Conservation of <i>sbp1</i> gene synteny.

(A) Genomic information corresponding to 30 to 50 kb of the left arm of P. falciparum chromosome 5 (top), the right arm of P. knowlesi chromosome 10 (middle) and the left arm of P. berghei chromosome 11 (bottom). Syntenic genes are highlighted in orange. To better represent synteny, the P. knowlesi genome neighborhood is inverted, but the original color coding of genes encoded on the top strand (blue) and bottom strand (red) and genome position (represented by number scales for each species) have been retained. (B) Schematics of Plasmodium SBP1 orthologs showing the overall length and general protein structures. Repeat regions are shown in orange and the single transmembrane regions in light blue. Recently reported SBP1 orthologs of P. ovale and P. malariae are also included. (C) The conservation of amino acid sequence within the transmembrane and adjacent regions. The transmembrane regions are predicted by TMHMM 2.0 and highlighted in light blue.</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 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

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 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