Substrate specificity of r<i>Cs</i>HK.

<p>^a from reference [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003641#pntd.0003641.ref017" target="_blank">17</a>].</p><p>Substrate specificity of r<i>Cs</i>HK.</p

Worm burden and EPG of rats in different groups.

<p>Results of analysis represent the mean ± SD, and the recovered worm numbers and EPG in groups were compared by Student’s <i>t</i>-test.</p><p>^a<i>p</i> > 0.05 and</p><p>^b<i>p</i> < 0.01 (compared with PBS group).</p><p>Worm burden and EPG of rats in different groups.</p

Effects of phosphate donors, effectors and EbSe on the enzyme kinetics of r<i>Cs</i>HK.

<p>The effect of 0~3 mM phosphate donors (ATP, CTP, GTP, ITP, TTP, and UTP) and fixed 3 mM glucose (A). The effect of 0~5 mM AMP (B), 0~10 mM PEP (C), 0~10 mM citrate (D), or 0~100 μM EbSe (E) and fixed 3 mM glucose with respect to ATP. The effect of 0~100 μM EbSe and fixed 3 mM ATP with respect to glucose (F).</p

ELISA of antibody titers and isotype of IgG induced by r<i>Cs</i>HK.

<p>(A) Antibody titers of IgG induced by r<i>Cs</i>HK. (B) IgG isotype induced by r<i>Cs</i>HK. * <i>p</i> < 0.01.</p

Immunolocalization of <i>Cs</i>HK in <i>C</i>. <i>sinensis</i> and in liver from infected rats.

<p>Mouse anti-r<i>Cs</i>HK serum and anti-mouse IgG were applied as primary antibody and secondary antibody, respectively. Serum from pre-immune mice was employed as primary antibody for a negative control. Panels H, L, P, R, U, V, W, and X are negative controls. Panels B, D, F, H, J, L, N, P, and R are under fluorescence microscope and the same parts (panels A, C, E, G, I, K, M, O, and Q) are under white light. Panels B, D, and F, localization of <i>Cs</i>HK in adult worms; panel J, localization of <i>Cs</i>HK in metacercariae. Panels S and T, localization of <i>Cs</i>HK in intrahepatic bile ducts of a <i>C</i>. <i>sinensis</i> infected rat. In panels S, T, U, V, W, and X, peroxidase staining shows as a yellow/rust colored deposit and Mayer’s hematoxylin counterstains the nuclei in light purple. White arrows highlight the regions of intrahepatic bile duct tissue and the tissue that stained positive for <i>Cs</i>HK. Original magnification: × 50 for panels M, N, O, P, Q and R; × 100 for panels A, B, C, D, E, F, G, H, S, U, and W; × 400 for panels I, J, K, L, T, V, and X. Bar = 800 μm. v, vitellarium; e, egg; vs, ventral sucker; tg, tegument; i, intestine; u, uterus; ts, testicle; o, ovary; p, pharynx; s, spermatheca; l, lumen; w, within the cells; <i>Cs</i>, <i>C</i>. <i>sinensis</i>; BE, biliary epithelium.</p

Rat anti-r<i>Cs</i>HK serum affects <i>C</i>. <i>sinensis</i> adult survival in vitro.

<p>(A) The median survival of <i>C</i>. <i>sinensis</i> adults in the blank control group, 1:40 pre-immune serum group, 1:80 pre-immune serum group, 1:160 pre-immune serum group, 1:40 anti-r<i>Cs</i>HK serum group, 1:80 anti-r<i>Cs</i>HK serum group, and 1:160 anti-r<i>Cs</i>HK serum group was 15, 8, 8, 9, 2, 3, and 3 days, respectively. There was no significant difference in survival rates among pre-immune serum groups at any dilution (<i>p</i> > 0.05). Significant differences were observed in the survival rates among the other groups (<i>p</i> < 0.05). (B) The enzymatic activity of <i>Cs</i>HK in homogenate of parasites collected from each group at 1, 3, 5, and 6 days of incubation. The enzymatic activity of <i>Cs</i>HK in adult worms incubated in medium with different dilutions of anti-r<i>Cs</i>HK serum declined significantly in a dose- and time-dependent manner. (C) As a control, there was no obvious change of the enzymatic activity of <i>Cs</i>PLA₂ in the worms.</p

