Molecular characterization of cathepsin B from Clonorchis sinensis excretory/secretory products and assessment of its potential for serodiagnosis of clonorchiasis
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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 Clonorchis sinensis (Cs CB) and to investigate its diagnostic value for human helminthiases.
The predicted amino acid sequence of the cathepsin B of C. sinensis shared 63%, 52%, 50% identity with that of Schistosoma japonicum, Homo sapiens and Fasciola hepatica, respectively. Sequence encoding proenzyme of Cs CB was overexpressed in Escherichia coli. Reverse transcription PCR experiments revealed that Cs CB transcribed in both adult worm and metacercaria of C. sinensis. Cs CB was identified as a C. sinensis excretory/secretory product by immunoblot assay, which was consistent with immunohistochemical localization showing that Cs CB was especially expressed in the intestine of C. sinensis adults. Both ELISA and western blotting analysis showed recombinant Cs CB could react with human sera from clonorchiasis and other helminthiases.
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.
KeywordsCysteine Protease Adult Worm Praziquantel Cysticercosis Schistosoma Japonicum
Clonorchis sinensis is the causative agent of clonorchiasis, a chronic liver infection of human acquired through consumption of raw or undercooked fish and shrimps with infectious metacercariae. Clonorchiasis is endemic in Asian countries and over 35 million people globally are infected C. sinensis, including an estimated 15 million in People's Republic of China . Recently, this infection has emerged in non-endemic regions and developed countries following growing international markets, improved transportation systems and demographic changes such as population movements . C. sinensis adults reside chronically in the biliary tract and cause periductal inflammation, fibrosis, pyogenic cholangitis, biliary calculi, cholecystitis, liver cirrhosis and pancreatitis . Like Opisthorchis viverrini, C. sinensis is one of the direct causes of cholangiocarcinoma announced by the International Agency for Research on Cancer (IARC) in 2009 . It is important to take some measures to control clonorchiasis due to its public health threat. Until now, the main prevention and control strategies for this parasite are treatment of individual patients with praziquantel, and interrupting transmission at the intermediate host level . However, there have been little effective measures to prevent this neglected tropical disease .
Cysteine proteinase is ubiquitous in all species [7, 8, 9]. In parasites, cysteine proteases have attracted much attention for their essential roles in parasite physiology as well as in host-parasite interactions through their modulation of various pathobiological events, including host tissue invasion, nutrient uptake, host immune evasion and molting [10, 11, 12, 13]. Research has been conducted to characterize the biochemical properties and pathophysiological roles of cysteine proteases from trematode parasites. The essential roles of cysteine proteases in parasite survival or growth make them attractive targets for vaccines or chemotherapeutic agents [14, 15, 16]. Several genes encoding C. sinensis cysteine proteases have been identified and partially characterized [17, 18, 19]. Lee et al.  reported that cathepsin F-like cysteine protease of C. sinensis is a good vaccine candidate against clonorchiasis. Li et al.  found that endogenous cysteine proteases of C. sinensis metacercariae are probably involved in the excystment process. Kang et al.  indicated that partially purified cysteine protease from excretory/secretory products (ESP) of C. sinensis adults exhibited significant cytotoxic effects against cultured cells. ESP of parasites have attracted more attention for their significant roles in the diagnosis, vaccine, drug target and host-parasite interactions etc. In vitro biochemical studies have predicted that ESP from liver flukes have definitive roles in feeding behavior, detoxification of bile components and immune evasion . Ju et al.  have identified legumain from ESP as a serodiagnostic antigen of clonorchiasis. In addition, several genes encoding C. sinensis cysteine proteases have also been identified and their value as diagnostic antigens for clonorchiasis was investigated [24, 25]. However, little is known about cathepsin B (CB) in C. sinensis except five distinct sequences deposited in Genbank.
