Plasmodium falciparum 3D7-G7 strain was cultured in O type fresh human erythrocytes as described previously in complete RPMI 1640 medium (Gibco) with 0.5% Albumax I (Invitrogen) and a gas phase maintained under 5% CO₂, 5%O₂ and 90% N₂ at 37°C (Zhang et al., 2014). Parasites were regularly synchronized with repeated 5% sorbitol treatments at the ring stage. For assays on pfap2-exp2-ty1-glms parasites, cultures were tightly synchronized to a 5-h window by purification of schizont stage using Percoll-sorbitol gradients (70% Percoll and 40% Percoll) followed by 5% sorbitol treatment 5 h later (Lu et al., 2021).

The pL6cs-ap2-exp2-ty1-glms and pL6cs-ap2-exp2-ty1-GFP plasmids were constructed as described previously (Ghorbal et al., 2014; Zhao et al., 2020). First, the guide RNA was annealed by complementary oligonucleotides and cloned into the pL6cs construct between XhoI and AvrII restriction enzyme sites. Then the C-terminal fragment of pfap2-exp2 with ty1-glms or ty1-gfp was cloned into AflII and AscI restriction sites. The constructed plasmids verified by sequencing were transformed into E. coli XL10 for amplification and purification. All primers used for construction are listed in Supplementary Table S1.

Transfections were performed in uninfected red blood cells using 100 μg of purified pL6cs-ap2-exp2-ty1-glms or pL6cs-ap2-exp2-ty1-GFP plasmids together with 100 μg of pUF1-Cas9-BSD plasmids followed by the addition of purified schizont stage parasites (Zhao et al., 2020). Subsequently, parasites were cultured in the presence of 2.5 nM WR99210 and 2 μg/ml BSD (Invitrogen) until live parasites were refound in Giemsa’s solution-stained thin blood smears 3 weeks later. The sequences at the designed integration sites were examined by PCR of genomic DNAs followed by DNA sequencing to confirm genetic editing. The pfap2-exp2-ty1-glms strain was further cloned out by limiting dilution cloning (Fan et al., 2020). Primers used for verification are provided in Supplementary Table S1.

Pfap2-exp2-ty1-glms parasites were tightly synchronized to a 5-h window. Ring-stage parasites were plated at 0.5% parasitemia in a 6-well plate with 2% hematocrit in the presence or absence of 5 mM glucosamine (GlcN). Parasitemia was monitored periodically by counting parasites from Giemsa-stained thin blood smears in the next two cycles (Liu et al., 2020).

Sample preparation for western blot analysis was performed as previously described (Liu et al., 2020). Briefly, schizonts were released from erythrocytes with 0.15% saponin, resuspended with an equal volume of 2 × SDS–polyacrylamide gel electrophoresis (PAGE) protein loading buffer, and then heated for 5 min at 100°C before storing at -80°C. Proteins were separated by 10% SDS-PAGE, and then transferred to Immobilon-P transfer membranes (Millipore). Subsequent antibody incubation and membrane wash followed standard procedures. The primary antibodies used in this study included mouse anti-ty1 (Sigma, SAB4800032) at 1:1,000 and rabbit anti-aldolase (Abcam, ab207494) at 1:2,000. The horseradish peroxidase (HRP) conjugated secondary antibodies were used at 1:5,000, including goat anti-mouse IgG (Abcam, ab97040) and goat anti-rabbit IgG (Abcam, ab205718). HRP signals were detected using the ECL western blotting kit (GE healthcare). Especially, pfap2-exp2-ty1-glms parasites were tightly synchronized to a 5-h window and ring-stage parasites were diluted at 0.5% parasitemia with 2% hematocrit in the presence or absence of 5 mM glucosamine. At the second generation, 200 μL of schizont-staged samples were collected for western blotting.

The immunofluorescence assay was performed to detect the localization of PfAP2-EXP2 as described previously (Jing et al., 2018). Parasites were harvested, released, fixed by 4% paraformaldehyde (Electron Microscopy Sciences) at room temperature for 10 min, and washed by PBS. Prepared samples were incubated with the primary antibodies against ty1 (Sigma, SAB4800032) or GFP (Abcam, ab290) at 1:500 to 1:1,000, followed by the secondary antibodies AlexaFluor 488 goat anti-mouse IgG (ThermoFisher Scientific, A11029) or AlexaFluor 568 goat anti-rabbit IgG (ThermoFisher Scientific, A11036) at 1:500. Preparations were visualized with a Nikon A1R microscope at 60-100 × magnification and images were acquired with NIS Elements software and processed using Adobe Photoshop.

