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Background: Biliary tract infection with the carcinogenic human liver fluke, Clonorchis sinensis, provokes chronic inflammation, epithelial hyperplasia, periductal fibrosis, and even cholangiocarcinoma. Complications are proportional to the intensity and duration of the infection. In addition to mechanical irritation of the biliary epithelia from worms, their excretory-secretory products (ESPs) cause chemical irritation, which leads to inflammation, proliferation, and free radical generation., Methods: A three-dimensional in vitro cholangiocyte spheroid culture model was established, followed by ESP treatment. This allowed us to examine the intrinsic pathological mechanisms of clonorchiasis via the imitation of prolonged and repetitive in vivo infection., Results: Microarray and RNA-Seq analysis revealed that ESP-treated cholangiocyte H69 spheroids displayed global changes in gene expression compared to untreated spheroids. In ESP-treated H69 spheroids, 185 and 63 probes were found to be significantly upregulated and downregulated, respectively, corresponding to 209 genes (p < 0.01, fold change > 2). RNA-Seq was performed for the validation of the microarray results, and the gene expression patterns in both transcriptome platforms were well matched for 209 significant genes. Gene ontology analysis demonstrated that differentially expressed genes were mainly classified into immune system processes, the extracellular region, and the extracellular matrix. Among the upregulated genes, four genes (XAF1, TRIM22, CXCL10, and BST2) were selected for confirmation using quantitative RT-PCR, resulting in 100% similar expression patterns in microarray and RNA-Seq., Conclusions: These findings broaden our understanding of the pathological pathways of liver fluke-associated hepatobiliary disorders and suggest a novel therapeutic strategy for this infectious cancer.

Clonorchiasis caused by Clonorchis sinensis is endemic in East Asia; approximately 15 million people have been infected thus far. To diagnose the infection, serodiagnostic tests with excellent functionality should be performed. First, 607 expressed sequence tags encoding polypeptides with a secretory signal were expressed into recombinant proteins using an in vitro translation system. By protein array-based screening using C. sinensis-infected sera, 18 antigen candidate proteins were selected and assayed for cross-reactivity against Opisthorchis viverrini-infected sera. Of the six antigenic proteins selected, four were synthesized on large scale in vitro and evaluated for antigenicity against the flukes-infected human sera using ELISA. CsAg17 antigen showed the highest sensitivity (77.1%) and specificity (71.2%). The sensitivity and specificity of the bacterially produced CsAg17-28GST fusion antigen was similar to those of CsAg17 antigen. CsAg17 antigen can be used to develop point-of-care serodiagnostic tests for clonorchiasis., Competing Interests: The authors have declared that no competing interests exist.

Cholangiocarcinoma (CCA) is a slow-growing but highly metastatic cancer. Its metastatic potential largely explains its high mortality rate. A recognized risk factor for CCA development is infection with the liver flukes Opisthorchis viverrini and Clonorchis sinensis. We previously reported that the excretory-secretory products (ESPs) of C. sinensis promoted the three-dimensional aggregation and invasion of CCA cells. In the present study, a quantitative real-time PCR array of extracellular matrix (ECM) and adhesion molecules was used to examine the regulatory mechanism of ESP-mediated CCA cell migration and invasion. In particular, the expression levels of integrin α isoforms and β4 were upregulated in response to ESPs. Increased expression of integrin β4 was probably correlated with activation of focal adhesion kinase (FAK) and the steroid receptor coactivator (Src) family kinase and the subsequent activation of two downstream focal adhesion molecules, paxillin and vinculin. Moreover, inhibition of FAK/Src activation reduced paxillin and vinculin phosphorylation and attenuated ESP-induced CCA cell migration and invasion. These findings suggest that the integrin β4-FAK/Src signaling axis may play a crucial role in clonorchiasis-associated CCA metastasis during tumor progression., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Parasitic diseases remain an unarguable public health problem worldwide. Liver fluke Clonorchis sinensis is a high risk pathogenic parasitic helminth which is endemic predominantly in Asian countries, including Korea, China, Taiwan, Vietnam, and the far eastern parts of Russia, and is still actively transmitted. According to the earlier 8th National Survey on the Prevalence of Intestinal Parasitic Infections in 2012, C. sinensis was revealed as the parasite with highest prevalence of 1.86% in general population among all parasite species surveyed in Korea. This fluke is now classified under one of the definite Group 1 human biological agents (carcinogens) by International Agency of Research on Cancer (IARC) along with two other parasites, Opisthorchis viverrini and Schistosoma haematobium. C. sinensis infestation is mainly linked to liver and biliary disorders, especially cholangiocarcinoma (CCA). For the purposes of this mini-review, we will only focus on C. sinensis and review pathogenesis and carcinogenesis of clonorchiasis, disease condition by C. sinensis infestation, and association between C. sinensis infestation and CCA. In this presentation, we briefly consider the current scientific status for progression of CCA by heavy C. sinensis infestation from the food-borne trematode and development of CCA. [BMB Reports 2016; 49(11): 590-597].

