Identification and characterization of Clonorchis sinensis cathepsin B proteases in the pathogenesis of clonorchiasis
- Wenjun Chen†1, 2,
- Dan Ning†1, 2, 3,
- Xiaoyun Wang†1, 2,
- Tingjin Chen†1, 2,
- Xiaoli Lv4,
- Jiufeng Sun3,
- De Wu3,
- Yan Huang1, 2,
- Jin Xu1, 2Email author and
- Xinbing Yu1, 2Email author
© Chen et al. 2015
Received: 20 November 2015
Accepted: 5 December 2015
Published: 21 December 2015
Human clonorchiasis is a prevailing food-borne disease caused by Clonorchis sinensis infection. Functional characterizations of key molecules from C. sinensis could facilitate the intervention of C. sinensis associated diseases.
In this study, immunolocalization of C. sinensis cathepsin B proteases (CsCBs) in C. sinensis worms was investigated. Four CsCBs were expressed in Pichia pastoris yeast cells. Purified yCsCBs were measured for enzymatic and hydrolase activities in the presence of various host proteins. Cell proliferation, wound-healing and transwell assays were performed to show the effect of CsCBs on human cells.
CsCBs were localized in the excretory vesicle, oral sucker and intestinal tract of C. sinensis. Recombinant yCsCBs from yeast showed active enzymatic activity at pH 5.0–5.5 and at 37–42 °C. yCsCBs can degrade various host proteins including human serum albumin, human fibronectin, human hemoglobin and human IgG. CsCBs were detected in liver tissues of mice and cancer patients afflicted with clonorchiasis. Various bioassays collectively demonstrated that CsCBs could promote cell proliferation, migration and invasion of human cancer cells.
Our results demonstrated that CsCBs can degrade various human proteins and we proved that the secreted CsCBs are involved in the pathogenesis of clonorchiasis.
Clonorchiasis is a food-borne parasitic disease caused by infection with Clonorchis sinensis (C. sinensis). Mammals are often infected with C. sinensis by consuming raw or uncooked fish or shrimp containing infectious metacercaria. Adult worms reside in the bile ducts of hosts and secreted products from C. sinensis eventually lead to clonorchiasis resulting in: cholangectasis, cholecystitis, cholelithiasis, hepatic fibrosis, and even liver cancer and bile duct cancer [1–3]. It is estimated that about 35 million people are afflicted with clonorchiasis, with most cases in Asian countries such as Korea, China and Vietnam [4, 5]. Food security problems caused by liver flukes have attracted high attention of public health, increasing the urgency of finding new approaches to prevent the spread of clonorchiasis. Clonorchiasis is listed among food-borne parasitic diseases requiring urgent control in China.
With the recent progress of the C. sinensis genome and transcriptome [6, 7], scientific researchers have expended much effort to elucidate the underlying mechanism of carcinogenic liver fluke associated hepatobiliary diseases [8, 9]. Molecular characterizations of key pathogenic molecules could speed up the interventions of C. sinensis infection. Cysteine proteases of helminthes have been widely characterized for their biological functions, including digestion, encystation, excystation, immune evasion and tissue invasion [10, 11]. Although cysteine proteases are abundant in C. sinensis transcriptome, limited information is available to illustrate the biological roles for C. sinensis in the host. Biological roles of C. sinensis cathepsin B proteases (CsCBs) have not been sufficiently investigated, although extensive studies demonstrate the importance of cathepsins in other organisms.
In our previous work , we performed preliminary functional characterizations of four C. sinensis cathepsin B cysteine proteases (CsCB1, CsCB2, CsCB3 and CsCB4). CsCBs were cloned into a prokaryotic expression vector (pET-28a) and expressed in the form of inclusion bodies in E. coli. Purified proteins from E. coli (eCsCBs) were identified as C. sinensis excretory/secretory products and could trigger immune responses. However, we failed to perform further functional characterizations of these cysteine proteases due to loss of enzyme activity during the renaturation procedure. In this study, the eukaryotic expressing system in yeast was constructed using homologous recombination to express four yCsCBs (CsCB1, CsCB2, CsCB3 and CsCB4) in Pichia pastoris X33 yeast cells. Recombinant yCsCBs showed enzymatic activities and hydrolase activities in degrading various host proteins. CsCB was detected in the liver tissues of mice and cancer patients afflicted with clonorchiasis. Recombinant CsCB could promote cell proliferation, migration and invasion of human cancer cells. Our results provide evidence to support the role of CsCBs in the pathogenesis of clonorchiasis.
