Functional Genes and Proteins of Clonorchis sinensis

Article information

Korean J Parasitol. 2009;47(Suppl):S59-S68
Publication date (electronic) : 2009 October 27
doi : https://doi.org/10.3347/kjp.2009.47.S.S59
1Department of Medical Environmental Biology and Research Center for Biomolecules and Biosystems, Chung-Ang University College of Medicine, Seoul 156-756, Korea.
2Department of Parasitology and Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-751, Korea.
Corresponding author (hongsj@cau.ac.kr)
Received 2009 October 06; Revised 2009 October 08; Accepted 2009 October 08.

Abstract

During the past several decades, researches on parasite genetics have progressed from biochemical and serodiagnostic studies to protein chemistry, molecular biology, and functional gene studies. Nowadays, bioinformatics, genomics, and proteomics approaches are being applied by Korean parasitology researchers. As for Clonorchis sinensis, investigations have been carried out to identify its functional genes using forward and reverse genetic approaches and to characterize the biochemical and biological properties of its gene products. The authors review the proteins of cloned genes, which include antigenic proteins, physiologic and metabolic enzymes, and the gene expression profile of Clonorchis sinensis.

INTRODUCTION

Clonorchis sinensis-infections are the most prevalent form of parasitic infections in Korea today. Controlled mass chemotherapy has been deployed based on praziquantel in endemic areas since the 1980's, and this campaign has reduced the infection rate down to 2.9% and the worm burden of those infected to a lower level [1]. Clonorchiasis patients suffer chronically from fatigue, jaundice, abdominal discomfort, and indigestion. C. sinensis-infection provokes inflammation, hyperplasia of the biliary epithelium and periductal fibrosis of intrahepatic bile ducts [2]. Furthermore, C. sinensis infection can initiate the development of cholangiocarcinoma in biliary epithelial cells in the presence of chronic inflammation and hyperplastic proliferation [3,4]. In fact, it has been shown epidemiologically that clonorchiasis increases the prevalence of cholangiocarcinoma in endemic areas [5]. To prevent cholangiocarcinoma development, early diagnosis and treatment are crucial.

Serodiagnosis is considered to support a diagnosis of clonorchiasis, and to obtain satisfactory serodiagnostic results, the antigenic proteins employed should be sensitive and specific. In fact, the searches conducted to identify better antigenic proteins from excretory-secretory (ES) and somatic proteins propelled biochemical and molecular biological research on C. sinensis.

At the host-parasite interface, C. sinensis mechanically stimulates adjacent biliary epithelium and biochemically even stimulates remote biliary epithelium. It is obvious that the ES products of C. sinensis contain components that provoke pathologic changes in biliary epithelium, and molecules produced by C. sinensis have been shown to stimulate inflammation and the productions of endogenous bioreactive radicals in epithelial cells, and these radicals not only cause DNA damage but inhibit the repair of DNA damage, and thus, promote mutation [3,5]. The molecules responsible and the pathologic progress occurring in and around the biliary tract have long been of research interest. Much research had been conducted to identify the molecules and signal pathway networks involved, and several factors in C. sinensis ES products that provoke intracellular signals have been identified [6,7].

Information on the functioning genes can be obtained cost-effectively by collecting and annotating expressed sequence tags during the developmental stages of parasites. In C. sinensis, expressed sequence tags were collected from the adults and metacercariae, and annotated and analyzed by referring to the transcriptome and genome information of Schistosoma mansoni, Schistosoma japonicum, and Opisthorchis viverrini and to genetic databases in the public domain [8-12]. Genetic information on functional genes at the transcriptome level can provide important practical information to proteomic researches and enable comprehensive life phenomena of C. sinensis to be elucidated by high throughput microarray analyses.

CHROMOSOMES AND GEOGRAPHICAL ISOLATES

C. sinensis has 2n = 56 chromosomes, where n consists of 8 large and 20 small chromosomes. C. sinensis geographical isolates collected in Korea and northeastern China (Liaoning Province) have all demonstrated the same chromosome number, but different karyotypes. Specifically, Korean isolates have 3 metacentric and 1 meta-/submetacentric pairs, whereas Chinese isolates have 2 and 2 pairs, respectively. The 2 isolates have same number, 16 and 8, of submetacentric and subtelomeric pairs [13].

Genetic relationships between geographical isolates have been studied by employing sequence analyses of nuclear ribosomal DNA (18S, internal transcribed spacer 1 and 2; ITS1 and ITS2) and mitochondrial DNA (cytochrome c oxidase subunit 1 [CO-1]). C. sinensis Korean isolates collected in Kimhae city were compared with Chinese isolates collected in Shenyang, Liaoning and Nanning, and Guangxi provinces. The geographical isolates were nearly identical in terms of nuclear ribosomal and mitochondrial DNA sequences, and revealed no more than a 1% sequence difference between Korean and Chinese isolates [14-17]. Isozyme electrophoresis had been used to study trematode systematics, and isozymes of the Korean and Chinese isolates show homozygous monomorphic banding patterns. Furthermore, alpha-glycerophosphate dehydrogenase produced a unique band pattern and was found to differentiate geographical isolates of C. sinensis [14]. However, in C. sinensis, intraspecific genetic variations among geographical isolates in the Sino-Korea region appear minimal.

As compared with O. viverrini, C. sinensis ITS2 revealed 95% identity and differences at 28 nucleotide points. Furthermore, the mitochondrial CO1 gene of O. viverrini was found to be 96% identical with that of C. sinensis. Even a different genus in the same family Opisthorchidae, namely, O. viverrini appears to be genetically close to C. sinensis [17].