Clonorchis sinensis is a carcinogenic human liver fluke. Its infection promotes persistent oxidative stress and chronic inflammation environments in the bile duct and surrounding liver tissues owing to direct contact with worms and their excretory-secretory products (ESPs), provoking epithelial hyperplasia, periductal fibrosis, and cholangiocarcinogenesis. We examined the reciprocal regulation of two ESP-induced redox-active proteins, NF-κB and peroxiredoxin 6 (Prdx6), during C. sinensis infection. Prdx6 overexpression suppressed intracellular free-radical generation by inhibiting NADPH oxidase2 and inducible nitric oxide synthase activation in the ESP-treated cholangiocarcinoma cells, substantially attenuating NF-κB-mediated inflammation. NF-κB overexpression decreased Prdx6 transcription levels by binding to two κB sites within the promoter. This transcriptional repression was compensated for by other ESP-induced redox-active transcription factors, including erythroid 2-related factor 2 (Nrf2), hypoxia inducible factor 1α (HIF1α), and CCAAT/enhancer-binding protein β (C/EBPβ). Distribution of immunoreactive Prdx6 and NF-κB was distinct in the early stages of infection in mouse livers but shared concomitant localization in the later stages. The intensity and extent of their immunoreactive staining in infected mouse livers are proportional to lesion severity and infection duration. The constitutive elevations of Prdx6 and NF-κB during C. sinensis infection may be associated with more severe persistent hepatobiliary abnormalities mediated by clonorchiasis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Clonorchis sinensis is a high-risk pathogenic helminth that strongly provokes inflammation, epithelial hyperplasia, periductal fibrosis, and even cholangiocarcinoma in chronically infected individuals. Chronic inflammation is associated with an increased risk of various cancers due to the disruption of redox homeostasis. Accordingly, the present study was conducted to examine the time course relationship between histopathological changes and the appearance of oxidative stress markers, including lipid peroxidation, enzymes involved in lipid peroxidation, and mutagenic DNA adducts in the livers of mice infected with C. sinensis, as well as proinflammatory cytokines in infected mouse sera. Histopathological phenotypes such as bile duct epithelial hyperplasia, periductal fibrosis, edema and inflammatory infiltration increased in infected livers in a time-dependent manner. Intense immunoreactivity of lipid peroxidation products (4-hydroxy-2-nonenal; malondialdehyde), cyclooxygenase-2, 5-lipoxygenase and 8-oxo-7,8-dihydro-2'-deoxyguanosine were concomitantly observed in these injured regions. We also found elevated expressions of cyclooxygenase-2 and 5-lipoxygenase in C. sinensis excretory-secretory product-treated cholangiocarcinoma cells. Moreover, the levels of proinflammatory cytokines such as TNF-α, ILβ-1 and IL-6 were differentially upregulated in infected sera. With regard to oxidative stress-mediated carcinogenesis, our findings suggest that C. sinensis infestation may disrupt host redox homeostasis, creating a damaging environment that favors the development of advanced hepatobiliary diseases such as clonorchiasis-associated cholangiocarcinoma., (Copyright © 2015 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

A complementary DNA (cDNA) encoding a glucose transporter of Clonorchis sinensis (CsGLUT) was isolated from the adult C. sinensis cDNA library. The open reading frame of CsGLUT cDNA consists of 1653 base pairs that encode a 550-amino acid residue protein. Hydropathy analysis suggested that CsGLUT possess 12 putative membrane-spanning domains. The Northern blot analysis result using poly(A)(+)RNA showed a strong band at ~2.1 kb for CsGLUT. When expressed in Xenopus oocytes, CsGLUT mediated the transport of radiolabeled deoxy-D-glucose in a time-dependent but sodium-independent manner. Concentration-dependency results showed saturable kinetics and followed the Michaelis-Menten equation. Nonlinear regression analyses yielded a Km value of 588.5 ± 53.0 μM and a Vmax value of 1500.0 ± 67.5 pmol/oocyte/30 min for [1,2-(3)H]2-deoxy-D-glucose. No trans-uptakes of bile acid (taurocholic acid), amino acids (tryptophan and arginine), or p-aminohippuric acid were observed. CsGLUT-mediated transport of deoxyglucose was significantly and concentration-dependently inhibited by radio-unlabeled deoxyglucose and D-glucose. 3-O-Methylglucose at 10 and 100 μM inhibited deoxyglucose uptake by ~50 % without concentration dependence. No inhibitory effects by galactose, mannose, and fructose were observed. This work may contribute to the molecular biological study of carbohydrate metabolism and new drug development of C. sinensis.

