Red Blood Cell Invasion by the Malaria Parasite Is Coordinated by the PfAP2-I Transcription Factor

Abstract

Obligate intracellular parasites must efficiently invade host cells in order to mature and be transmitted. For the malaria parasite Plasmodium falciparum, invasion of host red blood cells (RBCs) is essential. Here we describe a parasite-specific transcription factor PfAP2-I, belonging to the Apicomplexan AP2 (ApiAP2) family, that is responsible for regulating the expression of genes involved in RBC invasion. Our genome-wide analysis by ChIP-seq shows that PfAP2-I interacts with a specific DNA motif in the promoters of target genes. Although PfAP2-I contains three AP2 DNA-binding domains, only one is required for binding of the target genes during blood stage development. Furthermore, we find that PfAP2-I associates with several chromatin-associated proteins, including the Plasmodium bromodomain protein PfBDP1 and that complex formation is associated with transcriptional regulation. As a key regulator of red blood cell invasion, PfAP2-I represents a potential new antimalarial therapeutic target.

Keywords: ApiAP2 protein; ChIP-seq; DNA motif; Plasmodium falciparum; gene expression; host cell invasion; malaria; parasite; transcription factor; transcriptional regulation.

Figure 1. PfAP2-I is a nuclear protein that may bind a conserved TGCA DNA motif upstream of some invasion genes

A- Merozoite attached to the RBC surface with the micronemes, rhoptries, merozoite surface, plasma membrane (PM), inner and outer inner membrane complex (IMC) depicted. Proteins found in the glideosome, merozoite surface, microneme, rhoptry neck and rhoptry bulb are highlighted. B- The NGGTGCA DNA sequence motif is conserved in invasion-related gene promoters across P. falciparum (Pf), P. berghei (Pb), P. vivax (Pv) or P. knowlesi (Pk) (grey box). The four black boxes (from top to bottom) contain rhoptry promoters, the pfasp rhoptry neck promoter, merozoite surface (msp) promoters, and glideosome (gap) promoters (adapted from (Young et al., 2008)) (see also figure S1A). C- The PfAP2-I protein structure contains a putative ACDC domain, the three AP2 DNA-binding domains, an AT-hook and a nuclear localization signal (NLS) (see also figure S2). D- Live fluorescence microscopy of synchronized parasites shows that PfAP2-I-GFP (see figure S1B) localizes to the nucleus of trophozoite and schizont stage parasites but is not detected in ring stages. Hoechst was used as a nuclear marker. BF denotes bright field. E- Nuclear fractionation followed by Western blot of schizont-stage PfAP2-I-GFP parasites confirms nuclear localization of PfAP2-I-GFP. Anti-histone H3 and anti-aldolase were used as nuclear and cytosolic markers, respectively. C: cytosol, N: nucleus.

Figure 2. PfAP2-I-GFP binds upstream of invasion genes by ChIP

A- Heatmap (left) of 165 genomic loci from ChIP-seq replicate 1, +/− 5kb of each anti-GFP peak summit found in at least two of three replicates, shows enrichment of sequence reads (PfAP2-I bound) on ChIP/input samples (shown is the log2 transformed coverage). Profile plot from replicate 1 (top right) shows enrichment of anti-GFP ChIP-seq (red) compared to the control IgG ChIP-seq (black). The increase in signal is unrelated to GC content (bottom right). B- Plot showing the position of ChIP-seq peak summits relative to the ATG in the three replicates. C- Functional categorization of the 157 ChIP-Seq target genes based on literature review and GO term enrichment. Annotated examples of genes belonging to each category are shown (genes tested in D are highlighted with filled black circles). For the full list of gene targets and GO term results see Table S2. D- ChIP-qPCR of selected genes confirms PfAP2-I binding (blue bars) to ChIP-seq targets and no binding to the ama1 and rh4 promoters. Although not detected by ChIP-seq (see figure S3D), PfAP2-I associates with msp5 according to ChIP-qPCR. 3D7 wt parasites were used as a negative control (black bars). The results are shown as fold enrichment of ChIP performed with antibody versus non-immune IgG (n=3). Data are represented as mean ± SD (see also figure S3A).

