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Molecular characterization and expression of a cysteine protease from Clonorchis sinensis and its application for serodiagnosis of clonorchiasis

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Abstract

Cysteine proteases play essential roles in parasite physiology as well as in host–parasite interactions through their modulation of various biological and pathobiological events. In the present study, a full-length sequence encoding cysteine protease of Clonorchis sinensis (CsCP) was isolated from our adult cDNA library. The open reading frame contains 984 bp encoding 327 amino acids. The present amino acid sequence shared 68% identity with two known CsCP genes and 29–49% identity with that of other species. Bioinformatics analysis showed that conserved domains and characteristic amino acid residues of cysteine proteases were observed in this sequence. Real-time PCR experiments revealed that CsCP was consecutively transcribed in various developmental stages of the parasite, including adult worm, excysted juvenile, metacercaria and egg. Recombinant CsCP (rCsCP) could be probed by rat anti-CsCP serum, rabbit anti-excretory–secretory products (ESP) serum and serum from human infected with Clonorchis sinensis in Western blot. The result of immunolocalization showed that CsCP was mainly located in the oral sucker, excretory bladder and tegument of cercariae and metacercariae, as well as the intestine of adult worm. The rCsCP-based IgG and its isotypes were all detected in sera from human infected with C. sinensis by enzyme-linked immunosorbent assay, and the level of IgG1 is the highest. The receiver-operating characteristic (ROC) analysis was used to determine the most appropriate cut-off value that yielded the high sensitivity (86.96%) and specificity (70.42%). These results revealed that CsCP may play an important role in the biology of C. sinensis and could be a diagnostic candidate for clonorchiasis.

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References

  • Bruneau C, Bonin H (1983) Evidence for a disease specific antigen in circulating immune complexes in ankylosing spondylitis. Clin Exp Immunol 53(3):529–535

    PubMed  CAS  Google Scholar 

  • Carnevale S, Rodriguez MI, Guarnera EA, Carmona C, Tanos T, Angel SO (2001) Immunodiagnosis of fasciolosis using recombinant procathepsin L cystein proteinase. Diagn Microbiol Infect Dis 41(1–2):43–49

    Article  PubMed  CAS  Google Scholar 

  • Chen W, Wang X, Deng C, Lv X, Fan Y, Men J, Liang C, Yu X (2011) Molecular cloning and characterization of a novel ras-related protein (rap2) from Clonorchis sinensis. Parasitol Res 108(4):1021–1026

    Article  PubMed  Google Scholar 

  • Choi MH, Park IC, Li S, Hong ST (2003) Excretory–secretory antigen is better than crude antigen for the serodiagnosis of clonorchiasis by ELISA. Korean J Parasitol 41(1):35–39

    Article  PubMed  Google Scholar 

  • Choi BI, Han JK, Hong ST, Lee KH (2004) Clonorchiasis and cholangiocarcinoma: etiologic relationship and imaging diagnosis. Clin Microbiol Rev 17(3):540–552, table of contents

    Article  PubMed  Google Scholar 

  • Delcroix M, Sajid M, Caffrey CR, Lim KC, Dvorak J, Hsieh I, Bahgat M, Dissous C, McKerrow JH (2006) A multienzyme network functions in intestinal protein digestion by a platyhelminth parasite. J Biol Chem 281(51):39316–39329

    Article  PubMed  CAS  Google Scholar 

  • DuBois KN, Abodeely M, Sakanari J, Craik CS, Lee M, McKerrow JH, Sajid M (2008) Identification of the major cysteine protease of Giardia and its role in encystation. J Biol Chem 283(26):18024–18031

    Article  PubMed  CAS  Google Scholar 

  • Fan Y, Wang X, Deng C, Huang Y, Wang L, Chen W, Liang C, Li X, Wu Z, Yu X (2011) Molecular cloning, expression, and immunolocalization of the NAD(+)-dependent glycerol 3-phosphate dehydrogenase (GPD) from Clonorchis sinensis. Parasitol Res 109(3):621–626

    Article  PubMed  Google Scholar 

  • Hagan P, Blumenthal UJ, Dunn D, Simpson AJ, Wilkins HA (1991) Human IgE, IgG4 and resistance to reinfection with Schistosoma haematobium. Nature 349(6306):243–245

    Article  PubMed  CAS  Google Scholar 

  • Han YP, Li ZY, Li BC, Sun X, Zhu CC, Ling XT, Zheng HQ, Wu ZD, Lv ZY (2011) Molecular cloning and characterization of a cathepsin B from Angiostrongylus cantonensis. Parasitol Res 109(2):369–378