Asparaginyl endopeptidases (AEP: EC 3.4.22.34) are a family of cysteine proteases classified into the MEROPS clan CD, family C13. In this study, we characterized the biochemical and antigenic properties of an AEP of Clonorchis sinensis (CsAEP). The recombinant CsAEP showed hydrolytic activity at pH values ranging from acidic to neutral with optimum activity at pH 6.0. While the recombinant CsAEP was stable at neutral pHs, it was unstable at acidic pHs and resulted in loss of enzymatic activity. The recombinant enzyme was effectively inhibited by iodoacetic acid and N-ethylmaleimide, but not by E-64. The partially purified native CsAEP showed biochemical properties similar to the recombinant enzyme. Native CsAEP is likely to be cleaved into an N-terminal mature enzyme and a C-terminal fragment via autocatalytic activation at acidic pHs. Polyclonal antibody raised against the recombinant CsAEP recognized three forms of CsAEP, proenzyme, the N-terminal mature enzyme and the C-terminal fragment, in the worm extract (WE) of C. sinensis. However, only the C-terminal fragment was mainly found in the excretory and secretory (ES) products of the parasite. Strong CsAEP activity was found in the WE, but only a trace level of CsAEP activity was detected in the ES products of the parasite. CsAEP was expressed in various developmental stages of C. sinensis, from metacercariae to adults, and was found to be localized in the intestine of the parasite as well as in intestinal contents. Sera from rats experimentally infected with C. sinensis reacted with CsAEP beginning 4 weeks after infection. These results suggest that CsAEP is a gut-associated enzyme synthesized in the intestine of C. sinensis and subsequently secreted into the intestinal lumen of the parasite., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Paramyosin of Clonorchis sinensis (CsPmy) is a myofibrillar protein localized in subtegumental muscle, tegument, and the muscle layer surrounding the intestine of the parasite. Previously, we have identified that CsPmy reacted with sera of human clonorchiasis and this protein had a potential as a candidate antigen for serodiagnosis of clonorchiasis. However, we also found that CsPmy is able to bind to human immunoglobulin G (IgG) in non-specific manners, which can affect the diagnostic value of the protein. Here, we mapped CsPmy-specific IgG binding site on CsPmy to analyze the putative epitopes recognized by CsPmy-specific IgG in sera of human clonorchiasis. The fragmental expression of CsPmy followed by immunoblot analyses with sera from patients with clonorchiasis and non-specific human IgG revealed that the middle portion of CsPmy (CsPmyC: 301-600 amino acid residues) had epitopes responsible for CsPmy-specific IgG recognition. The precise CsPmy-specific IgG binding site was further narrowed down to a fragment (CsPmyC-2), which harbors 151 amino acid residues (375-525) of CsPmy. Specific antibodies for CsPmyC-2 were produced in rats after two-weeks of post-experimental infection. The CsPmyC-2 showed low levels of cross reactivity against the sera from patients with other helminth parasites. Our results suggested that CsPmyC-2 has real epitopes recognized by CsPmy-specific IgG in sera of human clonorchiasis and the fragment can be useful as a reliable serodiagnostic antigen to develop a serodiagnostic method for clonorchiasis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Clonorchis sinensis is a carcinogenic human liver fluke by which chronic infection is strongly associated with the development of cholangiocarcinoma. Although this cholangiocarcinoma is caused by both physical and chemical irritation from direct contact with adult worms and their excretory-secretory products (ESPs), the precise molecular events of the host-pathogen interactions remain to be elucidated. To better understand the effect of C. sinensis infection on cholangiocarcinogenesis, we profiled the kinetics of changes in cancer-related microRNAs (miRNAs) in human cholangiocarcinoma cells (HuCCT1) treated with C. sinensis ESPs for different periods. Using miRNA microarray chips containing 135 cancer-related miRNAs, we identified 16 miRNAs showing differentially altered expression following ESP exposure. Of these miRNAs, 13 were upregulated and 3 were downregulated in a time-dependent manner compared with untreated controls. Functional clustering of these dysregulated miRNAs revealed involvement in cell proliferation, inflammation, oncogene activation/suppression, migration/invasion/metastasis, and DNA methylation. In particular, decreased expression of let-7i, a tumor suppressor miRNA, was found to be associated with the ESP-induced upregulation of TLR4 mRNA and protein, which contribute to host immune responses against liver fluke infection. Further real-time quantitative PCR analysis using ESP-treated normal cholangiocytes (H69) revealed that the expressions of nine miRNAs (miR-16-2, miR-93, miR-95, miR-153, miR-195, miR-199-3P, let7a, let7i, and miR-124a) were similarly regulated, indicating that the cell proliferation and inhibition of tumor suppression mediated by these miRNAs is common to both cancerous and non-cancerous cells. These findings constitute further our understanding of the multiple cholangiocarcinogenic pathways triggered by liver fluke infection.

CsStefin-2, the second cysteine protease inhibitor of Clonorchis sinensis, was identified and characterized. CsStefin-2 is a cysteine protease inhibitor that belongs to family 1 stefins based on its phylogenetic and structural properties. However, CsStefin-2 had a QIVSG cystatin motif distinct from the common QVVAG cystatin motif that is well conserved in family 1 stefins. Mutagenesis analysis revealed that the two amino acid substitutions in the QIVSG cystatin motif of CsStefin-2 did not affect its inhibitory activity. Molecular modeling also indicated that no critical change was induced in the interaction between CsStefin-2 and its target enzyme. CsStefin-2 showed broad inhibitory activities against several cysteine proteases, including human cathepsins B and L, papain, and cathepsin Fs of C. sinensis (CsCFs), and effectively inhibited the autocatalytic maturation of CsCF-6. Native CsStefin-2 was assembled into a homo-tetramer, in which intermolecular disulfide bonds are not involved in the assembly of the tetramer. CsStefin-2 was expressed throughout the various developmental stages of the parasite and was localized in the intestinal epithelium, where CsCFs are actively synthesized. These results suggest that CsStefin-2 is the second active cysteine protease inhibitor of C. sinensis that shares functional redundancy with CsStefin-1 to modulate the activity and processing of CsCFs.

