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

Identification and Characterization of Paramyosin from Cyst Wall of Metacercariae Implicated Protective Efficacy against Clonorchis sinensis Infection

Human clonorchiasis has been increasingly prevalent in recent years and results in a threat to the public health in epidemic regions, motivating current strategies of vaccines to combat Clonorchis sinensis (C. sinensis). In this study, we identified C. sinensis paramyosin (CsPmy) from the cyst wall proteins of metacercariae by proteomic approaches and characterized the expressed recombinant pET-26b-CsPmy protein (101 kDa). Bioinformatics analysis indicated that full-length sequences of paramyosin are conserved in helminthes and numerous B-cell/T-cell epitopes were predicted in amino acid sequence of CsPmy. Western blot analysis showed that CsPmy was expressed at four life stages of C. sinensis, both cyst wall proteins and soluble tegumental components could be probed by anti-CsPmy serum. Moreover, immunolocalization results revealed that CsPmy was specifically localized at cyst wall and excretory bladder of metacercaria, as well as the tegument, oral sucker and vitellarium of adult worm. Both immunoblot and immunolocalization results demonstrated that CsPmy was highly expressed at the stage of adult worm, metacercariae and cercaria, which could be supported by real-time PCR analysis. Both recombinant protein and nucleic acid of CsPmy showed strong immunogenicity in rats and induced combined Th1/Th2 immune responses, which were reflected by continuous high level of antibody titers and increased level of IgG1/IgG2a subtypes in serum. In vaccine trials, comparing with control groups, both CsPmy protein and DNA vaccine exhibited protective effect with significant worm reduction rate of 54.3% (p<0.05) and 36.1% (p<0.05), respectively. In consistence with immune responses in sera, elevated level of cytokines IFN-γ and IL-4 in splenocytes suggested that CsPmy could induce combined cellular immunity and humoral immunity in host. Taken together, CsPmy could be a promising vaccine candidate in the prevention of C. sinensis regarding its high immunogenicity and surface localization

Immunohistochemical localization of <i>Cs</i>Pmy at adult worm and metacercaria.

<p>Adult worms and metacercariae of <i>C. sinensis</i> were fixed with 4% paraformaldehyde, embedded with paraffin and sliced into 3–5 µm in thick. The sections were blocked with normal goat serum overnight at 4°C, and then incubated with primary antibody (1∶200 dilutions) at room temperature for 2 h. After washing procedures, the sections were incubated with goat anti-rat IgG Alexa Fluor 594 (1∶400 dilutions) at room temperature for 1 h in dark. The images were captured under fluorescence microscope (ZEISS, Goettingen, Germany). Panel A–H, adult worm of <i>C. sinensis</i>. Pane I–L, metacercariae of <i>C. sinensis</i>. Pane A, B, E, F, I and J were sections treated with anti-pET26b-<i>Cs</i>Pmy serum. C, D, G, H, K and L were sections treated with naïve serum and imaged under the same conditions. Specific immunofluorescence was indicated in red (pane A, E and I), while no immunofluorescence was detected in pane C, G and K. Corresponding white light of parasite was panel B, D, F, H, J and L. <b>T</b>, tegument. <b>OS</b>, oral sucker. <b>V</b>, vitellarium. <b>CW</b>, cyst wall. <b>EB</b>, excretory bladder. Magnification for adult worm and metacercaria were ×100 and ×400, respectively.</p

Protective effect of in vaccination trials^a.

a<p>Protective effect was assessed by comparing the worm burden and EPG between pET-26b-<i>Cs</i>Pmy group and PBS group, as well as pcDNA-<i>Cs</i>Pmy group and pcDNA group, respectively. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033703#s3" target="_blank">Results</a> for analysis represented mean ± S.D., and the recovered worm numbers and EPG in groups were compared by Student's <i>t</i>-test.</p>(*)<p><i>p</i><0.05 and.</p>(**)<p><i>p</i><0.01 (compared to corresponding control).</p

IgG isotype induced by <i>Cs</i>Pmy measured by ELISA.