Parasites, animals and patient samples
C. sinensis worms (larva, juvenile and adult) were freshly isolated from artificially infected freshwater fishes or Sprague–Dawley rats as we previously described . Ethical Approval: Male Sprague–Dawley rats were purchased from the animal center at Sun Yat-sen University and raised in accordance with the National Institutes of Health animal care and ethical guidelines. BALB/c mice (8-weeks-old) were intragastrically infected with metacercariae to establish the C. sinensis infected mice model. Mice in the control group were treated with phosphate-buffered saline (PBS). The mice were sacrificed at 8 weeks after the infection and liver tissues were isolated for immunohistochemistry. Clonorchiasis-induced liver cancer specimens acquired from People’s Hospital of HengXian, Guangxi Zhuang Autonomous Region were pathologically diagnosed. Normal liver specimens were acquired from the first affiliated hospital of Sun Yat-Sen University. Ethical approval to use patients’ samples in this study was obtained from local hospitals and animal procedures were approved by the animal care and use committee of Sun Yat-sen University (Permit Numbers: SCXK (Guangdong) 2009–0011).
Immunolocalization of CsCBs in adult worm, cercaria and metacercaria
C. sinensis worms (larva, juvenile and adult) were used for the immunolocalization assay. Sectioned worms in paraffin wax were deparaffinized and incubated with previously prepared anti-CsCBs sera (1: 400 in dilution). Pre-immune rat serum was applied as a negative control. Subsequently, the sections were incubated with Cy3 conjugated goat anti-rat IgG secondary antibody (1: 400 in dilution, Alexa Fluor 594, Molecular Probe) at RT for 1 h and imaged using an Axio Imager Z1fluorescent microscope (ZEISS).
Homologous recombination of CsCBs in yeast
Primers used in this study
Expression and purification of yCsCBs
Selected transformants were cultured in a BMGY medium for 16–18 h until OD600 of 2–6, cells were harvested by centrifugation and re-suspended in a BMMY medium at an OD600 of 1.0. The expression of yCsCBs was induced by the daily addition of 0.5 % (v/v) methanol at 24, 48, 72 and 96 h, respectively. The culture filtrate of recombinant X33 cells was concentrated using ammonium sulfate. Concentrated supernatant was used for SDS-PAGE and Western blotting experiments to examine the extracellular expression of yCsCBs in yeast. After that, recombinant protein was induced for 96 h and purified by His-bind resin chromatography (Novagen) followed by dialysis in PBS (pH7.2). Protein concentration was determined using the BCA method and stored at −80 °C for enzymatic assays.
SDS-PAGE and Western blotting
Concentrated supernatant was subjected to 12 % SDS-PAGE stained with Coomassie brilliant blue. To further confirm extracellular expression of CsCBs in X33 cells, concentrated supernatant was also subjected to Western Blotting. Protein samples were transferred onto a PVDF membrane (Millipore) followed by incubation with different antibodies: mouse anti-His antibody (1: 500 in dilution, Life Technologies), mouse anti-c-Myc monoclonal antibody (1: 500 in dilution, Life Technologies) and rat anti-CsCB1 antibody (1: 800 in dilution), which was produced in our previous study. HRP-conjugated goat anti-mouse IgG or goat anti-rat IgG (1: 2,000 in dilution) was further incubated with each membrane, followed by enhanced chemiluminescence (ECL).
Enzyme activity assays
The enzyme activity of yCsCBs was assayed fluorometrically according to the previous report . Enzyme reactions were performed under different enzyme concentrations, pH values and temperatures, respectively. Typically, the measurements were performed at 37 °C for 1 h in a 100 μl mixture containing yCsCBs (0–20 μM), fluorescent Z-Phe-Arg-AMC/Z-Arg-Arg-AMC (20 μM, Bachem), 10 mM DTT, 0.05 % Brij-35 (AMRESCO), EDTANa2 (1 mM), and C2H3NaO2/Na3PO4/Tris–HCl (100 mM). The enzyme reaction was terminated by adding stop buffer (70 mM C2H4O2, 30 mM C2H3NaO2, 100 mM C2H2ClO2Na, pH 4.3). Fluorescent intensity was measured by plate reader at 348 nm.