The voltage-gated Ca(2+) channel β-subunit is a member of the membrane-associated guanylate kinase family and modulates kinetic properties of the Ca(2+) channels, such as their voltage-dependent activation and inactivation rates. Two cDNA clones were identified to encode each β-subunit isotype of the voltage-gated Ca(2+) channel of Clonorchis sinensis, CsCavβ1 and CsCavβ2, which consist of 606 and 887 amino acids, respectively. CsCavβ1 was found to be similar to the β-subunit containing two conserved serine residues that constitute the consensus protein kinase C phosphorylation site in the β-interaction domain (BID). CsCavβ2 had cysteine and alanine residues instead of the two serine residues conserved in BID and was homologous to variant β-subunit of Schistosoma mansoni and Schistosoma japonicum. CsCavβ1 and CsCavβ2 were almost equally expressed in the adults and metacercariae, but were more expressed in adult C. sinensis than in metacercariae. Collectively, our findings suggest that substitution of the two serine residues in BID of CsCavβ2 may render C. sinensis sensitive to praziquantel.

Microscopic examination of eggs of parasitic helminths in stool samples has been the most widely used classical diagnostic method for infections, but tiny and low numbers of eggs in stool samples often hamper diagnosis of helminthic infections with classical microscopic examination. Moreover, it is also difficult to differentiate parasite eggs by the classical method, if they have similar morphological characteristics. In this study, we developed a rapid and sensitive polymerase chain reaction (PCR)-based molecular diagnostic method for detection of Clonorchis sinensis eggs in stool samples. Nine primers were designed based on the long-terminal repeat (LTR) of C. sinensis retrotransposon1 (CsRn1) gene, and seven PCR primer sets were paired. Polymerase chain reaction with each primer pair produced specific amplicons for C. sinensis, but not for other trematodes including Metagonimus yokogawai and Paragonimus westermani. Particularly, three primer sets were able to detect 10 C. sinensis eggs and were applicable to amplify specific amplicons from DNA samples purified from stool of C. sinensis-infected patients. This PCR method could be useful for diagnosis of C. sinensis infections in human stool samples with a high level of specificity and sensitivity.

Leucine aminopeptidases (LAP; EC 3.4.11.1) are a group of metalloexopeptidases, which catalyze the sequential removal of leucine amino acids from the N-termini of the polypeptides or proteins. In this study, we identified two novel genes that encode LAPs of Clonorchis sinensis (CsLAP1 and CsLAP2) and characterized their biochemical and functional properties. Multiple sequence alignment of the deduced amino acid sequences of CsLAP1 and CsLAP2 with those of other organisms revealed that typical metal-binding coordinating and active site residues for LAPs were well conserved in CsLAP1 and CsLAP2. Recombinant CsLAP1 and CsLAP2 showed similar biochemical properties such as pH optima at pH 8.0 and stability at neutral pHs. Both enzymes were specifically inhibited by bestatin and showed preferential substrate specificity for Leu-MCA. However, the enzymes differed in that they required different metal ions for maximum activity. Expressions of CsLAP1 and CsLAP2 were detected throughout the various developmental stages of C. sinensis, and their transcription levels increased gradually in accordance with the maturation of the parasite. Both enzymes were identified in soluble worm extract of C. sinensis, but not in excretory and secretory products. Immunolocalization studies showed that both enzymes were co-localized to the intestinal epithelial cells and gastrodermis of the parasite. These results collectively suggest that CsLAP1 and CsLAP2 are synthesized in the intestinal epithelial and gastrodermal cells of C. sinensis and may be involved in the final digestion of peptides that hydrolyzed within intestinal lumen followed by absorbed into gastrodermal cells of the parasite., (© 2011 Elsevier B.V. All rights reserved.)

A large-scale survey was conducted to investigate the infection status of fresh water fishes with Clonorchis sinensis metacercariae (CsMc) in 3 wide regions, which were tentatively divided by latitudinal levels of the Korean peninsula. A total of 4,071 freshwater fishes were collected from 3 regions, i.e., northern (Gangwon-do: 1,543 fish), middle (Chungcheongbuk-do and Gyeongsangbuk-do: 1,167 fish), and southern areas (Jeollanam-do, Ulsan-si, and Gyeongsangnam-do: 1,361 fish). Each fish was examined by the artificial digestion method from 2003 to 2010. In northern areas, only 11 (0.7%) fish of 2 species, Pungtungia herzi and Squalidus japonicus coreanus from Hantan-gang, Cheolwon-gun, Gangwon-do were infected with av. 2.6 CsMc. In middle areas, 149 (12.8%) fish were infected with av. 164 CsMc. In southern areas, 538 (39.5%) fish were infected with av. 159 CsMc. In the analysis of endemicity in 3 regions with an index fish, P. herzi, 9 (6.2%) of 146 P. herzi from northern areas were infected with av. 2.8 CsMc. In middle areas, 34 (31.8%) of 107 P. herzi were infected with av. 215 CsMc, and in southern areas, 158 (92.9%) of 170 P. herzi were infected with av. 409 CsMc. From these results, it has been confirmed that the infection status of fish with CsMc is obviously different among the 3 latitudinal regions of the Korean peninsula with higher prevalence and burden in southern regions.