As members of the cysteine protease family, cathepsins have been assayed in the serodiagnosis of both human and animal in parasite infections. Cornelissen et al.  reported a specificity of 75.3% in naturally infected cattle using Fasciola hepatica cathepsin L as coating antigen. Carnevale et al.  found that recombinant pro-cathepsin-L was 100% specific in the diagnosis of human F. hepatica infection. Sripa et al.  indicated that Ov-CB-1 was acceptable in ELISA for the serodiagnosis of human opisthorchiasis with 67% and 81% of sensitivity and specificity, respectively. To find out whether CB could be applied for serodiagnosis in C. sinensis infection, we identified a gene encoding cathepsin B of C. sinensis (Cs CB) and investigated its diagnostic value for human helminthiases.
Sequence analysis of Cs CB gene sequence
Cloning, expression and purification of recombinant Cs CB (rCs CB) in E. coli
Identification of Cs CB as ESP by western blotting
Rat anti-rCs CB antibody titers were higher than 1:102,400 as determined by ELISA. In western blotting assay, both C. sinensis- infected rat serum and anti-Cs CB rat serum could react with the Cs CB while the normal rat serum could not. Comparing with the control group, sera from rats immunized with total ESP could recognize Cs CB. In addition, ESP has been shown to react with anti-Cs CB rat serum (data not shown). The results above indicated that Cs CB was a component of ESP (Figure 2B).
RT-PCR analysis of Cs CB at life-stage of C.sinensis
Immunohistochemical localization of CsCB in C. sinensis adults
Serodiagnosis of human helminthiases
Summary of difference between rCs CB ELISA and stool examinations results.
No. of samples from stool examination
No. of serum samples
No. positive of Cs CB ELISA
Cathepsins in general are of interest to parasitologists, as there is considerable evidence that they play a key role in the biology of parasites . In this study, a CB of C. sinensis was cloned and overexpressed in E. coli. It was classified as CB due to its sequence homology to cathepsin B protein and structure. The putative amino acid sequence shared 63%, 52% and 50% identities with cathepsin B from S. japonicum, H. sapiens and F. hepatica, respectively. Sequence analysis showed that Cs CB has typical catalytic residue of cysteine, histidine and asparagine, as well an occluding loop that is the signature of cathepsin Bs . A haemoglobinase motif which is shared by helminth blood-feeders could be found in this deduced sequence . Since C. sinensis generally feed on bile and epithelial cells rather than blood, however, it is thought that this motif may be an important tool for identifying potential hemoglobinases and contribute to haemoglobin degradation . The occluding loop is a distinctive feature of CBs; it is not only responsible for exopeptidase activity to CBs, but also governing the pH dependence of auto-activation. In F. hepatica, Beckham  reported that recombinant FhCatB1 did not auto-activate upon secretion by yeast but could be auto-activated in a low pH buffer. As it stands, Cs CB may develop auto-activation in an appropriate pH buffer, so further studies will be carried out to investigate the auto-activation and enzyme activity of this protease.
Cathepsin B proteases of parasitic helminths such as F. hepatica and Schistosoma mansoni have been well studied. In F. hepatica, for example, there are at least 10 genes encoding CB being identified by using proteomics and transcriptome analysis, among which six are components of ESP of immature flukes. RNAi directed to Fh CB2 reduced invasion of F. hepatica newly excysted juveniles through the intestine in vitro and vaccine with F. hepatica CB2 reduced fluke burdens and liver damage in rats following challenge, revealing that Fh CB2 was a key molecule in F. hepatica biology and drug therapy . In S. mansoni, Sm CB1 and Sm CB2 have been identified and the function of Sm CB1 was examined using RNAi silencing, which illustrated that Sm CB1 played a role in nutrient acquisition [35, 36]. Based on the key role of CB in the biology of parasites, we expect that Cs CB may play the same role in nutrition intake and immune invasion of host. In multicellular parasites such as trematodes, the intestine is a major source of secreted proteases and also a place for nutrition digestion and absorption. According to results of immunolocalization, Cs CB was distributed in the intestine of adult worm, indicating that Cs CB might be involved in digestion of host protein and nutrient uptake for this parasite itself. In our RT-PCR experiments, Cs CB could be detected in both adult worm and metacercaria of C. sinensis. As a secreted protease, these observations suggested that Cs CB may play an important role in the biology of this parasite.