Genome variation

C. sinensis has more than 100 copies of an uncorrupted long-terminal-repeat retrotransposon (CsRn1) distributed over its genome. The functional domains of Gag, proteinase, reverse transcriptase, Rnase H, and subdomains of integrase are strongly conserved in CsRn1, which has been predicted to be mobile based on structure considerations and from the presence of mRNA transcripts. CsRn1 belongs to the Ty3/gypsy-like long-terminal-repeat transposon family [18]. Multiple copies of the CsRn1 were clustered into 4 subsets. Sequential transposition of these subsets into the genome was evidenced by the differential sequence divergence and heterogeneous integration patterns of CsRn1. Insertions of CsRn1 appear preferentially at repetitive and agenic chromosomal regions. Furthermore, CsRn1 was reported to induce variations in the genome that may influence evolution of the C. sinensis [19].

EXPRESSED SEQUENCE TAGS

Information on genes expressed in parasites has been collected from diverse taxa, from individual genes, or in massive amounts by the high-throughput sequencing of transcriptomes. In the later case, the mRNAs extracted from an organism are converted into cDNAs to constitute a cDNA library, and sequenced from the 5'-end of each clone to produce expressed sequence tags (ESTs). Functional genes in a parasite can be profiled cost effectively by collecting ESTs under an experimental conditions or a developmental stage.

A total of 3,221 ESTs of C. sinensis has been registered in public dbEST databases (http://www.ncbi.nlm.nih.gov/dbEST); 2,802 ESTs from the adults and 415 from the metacercariae [8,9,20]. In adult C. sinensis, the genes abundantly expressed in decreasing order were; groups of metabolic enzymes, regulatory and signal proteins, structure and cytoskeletal proteins, transcription and translation machinery proteins, and proteases and inhibitors. C. sinensis adults utilize large amounts of exogenous glucose to produce energy and to provide metabolic intermediates for physiologic processes [21]. As a result, enzymes of the glycolytic pathway are abundantly expressed to drive energy production. Mitochondrial genes expressed at elevated levels also participate in energy production. Furthermore, genes of muscular components, such as limpet homologue, alpha-tubulins, and actin-binding protein, have been identified in second most expressed EST populations; this implies that adult flukes move actively to abrade and feed on biliary epithelia and host blood cells. Cysteine proteases were also found to be expressed at particularly high levels. The proteolytic activities of these proteases help the fluke abrade biliary epithelium and digest engulfed tissue debris and blood cells.

Vitelline precursor protein is abundantly expressed in the adult, which produce a large number of eggs to perpetuate life cycle. The vitelline precursor protein is produced in vitelline glands by yolk cells, which also surrounds germ cells. By stimulation of the Mehlis' gland, vitelline precursor protein is begun to be secreted and to form the eggshell [22].

In C. sinensis metacercariae, the genes abundantly expressed are those of structural and cytoskeletal proteins, energy and other metabolic enzymes, transcription and the translation machinery, the kinases and phosphatases, the DNA scaffold and binding proteins, and the proteases and their inhibitors [9]. The genes expressed higher in metacercariae than in the adult are those of structural and cytoskeletal proteins, kinases and phosphatases, and of the DNA scaffold and binding proteins. Of the annotated ESTs, 26.3% are of structural and cytoskeletal proteins, implying that the metacercariae are in rest phase and maintain minimal metabolism in fish hosts due to the cold environment.

DIFFERENTIALLY EXPRESSED GENES

Gamma-ray treatment of metacercariae prior to infection dose-dependently decreased the recovery rates of adult flukes from experimental animals. However, no chromosome aberrations were found among surviving adult flukes, other than a reduction in chromosome size in some flukes irradiated with 30 Gy [23]. C. sinensis metacercariae are relatively resistant to gamma irradiation [24]. In gamma-irradiated C. sinensis metacercariae, 19 genes were found to be up-regulated by annealing control primer (ACP)-based PCR, and the up-regulated genes were associated with energy metabolism, protein processing, and DNA repair. It was suggested that the upregulated genes orchestrate DNA and cellular damage repairs induced by gamma-irradiation in metacercariae [25].

Bile is a stimulant to organisms that colonize the mammalian intestine. C. sinensis metacercariae excyst in the duodenum, and newly excysted juveniles migrate, driven by bile-chemotaxis to the bile duct and there mature to adults [26,27]. The incubation of C. sinensis metacercariae in bile containing media induced the differential expressions of 16 genes, and the corresponding gene products were found to be associated with energy generation or the cell proliferation signal pathway. Bile induces C. sinensis metacercariae genes that participate in energy metabolism and modulate the regulatory signals of cell proliferation and of the growth and development of newly excysted juveniles [28].

ANTIOXIDANT ENZYMES

The glutathione S-transferases (GSTs) are a family of antioxidant enzymes that catalyze the conjugations of reduced glutathione to electrophilic radicals of a wide variety of substrates. This conjugation activity neutralizes endogenous and exogenous bio-reactive intermediates. Furthermore, GSTs also bind to hydrophobic substrates and transport the GST-substrate complexes to some cellular sites [29]. GSTs have been found ubiquitously in animals from protozoa to vertebrates and are classified as α, µ, π, θ, ζ, σ, and ω classes [30].

Four cytosolic GSTs have been identified in C. sinensis with estimated molecular mass of 24.3, 24.7 (2 clones with different peptide sequences) and 25.1 kDa [31-34]. The C. sinensis GSTs show enzymatic activity toward 1-chloro-2,4-dinitrobenzene (CDNB). GSTs with molecular masses of 24.3 and 24.7 kDa appear to be homologous in terms of peptide sequence and secondary structure with the 28 kDa GSTs of helminthes and vertebrates. These 3 GSTs were found to be sensitively inhibited by different inhibitors used for the classification. Using these biochemical and enzymatic properties, these 3 GSTs were grouped into the sigma class. The GST of 25.1 kDa revealed molecular and enzymatic properties homologous with the 26 kDa GSTs of invertebrate animals and was allocated to class µ GST [32].