Degradation of host proteins
We first investigated hydrolase activity of yCsCBs. Purified CsCBs from E. coli or from Pichia pastoris were loaded into a 12 % SDS-PAGE containing 0.1 % gelatin. The gel was washed with washing buffer (2.5 % Tritonx-100, 50 mM Tris–HCl, 5 mM CaCl2, pH 7.5), followed by incubation with Na3PO4 (100 mM, pH 7.5) at 37 °C for 24 h. The hydrolase activity of yCsCBs was visualized by Coomassie brilliant blue staining.
Second, we tested whether yCsCBs could degrade host proteins, given that CsCBs were proven components of secreted products of C. sinensis . Purified yCsCBs were incubated with host proteins at 37 °C for 2 h. Human serum albumin (MB-CHEM), human hemoglobin (MB-CHEM), human IgG (MB-CHEM), human fibronectin (Sigma) and bovine serum albumin (MB-CHEM) were used as the substrates. The assays were performed in a 200 μl mixture containing Na3PO4 (100 mM, pH 5.5), EDTANa2 (1 mM), DTT (10 mM), yCsCBs (20 μM) and various host proteins (1 mg). The reactions were terminated by adding a reducing sample buffer and analyzed by SDS-PAGE.
Inhibition effect on enzyme activity of yCsCBs
To confirm the specificity of enzyme activity from the above-mentioned assays, we performed enzymatic inhibition experiments by using different enzyme inhibitors purchased from Sigma. Briefly, yCsCBs (20 μM) were pre-incubated with or without protease inhibitors, including E-64 (20 μM), iodoacetic acid (10 μM), PMSF (2 mM), EDTA (2 mM), AEBSF (200 μM), TPCK (200 μM) and CA-074 methyl ester (1 μM). Z-Phe-Arg-AMC (20 μM) was added to the reactions after 30 min and incubated for another 1 h. Each assay was performed in triplicate and enzyme activity was measured by plate reader at 348 nm.
Immunohistochemistry of CsCB in infected mouse and patient
Next, we sought to investigate whether CsCBs are involved in the pathogenesis of clonorchiasis using the yCsCB4 protein. First, we performed an immunohistochemistry using an anti-CsCB4 antibody to see the localization of CsCB4 in liver tissues of clonorchiasis afflicting mice and patients. Generally, tissue samples were fixed in 10 % formalin and sectioned to 4 μm in thickness. The sections were routinely treated with ethanol and slides were immersed in a 0.3 % hydrogen peroxide solution for 20 min to block the endogenous peroxidase activity. The sections were then incubated overnight at 4 °C with rat anti-CsCB4 antibody (1: 100 in dilution). Sections were subsequently incubated with horseradish peroxidase (HRP) conjugated rat-specific secondary antibodies (1: 200 in dilution). Immunohistochemistry results were developed using diaminobenzidine (DAB) and counterstained with hematoxylin. The images were taken under a light microscope (Leica DMI3000B) and subsequently analyzed using ImagePro Plus software (Media Cybernetics, Roper, USA). The brown staining was indicated as Integrated Optical Density (IOD), and IOD/Area was indicated as a relative expression level of CsCB4 in liver tissues.
Cell proliferation analysis
The cell proliferation level induced by yCsCB4 was measured in two human cancer cell lines, human hepatocellular carcinoma cell line (MHCC-97H, ATCC) and human cholangiocarcinoma cell line (RBE, ATCC). MHCC-97H and RBE cells were grown in DMEM (Hyclone, USA) and RPMI-1640 (Gibco, USA), respectively and supplemented with 10 % fetal bovine serum (Gibco, USA) and 1 % penicillin/streptomycin (Gibco, USA). Cells were incubated at 37 °C in a humidified chamber under 5 % CO2. Cells at the logarithmic phase were plated into 96-well plates in triplicate and treated with yCsCB4 protein (1 μg/ml). Cell viability at 24 h was measured using Cell Counting Kit-8 (CCK-8) as previously described . For cell cycle analysis using flow cytometry, cells were incubated with yCsCB4 (1 μg/ml) for 24 h. Then the cells were trypsinized and fixed in 100 % ethanol at −20 °C overnight. Cell cycle distribution was determined by fluorescence activated cell sorting (FACS). Data was analyzed using the FlowJo software.