Proteases contained in ESP of parasites released to the environment, play key roles in bile duct malignancy and the subsequent development of cholangiocarcinoma [37, 38]. Our previous studies revealed that ESP of adult C. sinensis lysophospholipase and phospholipase A2 might be pathogenic factors of human hepatic fibrosis caused by infection of C. sinensis[39, 40], which could deepen our understanding of the pathogenesis of C. sinensis. CB has already been found in ESP of C. sinensis and some other helminths . Moreover, western blotting revealed that Cs CB was a component of ESP, and Cs CB could react with C. sinensis-infected rat serum. In ELISA results, rats injected with recombinant Cs CB developed high antibody titers. Our results showed that Cs CB might be involved in the pathogenesis of C. sinensis-related hepatobiliary diseases as a component of ESP with antigenicity and immunogenicity. Indeed, C. sinensis adults dwell in the bile duct of host, persistently released ESP resulting in mechanical damage and chemical stimulus which must have brought on infiltration of inflammatory cells surrounding bile ducts and adenomatous hyperplasia of biliary epithelia [42, 43, 44]. However, there remains much to do to elucidate the mechanism of C. sinensis causing hepatobiliary diseases. We have also approached the antigenicity and diagnostic value of Cs CB in human helminthiases. Both the ELISA and western blotting showed that Cs CB could react with sera from most of human helminthiases, but not with sera from healthy people. High identity in the predicted amino acid sequences of C. sinensis and other helminths could be responsible for the observed cross-reactivity. Though cross-reactivity existed, Cs CB could also be applied as a diagnostic candidate of clonorchiasis since treatment with praziquantel has the same efficacy on helminths.
A gene encoding cathepsin B protein of adult C. sinensis was cloned and expressed for the first time. Our findings revealed that Cs CB, which is expressed in the intestine and released outside the worm as a component of ESP, may play an important role in the biology of C. sinensis. Moreover, CB may play conserved roles in helminths for the high homology analyzed by bioinformatics. Further investigations are required to characterize the conserved functions of such important protease and the role as a potential vaccine candidate against C. sinensis infection.
Materials and methods
Sequence analysis of Cs CB gene sequence
A complete coding sequence encoding CB was isolated from GenBank (Accession no. EF102086.1) in NCBI http://www.ncbi.nlm.nih.gov/, the physicochemical properties and the functional domains in deduced amino acids were predicted by proteomics tools in ExPaSy web site http://www.expasy.org/. Based on the similarity, the homologous sequences of different species, including E. multilocularis (BAJ83491.1), F. hepatica(ABU62925.1), S. japonicum (CAX71086.1), H. sapiens (NP_001899.1), Paragonimus westermani (AAB93494.1) and Taenia solium (AAS00027.1) were identified using the basic local alignment search tool (BLAST) server. Multiple sequences alignments were performed using bioinformatics analysis software Vector NTI suite 8.0.
Cloning, expression, purification and refolding of Cs CB in E. coli
Gene sequence encoding Cs CB (signal peptide excluded) was amplified from cDNA of C. sinensis by polymerase chain reaction (PCR) using forward primer: 5'-CGCGGATCC GAGTATATTCCATCTTTCGA-3' and the reverse primer: 5'-GTCCTCGAG TCACAGTTTTGGATGACC-3' with BamH I/Xho I restriction enzyme sites (underlined). PCR was carried out for 30 cycles at 94°C for 45 s, 57°C for 45 s, and 72°C for 60 s, and the reaction continued for 10 min at 72°C after the last cycle. Purified PCR products were cloned into the His6 tag expression vector pET-28a(+) (Novagen; USA) with corresponding incision enzymes. The recombinant plasmid was transformed into E. coli for expression and insertion confirmed by digestion with restriction enzyme and DNA sequencing. Expression of recombinant Cs CB protein was induced by isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.2 mM for 3 h at 30°C. The bacterial cells were collected by centrifugation at 4°C, and the inclusion bodies containing the recombinant fusion protein were solubilized completely with 6 M urea in 20 mM Tris-HCl buffer (pH 8.0), followed by purification with His Bind Purification kit (Novagen; USA) and elution with 150 mM imidazole. Renaturation was carried out by stepwise diluting urea in dialysate buffer (20 mM Tris-HCl, 5 mM EDTA buffer, pH 8.0). Purified protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue, final recombinant protein concentration was estimated by Bradford assay using BSA method used as a standard.