In adult worms, 28 kDa GSTs play the major role in antioxidant activity rather than 26 kDa GST, because the 2 GSTs' molar ratio is 14:1 in the adult [31]. C. sinensis 28 and 26 kDa GSTs are localized in the tegument and mesenchymal tissues and in intra-uterine eggs [31,35,36]. Based on their enzymatic activities and localizations in C. sinensis adults, they are believed to play a role in the secondary defense system against exogenous and endogenous bioreactive compounds. Whereas other antioxidant enzymes, such as, superoxide dismutase and glutathione peroxidase are abundantly found in the teguments of trematodes and play a role in the primary defense system against exogenous bioreactive compounds and endogenous radicals resulting from hydroperoxidation [37]. The C. sinensis 28 and 26 kDa GSTs are localized to the reproductive system, such as, to ovary and sperm and intra-uterine eggs. These antioxidant enzymes could play a defense role against bioreactive species during the reproduction of C. sinensis [31-36,38].

Phospholipid hydroperoxide glutathione peroxidases, CsGPx1, CsGPx2, CsGPx3, and CsGPx4, were cloned and localized in the vitellocytes of vitelline glands and in the intrauterine premature eggs of adult C. sinensis. CsGPx proteins were co-localized with glutathione in vitellocytes and eggs, whereas thioredoxin was found principally between embryonic cell masses and eggshells [39]. In S. mansoni, glutathione peroxidase is present at low activity in the tegument of adult flukes, and thioredoxin is secreted from eggs [37]. Glutathione peroxidase, thioredoxins, and peroxiredoxins are crucial for the protection of developing embryos from reactive oxygen radicals derived from endogenous metabolism and from hosts [39,40].

Furthermore, recombinant C. sinensis 26 kDa GSTs with or without an oligopeptide of 14 or 48 amino acids at its N-terminus were crystallized and diffracted to 2.3 Å resolution. The crystallization system used for CsGST-peptide fusion proteins could also be applicable to studies of the crystallographic structures of small peptides [41].

PROTEASES

Proteases are ubiquitous enzymes that take part in diverse biological functions in organisms ranging from viruses to man. Recent advances in genomic analysis have revealed that proteases comprise approximately 2% of the total number of proteins in all types of organisms [42,43]. Proteases have been classified into functional groups based on whether they use the hydrolytic mechanisms used by serine, threonine, aspartate, metallo- or cysteine proteases [43]. Furthermore, they function not only as individual enzymes but often in cascades or networks [43]. Genomic and proteomic analysis of several major global helminth parasites have revealed that parasite-derived proteases are key virulence factors [44-52]. The proteases of helminth parasites play numerous indispensable roles in parasite physiology, such as, in protein processing and the turnover of parasite proteins, and in various pathogenic aspects, such as, the facilitation of parasite penetration or invasion into host tissue, the hydrolysis of host proteins for nutrient uptake, and host immune system modulation [46,53-59]. Accordingly, their activities are essential for parasite survival and growth, which suggests that they are attractive targets for vaccines or chemotherapeutic agents [53,60-63]. Therefore, the biochemical and functional characterizations of the proteases of helminthes and their medical applications have been well studied as host issues.

Analyses of the expressed sequence tags (ESTs) of C. sinensis have revealed that proteases constitute a large proportion of the protein population of the flukes [8,9], which implies their physiological significance. The cysteine proteases of C. sinensis are developmentally regulated and are essential for fluke survival in terms of the contributions they make to biological processes, such as, stage transition, nutrient uptake, and immune evasion [64,65]. Furthermore, the partially purified cysteine proteases from ES products of C. sinensis adult worms have been found to have cytotoxic effects on cultured cells [66,67]. In addition, the endogenous cysteine proteases of C. sinensis metacercariae appear to be involved in excystation of metacercariae [68].

Several genes encoding cysteine proteases have been isolated from C. sinensis [15,67,69-71], and phylogenetic analysis showed that these enzymes are most closely related to the mammalian cathepsin F enzymes [67,71]. It is not yet clear whether C. sinensis also expresses and secretes cathepsin L-like enzymes in addition to cathepsin F enzymes. Transcripts encoding cathepsin L-like enzymes are not listed among currently available C. sinensis ESTs, although transcripts encoding several other Family C1 proteases, such as, cathepsin B's have been found. The definitive biological functions of these cathepsin F enzymes are not yet clear, but a recent study demonstrated that they participate in nutrient uptake in C. sinensis. The biochemical analysis of CsCF-6, a recently identified cathepsin F of C. sinensis, suggests that the enzyme is a typical cathepsin F-like enzyme with broad substrate specificity against various human proteins [71]. The enzyme is mainly localized in the intestine of C. sinensis and is abundantly identified among ES products [71]. These findings implying that CsCF-6, which is synthesized in the intestinal epithelium of C. sinensis and secreted into the intestinal lumen of the parasite, digests various host proteins and might play an important role in nutrient uptake by C. sinensis. Proteomic analysis of the ES products of C. sinensis adults also found that a large number of cysteine proteases present as major components [72]. The fact that cysteine proteases are secreted into ES products suggests that act as digestive agents in the intestine. A recent study on cathepsin F of O. viverrini revealed enzyme in epithelial cells lining the bile ducts of infected animals, which suggested to possibility that it may stimulate biliary epithelium inflammation and proliferation and promote cholangiocarcinogenesis [57]. Hence, the extracorporeal roles of the cysteine proteases of C. sinensis warrant further investigation. Furthermore, their involvements in biological roles essential to parasite survival in the host make the helminth cysteine proteases likely targets of novel vaccines [53,73]. In addition, to the cathepsin F class cysteine proteases currently available C. sinensis EST sequences have revealed that the parasite possesses a large number of proteases of different classes or clades.

