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Due to the lack of an effective prophylactic intervention and diagnosis, human liver fluke Clonorchis sinensis continues to afflict a large human population, causing a chronic inflammatory bile duct disease. With an aim to identify target antigens for sensitive serodiagnosis, adenylate kinase 3 of C. sinensis (CsAK3) was successfully expressed in soluble form in Escherichia coli by fusion to an RNA-interacting domain derived from human Lys-tRNA synthetase and purified by Ni2+-affinity chromatography. Anti-CsAK3 serum was raised by immunization of mice, and Western blotting confirmed that CsAK3 was expressed in adult-stage C. sinensis. Histochemical analysis showed that CsAK3 was localized to the subtegumental tissue of C. sinensis and was excreted into the bile duct of the host. When tested against sera from various parasite-infected patients by enzyme-linked immunosorbent assay, the recombinant CsAK3 elicited a specific response to C. sinensis-infected sera. The results suggest that CsAK3, either alone or in combination with other antigens, could be used for improving the clinical diagnosis of clonorchiasis.

Venom allergen-like (VAL) proteins, members of the SCP/TAPS (sperm coating protein/Tpx-1/Ag5/PR-1/Sc7) protein superfamily, have been reported from several parasitic helminths. As little is known about their biological functions, a VAL protein of the Chinese liver fluke Clonorchis sinensis was cloned and characterized. A complementary DNA (cDNA) encoding a 25-kDa protein was identified from an EST database of C. sinensis. A BLAST search revealed that the protein shares 46% sequence identity with Schistosoma mansoni VAL 13 protein, and thus, the protein was named CsVAL13. Multiple sequence alignment indicated that the SCP/TAPS domain of CsVAL13 shares 39-46% sequence identity with VAL proteins from parasitic helminths. His and Tyr, which help to stabilize protein structure, were highly conserved across the VAL protein sequences. Phylogenetic analysis showed that the SCP/TAPS domain of the CsVAL13 sequence clusters together with other group 2 VAL protein sequences. In the homology-modeled structure of CsVAL13, an α-β-α sandwich and residues for a putative active site were highly conserved. Immune sera were obtained from BALB/c mice immunized with the recombinant CsVAL13 protein. Immunohistochemical localization using the immune sera revealed that CsVAL13 was distributed mainly in the tegument and intrauterine eggs of adult C. sinensis. These findings suggest that CsVAL13 may be involved in host-parasite interactions and immune stimulation on the surrounding host environments.

A cDNA clone encoding 8 kDa protein was retrieved from an EST pool of Chinese liver fluke Clonorchis sinensis. A deduced polypeptide of the cDNA clone was similar to 8 kDa Ca(2+)-binding proteins from other parasitic trematodes, and, thus, named as CsCa8, containing two EF-hand Ca(2+)-binding sites. Homology models predicted CsCa8 to be a single globular structure having four helices and molecular folds similar to Ca(2+)-binding state of other small Ca(2+)-binding proteins. Recombinant CsCa8 protein showed specific Ca(2+)-binding affinity and shifting in native gel mobility assay. Mouse immune sera raised against recombinant CsCa8 protein recognized native CsCa8 from adult C. sinensis worm extract. CsCa8 was localized in oral and ventral suckers, vitelline follicles and subtegumental tissues. These findings suggest that CsCa8 might be involved in cellular Ca(2+) signal transduction for muscle contraction and egg production.

In the course of examining EST of the liver fluke Clonorchis sinensis, a cDNA encoding the protein similar to tetraspanin 2 (TSP-2) of blood fluke schistosome was identified as CsTSP2. TSPs are a family of eukaryotic cell membrane-spanning proteins thought to anchor multiple proteins to one area of the cell membrane. Multiple sequence alignment of CsTSP2 revealed over 40% of identities with those of schistosomes. The CCC, PXSC, and CG motives characteristic to extracellular loop 2 (EC2) region of TSP-2 were well conserved. PCR-amplified EC2 of CsTSP2 (CsTSP2EC2) was subcloned into pEXP5 NT/TOPO bacterial expression plasmid vector. Recombinant CsTSP2EC2 protein (rCsTSP2EC2) fused with 6X His tag was overexpressed bacterially and purified by using Ni-NTA affinity chromatography under denaturing condition. The purified rCsTSP2EC2 was recognized specifically by the sera from human infected with C. sinensis. Considering biological role and specific immunity of TSPs, rCsTSP2EC2 might be a probable vaccine candidate against clonorchiasis. Protective effects of immunizing rCsTSP2EC2 should be further studied.