Cell migration and invasion assay
To further confirm the role of yCsCB4 in human cancer cell growth, wound-healing assays were performed to evaluate the effect of yCsCB4 on cell migration according to the previous method . MHCC-97H and RBE cells seeded in 6-well plates were grown to 80 % confluence and incubated with yCsCB4 (1 μg/ml) or PBS for 24 h. Cells were wounded by scratching with pipette tips. Wounds at 24 h were observed and photographed under a light microscope (Leica DMI3000B).
To evaluate the effect of yCsCB4 on cell invasion, we performed transwell assays according to the method described elsewhere . MHCC-97H and RBE cells were suspended in serum-free media and placed in 8 μm pores. These inserts were placed in wells with serum-containing media. Cells were incubated with yCsCB4 (1 μg/ml) or PBS for 24 h. Invasion assays were performed using matrigel-coated membranes (BD, USA). The migration assay was similar to the invasion assay, except that the upper side of the membranes was not coated with the matrigel. Cells attached to the lower surface of the membranes at 24 h were counted under a light microscope.
Experimental data were obtained from three independent experiments with a similar pattern; data are expressed as means ± standard deviation. All the data were analyzed by SPSS 13.0. Student’s t-test and ANOVA were used to analyze the data. P value <0.05 was considered statistically significant.
Immunolocalization of CsCBs in C. sinensis worms
Homologous recombination of CsCBs in yeast
Enzyme activity of yCsCB
Host proteins degradation by yCsCBs
Inhibition effect on enzyme activity of yCsCBs
Immunohistochemistry of CsCB in infected mice and liver cancer patients
Cell proliferation promoted by CsCB4
Cell migration and invasion triggered by CsCB4
Proteases are ubiquitous in nature and most organisms. In addition to their housekeeping functions, proteases are involved in the digestion of host proteins such as fibronectin, collagen and albumin, to facilitate migration and feeding in the host . Cathepsins are of particular interest to parasitologists because there is considerable evidence that cathepsins are involved in parasitism. All of the trematodes have been shown to contain genes encoding cathepsin B-like proteins. For example, in Fasciola hepatica, cathepsin B was identified as an important factor associated with invasion of the mammalian host  and cathepsin B was suggested as a potential digestive factor in newly excysted juvenile parasites . Cathepsin B was also identified as a stage and tissue-specific expression protease in Fasciola gigantica . In Schistosoma mansoni, secreted cathepsin B was proposed to interact with host molecules and thus be a vital factor in parasitism . In Angiostrongylus cantonensis, cathepsin B plays a potential role in the invasion of the central nervous system during parasite-host interactions . Thus, cathepsin B proteases clearly play an important role in the biology of trematode parasites. Cysteine proteases were abundant genes in C. sinensis genome and transcriptome. As the main components of C. sinensis excretory/secretory products, CsCBs were proved to be potential vaccine candidates and diagnostic markers [11, 12]. In this study, we constructed a eukaryotic expressing system by homologous recombination to express four CsCBs in yeast. Active yCsCBs were purified for biochemical and functional characterizations. The cellular effect of CsCBs on human cancer cells was observed using various cellular assays. Our results provide evidence to support the role of CsCBs in the pathogenesis of clonorchiasis.