ENZYMES OF ENERGY METABOLISM

Adult C. sinensis dwell in the bile duct, an anaerobic environment, and therefore, run anaerobic metabolism utilizing large amounts of exogenous glucose as a carbon source for energy metabolism. By utilizing the glycolytic pathway, glucose is converted into energy and provides metabolic intermediates for other physiologic pathways. Phosphoglycerate kinase (CsPGK), a glycolytic enzyme, has been cloned and produced as a enzymatically active recombinant protein in vitro. This enzyme was found to be localized extensively in the muscular tissues of oral and ventral suckers, ovary, testes, and tegument, and in intrauterine eggs [74,75]. Phosphoglycerate mutase, a glycolytic enzyme, of C. sinensis was found to catalyze the conversion of 3-phosphoglycerate to 2-phosphoglycerate in the present of cofactor; and its enzymatic activity was found to be inhibited by vanadate [76]. Lactate dehydrogenase (CsLDH) showed no inhibition by high concentration lactate and NAD+, but greatly inhibited by Cu+2, Fe+2, or Zn+2. Furthermore, Gossypol was found to inhibit CsLDH, and thus, was recognized as a potent candidate treatment for C. sinensis [77].

Cytosolic and mitochondrial malate dehydrogenases (CscMDH and CsmMDH) of C. sinensis share low amino acid sequence homology (22%), but show high MDH enzymatic activity, without lactate dehydrogenase activity or NADPH selectivity. However, these enzymes are differentially inhibited by 4,4'-bisdimethylamino diphenylcarbol. The CscMDH is more stable against heat and acidity than CsmMDH. The malate-aspartate shuttle pathway provides an important mean for the shuttle exchange of substrates and reducing equivalents between cytosol and mitochondria. Furthermore, cMDH plays a pivotal role on the cytosolic side of the malate-aspartate shuttle. mMDH is a key enzyme in the tricarboxylic acid cycle and in the malate-aspartate shuttle. Zheng et al. [78-80] have demonstrated that glycolytic enzymes are essential required for the survival and pathogenesis of C. sinensis [78-80].

MEMBRANE PROTEINS

The cytolytic pore-forming peptides are synthesized in the cells of a wide range of animals from protozoa to mammals and are secreted in recognition of their targets. The cytolytic peptides participate in primary defense against infective agents, as they have cytolytic effects on bacteria in food vacuoles and in the environment, and target host cellular components [81]. A pore-forming peptide, clonorin, has been cloned from adult C. sinensis, and was found to have 4 amphipathic α-helices and invariably conserved cysteine residues. Clonorin is expressed developmentally in the juvenile and adult stages, and distributes exclusively in the intestinal epithelium of adult flukes. Furthermore, clonorin has hemolytic dose-dependent activity, and is inhibited by specific immune sera. It has been suggested that clonorin could enhance the proteolytic digestion of cellular components in the intestine [82].

In addition, a pore-forming subunit of ATP-sensitive potassium channel (CsKir6.2) cloned from C. sinensis adult has 2 transmembrane domains and a GFG-motif in its pore-forming loop, which is a conserved feature of kir channels. Small amounts of the CsKir6.2 transcript have been detected in the adult stage [83].

ANTIGENIC PROTEINS

Human infections with C. sinensis have been confirmed by microscopic findings of eggs in stool samples. Serodiagnostic methods have been devised using antigenic proteins of C. sinensis in crude or molecularly defined forms as alternative or complementary diagnostic methods. Using crude antigenic preparations these methods showed higher sensitivities but low specificities. On the other hand, some molecularly defined and recombinant antigenic proteins of C. sinensis were found to have markedly improved specificities and limited sensitivities.

Antigenic proteins of C. sinensis have been largely found in ES products or purified from soluble extracts of worm lysates. These antigenic proteins are derived from the intestinal epithelium and its contents, namely, the excretory bladder, vitelline follicles, and tegument, as evidenced by immunohistochemical staining using infected or immunized animal sera [84,85]. During early stage infections (< 20 weeks), antigenic molecules were found to be proteins > 34 kDa derived from tegument, testes, or intra-uterine contents, and during the later stage, the major antigenic proteins were found to have molecular weights in the range 29-26 kDa and to be derived from the intestine, excretory, or reproductive organs [86].

Excretory-secretory (ES) antigens

A glycine-rich C. sinensis protein (GRCsP) containing 23 repetitions of AQPPKSGDGG was found to be localized in vitelline follicles. Furthermore, recombinant GRCsP protein showed high sensitivity and specificity by ELISA for clonorchiasis [87,88]. Furthermore, a proline-reach antigen (CsRPA) containing 15 GPDAPVPKSG repeats was found to have high sensitivity and specificity against clonorchiasis sera [89].

In addition, a 7 kDa antigenic protein was purified from the ES products of adult C. sinensis and localized to uterine contents and tegumental syncytium. This protein was found to be reactive to clonorchiasis sera but not to paragonimiasis sera [90]. An egg products protein of molecular weight 28 kDa was also found to show high reactivity to clonorchiasis patient sera, but it cross-reacted strongly with the sera of paragonimiasis, opisthorchiasis, and schistosomiasis patients, although it did not react with cysticercosis or sparganosis patient sera [91].

Myoglobin is an abundant protein in ES products, and is localized throughout parenchymal tissues other than those of the reproductive organs of adult C. sinensis. In the bile duct, myoglobin may play an oxygen-capturing role and slowly release this oxygen to metabolic pathways. Recombinant myoglobin has been reported to react to 50% of C. sinensis-infected rabbit sera and to 25% of clonorchiasis patient sera [92,93], whereas recombinant clonorin showed 100% specificity but low sensitivity for clonorchiasis patient sera. In experimental rabbits, clonorin-specific IgG antibody remarkably increased and remained high at 8 weeks to 1 year after C. sinensis infection [94].

