PfAP2-EXP2, an Essential Transcription Factor for the Intraerythrocytic Development of Plasmodium falciparum

Abstract

Plasmodium falciparum undergoes a series of asexual replications in human erythrocytes after infection, which are effective targets for combatting malaria. Here, we report roles of an ApiAP2 transcription factor PfAP2-EXP2 (PF3D7_0611200) in the intraerythrocytic developmental cycle of P. falciparum. PfAP2-EXP2 conditional knockdown resulted in an asexual growth defect but without an appreciable effect on parasite morphology. Further ChIP-seq analysis revealed that PfAP2-EXP2 targeted genes related to virulence and interaction between erythrocytes and parasites. Especially, PfAP2-EXP2 regulation of euchromatic genes does not depend on recognizing specific DNA sequences, while a CCCTAAACCC motif is found in its heterochromatic binding sites. Combined with transcriptome profiling, we suggest that PfAP2-EXP2 is participated in the intraerythrocytic development by affecting the expression of genes related to cell remodeling at the schizont stage. In summary, this study explores an ApiAP2 member plays an important role for the P. falciparum blood-stage replication, which suggests a new perspective for malaria elimination.

Keywords: ApiAP2 transcription factor PfAP2-EXP2; Plasmodium falciparum; asexual replication; cell remodeling; transcription regulation.

FIGURE 1

PfAP2-EXP2 transcription factor is required for the blood-stage parasite development. (A) Expression profiles of pfap2-exp2 in Plasmodium falciparum 3D7 strain throughout the intraerythrocytic developmental cycle according to RNA-seq transcriptomic analysis (Toenhake et al., 2018). (B) Incorporation of a pfap2-exp2-ty1-glms construct into the pfap2-exp2 locus of the wildtype 3D7-G7 line by homologous recombination. F1, R1, and R2 indicate locations of diagnostic PCR primers. (C) The glucosamine inducible knockdown transgenic parasite line was confirmed by diagnostic PCR using primers indicated in (B). (D) Western blotting proved effective knockdown of the pfap2-exp2 gene by the glucosamine (GlcN) addition in the 3D7/pfap2-exp2-ty1-glms transgenic parasite line. (E) Immunofluorescence assays using anti-ty1 revealed perinuclear distribution of PfAP2-EXP2 in the 3D7/pfap2-exp2-ty1-glms rings and schizonts. (F) Growth curves of 3D7-G7 (left panel) and the 3D7/pfap2-exp2-ty1-glms line (right panel) in the presence or absence of glucosamine (+/− GlcN). Comparisons between (+) GlcN and (−) GlcN treatments were performed with student’s t-tests. *** indicates p-value < 0.001.

FIGURE 2

Genome-wide occupancies of PfAP2-EXP2 at the schizont stage. (A) Generation of a pfap2-exp2 transgenic parasite line tagged with GFP at the C-terminus using the CRISPR/Cas9 gene editing system. (B) The GFP tagged PfAP2-EXP2 transgenic line was verified by diagnostic PCR using F1, R1, and R2 primers. (C) The expression of PfAP2-EXP2 in the 3D7/pfap2-exp2-ty1-gfp transgenic line was validated by western blotting. (D) Immunofluorescence assays using anti-GFP demonstrated perinuclear distribution of PfAP2-EXP2 in the 3D7/pfap2-exp2-ty1-gfp rings and schizonts. (E) Reads per kilobase of sequence range per Million mapped reads (RPKM) of ChIP, RPKM of input, and peaks in chromosomes 1 to 14 at the schizont stage detected by one of the two biological replicates of ChIP-seq for PfAP2-EXP2.

FIGURE 3

Characteristics of PfAP2-EXP2 occupancy. (A) Constitution of PfAP2-EXP2 target genes. Gene numbers are listed after functional categories. (B) A DNA motif discovered in heterochromatic binding sites. (C) PfAP2-EXP2 binding distribution at heterochromatic gene loci. (D) PfAP2-EXP2 binding distribution at euchromatic gene loci. (E) MPMP pathways and functional families enriched in PfAP2-EXP2 heterochromatic target genes at the schizont stage (BH adjusted enrichment p-values of <0.01).

FIGURE 4

Impacts of PfAP2-EXP2 on gene transcription. (A) The scheme of parasite collection for RNA-seq assays. (B) Transcriptome changes in the PfAP2-EXP2 knockdown rings, trophozoites, and schizonts, respectively (fold change of >2 and false discovery rate of <0.05). (C) Numbers of genes differentially expressed in the PfAP2-EXP2 knockdown rings, trophozoites, and schizonts, respectively. (D) MPMP pathways enriched in genes with expression downregulated by the PfAP2-EXP2 knockdown at the schizont stage (BH adjusted enrichment p-values of <0.01).

FIGURE 5

PfAP2-EXP2 binding sites at representative genes and their expression changes caused by the PfAP2-EXP2 knockdown at the schizont stage, including epf3 (A, B), epf4 (C), sbp1 (D), spatr (E), and trxl1 (F). For each gene locus, from the top to the bottom panels: log₂ fold enrichment (FE) of PfAP2-EXP2 binding, reads per kilobase per million mapped fragments (RPKM) in the 3D7/pfap2-exp2-ty1-glms line without glucosamine treatment, and RPKM in the 3D7/pfap2-exp2-ty1-glms line with glucosamine treatment.