The Putative Bromodomain Protein PfBDP7 of the Human Malaria Parasite Plasmodium Falciparum Cooperates With PfBDP1 in the Silencing of Variant Surface Antigen Expression

Abstract

Epigenetic regulation is a critical mechanism in controlling virulence, differentiation, and survival of the human malaria parasite Plasmodium (P.) falciparum. Bromodomain proteins contribute to this process by binding to acetylated lysine residues of histones and thereby targeting the gene regulatory machinery to gene promoters. A protein complex containing the P. falciparum bromodomain proteins (PfBDP) 1 and PfBDP2 (BDP1/BDP2 core complex) was previously shown to play an essential role for the correct transcription of invasion related genes. Here, we performed a functional characterization of a third component of this complex, which we dubbed PfBDP7, because structural modelling predicted a typical bromodomain fold. We confirmed that PfBDP7 is a nuclear protein that interacts with PfBDP1 at invasion gene promoters in mature schizont stage parasites and contributes to their transcription. Although partial depletion of PfBDP7 showed no significant effect on parasite viability, conditional knock down of either PfBDP7 or PfBDP1 resulted in the de-repression of variant surface antigens (VSA), which are important pathogenicity factors. This de-repression was evident both on mRNA and protein level. To understand the underlying mechanism, we mapped the genome wide binding sites of PfBDP7 by ChIPseq and showed that in early schizonts, PfBDP7 and PfBDP1 are commonly enriched in heterochromatic regions across the gene body of all VSA families, including genes coding for PfEMP1, RIFIN, STEVOR, and PfMC-2TM. This suggests that PfBDP7 and PfBDP1 contribute to the silencing of VSAs by associating with heterochromatin. In conclusion, we identified PfBDP7 as a chromatin binding protein that is a constitutive part of the P. falciparum BDP1/BDP2 core complex and established PfBDP1 and PfBDP7 as novel players in the silencing of heterochromatin regulated virulence gene families of the malaria parasite P. falciparum.

Keywords: P. falciparum; bromodomain; chromatin; malaria; variant surface antigens.

FIGURE 1

Structure prediction of PfBDP7. (A) Model of PfBDP7 with the putative bromodomain (BRD) from R299 to N394 highlighted in dark grey. (B) Alignment of P. falciparum bromodomains including the modelled PfBDP7 domain. Amino acids are shaded by degree of similarity. The sequence logo depicts conserved residues. Structural features (αZ, αA, αB, αC helices with inter-helical connecting loops ZA-, AB- and BC-loops) are indicated at the bottom. The conserved Y and D residues stabilizing the bromodomain fold, and the conserved N critical for Kac binding are indicated by red asterisks. (C) Structural model of the PfBDP7 bromodomain predicted by Phyre2. The structural features characteristic for the bromodomain fold are labelled. The conserved N370 is highlighted in yellow halos. (D) Phylogenetic tree of PfBDP7 orthologs identified in the Plasmodium genus. PF3D7_1124300 (PfBDP7) is labelled in red.

FIGURE 2

PfBDP7 expression during the P. falciparum asexual life cycle. (A) Scheme of SLI strategy for modification of the PfBDP7 genomic locus by integration of the BirA* biotin ligase and 5xTy sequences fused to a 2A skip peptide and the neomycin (Neo) selection cassette. Primer positions for diagnostic PCR are indicated in dark blue, with numbers according to Supplementary Table S1. (B) Diagnostic PCR screen for plasmid integration of the double transgenic parasite line PfBDP1HA::PfBDP7BirATy (wt = wild type, ctrl = control). (C) Western blot analysis of PfBDP7BirATy expression across the 48 h asexual parasite life cycle relative to PfBDP1HA and aldolase as a loading control. Synchronized parasite lysates were prepared in 6 h intervals. Relative intensities compared to aldolase were determined by densitometry and are indicated underneath each blot. (D) Immunofluorescence analysis of PfBDP1HA::PfBDP7BirATy double transgenic parasites using antibodies detecting the HA tag fused to PfBDP1 or the Ty tag fused to PfBDP7. Hoechst 33342 was used for DNA staining. The parasite age post invasion of the parasites is indicated on the left (h). Scale bars, 5 µm. (E) Cellular fractionation of PfBDP7Ty parasites in cytoplasmic (CP), salt soluble nuclear (Ns) and pellet (P) fractions. A total lysate of PfBDP7Ty parasites as positive control [TL (+)] was analyzed alongside. The Western Blots were probed with antibodies detecting Ty, aldolase, HP1 (heterochromatin protein 1) or H3 (histone 3).

FIGURE 3

Identification of PfBDP7 interaction partners. (A) Co-Immunoprecipitation of PfBDP7BirATy and PfBDP1HA from PfBDP1HA::PfBDP7BirATy parasites demonstrates that the two proteins are part of the same protein complex. The immunoprecipitation (IP) from micrococcal nuclease treated chromatin (input, IN) was performed with mouse anti-Ty, rat anti-HA or IgG antibodies as negative control. (B–F) PfBDP7 or PfBDP1 associated proteins were isolated by Co-IP and identified by Mass Spectrometry. Enrichment of peptides was calculated as % peptide counts of all peptides identified in each IP, biological replicates were averaged (N = 1-3, see Supplementary Table S3) and plotted against the negative controls. IP 3D7: anti-Ty IP in 3D7 wt parasites, IP IgG: non-immune IgG on PfBDP7Ty_GlmS parasites. (G) Enrichment of PfBDP7, PfBDP1, PfBDP2 and Pf3D7_0823200 peptides from individual IPs.