Lysophosphatidic acid phosphatase (LPAP) belongs to the acid phosphatase family and hydrolyzes lysophosphatidic acid, a bioactive phospholipid that enhances cell growth, fibroblast chemotaxis, and stimulates neurite retraction. LPAP was identified as an ES-antigen of adult C. sinensis and showed higher sensitivity and specificity than crude worm antigen preparations for the serodiagnosis of human clonorchiasis by ELISA [95]. On the other hand, lysophospholipase catalyses the hydrolysis of lysophospholipids to glycerophosphate derivatives and fatty acids, and fatty acid-binding protein plays a role in the intracellular transport of long-chain fatty acids. Lysophospholipase (25.2 kDa) and fatty acid-binding protein (15.2 kDa) cloned from C. sinensis adults was found to reactive positively toward clonorchiasis patient sera, but were unsatisfactory as a diagnostic reagent [96,97].

Cysteine proteases of C. sinensis are highly antigenic, and thus, their immunodiagnostic values as diagnostic antigens have been investigated [15,67,69,70,98]. The results obtained suggest that the cysteine proteases of C. sinensis are reliable serodiagnostic antigens for clonorchiasis [99], but the methodology required for serodiagnosis remains to be determined.

Furthermore, recombinant 26 and 28 kDa glutathione S-transferases have been reported to react with IgG antibodies in patient sera with low sensitivity, but considerations of the highest specificity expected favored further investigations on multi-antigen cocktails [100]. In addition, 2 glutathione S-transferases have been reported to increase C. sinensis-specific IgE antibody levels in clonorchiasis patient sera [31,38].

Tegumental proteins

The tegumental syncytium is the outermost surface of human-infecting trematodes, and thus, plays crucial functions at the host-parasite interface. In fact, this surface layer secrets defensive molecules that neutralize host-originated immune and bioreactive radicals. Tegumental proteins are currently the leading vaccine candidates for schistosomiases [101,102]. A tegumental protein of 20.8 kDa (CsTP20.8) has been cloned from adult and metacercariae cDNA libraries and localized to the outer surface membrane of C. sinensis. However, recombinant CsTP20.8 protein was considered of limited use for the serodiagnosis of clonorchiasis because it showed moderate sensitivity and high specificity. Oral administration of the CsTP20.8 protein provoked specific IgA production, detected in feces [103,104]. Furthermore, a tegumental protein of 31.8 kDa (CsTP31.8) was immunolocalized to the tegument of adult C. sinensis, and has been suggested to be an antigenic protein for the serodiagnosis of clonorchiasis [105].

PERSPECTIVES

C. sinensis thrives in bile juice, which is a biochemically formidable environment. However, bile juice constitutes a favorable environment for C. sinensis, and it also plays crucial roles as a growth stimulatory factor and physiological regulator. In the post-genomic era, genetic information on whole genomes and transcitomes provides an essential and fundamental platform, which facilitates approaches based on functional genomics and bioinformatics and provides an overview of the complexities of life phenomena, physico-metabolism, and responses to external stimuli. Bile-chemotaxis attracts C. sinensis juveniles into bile ducts, and bile components stimulate the activities and growths of these juvenile flukes. Accordingly, we consider that the biological significance of bile to C. sinensis deserves study in depth with respect; its influence on the biological aspects of cellular signaling networks, the genes involved, and the neurologic circuits that empower the chemotactic migration of juvenile flukes. Single antigenic proteins have been evaluated, but have been found to have low sensitivity, though high specificity, for the serodiagnosis of C. sinensis infections. Multiple antigen cocktails involving optimizations of specific antigenic epitopes offer an alternative approach. However, to achieve this target, a large number of proteins of high specificity must be identified using forward and reverse genetic approaches and molecular biological analyses. As C. sinensis is identified to be a biological agent for cholagiocarcinoma, researches should be pursued on carcinogenic material derived from C. sinensis and on molecular networks and genetic regulation of cholangiocarcinogenesis in the biliary system.