    Article  PubMed  Google Scholar 

  • Ikeda T, Oikawa Y, Nishiyama T (1996) Enzyme-linked immunosorbent assay using cysteine proteinase antigens for immunodiagnosis of human paragonimiasis. Am J Trop Med Hyg 55(4):435–437

    PubMed  CAS  Google Scholar 

  • Ju JW, Joo HN, Lee MR, Cho SH, Cheun HI, Kim JY, Lee YH, Lee KJ, Sohn WM, Kim DM, Kim IC, Park BC, Kim TS (2009) Identification of a serodiagnostic antigen, legumain, by immunoproteomic analysis of excretory–secretory products of Clonorchis sinensis adult worms. Proteomics 9(11):3066–3078

    Article  PubMed  CAS  Google Scholar 

  • Kaewpitoon N, Laha T, Kaewkes S, Yongvanit P, Brindley PJ, Loukas A, Sripa B (2008) Characterization of cysteine proteases from the carcinogenic liver fluke, Opisthorchis viverrini. Parasitol Res 102(4):757–764

    Article  PubMed  Google Scholar 

  • Keiser J, Utzinger J (2009) Food-borne trematodiases. Clin Microbiol Rev 22(3):466–483

    Article  PubMed  Google Scholar 

  • Kim EM, Verweij JJ, Jalili A, van Lieshout L, Choi MH, Bae YM, Lim MK, Hong ST (2009) Detection of Clonorchis sinensis in stool samples using real-time PCR. Ann Trop Med Parasitol 103(6):513–518

    Article  PubMed  CAS  Google Scholar 

  • Klinkert MQ, Bommert K, Moser D, Felleisen R, Link G, Doumbo O, Beck E (1991) Immunological analysis of cloned Schistosoma mansoni antigens Sm31 and Sm32 with sera of schistosomiasis patients. Trop Med Parasitol 42(4):319–324

    PubMed  CAS  Google Scholar 

  • Le TH, Van De N, Blair D, Sithithaworn P, McManus DP (2006) Clonorchis sinensis and Opisthorchis viverrini: development of a mitochondrial-based multiplex PCR for their identification and discrimination. Exp Parasitol 112(2):109–114

    Article  PubMed  CAS  Google Scholar 

  • Li S, Chung YB, Chung BS, Choi MH, Yu JR, Hong ST (2004) The involvement of the cysteine proteases of Clonorchis sinensis metacercariae in excystment. Parasitol Res 93(1):36–40

    Article  PubMed  Google Scholar 

  • Lightowlers MW, Rickard MD (1988) Excretory–secretory products of helminth parasites: effects on host immune responses. Parasitology 96(Suppl):S123–S166

    Article  PubMed  Google Scholar 

  • Lim MK, Ju YH, Franceschi S, Oh JK, Kong HJ, Hwang SS, Park SK, Cho SI, Sohn WM, Kim DI, Yoo KY, Hong ST, Shin HR (2006) Clonorchis sinensis infection and increasing risk of cholangiocarcinoma in the Republic of Korea. Am J Trop Med Hyg 75(1):93–96

    PubMed  Google Scholar 

  • Lun ZR, Gasser RB, Lai DH, Li AX, Zhu XQ, Yu XB, Fang YY (2005) Clonorchiasis: a key foodborne zoonosis in China. Lancet Infect Dis 5(1):31–41

    Article  PubMed  Google Scholar 

  • McKerrow JH, Caffrey C, Kelly B, Loke P, Sajid M (2006) Proteases in parasitic diseases. Annu Rev Pathol 1:497–536

    Article  PubMed  CAS  Google Scholar 

  • Na BK, Lee HJ, Cho SH, Lee HW, Cho JH, Kho WG, Lee JS, Song KJ, Park PH, Song CY, Kim TS (2002) Expression of cysteine proteinase of Clonorchis sinensis and its use in serodiagnosis of clonorchiasis. J Parasitol 88(5):1000–1006

    PubMed  CAS  Google Scholar 

  • Na BK, Kim SH, Lee EG, Kim TS, Bae YA, Kang I, Yu JR, Sohn WM, Cho SY, Kong Y (2006) Critical roles for excretory–secretory cysteine proteases during tissue invasion of Paragonimus westermani newly excysted metacercariae. Cell Microbiol 8(6):1034–1046

    Article  PubMed  CAS  Google Scholar 

  • Na BK, Kang JM, Sohn WM (2008) CsCF-6, a novel cathepsin F-like cysteine protease for nutrient uptake of Clonorchis sinensis. Int J Parasitol 38(5):493–502