<p>1 µg/well recombinant pET-26b-<i>Cs</i>Pmy protein was coated on the plates and blocked with 5% skimmed milk. Immune sera from week 2 to week 6 were diluted at 1∶400. Rat sera immunized with PBS and pcDNA were measured under the same conditions as negative controls. IgG (1∶20000 dilutions), IgG1 and IgG2a (1∶1000 dilutions) were used as secondary antibodies. (A) Immune responses induced by pET-26b-<i>Cs</i>Pmy. (B) Immune responses induced by pcDNA-<i>Cs</i>Pmy.</p

Identification of <i>Cs</i>Pmy by SDS-PAGE and Western blot analysis.

<p>Protein molecular weight marker (M), purified pET-26b-<i>Cs</i>Pmy protein (lane 1), TWE of adult worm (lane 2), TWE of metacercaria (lane 3), TWE of cercaria (lane 4), TWE of egg (lane 5), cyst wall proteins of metacercaria (lane 6), and soluble tegumental components of adult worm (lane 7). (A) 8% SDS-PAGE. (B) Western blot analysis. Corresponding proteins were subjected to 8% SDS-PAGE and immobilized onto the membrane, then the membrane was incubated with anti-pET-26b-<i>Cs</i>Pmy rat serum (1∶2000 dilutions) at room temperature for 2 h. Subsequently, the membrane was followed by incubation with rabbit anti-rat IgG HRP-conjugated secondary antibody (1∶2000 dilutions) at room temperature for 1 h. 2 µg of purified pET-26b-<i>Cs</i>Pmy protein and 10 µg of TWE were loaded per lane. SDS-PAGE was visualized by Coomassie Blues staining and the protein bands that might be native paramyosin in different life stages were indicated with arrows. Western blot was visualized by ECL method, the detected protein bands were around 100 kDa.</p

Multiple sequence alignment of deduced amino acid sequence of paramyosin among helminthes.

<p><i>C. s-</i>1 (JQ041818) represents the sequence from our <i>C. sinensis</i> metacercaria cDNA plasmid library. <i>C. s-</i>2 (ABN79674.1) represents the sequence submitted by the laboratory from Korea. <i>Paragonimus westermani</i> (<i>P. w</i>, AAY44740.1), <i>Schistosoma haematobium</i> (<i>S. h</i>, BAF62291.1), <i>Schistosoma japonicum</i> (<i>S. j</i>, AAA81003.1), <i>Schistosoma mansoni</i> (<i>S. m</i>, AAA29915.1), <i>Taenia solium</i> (<i>T. s</i>, AAK58494.1) and <i>Echinococcus granulosus</i> (<i>E. g</i>, CAA79849.1). Amino acids shared among helminthes were indicated in black, high conserved amino acids among helminthes were indicated in gray. B-cell and T-cell linear epitopes were indicated with full lines and dotted lines, respectively.</p

Expression and purification of recombinant pET-26b-<i>Cs</i>Pmy identified by 8% SDS-PAGE.

<p>(A) Expression of pET-26b-<i>Cs</i>Pmy. Protein molecular weight marker (M), lysate of <i>E. coli</i> with pET-26b(+) before induction with IPTG (lane 1) and after induction (lane 2), lysate of <i>E. coli</i> with pET-26b-<i>Cs</i>Pmy before induction with IPTG (lane 3) and after induction (lane 4), supernatant of induced <i>E. coli</i> with pET-26b-<i>Cs</i>Pmy (lane 5) and sediment (lane 6). (B) Denaturation of inclusion bodies containing pET-26b-<i>Cs</i>Pmy. Supernatant collected from inclusion bodies dissolved in 2 M urea (lane 1) and 6 M urea (lane 2). (C) Purification of pET-26b-<i>Cs</i>Pmy. Protein eluted with 40 mM imidazole (lane 1–3), 80 mM imidazole (lane 4–8), 100 mM imidazole (lane 9–12), 200 mM imidazole (lane 13–14). Proteins were visualized by Coomassie Blue staining, the protein bands were around 100 kDa.</p