References

1. Kim TS, Cho SH, Huh S, Kong Y, Sohn WM, Hwang SS, Chai JY, Lee SH, Park YK, Oh DK, Lee JK. Working Groups in National Institute of Health. Korea Association of Health Promotion. A nationwide survey on the prevalence of intestinal parasitic infections in the Republic of Korea, 2004. Korean J Parasitol 2009;47:37–47. 19290090.
2. Hong ST, Hong SJ. In : Arizino N, Chai JY, Nawa Y, Takahashi Y, eds. Clonorchis sinensis and clonorchiasis in Korea. Food-borne Helminthiasis in Asia 2005. Vol. 1Tokyo, Japan: Federation of Asian Parasitologists. p. 35–36.
3. Suttiprapa S, Loukas A, Laha T, Wongkham S, Kaewkes S, Gaze S, Brindley PJ, Sripa B. Characterization of the antioxidant enzyme, thioredoxin peroxidase, from the carcinogenic human liver fluke, Opisthorchis viverrini. Mol Biochem Parasitol 2008;160:116–122. 18538872.
4. Bouvard V, Baan R, Straif K, Grosse Y, Secretan B, El Ghissassi F, Benbrahim-Tallaa L, Guha N, Freeman C, Galichet L, Cogliano V. WHO International Agency for Research on Cancer Monograph Working Group. A review of human carcinogens-Part B: biological agents. Lancet Oncol 2009;10:321–322. 19350698.
5. Pak JH, Kim DW, Moon JH, Nam JH, Kim JH, Ju JW, Kim TS, Seo SB. Differential gene expression profiling in human cholangiocarcinoma cells treated with Clonorchis sinensis excretory-secretory products. Parasitol Res 2009;104:1035–1046. 19039603.
6. Kim YJ, Choi MH, Hong ST, Bae YM. Proliferative effects of excretory/secretory products from Clonorchis sinensis on the human epithelial cell line HEK293 via regulation of the transcription factor E2F1. Parasitol Res 2008;102:411–417. 18026993.
7. Kim YJ, Choi MH, Hong ST, Bae YM. Resistance of cholangiocarcinoma cells to parthenolide-induced apoptosis by the excretory-secretory products of Clonorchis sinensis. Parasitol Res 2009;104:1011–1016. 19066964.
8. Cho PY, Lee MJ, Kim TI, Kang SY, Hong SJ. Expressed sequence tag analysis of adult Clonorchis sinensis, the Chinese liver fluke. Parasitol Res 2006;99:602–608. 16705464.
9. Cho PY, Kim TI, Whang SM, Hong SJ. Gene expression profile of Clonorchis sinensis metacercariae. Parasitol Res 2008;102:277–282. 17924144.
10. Oliveira G. The Schistosoma mansoni transcriptome: an update. Exp Parasitol 2007;117:229–235. 17624328.
11. Fung MC, Lau MT, Chen XG. Expressed sequence tag (EST) analysis of a Schistosoma japonicum cercariae cDNA library. Acta Trop 2002;82:215–224. 12020895.
12. Laha T, Pinlaor P, Mulvenna J, Sripa B, Sripa M, Smout MJ, Gasser RB, Brindley PJ, Loukas A. Gene discovery for the carcinogenic human liver fluke, Opisthorchis viverrini. BMC Genomics 2007;8:189. 17587442.
13. Park GM, Im KI, Huh S, Yong TS. Chromosomes of the liver fluke, Clonorchis sinensis. Korean J Parasitol 2000;38:201–206. 11002660.
14. Park GM, Yong TS, Im KI, Lee KJ. Isozyme electrophoresis patterns of the liver fluke, Clonorchis sinensis from Kimhae, Korea and from Shenyang, China. Korean J Parasitol 2000;38:45–48. 10743360.
15. Park GM, Yong TS. Geographical variation of the liver fluke, Clonorchis sinensis, from Korea and China based on the karyotypes, zymodeme and DNA sequences. Southeast Asian J Trop Med Public Health 2001;32(suppl 2):12–16. 12041573.
16. Lee SU, Huh S. Variation of nuclear and mitochondrial DNAs in Korean and Chinese isolates of Clonorchis sinensis. Korean J Parasitol 2004;42:145–148. 15381864.
17. Park GM. Genetic comparison of liver flukes, Clonorchis sinensis and Opisthorchis viverrini, based on rDNA and mtDNA gene sequences. Parasitol Res 2007;100:351–357. 16902795.
18. Bae YA, Moon SY, Kong Y, Cho SY, Rhyu MG. CsRn1, a novel active retrotransposon in a parasitic trematode, Clonorchis sinensis, discloses a new phylogenetic clade of Ty3/gypsy-like LTR retrotransposons. Mol Biol Evol 2001;18:1474–1483. 11470838.
19. Bae YA, Kong Y. Evolutionary course of CsRn1 long-terminal-repeat retrotransposon and its heterogeneous integrations into the genome of the liver fluke, Clonorchis sinensis. Korean J Parasitol 2003;41:209–219. 14699262.
20. Lee JS, Lee J, Park SJ, Yong TS. Analysis of the genes expressed in Clonorchis sinensis adults using the expressed sequence tag approach. Parasitol Res 2003;91:283–289. 14574557.
21. Hahn SS, Hahn HJ, Seo BS. The uptake of C14 glucose by Clonorchis sinensis. Korean J Intern Med 1961;4:281–285.
22. Tang Y, Cho PY, Kim BS, Hong SJ. Molecular cloning and characterization of vitelline precursor protein B1 from Clonorchis sinensis. J Parasitol 2005;91:1374–1378. 16539019.
23. Park GM, Yong TS. Effects of gamma-irradiation on the infectivity and chromosome aberration of Clonorchis sinensis. Korean J Parasitol 2003;41:41–45. 12666729.
24. Lee SH, Park YH, Sohn WM, Hong ST, Chai JY. The effects of gamma irradiation on the survival and development of Clonorchis sinensis metacercariae. Korean J Parasitol 1989;27:187–195.
25. Kim TI, Cho PY, Song KJ, Li S, Hong SJ, Park SW, Chai JY, Shin EH. Gene expression of Clonorchis sinensis metacercaria induced by gamma irradiation. Parasitol Res 2008;102:1143–1150. 18224473.