Figure 3. PfAP2-I binding to the TGCA DNA motif is important for transcription

A- DNA motif heatmap of the trimmed ChIP-seq peaks +/−12bp surrounding the highest scoring motif (as discovered by DREME, figure S3B) shows that the GTGCA DNA motif is found within the majority of the peaks. Each row represents a peak summit and each column represents an individual nucleotide (See also figures S3B-C and S4). B- The mutations introduced to generate PfAP2-I-GFP::msp5MUT parasites (see also figures S3D-E). C- ChIP-qPCR showing that PfAP2-I binding to msp5 in PfAP2-I-GFP::msp5MUT parasites is decreased more than 7-fold versus PfAP2-I-GFP parasites. The data are represented as mean ± SD and n=3 (see figure S3F for original fold values). D- Microarray data using RNA extracted from PfAP2-I-GFP and PfAP2-I-GFP::msp5MUT parasites at eight time points beginning at 18hpi (see figure S3G) shows that when the TGCA motif is mutated upstream of msp5, levels of the msp5 transcript are reduced (see also figures S3H-I). The plot shows the average of two biological replicates (See Table S3 for complete microarray results). E- Western blot using specific antibodies against MSP5 show that the MSP5 protein level is decreased in PfAP2-I-GFP::msp5MUT (mut) versus PfAP2-I-GFP (wt) parasites but MSP4 and aldolase protein levels are unchanged at 36 and 45hpi. Densitometry values are shown below each blot.

Figure 4. Only PfAP2-I-D3 is required for DNA-binding during the IDC

A- The mutations introduced in the AP2 domains resulting in PfAP2-I-GFP-D1mut and PfAP2-I-GFP-D2mut parasites (see also figures S5A-D). B- ChIP-qPCR with PfAP2-I-D2mut parasites shows that PfAP2-I still binds to its target genes when D2 is mutated (blue bars). PfAP2-I-GFP parasites were used as control (black bars). Data are represented as mean ± SD and n=3. C- PBMs show that the DNA-binding of PfAP2-I AP2 domains D1, D2 and D3 are altered when the domains are mutated. Shown are both wt and mutant PBM results. Enrichment values below 0.45 represent low affinity DNA-binding (see Table S6 for position weight matrices for all AP2 domains).

Figure 5. PfAP2-I and PfBDP1 coimmunoprecipitate and regulate transcription of many of the same genes

A- List of PfAP2-I interaction partners with probability (pSAINT) of interaction as measured by SAINT of 0.9 or above (see also figure S6A and for full list of proteins detected see Table S4). B- Venn diagram comparing the peaks found by ChIP-seq for PfBDP1-HA (Josling et al., 2015) (purple) and PfAP2-I-GFP (this study) (light blue) shows that 115 genomic regions are bound by both proteins. See also Table S2. C-ChIP-qPCR of PfAP2-I-GFP, PfBDP1-HA and PfCHD1-GFP confirms that PfAP2-I does not bind genes only found in the PfBDP1 ChIP-seq dataset. Data are represented as mean ± SD and n=4 (see also Figure S6B, which shows some of the same data).

Figure 6. Model of transcription regulation by PfAP2-I and PfBDP1

Our model is that PfAP2-I must first bind the TGCA DNA motif within the promoters of invasion genes via its third AP2 domain (D3). Subsequently, PfBDP1 and PfCHD1 are recruited followed by RNA polymerase II thereby initiating transcription.

Figure 7. PfAP2-I binding precedes PfBDP1 binding to the target gene promoters

A- ChIP-qPCR demonstrates that PfAP2-I-GFP, but not PfBDP1-HA, is already bound to its target promoters at 22hpi. Data are represented as mean ± SD and n=2. The schizont data is the same as in Figure S6B (For PfBDP1 ChIP positive control see Figure S7A). B- In the presence of Shld-1, there are wildtype levels of PfBDP1, but in the absence of Shld-1, PfBDP1-DD is degraded and target gene transcription cannot be initiated (Josling et al., 2015). PfAP2-I may or may not remain associated to the TGCA DNA motif in the absence of PfBDP1. C- ChIP-qPCR shows that in PfAP2-I-GFP::PfBDP1-HA-DD parasites (see Figure S7), PfAP2-I-GFP remains bound to the target gene promoters even when PfBDP1 is knocked down (- Shld-1). Data are represented as mean ± SD and n=3. rh4 was used as negative control for DNA-binding.