At this time, CsCBs were expressed in soluble form with enzyme activity because of the advantages of the methylotropic yeast, Pichia pastoris, and the shuttle vector pPICZαB [25, 26]. This shuttle vector facilitated our transformation operation from the E. coli system to the Pichia pastoris system. yCsCBs showed active enzyme activity with a wide range of pHs, while peak enzymatic activity was assayed at pH 5.0–5.5, suggesting that yCsCBs were functional enzymes under acidic conditions. The hypothesis that CsCBs are acidic enzymes located in gut of flukes could be supported by previous reports [23, 27, 28], considering the fact that the pH of the gut lumen of Fasciola hepatica has been suggested to be pH 5.5 . Enzymatic activities of yCsCBs could be completely inhibited by cathepsin B specific inhibitors or cysteine protease specific inhibitors, while serine protease specific inhibitors and trypsin specific inhibitor showed only a weak inhibition effect. These results helped us confirm that our obtained yCsCBs belong to the typical cathepsin B cysteine protease family. In addition to typical enzymatic activities, yCsCBs could degrade all tested host proteins such as human serum albumin, human fibronectin, human hemoglobin and human IgG. Those host proteins have been used in other parasites to test the digestive effect of cathepsins . For instance, FhCB2 could cleave serum albumin and IgG, indicating a role in the digestion of protein substrates for nutritional purposes . Recombinant FgCB3 was recently shown to digest fibronectin, consistent with a role in digesting connective tissue and host invasion . The digestive effect of yCsCBs supports our hypothesis that CsCBs serve as key virulence factors for C. sinensis, it is most likely that CsCBs are involved in the pathogenesis of clonorchiasis. The biological role of CsCB could be implied by immunolocalization results, showing that CsCBs were localized in the excretory vesicle, oral sucker and intestinal tract of C. sinensis worms. Immunolocalization of CsCB is similar to C. sinensis cathepsin F, which is also a secreted protein in the intestine of C. sinensis . These two enzymes were expressed throughout developmental stages of the parasite. Given that CsCBs and CsCFs are from the same protease family, it is reasonable to assume they are synthesized in epithelial cells lining the parasite intestine followed by secretion into the intestinal lumen of the parasite, to play a role for nutrient uptake in the parasite [33–35].
As the key component of secreted products, many proteins have been connected with hepatobiliary diseases observed in individuals infected with liver flukes [36, 37]. It was suggested that secreted products released by liver flukes could lead to pathologic changes in biliary epithelial cells [38, 39]. Human cells exposed to ESPs from liver flukes (C. sinensis, Fasciola hepatica, and Opisthorchis viverrini) showed diverse pathophysiological responses including proliferation, apoptosis and inflammation [40–42]. In human diseases, experimental and clinical evidence have linked cathepsin B with tumor invasion and metastasis. Cathepsin B expression increases in many human cancers at mRNA, protein and activity levels . In this study, we found that CsCB4 was detected in liver tissues from infected mice or liver cancer patients induced by clonorchiasis. To gain a better understanding of CsCBs-associated human diseases, we measured the biological effects of yCsCB4 protein on human cancer cells. The results from different approaches demonstrated that yCsCB4 could promote cell proliferation, cell migration and cell invasion of human hepatocellular carcinoma cells and human cholangiocarcinoma cells. Our observed results could be supported by our previous report that severin protein from CsESPs had an anti-apoptotic role in hepatocarcinoma PLC cells . Given that four CsESPs have similar biochemical properties, it is conceivable that CsCBs are involved in the pathogenesis of clonorchiasis during C. sinensis infection. However, further investigations are required in order to identify precise mechanisms to provide therapeutic strategies for clonorchiasis. With RNA interference applications in helminth [45, 46], it is feasible to perform a CsCBs-mediated intervention in C. sinensis associated diseases.
In summary, we expressed and purified four CsCBs in yeast and demonstrated that CsCBs can degrade various human proteins. CsCBs could be detected at a high expression level in clonorchiasis-induced liver cancer tissues. In addition, our results indicate that CsCBs could confer proliferative and invasive role in human cancer cells. The present study supports the involvement of CsCBs in the pathogenesis of clonorchiasis.
This work was supported by Science and Technology Plan in Guangdong Province (No.: 2012ZX10004-220, 2013B010404010, 2014B020203001), National Key Basic Research and Development Project (973 project; No. 2010CB530000) and National Natural Science Foundation of China (No. 81171602) to XBY. This work was supported in part by National Natural Science Foundation of China (No. 81101270) to YH. We appreciate Prof. Lifang Jiang (Department of Microbiology in Zhongshan School of Medicine at Sun Yat-sen University) for providing us shuttle vector pPICZαB and Pichia pastoris X33 cells.
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