogenesis.

Extended Data Fig. 1. Features of PfAP2-P DNA binding protein.

a, Expression of pfap2-p (thick black line) and the top 40 down-regulated genes (grey lines) in P. falciparum line II3 over a 48-hour IDC. b, The primary structure of PfAP2-P, with a nuclear localization signal (NLS) and a single AP2 DNA binding domain. Both NLS and DNA binding domain are encoded by exon 2 of the gene. c, A phylogenetic distribution of AP2 genes in the genus Plasmodium and related alveolates. d, IFA shows that PfAP2-P (HA tagged) localizes to the parasite nucleus at various IDC developmental stages (representative of two independent experiments). Scale bar 2μM. e, RNA-seq data from different IDC time-points mapped to the pfap2-p locus; there is a drastic reduction in RNA-seq reads mapping to the second exon 16 hours after the addition of rapamycin that is at 20 h.p.i. f, Schematic showing rapamycin treatment schedule to disrupt either first or second peak of pfap2-p expression. Panel f created with Biorender.com.

Extended Data Fig. 2. Truncation of pfap2-p affects merozoite development.

a, Uncropped images of figure presented as Fig. 1i. b, Uncropped images of figure presented as Fig. 1j. Scale bar 2μM.

Extended Data Fig. 3. Truncation of pfap2-p affects parasite’s transcriptome.

a, b, Volcano plots showing significantly differentially expressed genes in rapamycin-treated compared to control parasites at 16 h.p.i. (a) and 40 h.p.i. (b). c, non-differentially expressed genes and up-regulated genes (n = 658) in treated [RAPA (+)] compared to control [RAPA (-)] parasites at 40 h.p.i., which had been reported to express at least 4-fold higher in schizont-stage parasites (>35 h.p.i.) compared to early-stage parasites (<35 h.p.i.); error bar is mean with ± s.d. d, RNA-seq reads coverage from 8 and 40 h.p.i. parasites of pf-ap2-p locus. e-f Volcano plots showing significantly differentially expressed genes in rapamycin-treated compared to control parasites at 8 h.p.i. (e) and 30 h.p.i. (f).

Extended Data Fig. 4. Truncation of pfap2-p deregulates malaria pathogenesis-associated genes.

a, Expression at 16 h.p.i. of members of the gene families: rifin, stevor and Pfmc-2tm that encode antigenically variant proteins. b, Gene-ontology (GO) enrichment analysis of all genes down-regulated in rapamycin-treated compared with control parasites at 40 h.p.i. Shown is the number of genes down-regulated in treated parasites out of the total number of genes assigned to that specific GO term. c, Heatmap of the expression of all known and putative P. falciparum kinases downregulated following rapamycin treatment compared to controls, at 40 h.p.i. d, Violin plots of all surfin, rifin, stevor and pfmc-2tm expression per cell in treated [RAPA (+)] and control [RAPA (−)] parasites at 16 h.p.i. Violin plot shows the median, Q1–Q3 (box), distribution of values (violin). n = 3,992, 2,747, 3,629 and 3,591 for 16 and 40 h.p.i. control and RAPA-treated cells respectively. The P-values were calculated using unpaired two-samples Mann Whitney Wilcoxon Test (two-sided) with continuity correction was used. e, FACS gating strategy for surface PfEMP1 expression detected by IgG binding from pooled serum of malaria-infected individuals. Cells were incubated either without (HS-) or with (HS+) pooled serum from malaria-infected individuals. The percentage of Human IgG+ iRBCs is the mean of three biological replicates. A total of 7,500 Sybr green positive events (iRBCs) per sample were analyzed.

Extended Data Fig. 5. PfAP2-P is a repressor of early gametocyte marker genes.

a, Gene ontology enrichment analysis of up-regulated genes in ∆pfap2-p parasites at 40 h.p.i. b, Differential expression in ∆pfap2-p parasites at 40 h.p.i. and 16 h.p.i. of genes that are known or putative early gametocyte markers. c, Differential expression of selected genes in RAPA-treated and control parasites (n = 3 biological replicates) measured using qRT-PCR, to validate RNA-seq data; error bar is mean with ± standard error of the mean.

