Molecular characterization of cathepsin B from Clonorchis sinensis excretory/secretory products and assessment of its potential for serodiagnosis of clonorchiasis

<p>Abstract</p> <p>Background</p> <p>Cathepsin cysteine proteases play multiple roles in the life cycle of parasites such as food uptake, immune invasion and pathogenesis, making them valuable targets for diagnostic assays, vaccines and drugs. The purpose of this study was to identify a cathepsin B of <it>Clonorchis sinensis </it>(<it>Cs</it>CB) and to investigate its diagnostic value for human helminthiases.</p> <p>Results</p> <p>The predicted amino acid sequence of the cathepsin B of <it>C. sinensis </it>shared 63%, 52%, 50% identity with that of <it>Schistosoma japonicum</it>, <it>Homo sapiens </it>and <it>Fasciola hepatica</it>, respectively. Sequence encoding proenzyme of <it>Cs</it>CB was overexpressed in <it>Escherichia coli</it>. Reverse transcription PCR experiments revealed that <it>Cs</it>CB transcribed in both adult worm and metacercaria of <it>C. sinensis</it>. <it>Cs</it>CB was identified as a <it>C. sinensis </it>excretory/secretory product by immunoblot assay, which was consistent with immunohistochemical localization showing that <it>Cs</it>CB was especially expressed in the intestine of <it>C. sinensis </it>adults. Both ELISA and western blotting analysis showed recombinant <it>Cs</it>CB could react with human sera from clonorchiasis and other helminthiases.</p> <p>Conclusions</p> <p>Our findings revealed that secreted CsCB may play an important role in the biology of C. sinensis and could be a diagnostic candidate for helminthiases.</p

Molecular identification of Clonorchis sinensis and discrimination with other opisthorchid liver fluke species using multiple Ligation-depended Probe Amplification (MLPA)

<p>Abstract</p> <p>Background</p> <p>Infections with the opisthorchid liver flukes <it>Clonorchis sinensis</it>, <it>Opisthorchis viverrini</it>, and <it>O. felineus </it>cause severe health problems globally, particularly in Southeast Asia. Early identification of the infection is essential to provide timely and appropriate chemotherapy to patients.</p> <p>Results</p> <p>In this study we evaluate a PCR-based molecular identification method, Multiplex Ligation-dependent Probe Amplification (MLPA), which allows rapid and specific detection of single nucleotide acid differences between <it>Clonorchis sinensis</it>, <it>Opisthorchis viverrini </it>and <it>O. felineus</it>. Three probe pairs were derived from the Internally Transcribed Spacer 1 (ITS1) of three opisthorchid liver flukes using a systematic phylogenetic analysis. Specific loci were detected in all three species, yielding three amplicons with 198,172 and 152 bp, respectively, while no cross reactions were observed. A panel of 66 <it>C. sinensis </it>isolates was screened using MLPA. All species were positively identified, and no inhibition was observed. The detection limit was 10³copies of the ITS gene for the three liver flukes, or about 60 pg genomic DNA for <it>Clonorchis sinensis</it>. Amplification products can be detected by electrophoresis on agarose gel or in a capillary sequencer. In addition, genomic DNA of <it>Clonorchis sinensis </it>in fecal samples of infected rats was positively amplified by MLPA.</p> <p>Conclusion</p> <p>The flexibility and specificity make MLPA a potential tool for specific identification of infections by opisthorchid liver flukes in endemic areas.</p

Sequence Analysis and Molecular Characterization of <i>Clonorchis sinensis</i> Hexokinase, an Unusual Trimeric 50-kDa Glucose-6-Phosphate-Sensitive Allosteric Enzyme

<div><p>Clonorchiasis, which is induced by the infection of <i>Clonorchis sinensis</i> (<i>C. sinensis</i>), is highly associated with cholangiocarcinoma. Because the available examination, treatment and interrupting transmission provide limited opportunities to prevent infection, it is urgent to develop integrated strategies to prevent and control clonorchiasis. Glycolytic enzymes are crucial molecules for trematode survival and have been targeted for drug development. Hexokinase of <i>C. sinensis</i> (<i>Cs</i>HK), the first key regulatory enzyme of the glycolytic pathway, was characterized in this study. The calculated molecular mass (Mr) of <i>Cs</i>HK was 50.0 kDa. The obtained recombinant <i>Cs</i>HK (r<i>Cs</i>HK) was a homotrimer with an Mr of approximately 164 kDa, as determined using native PAGE and gel filtration. The highest activity was obtained with 50 mM glycine-NaOH at pH 10 and 100 mM Tris-HCl at pH 8.5 and 10. The kinetics of r<i>Cs</i>HK has a moderate thermal stability. Compared to that of the corresponding negative control, the enzymatic activity was significantly inhibited by praziquantel (PZQ) and anti-r<i>Cs</i>HK serum. r<i>Cs</i>HK was homotropically and allosterically activated by its substrates, including glucose, mannose, fructose, and ATP. ADP exhibited mixed allosteric effect on r<i>Cs</i>HK with respect to ATP, while inorganic pyrophosphate (PPi) displayed net allosteric activation with various allosteric systems. Fructose behaved as a dose-dependent V activator with the substrate glucose. Glucose-6-phosphate (G6P) displayed net allosteric inhibition on r<i>Cs</i>HK with respect to ATP or glucose with various allosteric systems in a dose-independent manner. There were differences in both mRNA and protein levels of <i>Cs</i>HK among the life stages of adult worm, metacercaria, excysted metacercaria and egg of <i>C. sinensis</i>, suggesting different energy requirements during different development stages. Our study furthers the understanding of the biological functions of <i>Cs</i>HK and supports the need to screen for small molecule inhibitors of <i>Cs</i>HK to interfere with glycolysis in <i>C. sinensis</i>.</p></div

