Fragment Molecular Orbital Study of the Interaction between Sarco/Endoplasmic Reticulum Ca²⁺-ATPase and its Inhibitor Thapsigargin toward Anti-Malarial Development

Plasmodium falciparum, the causative agent of malignant malaria, is insensitive to thapsigargin (TG), a well-known inhibitor of the human sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). To understand the key factor causing the difference of the sensitivity, the molecular interaction of TG and each SERCA was analyzed by the fragment molecular orbital (FMO) method. While the major component of the interaction energy was the nonpolar interaction, the major difference in the molecular interaction arose from the polar interaction, namely, the hydrogen bonding interaction with a hydroxyl group of TG. Additionally, we successfully confirmed these FMO calculation results by measuring the inhibitory activity of a synthesized TG derivative. Our calculations and experiments indicated that, by replacing the hydroxyl group of TG with another functional group, the sensitivities of TG to human and P. falciparum SERCAs can be reversed. This study provides important information to develop antimalarial compounds targeting P. falciparum SERCA

Three phase processes of the red blood cell (RBC) invasion by <i>Plasmodium yoelii</i>.

<p>Time-lapse imaging of RBC invasion was captured every 0.1 sec with transmitted light for <i>P. yoelii</i> 17XL (A), <i>P. yoelii</i> 17X1.1 (B), and <i>Plasmodium falciparum</i> 3D7 line (C). First “Pre-invasion” phase started from the initial attachment between the merozoite (0 second, arrow head) and RBC plasma membrane, followed by the RBC deformation, and apical reorientation of the merozoite (rightmost column of “Pre-invasion” phase). Second “Invasion” phase consisted of the internalization of a merozoite into RBC and a rapid rotary movement of the internalized merozoite (arrow). Final “Echinocytosis” phase was defined as RBC being deformed to spike-like shape. The bars represent 5 µm.</p

Time-lapse imaging for the rupture of schizont-infected red blood cells.

<p>Images were captured every 0.1 sec with transmitted light for <i>Plasmodium yoelii</i> 17XL (A), <i>P. yoelii</i> 17X1.1 (B), and <i>Plasmodium falciparum</i> 3D7 line (C). The bars represent 5 µm.</p

Kinetic difference in red blood cell (RBC) invasion between <i>Plasmodium</i> species.

<p>The median time for each step are shown as a box plot with whiskers from minimum to maximum. The interquartile range shows as box with the median marked as a horizontal line, minimum and maximum from lower and upper quartile represent error bar. <i>P</i> values were determined using the Mann-Whitney U test. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050780#pone.0050780.s001" target="_blank">Table S1</a> for detail values.</p

Morphological change of the <i>Plasmodium yoelii</i> merozoite after released from red blood cell (RBC).

<p>The major axis (A), minor axis (B), longitudinal cross section area (C), and circularity (D) were measured every 10 sec from RBC rupture to pre-invasion for invasive merozoites (n = 9–12). The average and the error representing one standard deviation were plotted in the line charts. Circularity was calculated using the following formula: Circularity = 4πArea/Perimeter². A value of 1 indicates a perfect circle and the value of 0 indicates an increasingly elongated polygon. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050780#pone.0050780.s001" target="_blank">Table S1</a> for detail values. (E) Time-lapse sequence of merozoite release of <i>P. yoelii</i> 17XL was recorded every 0.1 sec. Arrowhead indicates same invasive merozoite in the sequence and the arrow indicates an attachment of an immature flat elongated oval merozoite. A mature spherical invasive merozoite attached to the RBC and deformed RBC (Pre-invasion) at 180 sec. The bar represents 5 µm.</p

MOESM3 of Plasmodium Rab5b is secreted to the cytoplasmic face of the tubovesicular network in infected red blood cells together with N-acylated adenylate kinase 2

Additional file 3. Multiple-alignment of amino acids sequences of Plasmodium Rab5b and Toxoplasma Rab5b. The GTP-binding box (red boxes) and the effector domain (light green box) are shown. Amino acids in the blue box indicate N-terminal myristoyl and palmitoyl modification sites. Toxoplasma gondii Rab5b possesses atypical insertion sequences at amino acid positions 165-182, which are not present in Plasmodium Rab5b

MOESM1 of Plasmodium Rab5b is secreted to the cytoplasmic face of the tubovesicular network in infected red blood cells together with N-acylated adenylate kinase 2