Extended Data Fig. 6. PfAP2-P binds to the putative promoter regions of var genes.

a, Enrichment of PfAP2-P bound reads (from replicate 1) around the ± 5 kb region of peak summits, from 16 h.p.i. (left panel) and 40 h.p.i. (right panel) parasite samples. b, The position of ChIP-seq peak summits (common between two biological replicates) relative to the predicted ATG translational start codon, in 16 h.p.i. (red) and 40 h.p.i. (blue) parasites. c, Three independent PfAP2-P ChIP experiments followed by qPCR, were performed to validate ChIP-seq data, using selected PfAP2-P-bound promoter regions of genes from samples at 40 h.p.i. The bar-plot shows percent input (% Input) enrichment of PfAP2-P on target genes (mean ± s.d. of three independent experiments). IgG was used as the mock-treated control. P-values were calculated using a two-tailed t-test. d, Input subtracted ChIP peaks of PfAP2-P in chromosome 4 and 7 as representatives in both 16 and 40 h.p.i. stages. Also, zoomed in PfAP2-P bound central chromosomal and sub-telomeric heterochromatin regions are shown. X-axis shows the genomic position and numbers on the right show the enrichment score. e, Schematic diagram of the chromosomal position of var genes with promoters bound by PfAP2-P at either 16 h.p.i. (green), 40 h.p.i. (red) or at both stages (black).

Extended Data Fig. 7. PfAP2-P, PfAP2-I and PfAP2-G binds to many common genomic regions.

a, Occupancy of PfAP2-P in the promoter region of pfap2-I at 16 and 40 h.p.i. b, Occupancy of PfAP2-P and PfAP2-I in the promoter region of pfap2-p at 16 and 40 h.p.i. c, Schematic showing probable gene regulatory network between PfAP2-P and PfAP2-I. Orange arrows indicate the binding of protein to its gene promoter. d, Comparison of genes with promoters bound by PfAP2-P (red), PfAP2-I (green) and PfAP2-G (light blue) at 40 h.p.i. e, Input-subtracted ChIP-seq read coverage for exemplar genes with promoters that are either bound by all three AP2 proteins (left panel), uniquely by PfAP2-P, by both PfAP2-P and PfAP2-I, or by both PfAP2-P and PfAP2-G (right panel). Arrows show the direction of gene transcription. X-axis shows the genomic position and numbers on the right show the enrichment score.

Extended Data Fig. 8. PfAP2-P binds to the putative promoter region of invasion-associated genes.

Occupancy of PfAP2-P in the promoter region of different invasion-associated genes. The ChIP tracks show two replicates with input subtracted from the PfAP2-P ChIP data. Arrows indicate direction of transcription. X-axis shows the genomic position and numbers on the right show the enrichment score.

Extended Data Fig. 9. Status of known histone marks in the heterochromatic regions of pfap2-p truncated parasites.

a-b, ChIP-seq profiles of various histone marks (H3K4me3, H3K9ac and H3K9me3) in the chromosome 7 (a) and in the promoter and gene body of a var gene (b) as examples. For ChIP-seq chromatin was sampled at 16 and 40 h.p.i. corresponding to the two peaks of pfap2-p expression. We have also provided the binding profile of PfAP2-P at both 16 and 40 h.p.i. for comparison. D;DMSO treated mock control; R: Rapamycin treated pfap2-p truncated parasites. Each track is input subtracted ChIP tracks.

Extended Data Fig. 10. Depletion of PfAP2-P increases chromatin accessibility.

a, ICE-normalized contact count heatmaps at 10 kb resolution of intrachromosomal interactions for the 14 chromosomes are given for both the 16 h.p.i. and 40 h.p.i. time points. Heatmaps represent a single chromosome of the wild type (left), Δpfap2-p (center), and log2 fold change differential interactions (right). The data for the two biological replicates were merged using a weighted average based on the total read count and then counts-per-million normalized prior to generating heatmaps. The scale of the legend was also normalized to improve comparisons between WT and Δpfap2-p. For each bin i, all interactions within i +/−2 are set to 0 (see white line at diagonal) to enhance visualization of remaining bins due to intra-bin and very short-range contacts being significantly higher b, Whole-genome interchromosomal contact count heatmaps at 16 h.p.i. (top) and 40 h.p.i. (bottom) for the WT and Δpfap2-p. Chromosomes are sorted from left to right and bottom to top. Intrachromosomal interactions are removed. c, Number of interactions between var gene containing bins. d, Whole-genome 3D chromatin models at 16 h.p.i. for the WT (left) and Δpfap2-p (right). Centromeres (blue), telomeres (green), and var genes (red) are enhanced to display differences between the two samples.