Pfap2-exp2-ty1-glms parasites were tightly synchronized to a 5-h window with or without 5 mM glucosamine treatment as described above. Samples were collected in TRIzol at ring (10–15 hpi, one biological replicate), trophozoite (25–30 hpi, two biological replicates) and schizont (40–45 hpi, three biological replicates) stages of the next cycle, respectively. Total RNA was extracted according to the manufacturer’s protocol (Zymo Research). Library preparation for strand-specific RNA-seq was first carried out by poly(A) selection with the KAPA mRNA Capture Beads (KAPA) and fragmentation to about 300–400 nucleotides (nt) in length. All subsequent steps were performed according to the KAPA Stranded mRNA-Seq Kit (KK8421). Libraries were sequenced on an Illumina HiSeq Xten system to generate 150 bp pair-end reads (Liu et al., 2019; Lu et al., 2021).

ChIP-seq assays were carried out in two biological replicates as previously described with minor modifications (Lopez-Rubio et al., 2013; Santos et al., 2017; Josling et al., 2020). Synchronized parasites were harvested at schizont stage and cross-linked immediately with 1% paraformaldehyde (Sigma) by rotating for 10 min at 37°C, then quenched with 0.125 M glycine for 5 min on ice. The parasites were resuspended with 50 ml of PBS and lysed with 0.15% saponin for 5 min on ice. The released nuclei were washed several times with PBS, then sonicated using an M220 sonicator (Covaris) at 5% duty factor, 200 cycles per burst, and 75 W of peak incident power to generate 100–500 bp fragments. The samples were diluted ten-fold with dilution buffer and precleared with Protein A/G magnetic beads (Thermo) for 2 hours at 4°C. The precleared chromatin supernatants, a small part of which were aliquoted as input controls, were incubated overnight at 4°C with 0.5 μg of antibodies against GFP (Abcam, ab290) and 20 μL of Protein A/G magnetic beads. The immunoprecipitates were washed with low salt wash buffer, high salt wash buffer, LiCl wash buffer, and TE buffer, then eluted with Elution Buffer. To reverse cross-link, the eluted samples were incubated overnight at 45°C and treated with RNase A at 37°C for 30 min and Proteinase K at 45°C for 2 hours. Finally, DNA was extracted using the MinElute PCR purification kit (Qiagen, 28006). In library preparation, 1.5 ng of ChIP-DNAs were end-repaired (Epicentre No. ER81050), added with protruding 3’ A base (NEB No. M0212L), and ligated with adapters (NEB No. M2200L). The Agencourt AMPure XP beads (Beckman Coulter) were then used for size selection and purification. Libraries were amplified using the KAPA HiFi PCR Kit (KAPA Biosystems, KB2500) with the following program: 1 min at 98°C; 12 cycles of 10 s at 98°C and 1 min at 65°C; finally, extension 5 min at 65°C. Library sequencing was conducted on an Illumina HiSeq Xten platform and generated 150 bp pair-end reads.

To remove residual adapters and low-quality bases, read trimming was conducted with Trimmomatic (Bolger et al., 2014) using a 4 bp window and average window quality above 15. Clipped reads with a minimum length of 50 bp and average read quality above 20 were mapped to the P. falciparum 3D7 genome build 47 using Bowtie2 (Langmead and Salzberg, 2012) and default parameters. Peaks were identified using the--call-summits option of the MACS2 callpeak function (Zhang et al., 2008) and a q-value cutoff of 0.05. Log2-transformed ChIP/input fold enrichment signals were calculated with the MACS2 bdgcmp function and visualized with Gviz (Hahne and Ivanek, 2016).

GenomicRanges (Lawrence et al., 2013) assigned peaks to nearby target genes if they overlapped 5′ UTRs (<3 kb upstream of the translation start sites), gene body, and 3’ UTRs (<0.5 kb downstream of the translation stop sites). Functions enriched in the target genes were analyzed using malaria parasite metabolic pathways (MPMP) (Ginsburg, 2006), functional gene families, and clusterProfiler (Yu et al., 2012) [Benjamini-Hochberg (BH) adjusted p-value of <0.01].