Semi-quantitative reverse transcription-PCR (RT-PCR) analysis of Cs CB
In order to determine the mRNA transcriptional level of Cs CB in various stages of the parasite, total RNA of adult worms and metacercariae were extracted and quantitated by nucleic acid/protein analyzer (Beckman Coulter; USA). Reverse transcription reactions were carried out by transforming equal amounts of total RNA (1 μg each) to cDNA using RT-PCR Kit (TaKaRa; PR China). RT-PCR experiments were employed to amplify the transcripts of Cs CB from cDNA of adult worms and metacercariae, respectively, and C. sinensis β-actin (GenBank accession no. EU109284) was used as a positive control. The forward and reverse primer for Cs CB were 5'-GGATTCGGCCTGGAAAAAC-3', 5'-CAGTTTTGGATGACCAGCAT-3' and for β-actin were 5'-GGTGACGCTGAAGTATCCTATTGA-3', 5'-CCAAAGCATAGCCCTCGTAGAT-3', respectively. The programs for PCR were the same as described above. PCR products from two life stages were quantified by Tanon Gis software (Tanon 4100; PR China) normalized by C. sinensis β-actin and analyzed by Student's t test (Software package SPSS16.0), P-value of < 0.05 indicated statistical significance.
Preparation for the antisera of recombinant protein and C. sinensis excretory/secretory product (ESP)
Living adults of C. sinensis were collected and cultured in RPMI-1640 (Gibco; USA) at 37°C under 5% CO2 for 6 h. The culture was centrifuged at 12,000× g at 4°C for 30 min to remove insoluble debris. The supernatant was dialyzed in PBS for 12 h and concentrated with sucrose. Six-week-old male Sprague-Dawley rats were purchased for animal experiments under the Guide for the Care and Use of Laboratory Animals. Both the recombinant CB and ESP were emulsified with complete Freund's adjuvant and immunized subcutaneously to SD rats. Each rat was given 200 μg rCs CB or ESP at the first injection, and 100 μg protein (emulsified with incomplete Freund's adjuvant) was given for 2 booster injections at 2-week intervals; antisera were collected before each injection and serum antibody responses to rCs CB and ESP were tested by ELISA.
SDS-PAGE and western blotting
The recombinant protein (1 μg per lane) was subjected to SDS-PAGE and electrotransferred onto polyvinylidene difluoride membrane (PVDF, Whatman; USA) at 100 v for 1 h. The membrane was blocked with 5% skim milk in phosphate buffered saline tween-20 (PBST, pH 7.4) at 37°C for 2 h, washed 5 times with PBST, then incubated with different antisera (antisera against the recombinant Cs CB protein, antisera against the ESP, sera from C. sinensis-infected rats and normal rats, respectively, 1:200 dilutions for all sera) at 37°C for 2 h. After washing, the membrane was incubated with rabbit anti-rat IgG HRP-conjugated horse radish peroxidase (1:2,000 dilution; Boster; PR China) at 37°C for 1 h. Diaminobenzidine (DAB) substrate solution (Invitrogen, USA) was used to visualize the reaction according to the manufacturer's instructions.