Clonorchis sinensis is the causative agent of the life-threatening disease endemic to China, Korea, and Vietnam. It is estimated that about 15 million people are infected with this fluke. C. sinensis provokes inflammation, epithelial hyperplasia, and periductal fibrosis in bile ducts, and may cause cholangiocarcinoma in chronically infected individuals. Accumulation of a large amount of biological information about the adult stage of this liver fluke in recent years has advanced our understanding of the pathological interplay between this parasite and its hosts. However, no developmental gene expression profiles of C. sinensis have been published. In this study, we generated gene expression profiles of three developmental stages of C. sinensis by analyzing expressed sequence tags (ESTs). Complementary DNA libraries were constructed from the adult, metacercaria, and egg developmental stages of C. sinensis. A total of 52,745 ESTs were generated and assembled into 12,830 C. sinensis assembled EST sequences, and then these assemblies were further categorized into groups according to biological functions and developmental stages. Most of the genes that were differentially expressed in the different stages were consistent with the biological and physical features of the particular developmental stage; high energy metabolism, motility and reproduction genes were differentially expressed in adults, minimal metabolism and final host adaptation genes were differentially expressed in metacercariae, and embryonic genes were differentially expressed in eggs. The higher expression of glucose transporters, proteases, and antioxidant enzymes in the adults accounts for active uptake of nutrients and defense against host immune attacks. The types of ion channels present in C. sinensis are consistent with its parasitic nature and phylogenetic placement in the tree of life. We anticipate that the transcriptomic information on essential regulators of development, bile chemotaxis, and physico-metabolic pathways in C. sinensis that presented in this study will guide further studies to identify novel drug targets and diagnostic antigens.

A gene encoding a serine protease inhibitor of Clonorchis sinensis (CsSERPIN) was identified and characterized. CsSERPIN contained an open reading frame of 1158bp that encoded 385 amino acid residues. Sequence analysis of the primary structure of CsSERPIN revealed that it had essential structural motifs including a reactive central loop (RCL), which well conserved in the serine protease inhibitor (serpin) superfamily. CsSERPIN was classified as a member of the ovalbumin-type serpin family on the basis of phylogenetic analysis and the absence of a classical N-terminal signal peptide. Recombinant CsSERPIN showed an inhibitory effect on chymotrypsin in a dose-dependent manner, but did not effectively inhibit trypsin, thrombin, elastases or cathepsin G. Optimal pH values of CsSERPIN were between 7.0 and 9.0, as evidenced by the rapid loss of inhibitory activity under acidic conditions. CsSERPIN was expressed at various developmental stages of the parasite, from eggs to adult worms, but its expression level was higher in eggs and adult worms than in metacercariae and juvenile worms. CsSERPIN was identified in the soluble extract of the parasite, but not in the excretory and secretory products (ESP) or insoluble extract of the parasite. Immunolocalization analysis of CsSERPIN showed that it mainly localized to the eggs and vitelline glands of the adult worm. These results suggest that intracellular CsSERPIN may be possibly involved in maintaining the physiology of eggs as well as in egg production of C. sinensis by regulating endogenous serine proteases., (Copyright 2010 Elsevier B.V. All rights reserved.)

Clonorchiasis is an infection associated with bile duct malignancy and subsequent development of cholangiocarcinoma. This disease is mainly caused by Clonorchis sinensis worms and their excretory-secretory products (ESP). However, the precise molecular mechanisms of carcinogenesis remain to be determined. Previously, we established differential gene expression profiles from microarrays containing 23,920 human genes of known function in a human cholangiocarcinoma cell line, HuCCT1, treated with ESP. Among the upregulated genes, we focused on minichromosome maintenance protein 7 (Mcm7), which is implicated in various cancer types, and analyzed transcriptional regulation mediated by ESP to further elucidate its role in cholangiocarcinoma development. Global histone acetylation levels were increased in ESP-treated cells, along with histone acetyltransferase (HAT) protein expression. Detailed promoter analysis using reporter and chromatin immunoprecipitation assays revealed that transcriptional activation of Mcm7 is mediated by HAT recruitment to the promoter region upon C. sinensis ESP treatment. These findings contribute to clarification of the intrinsic mechanism underlying the cellular carcinogenesis process stimulated by Mcm7 in C. sinensis-treated host cells., (2010 Elsevier B.V. All rights reserved.)

