SICA-mediated cytoadhesion of Plasmodium knowlesi-infected red blood cells to human umbilical vein endothelial cells

Zoonotic malaria due to Plasmodium knowlesi infection in Southeast Asia is sometimes life-threatening. Post-mortem examination of human knowlesi malaria cases showed sequestration of P. knowlesi-infected red blood cells (iRBCs) in blood vessels, which has been proposed to be linked to disease severity. This sequestration is likely mediated by the cytoadhesion of parasite-iRBCs to vascular endothelial cells; however, the responsible parasite ligands remain undetermined. This study selected P. knowlesi lines with increased iRBC cytoadhesion activity by repeated panning against human umbilical vein endothelial cells (HUVECs). Transcriptome analysis revealed that the transcript level of one gene, encoding a Schizont Infected Cell Agglutination (SICA) protein, herein termed SICA-HUVEC, was more than 100-fold increased after the panning. Transcripts of other P. knowlesi proteins were also significantly increased, such as PIR proteins exported to the iRBC cytosol, suggesting their potential role in increasing cytoadhesion activity. Transgenic P. knowlesi parasites expressing Myc-fused SICA-HUVEC increased cytoadhesion activity following infection of monkey as well as human RBCs, confirming that SICA-HUVEC conveys activity to bind to HUVECs

rPkSBP1-positive membranous structures in the monkey erythrocytes infected with PkSBP1-transgenic H-DMU line.

<p>Representative micrographs of immunogold-labeled rPkSBP1. Gold particles were visible at slit-like clefts (<b>A</b>) and oblong vesicular clefts (<b>B</b>) in the erythrocyte cytoplasm infected with PkSBP1-transgenic line. c, clefts; EM, erythrocyte membrane; P, parasite. Scale bar represents 500 nm.</p

Expression and localization of rPkSBP1 in infected monkey erythrocytes.

<p><b>(A)</b> Schematic of <i>P</i>. <i>knowlesi</i> rPkSBP1 expression construct (not to scale). Two myc epitopes (2myc) were fused at the C-terminus of full-length PkSBP1 open reading frame (PkSBP1 ORF) and expressed using the <i>P</i>. <i>falciparum</i> CRT 5' region (PfCRT 5') as a promoter. <b>(B)</b> Representative IFAT images of PkSBP1-transgenic <i>P</i>. <i>knowlesi</i> H-DMU line with anti-myc antibody (α-myc, green). α-myc-stained rPkSBP1 images were merged with DAPI nucleus-staining (blue) and differential interference contrast image (merge). The top panel is a negative control reacted with normal mouse IgG. R, ring; T, trophozoite; S, schizont stages. Scale bar represents 5 μm. <b>(C)</b> Western blotting of wild type parental <i>P</i>. <i>knowlesi</i> H-DMU line (WT) and PkSBP1-transgenic line (TG) with anti-myc antibody. Parasite proteins were sequentially extracted by freeze-thawing (FT), followed by extraction with 1% Triton X-100 (Tx), then with 2% SDS. Parasite protein cross-reacting by an antibody against <i>P</i>. <i>berghei</i> HSP70 serves as a loading control (bottom).</p

PkSBP1 delineates <i>P</i>. <i>knowlesi</i> host modifications.

<p>Schematic representation of host erythrocyte modifications revealed by studying the localization of SBP1 ortholog. <i>P</i>. <i>knowlesi</i> infection in both monkey and human erythrocytes induces membranous structures onto which PkSBP1 localizes. Involvement of tether structures (white bars) adjoining these membranes to the host cell cytoskeleton is possible [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164272#pone.0164272.ref021" target="_blank">21</a>] but was not investigated in this study. PfSBP1 interacts with erythrocyte membrane protein 4.1 and spectrin, as described for <i>P</i>. <i>falciparum</i> (yellow and orange shapes) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164272#pone.0164272.ref016" target="_blank">16</a>]. PPM, parasite plasma membrane; PV, parasitophorous vacuole; PVM, parasitophorous vacuole membrane.</p

rPkSBP1 is exported to <i>P</i>. <i>falciparum</i> Maurer's clefts and <i>P</i>. <i>knowlesi</i> ‘Sinton and Mulligan’ stipplings.

<p><b>(A)</b> Human erythrocytes infected with PkSBP1-transgenic <i>P</i>. <i>falciparum</i> co-stained with anti-myc antibody (green) and PfSBP1 (red). Merged image of rPkSBP1, PfSBP1, DAPI nucleus-staining (blue), and differential interference contrast (DIC) image are shown (merge). Top panel was labeled with anti-myc antibody (α-myc) and normal rabbit IgG, middle panel was labeled with normal mouse IgG and rabbit anti-PfSBP1 antibody, and bottom panel was labeled with mouse anti-myc and rabbit anti-PfSBP1 antibodies. <b>(B)</b> Colocalization of rPkSBP1 puncta (green) and Giemsa-stained ‘Sinton and Mulligan’ stipplings in monkey erythrocytes infected with PkSBP1-transgenic <i>P</i>. <i>knowlesi</i> H-DMU line. Merged image of rPkSBP1 and Giemsa-stained image are shown (merge). Scale bar represents 5 μm. Nuclei were stained with DAPI (blue).</p

Expression and localization of rPkSBP1 in the human erythrocytes infected with PkSBP1-transgenic <i>P</i>. <i>knowlesi</i> H_hu-HSPH line.