Immunolocalization of Cs CB protein in C. sinensis adult worm
C. sinensis adult worms fixed with 10% neutral formalin were embedded with paraffin and sliced into 3-5 μm thickness. After deparaffinating with dimethyl benzene and gradient alcohol, all sections were blocked with normal goat serum at 4°C overnight, then incubated with rat anti-Cs CB serum for 2 h at room temperature (RT), the sera from normal rats were used as negative control, the serum was diluted at 1: 200 for all sections. The sections were subsequently incubated with goat anti-rat IgG labeled with red-fluorescent Cy3 (1: 400 dilutions with 0.1% BSA in PBS, Jackson, USA) for 1 h at RT in dark and imaged using fluorescent microscope (ZEISS; Germany).
Source of serum samples
A total of 276 human serum samples were employed in order to approach the antigenicity and diagnostic value of Cs CB in human helminthiases. 80 sera from clonorchiasis patients were collected from endemic areas in Guangxi Province and were egg-positive proved by microscopic examination of stool samples. Other human sera including single infection of E. multilocularis (22), F. hepatica (42), S. japonicum (41), P. westermani (21), T. solium (20) and 50 samples of serum from healthy humans were provided and diagnosed by Centers for Disease Control and Prevention of Nanning, Jiangsu, Gansu and Fujian provinces. Sera were retrieved and stored in aliquots at -20°C until use.
Serodiagnosis of human helminthiases by ELISA and western blotting
The optimal antigen concentration and serum dilution required for ELISA were determined by checker board titration. Briefly, 96-well microtiter plates (Costar, USA) were coated with 100 μl 1 μg/ml, 3 μg/ml, 5 μg/ml, 7 μg/ml rCs CB (in 0.05 mol/l NaHCO3 buffer, pH 9.6) and incubated at 4°C overnight. After three washings with PBST, the microplates were blocked with 5% skimmed milk (w/v, in PBST containing 0.1% BSA) for 2 h at 37°C. Following another washing procedure, the plates were incubated with human helminthiases sera (1:50, 1:100, 1:200, 1:400 dilutions for each coating concentration in PBST containing 0.1% BSA) for 2 h at 37°C. Goat anti-human IgG (1:10,000 dilutions in 0.1% BSA-PBST recommended by producer; Invitrogen, USA) were employed as the secondary antibody. Subsequent reactions were developed with 3', 3', 5', 5'-tetramethyl benzidine (TMB; BD Biosciences; USA) and stopped with 2 M H2SO4. All assays were tested in triplicate and repeated twice. The absorbance value was measured at 450 nm.
In western blotting assay, rCs CB was subjected to 12% SDS-PAGE and electrotransferred onto PVDF membrane (Whatman; USA). The membrane was incubated with different human helminthiases sera (clonorchiasis, echinococciosis, fascioliasis, schistosomiasis, paragonimiasis, and cysticercosis, respectively) which showed high OD450 values by ELISA test at 37°C for 2 h, human healthy serum was used as negative control, all sera were 1:400 diluted in PBST containing 0.1% BSA. Goat anti-human IgG HRP-conjugated horse radish peroxidase (1:2,000 dilution; Boster; PR China) was the secondary antibody to react with the membrane and DAB substrate solution was used to visualize the reaction.
Software package SPSS16.0 was used in the present study for all statistical analysis. Briefly, Student's t test was used to analyze the measurement data among groups. P value of < 0.05 was considered statistically significant. The optimal cut-off value was calculated based on ROC curve analysis which correlated with sensitivity and 1-specificity . ROC curve, area under the curve (AUC) and scatter plot were carried out using SPSS16.0. The sensitivity and specificity were calculated using microscopic examination of stool samples as gold standard method.
Centers for Disease Control and Prevention of Nanning, Jiangsu, Gansu and Fujian provinces granted ethical approval for human sera used in the present study. All animal experiments were approved by institute's ethical committee of Sun Yat-sen University.
This work is supported by the National S & T Major Program (Grant No. 2008ZX10004-011) and Development Program of China (973 program; no. 2010CB530000).
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