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

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

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

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

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 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