Blockage of Spontaneous Ca²⁺ Oscillation Causes Cell Death in Intraerythrocitic <em>Plasmodium falciparum</em>

<div><p>Malaria remains one of the world’s most important infectious diseases and is responsible for enormous mortality and morbidity. Resistance to antimalarial drugs is a challenging problem in malaria control. Clinical malaria is associated with the proliferation and development of <em>Plasmodium</em> parasites in human erythrocytes. Especially, the development into the mature forms (trophozoite and schizont) of <em>Plasmodium falciparum</em> (<em>P. falciparum</em>) causes severe malaria symptoms due to a distinctive property, sequestration which is not shared by any other human malaria. Ca²⁺ is well known to be a highly versatile intracellular messenger that regulates many different cellular processes. Cytosolic Ca²⁺ increases evoked by extracellular stimuli are often observed in the form of oscillating Ca²⁺ spikes (Ca²⁺ oscillation) in eukaryotic cells. However, in lower eukaryotic and plant cells the physiological roles and the molecular mechanisms of Ca²⁺ oscillation are poorly understood. Here, we showed the observation of the inositol 1,4,5-trisphospate (IP₃)-dependent spontaneous Ca²⁺ oscillation in <em>P. falciparum</em> without any exogenous extracellular stimulation by using live cell fluorescence Ca²⁺ imaging. Intraerythrocytic <em>P. falciparum</em> exhibited stage-specific Ca²⁺ oscillations in ring form and trophozoite stages which were blocked by IP₃ receptor inhibitor, 2-aminoethyl diphenylborinate (2-APB). Analyses of parasitaemia and parasite size and electron micrograph of 2-APB-treated <em>P. falciparum</em> revealed that 2-APB severely obstructed the intraerythrocytic maturation, resulting in cell death of the parasites. Furthermore, we confirmed the similar lethal effect of 2-APB on the chloroquine-resistant strain of <em>P. falciparum</em>. To our best knowledge, we for the first time showed the existence of the spontaneous Ca²⁺ oscillation in <em>Plasmodium</em> species and clearly demonstrated that IP₃-dependent spontaneous Ca²⁺ oscillation in <em>P. falciparum</em> is critical for the development of the blood stage of the parasites. Our results provide a novel concept that IP₃/Ca²⁺ signaling pathway in the intraerythrocytic malaria parasites is a promising target for antimalarial drug development.</p> </div

Inhibition of intraerythrocytic <i>P. falciparum</i> development by 2-APB.

<p>(A) The FCR-3 strain was cultured for 40 h of the intraerythrocytic development cycle. Cultures were terminated at 20, 30 and 40 h of the assay after synchronization, and thin smears of erythrocytes were prepared for parasite counting. Representative results of 3 independent experiments are shown. (B) Morphology of intraerythrocytic parasites cultured with DMSO or 100 µM 2-APB at 20, 30 and 40 h of the assay after synchronization. (C) 100 µM 2-APB significantly decreased the area, perimeter and maximum diameter of intraerythrocytic parasites at 15, 30 and 40 h of the assay after synchronization. Columns and error bars represent the mean + S.D. Fifty parasites were measured at each time point. The <i>P</i> values compared with DMSO controls are given below each figure (two-tailed unpaired <i>t</i> test with Welch’s correction). (D) Cultures were terminated at 40 and 70 h of the assay after synchronization for parasite counting. Culture medium with DMSO or 2-APB was replaced at 40 h. Representative results of 3 independent experiments are shown. (E) The chloroquine-resistant strain K1 was cultured for 72 h of the intraerythrocytic development cycle. Cultures were terminated at 24, 48 and 72 h of the assay after synchronization, and thin smears of erythrocytes were prepared for parasite counting. Culture medium with DMSO or 2-APB was replaced at 24 and 48 h. Representative results of 3 independent experiments are shown. Parasiaemia of ring form (Rf), trophozoites (T), early schizonts (ES) and late schizonts (LS) is shown as mean + S.D. of 3 independent counts of a single well (A) or 3 wells (D, E). Stages with parasitaemia of less than 0.1% are not shown (A, D and E).</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

Life cycle of <i>Plasmodium. falciparum.</i>

<p>Schematic illustration of the life cycle of <i>P. falciparum</i>. The blood stages on which this study is focused are shown in detail.</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

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

Cytosolic calcium (Ca²⁺) dynamics in the early ring forms (ERf) (A) and early trophozoites (ET) (B) and effects of 2-aminoethyl diphenylborinate (2-APB).

<p>Each colour represents cytosolic Ca²⁺ dynamics acquired from individual parasites in the presence (right columns) or absence (left columns) of 100 µM 2-APB. Embedded images in left panels are representative images of Fluo-4-loaded <i>P. falciparum</i> during each intraerythrocytic stage (indicated by arrowheads). Scale bars, 5 µm.</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

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