FIGURE 4

Conditional knock down of PfBDP7 does not affect parasite growth. (A) Scheme of SLI strategy for modification of the PfBDP7 genomic locus by integration of 5 × Ty epitope tags and a GlmS ribozyme downstream of the 2A skip peptide and the neomycin (Neo) selection cassette. Primer positions for diagnostic PCR are indicated in dark blue, with numbers according to Supplementary Table S1. (B) Diagnostic PCR screen for plasmid integration in the PfBDP7Ty_GlmS parasite line and (wt = wild type, ctrl = control primers targeting the pfs16 gene). (C) qPCR analysis demonstrates efficient mRNA knock down in parasites treated with 2.5 mM glucosamine over two IDCs. Expression data were normalized to seryl-tRNA synthetase and the enrichment relative to untreated parasites was calculated. Mean and SD of N = 3, unpaired t-test. (D) Glucosamine treatment significantly reduces PfBDP7 protein expression. Tightly synchronized ring stage PfBDP7Ty_GlmS parasites (0–6 hpi) were treated for 24 or 72 h with increasing concentrations of glucosamine (GlcN) ranging from 0 to 2.5 mM, and protein expression was monitored by western blot with a Ty and a aldolase antibodies. Densitometry results are mean and SD of N = 3 replicates, unpaired t-test. (E) Parasite growth of PfBDP7Ty_GlmS and 3D7 control parasites treated with increasing concentrations of GlcN was examined after 24, 48, and 72 h by flow cytometry. After 24 h, the cultures were split 1:5 to prevent overgrowth. Parasite development was monitored in parallel by Giemsa staining. Representative images of cultures treated with 0 or 2.5 mM GlcN are shown.

FIGURE 5

Conditional depletion of PfBDP1 or PfBDP7 results in increased variant surface antigen expression and decreased invasion gene expression. (A) Western Blot of PfBDP1HADD trophozoite stage parasite lysates cultivated for 24 h in the presence (+, wt) or absence (−, KD) of Shield1. Antibodies were directed against A-type RIFIN variant RIF40 (Petter et al., 2008), STEVOR variant PF3D7_0300400 (Bachmann et al., 2015); the semi-conserved (SC) or the C-terminus (CT) of PfMC-2TM (Bachmann et al., 2015). One representative example of N = 3 biological replicates is shown. (B) qPCR analysis of PfBDP7Ty_GlmS parasites cultivated with 0 or 1.25 mM glucosamine (GlcN) (mean and SD of N = 3). Glucosamine was added to synchronized ring stage parasites (0–8 hpi) and RNA was harvested in the next cycle at 24–32 hpi and 32–40 hpi. cDNA was quantified with degenerate primers amplifying conserved motifs in members of the, rifA, rifB, stevor, or pfmc-2tm families; or specific primers amplifying var2csa and pfbdp7 and signals were normalised to hsp70. t-test, *p < 0.05, **p < 0.01. (C) Western Blot probing small variant surface antigen expression in lysates of trophozoite stage PfBDP7Ty_GlmS parasites cultivated with 0 or 1.25 mM GlcN. Treatment commenced in ring stage parasites and lysates were prepared after 24 h (D) qPCR analysis of PfBDP7Ty_GlmS parasites with primers specific for the invasion genes msp1, msp2 and msp9 as well as for hsp86 and seryl-tRNA synthetase (strs) as additional controls.

FIGURE 6

PfBDP7 interacts with PfBDP1 across the genome. Chromatin immunoprecipitation was performed on PfBDP7Ty_GlmS and PfBDP1HA::PfBDP7BirATy parasites in early and mature schizont stage parasites. (A) Chart depicting % of PfBDP7 and PfBDP1 peaks localized in the specified genomic features in PfBDP1HA::PfBDP7BirATy mature schizont stage parasites. Ups = upstream, down = downstream. The relative orientation of genetic elements is indicated in the scheme. (B) Venn diagram visualizing overlap of genes closest to PfBDP1 and PfBDP7 peaks in early and mature PfBDP1HA::PfBDP7BirATy schizont stage parasites. (C) Gene ontology (GO) analysis of genes closest to common PfBDP1 and PfBDP7 peaks in mature schizonts. The most significant, non-redundant GO terms concerning “cellular component” are presented. (D) Enrichment profiles [log 2 (ChIP/Input)] of PfBDP7 and PfBDP1 in selected invasion genes. Green: PfBDP7Ty (from PfBDP7Ty_GlmS parasite ChIP); blue: PfBDP7BirATy and red: PfBDP1HA (from PfBDP1HA::PfBDP7BirATy parasite ChIP). (E) Line plots of log2 (ChIP/Input) ratios for PfBDP7 and PfBDP1 in mature PfBDP1HA::PfBDP7BirATy schizont stage parasites. Euchromatin associated genes were ranked into top, medium, bottom or non-expressed genes (silent) by schizont stage expression. Heterochromatin (HC) associated genes were plotted separately (right panels, pink lines).

FIGURE 7

PfBDP7 and PfBDP1 are enriched across VSA gene bodies in early schizonts. (A) Log2 (ChIP/input) enrichment profiles of PfBDP7 and PfBDP1 in early schizonts across chromosome 4 depicts accumulation of ChIP signals in heterochromatic regions (highlighted in yellow) containing VSA genes. Green: PfBDP7Ty_Glms; blue: PfBDP7BirATy; red: PfBDP1HA. (B) Line plots of average PfBDP1 and PfBDP7 enrichment across VSA families var, rif, stevor and pfmc-2tm (blue) and all genes (green) in PfBDP1HA::PfBDP7BirATy early schizonts.