We report here a human case probably mixed-infected with Clonorchis sinensis and Fasciola sp. who was diagnosed by computed tomography (CT) scan, serological findings, and/or fecal examination. The patient was a 43-year-old Korean female and was admitted to Kyung Hee University Hospital with the complaints of fever and abdominal pain. On admission, marked eosinophilia was noted in her peripheral blood. CT scan showed specific lesions for clonorchiasis and fascioliasis in the liver, along with lesions suggestive of amebic abscess. Micro-ELISA revealed positive results for the 2 helminthic infections. Eggs of C. sinensis and trophozoites of Entamoeba histolytica were observed in the stool. Treatment with praziquantel followed by metronidazole and tinidazole reduced abnormalities in the liver and eosinophilia. This is the first case report of a possible co-infection with 2 kinds of liver flukes in the Republic of Korea.

WD40-repeat proteins have four to eight repeating units flanked by Gly-His (GH) and Trp-Asp (WD) at both termini and folds into a beta-propeller. A polypeptide deduced from a Clonorchis sinensis cDNA clone analyzed to have seven WD40-repeats and predicted to form a beta-propeller (CsWD1). The CsWD1 protein was expressed stage-specifically in the metacercariae and localized in the tegumental syncytium. The CsWD1 protein is suggested to serve as a platform for interacting partner proteins in the tegumental syncytium of C. sinensis metacercariae.

Elongation factor-1 (EF-1) plays a primary role in protein synthesis, e.g., in the regulation of cell growth, aging, motility, embryogenesis, and signal transduction. The authors identified a clone CsIH23 by immunoscreening a Clonorchis sinensis cDNA library. The cDNA of CsIH23 was found to have a putative open reading frame containing 461 amino acids with a predicted molecular mass of 50.5 kDa. Its polypeptide sequence was highly homologous with EF-1alpha of parasites and vertebrate animals. CsIH23 polypeptide contained three GTP/GDP-binding sites, one ribosome-binding domain, one actin-binding domain, one tRNA-binding domain, and two glyceryl-phosphoryl-ethanolamine attachment sites. Based on these primary and secondary structural similarities, it was concluded that CsIH23 cDNA encodes C. sinensis EF-1alpha (CsEF-1alpha). In a molecular phylogenic tree, CsEF-1alpha clustered with the EF-1alpha of helminthic parasites. Subsequently, CsEF-1alpha recombinant protein was bacterially overexpressed and purified by Ni-NTA affinity column chromatography. Immunoblotting using CsEF-1alpha recombinant protein produced positive signals for all serum samples tested from clonorchiasis, opisthorchiasis viverinii, and paragonimiasis westermani patients and normal healthy controls. These findings suggest that recombinant CsEF-1alpha is of limited usefulness as serodiagnostic antigen for clonorchiasis.

The pond smelt Hypomesus olidus and minnow Zacco platypus were collected from the Soyang and Daechung Lakes in January 2003, and their metacercarial infections was examined by the muscle compression and artificial digestion techniques. In the Soyang Lake, 161 metacercariae of Clonorchis sinensis (0.35 per fish) were harvested from 459 pond smelts examined. Also, 13 metacercariae of C. sinensis (0.43 per fish), 1 of Metagonimus sp., 4 of Echinostoma sp., 148 of Centrocestus armatus and 44 unidentified species were collected from 30 minnows. In the Daechung Lake, 369 metacercariae of C. sinensis (3.69 per fish) and 51 unidentified species were recovered from 100 pond smelts. The metacercariae of C. sinensis were fed to experimental rats, in which the adult flukes were identified. The pond smelts and minnows collected from the Soyang and Daechung Lakes were verified to be the second intermediate hosts and the sources of human C. sinensis infection.

We purified the 17 kDa protein abundant in Clonorchis sinensis crude extracts. The N-terminal amino acid sequence of this protein was determined and an oligonucleotide probe synthesized. Using this probe, the cDNA encoding the protein was cloned and sequenced from the C. sinensis cDNA library. It was found to consist of a total of 150 amino acids and to have 41% conserved homology with the myoglobin of the trematodes Paramphistomum epiclitum and Isoparorchis hypselobagri. The gene product over-expressed in the bacterial system was purified and identified as the same molecule in the adult worms. BALB/c mouse sera raised against the adult 17 kDa protein revealed that this myoglobin was distributed throughout the parenchymal tissues except for the eggs and reproductive organs and that the protein may be involved in the survival of C. sinensis in the oxygen-depleted environment of the host.