Cysteine proteases of helminth parasites play essential roles in parasite physiology as well as in a variety of important pathobiological processes. In this study, we identified a multigene family of cathepsin F cysteine proteases in Clonorchis sinensis (CsCFs). We identified a total of 12 CsCF genes through cDNA cloning using degenerate PCR primers followed by RACE. Sequence and phylogenetic analysis of the genes suggested they belonged to the cathepsin F-like enzyme family and further clustered into three different subfamilies. Enzymatic and proteomic analysis of C. sinensis excretory and secretory products (ESP) revealed that multiple isoforms of CsCF were the major proteins present in the ESP and the proteolytic activity of the ESP is mainly attributable to the enzymes. Comparative analysis of representative enzymes for each subfamily, CsCF-4, CsCF-6, and CsCF-11, showed that they share similar biochemical properties typical for cathepsin F-like enzymes, but significant differences were also identified. The enzymes were expressed throughout various developmental stages of the parasite and the transcripts increased gradually in accordance with the maturation of the parasite. Immunolocalization analysis of CsCFs showed that they were mainly localized in the intestine and intestinal contents of the parasite. These results collectively suggested that CsCFs, which are apparently synthesized in the epithelial cells lining the parasite intestine and secreted into the intestinal lumen of the parasite, might have a cooperative role for nutrient uptake in the parasite. Furthermore, they were eventually secreted into outside of the parasite and may perform additional functions for host-parasite interactions.

Severe Clonorchis sinensis infection is a significant risk factor for malignant changes in bile ducts and surrounding liver tissues occurring as a result of direct contact with C. sinensis worms and their excretory-secretory products (ESP). However, the intrinsic molecular mechanisms involved in these processes remain obscure. To determine the effects of C. sinensis infection on protein expression in host bile duct epithelium, we examined proteomic profile changes in the human cholangiocarcinoma cell line (HuCCT1) treated with ESP at 24 h. Using a combination of 2-DE, quantitative image and MALDI-TOF MS analysis, we identified 83 proteins that were translationally modulated in response to ESP, among which 49 were up-regulated and 34 down-regulated. These proteins were classified under various biological categories, including metabolism, cell structure and architecture, proteolysis, protein modification, transport, signal transduction, and reactive oxygen species (ROS) detoxification. In particular, ESP induced the expression of redox-regulating proteins, including peroxiredoxins (Prdx 2, 3, and 6) and thioredoxin 1 (Trx 1), possibly via intracellular ROS generation. Application of the proteomic approach to identify ESP response proteins should be a prerequisite before further investigation to clarify the molecular pathways and mechanisms involved in C. sinensis infection of host cells.

Clonorchis sinensis, the parasite that causes clonorchiasis, is endemic in many Asian countries, and infection with the organism drives changes in the liver tissues of the host. However, information regarding the molecular events in clonorchiasis remains limited, and little is currently known about host-pathogen interactions in clonorchiasis. In this study, we assessed the gene expression profiles in mice livers via DNA microarray analysis 1, 2, 4, and 6 weeks after induced metacercariae infection. Functional clustering of the gene expression profile showed that the immunity-involved genes were induced in the livers of the mice at the early stage of metacercariae infection, whereas immune responses were reduced in the 6-week liver tissues after infection in which the metacercariae became adult flukes. Many genes involved in fatty acid metabolism, including Peci, Cyp4a10, Acat1, Ehhadh, Gcdh, and Cyp2 family were downregulated in the infected livers. On the other hand, the liver tissues infected with the parasite expressed Wnt signaling molecules such as Wnt7b, Fzd6, and Pdgfrb and cell cycle-regulating genes including cyclin-D1, Cdca3, and Bcl3. These investigations constitute an excellent starting point for increased understanding of the molecular mechanisms underlying host-pathogen interaction during the development of C. sinensis in the host liver.

Clonorchis sinensis, the Chinese liver fluke, is the causative agent of clonorchiasis as well as liver and biliary diseases. The excretory-secretory products (ESPs) of the parasites play important roles in host-parasite interactions. In this study, we have investigated the proteome of ESPs obtained from C. sinensis adult worms. Although the full genome database of C. sinensis is not yet available, we have successfully identified 62 protein spots using 2-DE-based mass analysis and EST database of C. sinensis. The proteins identified include detoxification enzymes, such as glutathione S-transferase and thioredoxin peroxidase, myoglobin and a number of cysteine proteases that are expressed abundantly. In order to identify potential targets for the diagnosis and therapy of clonorchiasis, we conducted immunoblot analysis of the ESPs proteome using the sera obtained from clonorchiasis patients and identified legumains and cysteine proteases as antigens present in the ESPs. Although the cysteine proteases were previously reported to elicit antigenicity, the legumains are found herein for the first time as a serological antigen of C. sinensis. To confirm these findings, we expressed recombinant legumain in Escherichia coli and verified that recombinant legumain also functions as a potent antigen against the sera of clonorchiasis patients. Our results illustrate the validity of immuno-proteomic approaches in the identification of serodiagnostic antigens in the parasites.