<p><b>(A)</b> Representative fluorescence image showing localization of rPkSBP1 stained with anti-myc antibody (rPkSBP1, green) as puncta within the infected erythrocyte cytoplasm. rPkSBP1 signal was merged with erythrocyte membrane stained with anti-human CD235a (α-GlyA, red) and DAPI nucleus-staining (blue) (merge). DIC, differential interference contrast. Scale bar represents 5 μm. <b>(B)</b> Colocalization of rPkSBP1 puncta (green) and Giemsa stained ‘Sinton and Mulligan’ stipplings in the human erythrocyte infected with PkSBP1-transgenic <i>P</i>. <i>knowlesi</i> H_hu-HSPH line. Merged image of rPkSBP1 and Giemsa-stained image are shown (merge). Scale bar represents 5 μm. <b>(C)</b> Transmission electron micrographs of two representative erythrocytes infected with PkSBP1-transgenic <i>P</i>. <i>knowlesi</i> H_hu-HSPH line. Slit-like clefts (left) and oblong vesicular clefts (right) were observed. c, clefts; cv, caveola; EM, erythrocyte membrane; P, parasite. Scale bar represents 500 nm.</p

Expression and localization of rPk2TM-a in the monkey erythrocytes infected with Pk2TM-a-transgenic <i>P</i>. <i>knowlesi</i> H-DMU line.

<p><b>(A)</b> Schematic of the expression cassette of the <i>P</i>. <i>knowlesi</i> rPk2TM-a (not to scale). Two myc epitopes (2myc) were fused at the C-terminus of full-length Pk2TM-a open reading frame (Pk2TM-a ORF) and expressed using the <i>P</i>. <i>falciparum</i> CRT 5' region (PfCRT 5') as a promoter. Plasmid backbone is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164272#pone.0164272.g002" target="_blank">Fig 2A</a>. (<b>B</b>) Western blotting of wild type parental <i>P</i>. <i>knowlesi</i> H-DMU line (WT) and Pk2TM-a-transgenic <i>P</i>. <i>knowlesi</i> H-DMU line (TG) with anti-myc antibody (α-myc). Parasite proteins were sequentially extracted by freeze-thawing (FT), followed by extraction with 1% Triton X-100 (Tx), then with 2% SDS. Parasite protein cross-reacting by an antibody against <i>P</i>. <i>berghei</i> HSP70 serves as a loading control (bottom). <b>(C)</b> Representative IFAT images of Pk2TM-a-transgenic <i>P</i>. <i>knowlesi</i> parasites with anti-myc antibody (α-myc, green). α-myc-stained rPk2TM-a images were merged with DAPI nucleus-staining (blue) and differential interference contrast image (merge). The top panel is a negative control. R, ring; T, trophozoite; S, schizont stages. Scale bar represents 5 μm. <b>(D)</b> Representative transmission electron micrographs of immunogold labeled Pk2TM. Slit-like clefts (left) and oblong vesicular clefts (right) showed gold particles in the erythrocyte cytoplasm infected with Pk2TM-a-transgenic line. c, clefts; EM, erythrocyte membrane; P, parasite. Scale bar represents 500 nm.</p

A type II protein arginine methyltransferase regulates merozoite invasion in Plasmodium falciparum

Abstract Protein arginine methyltransferases (PRMTs) regulate many important cellular processes, such as transcription and RNA processing in model organisms but their functions in human malaria parasites are not elucidated. Here, we characterize PfPRMT5 in Plasmodium falciparum, which catalyzes symmetric dimethylation of histone H3 at R2 (H3R2me2s) and R8, and histone H4 at R3 in vitro. P fPRMT5 disruption results in asexual stage growth defects primarily due to lower invasion efficiency of the merozoites. Transcriptomic analysis reveals down-regulation of many transcripts related to invasion upon PfPRMT5 disruption, in agreement with H3R2me2s being an active chromatin mark. Genome-wide chromatin profiling detects extensive H3R2me2s marking of genes of different cellular processes, including invasion-related genes in wildtype parasites and PfPRMT5 disruption leads to the depletion of H3R2me2s. Interactome studies identify the association of PfPRMT5 with invasion-related transcriptional regulators such as AP2-I, BDP1, and GCN5. Furthermore, PfPRMT5 is associated with the RNA splicing machinery, and PfPRMT5 disruption caused substantial anomalies in RNA splicing events, including those for invasion-related genes. In summary, PfPRMT5 is critical for regulating parasite invasion and RNA splicing in this early-branching eukaryote