A recombinant pore-forming peptide of Clonorchis sinensis, clonorin, was evaluated for serodiagnostic reagent of clonorchiasis by enzyme-linked immunosorbent assay detecting IgG antibody. Recombinant clonorin showed 100% specificity and low sensitivity for sera of human clonorchiasis. In contrast, C. sinensis crude antigen revealed lower specificity and higher sensitivity than recombinant clonorin did. In sera of experimental rabbits, clonorin-specific IgG antibody was increased remarkably 8 weeks after the infection and retained around level of OD(490)=0.2 for 1 year. With excellent antigenic specificity, it is suggested that the recombinant clonorin can be used as an ingredient of the cocktail antigen for serodiagnosis of clonorchiasis from early stages of the infection.

A mu-class glutathione S-transferase (Cs26GST) of molecular mass 26 kDa was characterized from Clonorchis sinensis. In adult C. sinensis, the distribution of the Cs26GST was investigated by immuno-histochemistry and electron microscopy. Cs26GST was localized to the tegument and parenchyma. Immunogold labeling was strong in the tegumental cell bodies and moderate in the tegument and ova in the oviduct. It is suggested that Cs26GST plays a role in the metabolism and fecundity of C. sinensis.

Peptides pore-forming in cell membrane have been identified from a wide range of animals. A putative pore-forming peptide deduced from a cDNA clone of Clonorchis sinensis (clonorin) was predicted to consist of four amphipathic alpha-helices. Clonorin contained six invariably conserved cysteine residues, identified to form three disulfide bonds. These predicted structural features are highly homologous with pore-forming peptides, the amoebapores. Recombinant clonorin showed hemolytic activity toward rabbit erythrocytes. The hemolytic activity of C. sinensis extract increased dose-dependently and was inhibited by anti-clonorin immune sera. The clonorin was expressed developmentally in juvenile and adult flukes and localized in the intestinal epithelium of adult flukes. It is proposed that, through lysing host cellular components, clonorin could enhance proteolytic digestion in the intestine of C. sinensis.

A cDNA clone encoding 8 kDa protein was retrieved from an EST pool of Chinese liver fluke Clonorchis sinensis. A deduced polypeptide of the cDNA clone was similar to 8 kDa Ca 2+-binding proteins from other parasitic trematodes, and, thus, named as CsCa8, containing two EF-hand Ca 2+-binding sites. Homology models predicted CsCa8 to be a single globular structure having four helices and molecular folds similar to Ca 2+-binding state of other small Ca 2+-binding proteins. Recombinant CsCa8 protein showed specific Ca 2+-binding affinity and shifting in native gel mobility assay. Mouse immune sera raised against recombinant CsCa8 protein recognized native CsCa8 from adult C. sinensis worm extract. CsCa8 was localized in oral and ventral suckers, vitelline follicles and subtegumental tissues. These findings suggest that CsCa8 might be involved in cellular Ca 2+ signal transduction for muscle contraction and egg production. [ABSTRACT FROM AUTHOR]

A cDNA clone encoding 8 kDa protein was retrieved from an EST pool of Chinese liver fluke Clonorchis sinensis. A deduced polypeptide of the cDNA clone was similar to 8 kDa Ca 2+-binding proteins from other parasitic trematodes, and, thus, named as CsCa8, containing two EF-hand Ca 2+-binding sites. Homology models predicted CsCa8 to be a single globular structure having four helices and molecular folds similar to Ca 2+-binding state of other small Ca 2+-binding proteins. Recombinant CsCa8 protein showed specific Ca 2+-binding affinity and shifting in native gel mobility assay. Mouse immune sera raised against recombinant CsCa8 protein recognized native CsCa8 from adult C. sinensis worm extract. CsCa8 was localized in oral and ventral suckers, vitelline follicles and subtegumental tissues. These findings suggest that CsCa8 might be involved in cellular Ca 2+ signal transduction for muscle contraction and egg production. [ABSTRACT FROM AUTHOR]