Additional file 1. Structures of constructs used in this study. Plasmids used for the analysis of P. berghei (a) or P. falciparum (b). For coexpression of PbRab5b-mAG and RFP, a fusion fragment comprising the promoter region of the gene encoding the P. falciparum chloroquine resistance transporter (CRT) promoter [1], TagRFP amplified from the pTagRFP-C plasmid (Evrogen), and PbDT were PCR-amplified using overlapping oligonucleotides, and the In Fusion HD cloning kit was used to insert the DNA fragment into the XhoI site of the PbRab5b-mAG plasmid (PbRab5b-mAG+RFP). For single crossover transfection of constitutive active PbRab5bQ91L mutant, an upstream sequence encompassing nucleotide positions at –1,500 bp to –500 bp of PbRab5b was inserted into HindIII site of the plasmid for double crossover (PbRab5bQ91L-mAG for single crossover). Construction of PbRab5b-mAG, PbRab5b Chimeric, GOI-YFP-DD, RFP, GOI-RFP was described in the Materials section of the main manuscript.Reference[1] van Dooren GG, Marti M, Tonkin CJ, Stimmler LM, Cowman AF, McFadden GI. (2005) Development of the endoplasmic reticulum, mitochondrion and apicoplast during the asexual life cycle of Plasmodium falciparum. Mol Microbiol; 57:405-19

MOESM4 of Plasmodium Rab5b is secreted to the cytoplasmic face of the tubovesicular network in infected red blood cells together with N-acylated adenylate kinase 2

Additional file 4. Peripheral localization of PbRab5b-mAG in trophozoite stage of parasites. (a) Fluorescence image of PbRab5b-mAG in trophozoite-stage parasites. Transgenic parasites expressing PbRab5b-mAG under the regulation of PbRab5b promoter were fixed with PFA, and faint cytosolic fluorescence of mAG were detected (green). Nuclei were stained with DAPI (blue). In control wild-type parasites, mAG fluorescence signal was not detected. Bar, 10 μm. (b) Magnified images of trophozoites expressing both PbRab5b-mAG and TagRFP. Trophozoite stage of parasites expressing PbRab5b-mAG (green) and cytosolic TagRFP (red) were fixed and the mAG and TagRFP fluorescence signals were obtained. Histograms of the green and red intensities along the white arrow are shown in the right graph. Black arrowheads indicate regions where stronger PbRab5b-mAG signal were detected compared to the TagRFP signal. Bar, 5 μm. (c) Peripheral localization of PbRab5bQ91L-mAG expressed under the regulation of PbRab5b promoter in trophozoite-stage parasites. A constitutively active PbRab5bQ91L-mAG mutant, in which Gln at aa 91 was replaced with Leu [1,2] was integrated into the upstream of PbRab5b genomic locus by single crossover method. Fluorescence of PbRab5bQ91L-mAG mutant protein was accumulated at the periphery of the parasite (green). Nuclei were stained with DAPI (blue). Bar, 5 μm.References[1] Li G, Barbieri MA, Colombo MI, Stahl PD (1994) Structural features of the GTP-binding defective Rab5 mutants required for their inhibitory activity on endocytosis. J Biol Chem 269: 14631-14635.[2] Stenmark H, Parton RG, Steele-Mortimer O, Lutcke A, Gruenberg J, et al. (1994) Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis. EMBO J 13: 1287-1296

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

Schematic images of the centromere region of <i>P</i>. <i>vivax</i> and <i>P</i>. <i>yoelii</i> used in this study and the design of the plasmids to evaluate centromere function.

(A) The P. vivax centromere sequence was selected from chromosome 11 (CM000452.1). DNA fragments of approximately 1.9 and 1.5 kb size were used for plasmid construction and named PvCEN11S2 and PvCEN11S3, respectively. The P. yoelii centromere sequence was selected from chromosome 5 (DQ054838.1) and approximately 1.8 kb DNA fragment was used for the plasmid, named PyCEN5. These sequences include repeat sequence motifs (white arrowhead). (B) Centromere plasmids evaluated in this study (PvCEN11S2-H86HD-TG, PvCEN11S3-H86HD-TG, and PyCEN5-H86HD-TG) and a control plasmid without a centromere region (pNoCEN). CEN, centromere region; 5' PfHSP86, the 5' untranslated region (UTR) of P. falciparum heat shock protein 86; hDHFR, human dihydrofolate reductase open reading frame; 3TyGFP, triple Ty1 tag and green fluorescent protein open reading frame; 3' PbDT, 3' UTR of P. berghei dihydrofolate reductase-thymidine kinase; and 5' PvHSP86, 5' UTR of P. vivax heat shock protein 86.</p