Sir2a regulates rDNA transcription and multiplication rate in the human malaria parasite Plasmodium falciparum

Abstract

The Plasmodium falciparum histone deacetylase Sir2a localizes at telomeric regions where it contributes to epigenetic silencing of clonally variant virulence genes. Apart from telomeres, PfSir2a also accumulates in the nucleolus, which harbours the developmentally regulated ribosomal RNA genes. Here we investigate the nucleolar function of PfSir2a and demonstrate that PfSir2a fine-tunes ribosomal RNA gene transcription. Using a parasite line in which PfSir2a has been disrupted, we observe that histones near the transcription start sites of all ribosomal RNA genes are hyperacetylated and that transcription of ribosomal RNA genes is upregulated. Complementation of the PfSir2a-disrupted parasites restores the ribosomal RNA levels, whereas PfSir2a overexpression in wild-type parasites decreases ribosomal RNA synthesis. Furthermore, we observe that PfSir2a modulation of ribosomal RNA synthesis is linked to an altered number of daughter merozoites and the parasite multiplication rate. These findings provide new insights into an epigenetic mechanism that controls malaria parasite proliferation and virulence.

Figure 1. PfSir2a is a negative regulator of rDNA transcription.

(a) Schematic of a P. falciparum rRNA gene unit. It consists of a ~9 kb transcribed region coding for a 45S rRNA precursor, which is processed to mature 18S, 5.8S and 28S rRNAs. The parasite has four rDNA unit types, which are different in sequence, physically separated in the genome, and differentially transcribed over the developmental cycle. The graph shows the rRNA transcriptional profile in parasites synchronized on ring-stage, the stage at which Pol I transcription is greatest (mean±s.e.m., n=4). The arrow indicates the transcription start site and the direction of transcription. The bar indicates the position of the primers used for transcript and ChIP analysis. These primers independently detect the pre-rRNA transcripts from the four rRNA genes types (A1, A2, S1 and S2). (b) Fold enrichment of H3K9ac on the individual rRNA genes obtained by ChIP analysis of PfSir2a-disrupted parasites (3D7ΔSir2a, grey) relative to wild-type parasites (3D7, black) (mean±s.e.m., n=3). (c) Relative transcription of the four rRNA genes in 3D7ΔSir2a parasites (grey) compared with the wild-type parasites (black) (mean±s.e.m., n=4, P<0.05). (d) Representative sections of the ultrastructure from trophozoite stages of wild-type (3D7) and 3D7ΔSir2a parasites. The inset depicts the cytoplasmic ribosomal content. Scale bar, 1 μm in the main figure and 100 nm in the inset. R, red blood cell; C, parasite cytoplasm; N, parasite nucleus. (e) Quantification of the ribosomal content (mean±s.d., n=2, P<0.0001). At least 20 cells were analysed for each of the two independent parasite preparations.

Figure 2. PfSir2a disruption increases parasite intraerythrocytic growth rate.

(a) Growth rate of the 3D7ΔSir2a parasites (grey squares) compared with that of wild-type 3D7 (black diamonds). The parasitemia (% of infected erythrocytes) on day 0 was set at 0.01% for both parasite lines and followed for three cycles (mean±s.e.m., n=4, P<0.01). (b) Representative time course of 3D7 (black) and 3D7ΔSir2a parasite (grey) blood-stage cycles. The cycle time is the distance between the two peaks of schizonts; zero time corresponds to the first peak of mature schizonts. The average cycle duration for 3D7 and 3D7ΔSir2a was 47.74±1.2 and 45.96±0.9 h, respectively, (mean±s.d., n=3, P=0.2). (c) Frequency distribution of the number of merozoites formed after schizogony in populations of wild-type 3D7 (black) and 3D7ΔSir2a parasites (grey) (mean±s.e.m., n=5, P<0.001). Approximately 50–60 mature schizonts with clear separated merozoites and a single pigmented digestive vacuole were counted for each of the five independent experiments.

Figure 3. Complementation of 3D7ΔSir2a and PfSir2a overexpression.

(a) Schematic of the plasmid used for PfSir2a episomal expression (pLN_PfSir2a::mCherry). The arrow indicates the direction of transcription. (b) Immunofluorescence assay in paraformaldehyde fixed ring-stage parasites with anti-PfSir2a antibodies (red) in wild-type parasites (first row) and anti-mCherry antibodies (red) in 3D7ΔSir2a+Sir2a::mCherry parasites (second row). Nuclei of ring-stage parasites were stained with Dapi (blue). Scale bar, 1 μm. (c) Relative transcription of individual rRNA genes in 3D7ΔSir2a (grey), complemented 3D7ΔSir2a (3D7ΔSir2a+Sir2a::mCherry; red line) and wild-type overexpressing Sir2a (3D7+Sir2a::mCherry; red) compared with the wild-type parasites (3D7; black) (mean±s.e.m., n=3). P values calculated relative to the wild-type 3D7 are as follows: 3D7ΔSir2a+Sir2a::mCherry, NS; 3D7ΔSir2a, P<0.05; 3D7+Sir2a::mCherry, P<0.05 for the A1 rRNA gene.

Figure 4. Parasite multiplication rate altered with varying levels of PfSir2a.

(a) Growth rate of the complemented 3D7ΔSir2a (open red circles) and PfSir2a overexpressing parasites (red diamonds) compared with that of 3D7ΔSir2a (grey triangles) and 3D7 (black squares) parasites. As in Fig. 2b, the initial parasitemia was set at 0.01% for all parasite lines (mean±s.e.m., n=5). A summary of the parasite multiplication rate after two cycles and the average number of merozoites per schizont (segmented stage) in the various transgenic parasite lines, including the transfection controls is shown in Table 1. (b) Schematic representation of the observed inverse correlation between the PfSir2a levels and the rDNA transcription and parasite proliferation. Our data predict that the lower is the PfSir2a silencing activity the higher is the rDNA transcription and parasite multiplication rates, and vice-versa.