26. Rim HJ. Clonorchiasis: an update. J Helminthol 2005;79:269–281. 16153321.
27. Li S, Kim TI, Yoo WG, Cho PY, Kim TS, Hong SJ. Bile components and amino acids affect survival of the newly excysted juvenile Clonorchis sinensis in maintaining media. Parasitol Res 2008;103:1019–1024. 18587668.
28. Kim TI, Cho PY, Yoo WG, Li S, Hong SJ. Bile-induced genes in Clonorchis sinensis metacercariae. Parasitol Res 2008;103:1377–1382. 18682984.
29. Boyer TD. The glutathione S-transferases: an update. Hepatology 1989;9:486–496. 2646197.
30. Chiumiento L, Bruschi F. Enzymatic antioxidant systems in helminth parasites. Parasitol Res 2009;105:593–603. 19462181.
31. Kang SY, Ahn IY, Park CY, Chung YB, Hong ST, Kong Y, Cho SY, Hong SJ. Clonorchis sinensis: Molecular cloning and Characterization of 28 kDa glutathione S-transferase. Exp Parasitol 2001;97:186–195. 11384162.
32. Hong SJ, Lee JY, Lee DH, Sohn WM, Cho SY. Molecular cloning and characterization of a mu-class glutathione S-transferase from Clonorchis sinensis. Mol Biochem Parasitol 2001;115:69–75. 11377741.
33. Wu Z, Hu X, Wu D, Xu J, Chen S, Wu Z, Yu X. Clonorchis sinensis: molecular cloning and functional expression of a novel cytosolic glutathione transferase. Parasitol Res 2007;100:227–232. 17048005.
34. Wu Z, Wu D, Hu X, Xu J, Chen S, Wu Z, Yu X. Molecular cloning and characterization of cDNA encoding a novel cytosolic glutathione transferase from Clonorchis sinensis. Parasitol Res 2006;98:534–538. 16416295.
35. Hong SJ, Yu JR, Kang SY. Ultrastructural localization of 28 kDa glutathione S-transferase in adult Clonorchis sinensis. Korean J Parasitol 2002;40:173–176. 12509100.
36. Hong SJ, Kim TY, Kang SY, Yu JR, Song KY, Cho SY. Clonorchis sinensis: immunolocalization of 26 kDa glutathione S-transferase in adult worms. Exp Parasitol 2002;102:191–193. 12856316.
37. Mei H, LoVerde PT. Schistosoma mansoni: The developmental regulation and immunolocalization of antioxidant enzymes. Exp Parasitol 1997;86:69–78. 9149242.
38. Hong SJ, Kim YT, Gan XX, Shen LY, Sukontason K, Sukontason K, Kang SY. Clonorchis sinensis: glutathione S-transferase as a serodiagnostic antigen for detection IgG and IgE antibodies. Exp Parasitol 2002;101:231–233. 12594964.
39. Cai GB, Bae YA, Kim SH, Sohn WM, Lee YS, Jiang MS, Kim TS, Kong Y. Vitellocyte-specific expression of phospholipid hydroperoxide glutathione peroxidases in Clonorchis sinensis. Int J Parasitol 2008;38:1613–1623. 18588894.
40. Alger HM, Sayed AA, Stadecker MJ, Williams DL. Molecular and enzymatic characterization of Schistosoma mansoni thioredoxin. Int J Parasitol 2002;32:1285–1292. 12204228.
41. Han YH, Chung YH, Kim TY, Hong SJ, Choi JD, Chung YJ. Crystallization of Clonorchis sinensis 26kDa glutathione S-transferase and its fusion proteins with peptides of different lengths. Acta Crystallogr D Biol Crystallogr 2001;57:579–581. 11264588.
42. Rawlings ND, Barrett AJ. MEROPS: the peptidase database. Nucleic Acids Res 1999;27:325–331. 9847218.
43. Barrett AJ. Bioinformatics of proteases in the MEROPS database. Curr Opin Drug Discov Devel 2004;7:334–341.
44. Knudsen GM, Medzihradszky KF, Lim KC, Hansell E, McKerrow JH. Proteomic analysis of Schistosoma mansoni cercarial secretions. Mol Cell Proteomics 2005;4:1862–1875. 16112986.
45. Curwen RS, Ashton PD, Sundaralingam S, Wilson RA. Identification of novel proteases and immunomodulators in the secretions of schistosome cercariae that facilitate host entry. Mol Cell Proteomics 2006;5:835–844. 16469760.
46. Dvorak J, Mashiyama ST, Braschi S, Sajid M, Knudsen GM, Hansell E, Lim KC, Hsieh I, Bahgat M, Mackenzie B, Medzihradszky KF, Babbitt PC, Caffrey CR, McKerrow JH. Differential use of protease families for invasion by schistosome cercariae. Biochimie 2008;90:345–358. 17936488.
47. Hansell E, Braschi S, Medzihradszky KF, Sajid M, Debnath M, Ingram J, Lim KC, McKerrow JH. Proteomic analysis of skin invasion by blood fluke larvae. PLoS Negl Trop Dis 2008;2:e262. 18629379.
48. Moreno Y, Geary TG. Stage- and gender-specific proteomic analysis of Brugia malayi excretory-secretory products. PLoS Negl Trop Dis 2008;2:e326. 18958170.
49. Stack CM, Caffrey CR, Donnelly SM, Seshaadri A, Lowther J, Tort JF, Collins PR, Robinson MW, Xu W, McKerrow JH, Craik CS, Geiger SR, Marion R, Brinen LS, Dalton JP. Structural and functional relationships in the virulence-associated cathepsin L proteases of the parasitic liver fluke, Fasciola hepatica. J Biol Chem 2008;283:9896–9908. 18160404.
50. Robinson MW, Tort JF, Lowther J, Donnelly SM, Wong E, Xu W, Stack CM, Padula M, Herbert B, Dalton JP. Proteomics and phylogenetic analysis of the cathepsin L protease family of the helminth pathogen Fasciola hepatica: expansion of a repertoire of virulence-associated factors. Mol Cell Proteomics 2008;7:1111–1123. 18296439.