Clonorchiasis is associated with bile duct malignancy and the subsequent development of cholangiocarcinoma. Although this is likely caused by adult Clonorchis sinensis and its excretory-secretory products (ESP), the precise molecular mechanisms remain obscure. To evaluate the effect of C. sinensis infection on differential gene expression in host hepatocytes, we examined the kinetics of changes in gene expression in the human cholangiocarcinoma cell line HuCCT1 treated with ESP at different times. Using complementary DNA microarrays containing 23,920 human genes of known function, we initially identified 435 genes altered by ESP treatment. Of these, 31 were up-regulated and 35 were down-regulated more than twofold in a time-dependent manner following ESP treatment. Clustering of these genes by function revealed that several were involved in the cell cycle, oncogenesis, protein modification, immunity, signal transduction, cell structure, and developmental processes. These findings should provide a fundamental basis for further analysis of the molecular pathways and mechanisms involved in C. sinensis infection of host cells.

The accuracies of relative gene expressions as determined by quantitative real-time polymerase chain reaction are largely dependent on the variabilities of the reference genes used. Validation of the stabilities of reference genes under experimental conditions is an essential initial step for comparative studies on the expression levels of target genes in experimental groups. Using three total RNA samples extracted independently from Clonorchis sinensis metacercariae and adults, we determined the gene expression stabilities of eight reference gene candidates and the relative transcript levels of three target genes using the geNorm program. The reference genes found to be stably expressed in metacercariae and adults were phosphoglycerate kinase, beta-actin, and calcyphosine; reference genes found to be stably expressed under gamma-irradiated and non-irradiated conditions were succinate dehydrogenase, small nuclear ribonucleoprotein, and beta-actin; and those stably expressed regardless of bile treatment were small nuclear ribonucleoprotein, phosphoglycerate kinase, and succinate dehydrogenase. According to our data, the expression levels of target genes are dependent on normalization factors, such as the C (T) values of single reference genes and the geometric mean of the C (T) values of three reference genes. When comparing C. sinensis gene expressions, we propose to employ the geometric mean of the C (T) values of more than three reference genes validated in the same experimental setting.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a major antioxidant enzyme and may protect against lipid hydroperoxidation in biomembranes. We isolated full-length cDNA sequences encoding four different PHGPxs from a causative agent of cholangiocarcinoma, Clonorchis sinensis (CsGPx1, CsGPx2, CsGPx3 and CsGPx4). These sequences contained an in-frame TGA codon for selenocysteine (Sec) and a concurrent Sec insertion sequence in their 3'-untranslated regions. The open reading frames were composed of six exons in the chromosomal segments of CsGPx1 (7705bp), CsGPx2 (5871bp) and CsGPx3 (3867bp) and five exons in CsGPx4 (5655bp). The positions of these introns were tightly conserved between the trematode and vertebrate PHGPx genes. Oxidative stimulation of viable worms with H(2)O(2) or paraquat resulted in 1.5- to 2-fold induction of the GPx activity. The CsGPx proteins were specifically localised in vitellocytes within vitelline follicles and premature eggs in the proximal uterus. In the eggs, glutathione, an electron donor for GPx, was co-localised with the CsGPx proteins, while thioredoxin, which is preferred by peroxiredoxin, was principally detected in the extracellular space between the embryonic cell mass and an eggshell. Our data may suggest a concerted or a specialised function between a thioredoxin-dependent enzyme(s) and GPx in protecting against H(2)O(2)-derived damage during maturation of the embryo and formation of the eggshell, in these catalase-lacking trematode parasites. The uniquely conserved genomic organisation and Sec-dependency amongst trematode and vertebrate PHGPx homologues will also provide insight into the evolutionary episode and functional/biochemical diversification of GPx proteins.

Clonorchis sinensis thrives on bile juice. The effects of bile and bile acids on newly excysted juvenile C. sinensis (CsNEJ) were studied in terms of survival. Survival of CsNEJs maintained in 1x Locke's solution, Dulbecco's modified Eagle's medium, NCTC 109, Eagle's, RPMI 1640, and 0.1% glucose was high, but dropped rapidly in 2x Locke's, 0.85% NaCl, and phosphate-buffered saline. Most amino acids in the media favored CsNEJ survival; however, aspartic and glutamic acids and adenine reduced survival. Survival was also significantly lower in media containing more than 0.1% bile. CsNEJs preconditioned in low bile media survived longer in higher bile media. All bile acids and conjugated bile salts were found to favor CsNEJ survival, except for lithocholic acid (LCA) which was toxic. NCTC 109 medium was found to be optimal for the in vitro maintenance of CsNEJs and 1x Locke's solution to be suitable for analyzing the biological effects of bioactive compounds and molecules. Based on these results, we propose that bile acids enhance activity of CsNEJs, but LCA deteriorate CsNEJs.