    Article  PubMed  CAS  Google Scholar 

  • Nagano I, Pei F, Wu Z, Wu J, Cui H, Boonmars T, Takahashi Y (2004) Molecular expression of a cysteine proteinase of Clonorchis sinensis and its application to an enzyme-linked immunosorbent assay for immunodiagnosis of clonorchiasis. Clin Diagn Lab Immunol 11(2):411–416

    PubMed  CAS  Google Scholar 

  • Parvathi A, Sanath Kumar H, Kenchanna Prakasha B, Lu J, Xu X, Hu W, Feng Z, Karunasagar I (2007) Clonorchis sinensis: development and evaluation of a nested polymerase chain reaction (PCR) assay. Exp Parasitol 115(3):291–295

    Article  PubMed  CAS  Google Scholar 

  • Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29(9):e45

    Article  PubMed  CAS  Google Scholar 

  • Rahman SM, Bae YM, Hong ST, Choi MH (2011) Early detection and estimation of infection burden by real-time PCR in rats experimentally infected with Clonorchis sinensis. Parasitol Res 109(2):297–303

    Article  PubMed  Google Scholar 

  • Robinson MW, Dalton JP, Donnelly S (2008) Helminth pathogen cathepsin proteases: it's a family affair. Trends Biochem Sci 33(12):601–608

    Article  PubMed  CAS  Google Scholar 

  • Sajid M, McKerrow JH (2002) Cysteine proteases of parasitic organisms. Mol Biochem Parasitol 120(1):1–21

    Article  PubMed  CAS  Google Scholar 

  • Schur PH (1987) IgG subclasses—a review. Ann Allergy 58(2):89–96, 99

    PubMed  CAS  Google Scholar 

  • Smooker PM, Jayaraj R, Pike RN, Spithill TW (2010) Cathepsin B proteases of flukes: the key to facilitating parasite control? Trends Parasitol 26(10):506–514

    Article  PubMed  CAS  Google Scholar 

  • Wang X, Chen W, Li X, Zhou C, Deng C, Lv X, Fan Y, Men J, Liang C, Yu X (2011) Identification and molecular characterization of a novel signaling molecule 14-3-3 epsilon in Clonorchis sinensis excretory/secretory products. Parasitol Res. doi:10.1007/s00436-011-2642-7

  • Wynn TA, Hoffmann KF (2000) Defining a schistosomiasis vaccination strategy — is it really Th1 versus Th2? Parasitol Today 16(11):497–501

    Article  PubMed  CAS  Google Scholar 

  • Yoo WG, Kim TI, Li S, Kwon OS, Cho PY, Kim TS, Kim K, Hong SJ (2009) Reference genes for quantitative analysis on Clonorchis sinensis gene expression by real-time PCR. Parasitol Res 104(2):321–328

    Article  PubMed  Google Scholar 

  • Zhao QP, Moon SU, Lee HW, Na BK, Cho SY, Kong Y, Jiang MS, Li AH, Kim TS (2004) Evaluation of Clonorchis sinensis recombinant 7-kilodalton antigen for serodiagnosis of clonorchiasis. Clin Diagn Lab Immunol 11(4):814–817

    PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by Development Program of China (973 program; No. 2010CB530000) and the Sun Yat-sen University innovative talents cultivation program for excellent tutors.

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All authors declare that they have no competing interests.

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Authors and Affiliations

  1. Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, 510080, China

    Xiaoli Lv, Wenjun Chen, Xiaoyun Wang, Xuerong Li, Jiufeng Sun, Chuanhuan Deng, Jingtao Men, Yanli Tian, Chenhui Zhou, Huali Lei, Chi Liang & Xinbing Yu

  2. Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, Guangdong, 510080, China

    Xiaoli Lv, Wenjun Chen, Xiaoyun Wang, Xuerong Li, Jiufeng Sun, Chuanhuan Deng, Jingtao Men, Yanli Tian, Chenhui Zhou, Huali Lei, Chi Liang & Xinbing Yu

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  1. Xiaoli Lv
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  2. Wenjun Chen
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  3. Xiaoyun Wang
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  4. Xuerong Li
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  9. Chenhui Zhou
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  10. Huali Lei
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  11. Chi Liang
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  12. Xinbing Yu
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Correspondence to Xinbing Yu.

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X. Lv and W. Chen contributed equally to this study.

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Lv, X., Chen, W., Wang, X. et al. Molecular characterization and expression of a cysteine protease from Clonorchis sinensis and its application for serodiagnosis of clonorchiasis. Parasitol Res 110, 2211–2219 (2012). https://doi.org/10.1007/s00436-011-2751-3

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