Peaks were extended +/− 250 bp around summits. Those detected in both biological replicates were reserved for discovery of PfAP2-EXP2 DNA binding motifs between 6 bp and 10 bp using DREME (Bailey, 2011) as compared with random genomic regions of 500 bp.

RNA-seq reads were trimmed as described in the ChIP-seq analysis, then aligned to the 3D7 genome using HISAT2 (Kim et al., 2015) with the guide by the gene annotation and default parameters except--max-intronlen 5,000 --dta--rna-strandness RF. StringTie (Pertea et al., 2016) counted reads mapped to genes. Subsequently, edgeR (Robinson et al., 2010) analyzed differential gene expression (fold change of >2 and false discovery rate of <0.05). Over-representation analyses of MPMP pathways were performed on the differentially expressed genes using clusterProfiler (BH adjusted p-value of <0.01).

Based on time-series expression profiling of intraerythrocytic developmental cycle in Plasmodium falciparum 3D7 strain (Toenhake et al., 2018), the mRNA level of pfap2-exp2 reaches its peak at trophozoite and schizont stages (Figure 1A). To gain an insight into the function of PfAP2-EXP2, we first attempted to directly disrupt its gene using the CRISPR/Cas9 knockout system, but it failed after five independent transfections with two different sgRNAs (data not shown). We speculated that PfAP2-EXP2 might be essential for parasite development during the asexual replications. Afterwards, a conditional gene knockdown strategy was adopted, which introduced a glucosamine inducible glms riboswitch element into the 3’ end of pfap2-exp2 gene (Figure 1B) (Liu et al., 2020; Carrington et al., 2021). After drug selection and limiting dilution cloning, we successfully obtained a 3D7/pfap2-exp2-ty1-glms transgenic knockdown parasite line. Then the genomic DNA of the transgenic parasite line was collected, and two sets of primers were used for conventional PCR detection to verify the correct editing of the pfap2-exp2 locus (Figure 1C). Furthermore, to examine knockdown efficiency, tightly synchronized ring stage 3D7/pfap2-exp2-ty1-glms parasites within 5 h window were treated with or without 5 mM glucosamine (GlcN) and harvested at late trophozoite and early schizont stages of the next generation. PfAP2-EXP2 protein level dramatically decreased upon exposure to the glucosamine (Figure 1D), indicating effective knockdown of the pfap2-exp2 gene by the glms riboswitch system.

PfAP2-EXP2 was detected at the nuclear periphery in both rings and schizonts (Figure 1E), suggesting its involvement in transcriptional regulation the same as other ApiAP2 members (Josling et al., 2020; Singh et al., 2021). To investigate the effect of PfAP2-EXP2 on parasite viability, the conditional knockdown parasite line was tightly synchronized to a 5-h window and maintained with or without glucosamine. Repeated monitoring was performed by Giemsa-stained thin blood smears over three consecutive generations. Parental strain 3D7-G7 was also included in the growth curve analysis as a wild type control (Liu et al., 2020). Partial loss of PfAP2-EXP2 led to an approximate 40% reduction in the parasite growth rate (student’s t-test p-value < 0.001, Figure 1F). We examined the thin blood smears of the last cycle and found that the pfap2-exp2-ty1-glms parasites treated by the glucosamine still retained normal morphology. In conclusion, PfAP2-EXP2 plays an important role in the asexual proliferation of P. falciparum, but does not affect its morphology.

Given the perinuclear distribution of PfAP2-EXP2, ChIP-seq assays were performed to explore its role in transcriptional regulation. To achieve this, we first constructed a C-terminal GFP tagged PfAP2-EXP2 transgenic parasite line using the CRISPR/Cas9 gene editing system (Figure 2A). The newly developed pfap2-exp2-ty1-gfp strain was verified by diagnostic PCR (Figure 2B). Western blotting validated the expression of PfAP2-EXP2 in this transgenic parasite line (Figure 2C). Moreover, immunofluorescence assays confirmed the perinuclear localization of PfAP2-EXP2 in rings and schizonts (Figure 2D).