A gene encoding cysteine proteinase from Clonorchis sinensis has been cloned and expressed in Escherichia coli. The cysteine proteinase cDNA fragment was amplified by reverse transcription-polymerase chain reaction using degenerate oligonucleotide primers derived from conserved active site of cysteine proteinases. The 5' and 3' regions of the gene were amplified using rapid amplification of cDNA ends. The cloned gene has an open reading frame of 696 bp and deduced amino acid sequence of 232. Sequence analysis and alignment showed significant homologies with the eukaryotic cysteine proteinases and conservation of the Cys, His, and Asp residues that form the catalytic triad. Analysis of the expressed protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the molecular weight of the protein was approximately 28.5 kDa. Proteolytic activity of the expressed protein was inhibited by cysteine proteinase inhibitors such as L-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane, iodoacetic acid, and leupeptin. The expressed protein showed biochemical properties similar to those of cysteine proteinases of other parasites. The expressed protein strongly reacted with the sera from patients with clonorchiasis but not with the sera from patients with paragonimiasis, fascioliasis, cysticercosis, and sparganosis, or with sera from normal human controls. These results suggest that the expressed protein may be valuable as a specific diagnostic material for the immunodiagnosis of clonorchiasis.

A 7-kDa protein was purified from extracts of adult Clonorchis sinensis by a combination of ammonium sulfate precipitation, anion exchange chromatography, cation exchange chromatography, gel-filtration chromatography, and reversed-phase FPLC. The 7-kDa protein exists in the excretory-secretory products of adult C. sinensis, but not in extracts of adult Paragonimus westermani. Also, the 7-kDa protein reacted with the sera of patients with clonorchiasis but not with paragonimiasis or normal human sera. To observe the localization of the 7-kDa protein in the tissue of adult C. sinensis, an immunogold labeling method was followed using anti-7-kDa antibody. The gold particles were observed in the basal layer below the tegumental syncytium, in the interstitial matrix of the parenchyma, and in the content of the uterus. The 7-kDa cDNA was obtained through reverse transcription-polymerase chain reaction using a primer designed from N-terminal sequence analysis. Rapid amplification of cDNA ends (5'-RACE) was used to obtain the complete protein coding sequence. The sequence encodes a 90-amino acid polypeptide. The deduced amino acid sequence of the 7-kDa protein revealed no homology with proteins of different organisms reported so far. These results suggest that the 7-kDa protein is a fluid antigen and may be valuable as a tool for the immunodiagnosis of clonorchiasis.

Abstract: Objectives: The prevalence of Clonorchis sinensis infection was investigated among residents of the five major river basins, that is, Hangang, Nakdonggang, Seomjingang, Yeongsangang, and Geumgang River basins in Korea. Methods: From January to December 2007, a total of 31,268 stool samples were collected from 29 localities and examined by the formalin-ether sedimentation technique. Results: Intestinal parasite eggs and/or protozoan cysts were detected from 2957 (9.5%) inhabitants. Number of residents harbouring helminth eggs in the faeces was 2542 (8.1%) for C. sinensis, 255 (0.8%) for Heterophyes spp., 36 (0.1%) for Echinostoma spp., 30 (0.1%) for Trichuris trichiura, 8 (0.03%) for Ascaris lumbricoides, 7 (0.02%) for Gymnophalloide seoi, and 50 (0.02%) for Trichostrongylus orientalis. Number of residents harbouring protozoan cysts in the faeces was 133 (1.3%) for Entamoeba spp. and 50 (0.2%) for Giardia lamblia. The positive rates of C. sinensis in Nakdonggang, Seomjingang, Yeongsangang, Geumgang, and Hangang River basins were 12.2%, 9.5%, 3.3%, 3.0%, and 1.0%, respectively. The egg positive rate of C. sinensis was higher in male (10.6%) than in female (6.1%), and the age group of 50s had the highest positive rate (10.4%). Conclusion: The result of this study revealed little decrease in positive rate of C. sinensis compared with the result of southern endemic areas of Korea in 2006. [Copyright &y& Elsevier]

Clonorchis sinensis, a high-risk pathogenic human liver fluke, provokes various hepatobiliary complications, including epithelial hyperplasia, inflammation, periductal fibrosis, and even cholangiocarcinogenesis via direct contact with worms and their excretory–secretory products (ESPs). These pathological changes are strongly associated with persistent increases in free radical accumulation, leading to oxidative stress-mediated lesions. The present study investigated C. sinensis infection- and/or carcinogen N-nitrosodimethylamine (NDMA)-associated fibrosis in cell culture and animal models. The treatment of human cholangiocytes (H69 cells) with ESPs or/and NDMA increased reactive oxidative species (ROS) generation via the activation of NADPH oxidase (NOX), resulting in augmented expression of fibrosis-related proteins. These increased expressions were markedly attenuated by preincubation with a NOX inhibitor (diphenyleneiodonium chloride) or an antioxidant (N-acetylcysteine), indicating the involvement of excessive NOX-dependent ROS formation in periductal fibrosis. The immunoreactive NOX subunits, p47phox and p67phox, were observed in the livers of mice infected with C. sinensis and both infection plus NDMA, concomitant with collagen deposition and immunoreactive fibronectin elevation. Staining intensities are proportional to lesion severity and infection duration or/and NDMA administration. Thus, excessive ROS formation via NOX overactivation is a detrimental factor for fibrogenesis during liver fluke infection and exposure to N-nitroso compounds. [ABSTRACT FROM AUTHOR]