Additional file 1: Figure S1. of Sequence analysis and characterization of pyruvate kinase from Clonorchis sinensis, a 53.1-kDa homopentamer, implicated immune protective efficacy against clonorchiasis

Putative tertiary modelling of CsPK. K+ and Mg2+ ions are shown as grey and black spheres, respectively. The N-terminal domain is shown in black. a Ribbon drawing of superposed structure models of CsHK (darker tone) and truncated TgPK1 (lighter tone). The A, B, and C domains of CsHK are shown in blue, red and green, respectively. The catalytic site at the interface of domains A and B and the allosteric site in domain C are highlighted. b Ribbon representation of F16BP (red stick) binding sites of human PK-M2 (lighter tone). S434, S437 (yellow stick), W482 (magenta stick), and R489 (orange stick), which interact with the phosphate moieties, are indicated. The putative corresponding structure of CsPK (darker tone) is shown in panel c. In the active site signature of PK, I267 and S269 (dark red sticks) are replaced by L205 and A207 (blue-violet sticks) in CsHK. Oxalate is indicated as a ball model. d Ribbon representation of the K+-PK-MgIIoxalate-MgIIATP complex closed active site (rabbit PK-M1). ATP, oxalate, and significant residues are shown as red, magenta, and yellow sticks, respectively. e Ribbon drawing of the superposition between the A domains of CsPK with the closed rabbit PK-M1 in complex with ATP and oxalate (dark and lighter tones, respectively). The corresponding significant residues of CsPK are shown in orange (stick). K+ and Mg2+ ions, ATP and oxalate, are shown for reference, with their positions derived from a superposition with 1A49. f Ribbon drawing of superposed structural models of CsPK (darker tone) and LmPYK-suramin (lighter tone) complexed with glycerol (magenta stick) and suramin (an inhibitor of T. brucei glycolytic enzymes, red stick). g Enlargement of the active site of the LmPYK-suramin structure. Significant residues are coloured yellow (stick). The putative corresponding structure of CsPK is shown in panel h. The corresponding significant residues of CsPK are coloured orange (stick). (TIFF 2964 kb

Summary of the <i>C. sinensis</i> genome assembly.

<p>Ver. 2 and Ver. 1 indicate the upgraded genome and the previously published genome, respectively.</p>*<p>N50 and N90 size of contigs or scaffolds were calculated by ordering all sequences and then adding the lengths from the longest to the shortest until the summed length exceeded 50% and 90% of the total length of all sequences.</p

Evolutionary analysis of the signal peptide peptidase gene.

<p>Footnote: (A) Phylogenetic tree constructed from the SPP of different species including <i>C. sinensis</i> (csin112590), <i>O. viverrini</i> (Ov_Contig2040), <i>F. hepatica</i> (Contig19227), <i>Fasciola gigantica</i> (Fh_Contig1), <i>S. japonicum</i> (gi:256082245), <i>S. mansoni</i> (gi:226482537), <i>Echinococcus multilocularis</i> (gi:194212416), <i>Bursaphelenchus xylophilus</i> (BUX_s00862.17) and <i>Plasmodium falciparum</i> (PF3D7_1457000). Using a sliding window method, dN/dS was calculated in <i>C. sinensis vs. O. viverrini</i> (B), <i>Fasciola gigantica vs. Fasciola hepatica</i> (C), and <i>Equus caballus vs. Homo sapiens</i> (D). The window size was 60 nt and the step size was 9 nt. dN, dS and ω (dN/dS) were shown in blue, red and green lines, respectively. Bars in dark green represented transmembrane domains, and bars in brown represented peptidase A22B.</p

The <i>C. sinensis</i> metabolic pathway.

<p>The adult <i>C. sinensis</i> worm makes full use of bile fatty acids, sugars and amino acids as energy sources. All genes present in the citrate cycle and oxidative phosphorylation pathways were highly expressed, implying that the adult worm could obtain large amounts ATPs from aerobic respiration. (A) Fatty acid elongation. (B) Fatty acid metabolism. (C) Citrate cycle (TCA cycle). (D) Acetate:succinate cycle. (E) Fumarate reduction pathway. (F) Glycolysis/Gluconeogenesis. (G) Amino acid metabolism related to energy metabolism. (H) Oxidative phosphorylation.</p