Then, to investigate transcriptional regulation by PfAP2-EXP2, ChIP-seq assays were performed on the pfap2-exp2-ty1-gfp schizonts using antibodies against GFP, which showed good reproducibility with a correlation higher than 0.9 (Pearson correlation coefficient = 0.96, Figure 2E and Supplementary Figure S1). PfAP2-EXP2 binding sites were observed near a total of 233 genes, including 136 heterochromatic genes and 97 euchromatic genes (Figure 3A; Supplementary Table S2), which suggested a strong preference of this ApiAP2 factor for targeting heterochromatic genes (BH adjusted hypergeometric test p-value = 3.09e-81 and enrichment ratio of heterochromatin targeting to euchromatin targeting = 17). In addition, no motifs were detected in euchromatic PfAP2-EXP2 binding sites, which is consistent with the previous report on motif characterization using protein binding microarrays (Campbell et al., 2010). Intriguingly, a specific DNA motif CCCTAAACCC was found at the heterochromatic PfAP2-EXP2 binding sites (Figure 3B). Thus, PfAP2-EXP2 might regulate heterochromatic and euchromatic genes by different mechanisms. PfAP2-EXP2 is mostly bound to the gene body regions of both heterochromatic and euchromatic genes (Figures 3C,D). A notable part of occupancies was also observed at 5’ UTR regions. Malaria Parasite Metabolic Pathway (MPMP) and functional family analyses of heterochromatic target genes revealed that PfAP2-EXP2 might participate in regulating multiple crucial pathways for parasite growth and development, such as HP1 enrichment values, structure of telomere and sub-telomeric regions, rosette formation between normal and infected RBCs, interactions between modified host cell membrane and endothelial cell, and candidate genes related to virulence (Figure 3E; Supplementary Table S3). No MPMP pathways were found to be enriched in PfAP2-EXP2 euchromatic target genes.

To inspect roles of PfAP2-EXP2 in the asexual cycle of P. falciparum, we performed transcriptome analyses on the pfap2-exp2-ty1-glms parasites. Parasites were tightly synchronized to a 5-h window and cultured in the presence or absence of glucosamine (Figure 4A). After reinvasion, they were collected for RNA-seq at three time points, including 10–15 hpi, 25–30 hpi and 40–45 hpi (Figure 4A). The global transcriptome did not change much in the rings and trophozoites after PfAP2-EXP2 knockdown, and only less than 15 genes showed expression changes (Figures 4B,C; Supplementary Tables S4, S5). However, appreciable effects of PfAP2-EXP2 on gene transcription were observed at the schizont stage (Figures 4B,C; Supplementary Table S6). A total of 229 genes were altered on the expression level, out of which, 228 genes were downregulated by more than two times. Therefore, PfAP2-EXP2 regulates gene transcription mainly at the schizont stage, which is consistent with its peak expression from mid to late schizont stage. Furthermore, the genes downregulated in the PfAP2-EXP2 knockdown line included genes coding for exported proteins, invasion genes and genes involved in transcriptomic response of DHA treated K1 strain trophozoites (Figure 4D; Supplementary Table S7; Supplementary Figure S2), the first two functional groups of which are especially essential for parasite growth (Cowman et al., 2017; Frénal et al., 2017; Warncke and Beck, 2019).

Given that knockdown of pfap2-exp2 led to defects in the growth of asexual cycles, we focused on its impacts on exported proteins that are critical for cell remodeling. Ten genes coding for exported proteins were directly targeted by PfAP2-EXP2 with more than 50% expression reduction by the PfAP2-EXP2 knockdown as well, including EPF3 (Figures 5A,B), EPF4 (Figure 5C), and variant gene clusters. In addition, PfAP2-EXP2 directly activated skeleton-binding protein 1 (SBP1) (Figure 5D), secreted protein with altered thrombospondin repeat domain (SPATR) (Figure 5E), and thioredoxin-like protein 1 (TrxL1) (Figure 5F). SPATR protein is expressed around the rhoptries at the asexual erythrocytic stage and related to merozoite invasion (Chattopadhyay et al., 2003; Huynh et al., 2014). SBP1 plays an important role in transporting molecules to the surface of infected erythrocytes (Kats et al., 2015; Iriko et al., 2020). Even though the exact function of TrxL1 in Plasmodium falciparum is poorly understood, its orthologue in Toxoplasma gondii is a subunit of a microtubule-associated complex which regulates the cellular cytoskeleton in the cells (Liu et al., 2013). Thus, we speculated that PfAP2-EXP2 might affect the growth of Plasmodium falciparum in the asexual cycle through regulating cell remodeling process.