Low Divergence of <i>Clonorchis sinensis</i> in China Based on Multilocus Analysis

<div><p><i>Clonorchis sinensis</i>, an ancient parasite that infects a number of piscivorous mammals, attracts significant public health interest due to zoonotic exposure risks in Asia. The available studies are insufficient to reflect the prevalence, geographic distribution, and intraspecific genetic diversity of <i>C. sinensis</i> in endemic areas. Here, a multilocus analysis based on eight genes (ITS1, <i>act</i>, <i>tub</i>, <i>ef-1a</i>, <i>cox1</i>, <i>cox3</i>, <i>nad4</i> and <i>nad5</i> [4.986 kb]) was employed to explore the intra-species genetic construction of <i>C. sinensis</i> in China. Two hundred and fifty-six <i>C. sinensis</i> isolates were obtained from environmental reservoirs from 17 provinces of China. A total of 254 recognized Multilocus Types (MSTs) showed high diversity among these isolates using multilocus analysis. The comparison analysis of nuclear and mitochondrial phylogeny supports separate clusters in a nuclear dendrogram. Genetic differentiation analysis of three clusters (A, B, and C) showed low divergence within populations. Most isolates from clusters B and C are geographically limited to central China, while cluster A is extraordinarily genetically diverse. Further genetic analyses between different geographic distributions, water bodies and hosts support the low population divergence. The latter haplotype analyses were consistent with the phylogenetic and genetic differentiation results. A recombination network based on concatenated sequences showed a concentrated linkage recombination population in <i>cox1</i>, <i>cox3, nad4</i> and <i>nad5</i>, with spatial structuring in ITS1. Coupled with the history record and archaeological evidence of <i>C. sinensis</i> infection in mummified desiccated feces, these data point to an ancient origin of <i>C. sinensis</i> in China. In conclusion, we present a likely phylogenetic structure of the <i>C. sinensis</i> population in mainland China, highlighting its possible tendency for biogeographic expansion. Meanwhile, ITS1 was found to be an effective marker for tracking <i>C. sinensis</i> infection worldwide. Thus, the present study improves our understanding of the global epidemiology and evolution of <i>C. sinensis</i>.</p></div

mRNA and protein levels of <i>Cs</i>HK at life stages of <i>C. sinensis</i>.

<p>(A) Real-time PCR analysis. The mRNA of β-actin of <i>C. sinensis</i> was used as an internal control. Semiquantitative analysis as performed by the comparative 2^−ΔΔCt method. There was no significant difference between the adult worm and metacercaria (<i>p</i>>0.05). There were statistical differences in the mRNA levels of <i>Cs</i>HK not only between adult worm and encysted metacercaria (<i>p</i><0.05) but also between metacercaria and encysted metacercaria (<i>p</i><0.05). The mRNA level of <i>Cs</i>HK in the egg was higher than that in the adult worm (13.80-fold, <i>p</i><0.01), metacercaria (13.29-fold, <i>p</i><0.01) and excysted metacercaria (8.72-fold, <i>p</i><0.01). (B) Western blotting analysis. 50 µg of total proteins of each stage were blotted with mouse anti-r<i>Cs</i>HK serum (1∶200 dilution). Specific protein bands around 50 kDa were probed. There was no corresponding band in negative lanes (not shown). (C) The relative quantitation of protein levels were analyzed by Tanon Gis software. The protein level of <i>Cs</i>HK in the egg was the highest, followed by those of excysted metacercaria, metacercaria, and adult worm. The protein levels were consistent with the mRNA levels. The experiments were repeated three times. (#, <i>p</i>>0.05; *, <i>p</i><0.05; **, <i>p</i><0.01).</p

Determination of the Mr of r<i>Cs</i>HK.

<p>(A). M, the protein bands with known molecular mass (HMW Native, GE Healthcare, USA) were stained with Coomassie blue in descending order: thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa), and bovine serum albumin (67 kDa). a, Fresh purified r<i>Cs</i>HK. b, r<i>Cs</i>HK stored for 4 weeks at −80°C with four cycles of freezing and thawing. (B). The Mr of r<i>Cs</i>HK as determined by 8% native PAGE. The HRm values of the molecular markers were plotted against their Mr. The equation and curve were drawn. The Mr of r<i>Cs</i>HK was calculated using the obtained equation. (C). Elution profile of r<i>Cs</i>HK in gel filtration chromatography. (D). Mr of r<i>Cs</i>HK as determined by the elution profile. The calibration curve between the elution volume (Ve) and the log molecular mass (kDa) of standard marker proteins was obtained by AKTA FPLC using a sepharose 12 10/300 GL gel filtration column. The Ve of r<i>Cs</i>HK was obtained, and the Mr of r<i>Cs</i>HK was calculated using the deduced equation.</p

Enzyme activity analysis of r<i>Cs</i>HK in reaction system A.

<p>(A) Optimal pH analysis for r<i>Cs</i>HK. Different buffers were used to generate a pH range from 6.0 to 13.0 (50 mM glycine-NaOH and 100 mM Tris-Cl). (B) Enzymatic activity of r<i>Cs</i>HK at 20°C–60°C. (C) Thermal stability of the enzyme measured at time points at 50°C. (D) The effects of divalent cations (Mg²⁺, Mn²⁺, Ca²⁺, Zn²⁺, or Cu²⁺) on the enzymatic activity of r<i>Cs</i>HK. (E) The effect of praziquantel (PZQ). Equal volume of anhydrous ethanol as a control. (F) The effect of mouse anti-r<i>Cs</i>HK serum. The serum from a pre-immune mouse was used as a negative control. The dilutions of the sera ranged from 1∶640 to 1∶5.</p