51. Berriman M, Haas BJ, LoVerde PT, Wilson RA, Dillon GP, Cerqueira GC, Mashiyama ST, Al-Lazikani B, Andrade LF, Ashton PD, Aslett MA, Bartholomeu DC, Blandin G, Caffrey CR, Coghlan A, Coulson R, Day TA, Delcher A, DeMarco R, Djikeng A, Eyre T, Gamble JA, Ghedin E, Gu Y, Hertz-Fowler C, Hirai H, Hirai Y, Houston R, Ivens A, Johnston DA, Lacerda D, Macedo CD, McVeigh P, Ning Z, Oliveira G, Overington JP, Parkhill J, Pertea M, Pierce RJ, Protasio AV, Quail MA, Rajandream MA, Rogers J, Sajid M, Salzberg SL, Stanke M, Tivey AR, White O, Williams DL, Wortman J, Wu W, Zamanian M, Zerlotini A, Fraser-Liggett CM, Barrell BG, El-Sayed NM. The genome of the blood fluke Schistosoma mansoni. Nature 2009;460:352–358. 19606141.
52. Liu F, Zhou Y, Wang ZQ, Lu G, Zheng H, Brindley PJ, McManus DP, Blair D, Zhang QH, Zhong Y, Wang S, Han ZG, Chen Z. The Schistosoma japonicum genome reveals features of host-parasite interplay. Schistosoma japonicum Genome Sequencing and Functional Analysis Consortium. Nature 2009;460:345–351. 19606140.
53. Dalton JP, Neill SO, Stack C, Collins P, Walshe A, Sekiya M, Doyle S, Mulcahy G, Hoyle D, Khaznadji E, Moire N, Brennan G, Mousley A, Kreshchenko N, Maule AG, Donnelly SM. Fasciola hepatica cathepsin L-like proteases: biology, function, and potential in the development of first generation liver fluke vaccines. Int J Parasitol 2003;33:1173–1181. 13678633.
54. Williamson AL, Brindley PJ, Loukas A. Hookworm cathepsin D aspartic proteases: contributing roles in the host-specific degradation of serum proteins and skin macromolecules. Parasitology 2003;126:179–185. 12636356.
55. Delcroix M, Sajid M, Caffery CR, Kim KC, Dvorak J, Hsieh I, Bahgat M, Dissous C, McKerrow JH. A multienzyme network functions in intestinal protein digestion by a platyhelminth. J Biol Chem 2006;281:39316–39329. 17028179.
56. Na BK, Kim SH, Lee EG, Kim TS, Bae YA, Kang I, Yu JR, Sohn WM, Cho SY, Kong Y. Critical roles for excretory-secretory cysteine proteases during tissue invasion of Paragonimus westermani newly excysted metacercariae. Cell Microbiol 2006;8:1034–1046. 16681843.
57. Pinlaor P, Kaewpitoon N, Laha T, Sripa B, Kaewkes S, Morales ME, Mann VH, Parriott SK, Suttiprapa S, Robinson MW, To J, Dalton JP, Loukas A, Brindley PJ. Cathepsin F Cysteine Protease of the Human Liver Fluke, Opisthorchis viverrini. PLoS Negl Trop Dis 2009;3:e398. 19308250.
58. Ranjit N, Zhan B, Hamilton B, Stenzel D, Lowther J, Pearson M, Gorman J, Hotez P, Loukas A. Proteolytic degradation of hemoglobin in the intestine of the human hookworm Necator americanus. J Infect Dis 2009;199:904–912. 19434933.
59. Suttiprapa S, Mulvenna J, Huong NT, Pearson MS, Brindley PJ, Laha T, Wongkham S, Kaewkes S, Sripa B, Loukas A. Ov-APR-1, an aspartic protease from the carcinogenic liver fluke, Opisthorchis viverrini: functional expression, immunolocalization and subsite specificity. Int J Biochem Cell Biol 2009;41:1148–1156. 18996218.
60. Redmond DL, Knox DP. Protection studies in sheep using affinity-purified and recombinant cysteine proteinases of adult Haemonchus contortus. Vaccine 2004;22:4252–4261. 15474716.
61. Loukas A, Bethony JM, Mendez S, Fujiwara RT, Goud GN, Ranjit N, Zhan B, Jones K, Bottazzi ME, Hotez PJ. Vaccination with recombinant aspartic hemoglobinase reduces parasite load and blood loss after hookworm infection in dogs. PLoS Med 2005;2:e295. 16231975.
62. Abdulla MH, Lim KC, Sajid M, McKerrow JH, Caffrey CR. Schistosomiasis Mansoni: novel chemotherapy using a cysteine protease inhibitor. PLoS Med 2007;4:e14. 17214506.
63. Villa-Mancera A, Quiroz-Romero H, Correa D, Ibarra F, Reyes-Perez M, Reyes-Vivas H, Lopez-Velazquez G, Gazarian K, Gazarian T, Alonso RA. Induction of immunity in sheep to Fasciola hepatica with mimotopes of cathepsin L selected from a phage display library. Parasitology 2008;135:1437–1445. 18812010.
64. Song CY, Dresden MH, Rege AA. Clonorchis sinensis: purification and characterization of a cysteine proteinase from adult worms. Comp Biochem Physiol B 1990;97:825–829. 2085964.
65. Song CY, Rege AA. Cysteine proteinase activity in various developmental stages of Clonorchis sinensis: a comparative analysis. Comp Biochem Physiol B 1991;99:137–140. 1959323.
66. Park H, Ko MY, Paik MK, Soh CT, Seo JH, Im KI. Cytotoxicity of a cysteine proteinase of adult Clonorchis sinensis. Korean J Parasitol 1995;33:211–218. 8528628.
67. Kang TH, Yun DH, Lee EH, Chung YB, Bae YA, Chung JY, Kang I, Kim J, Cho SY, Kong Y. A cathepsin F of adult Clonorchis sinensis and its phylogenetic conservation in trematodes. Parasitology 2004;128:195–207. 15030007.
68. Li S, Chung YB, Chung BS, Choi MH, Yu JR, Hong ST. The involvement of the cysteine proteases of Clonorchis sinensis metacercariae in excystment. Parasitol Res 2004;93:36–40. 15052470.
69. Na BK, Lee HJ, Cho SH, Lee HW, Cho JH, Kho WG, Lee JS, Lee JS, Song KJ, Park PH, Song CY, Kim TS. Expression of cysteine proteinase of Clonorchis sinensis and its use in serodiagnosis of clonorchiasis. J Parasitol 2002;88:1000–1006. 12435144.
70. Nagano I, Pei F, Wu Z, Wu J, Cui H, Boonmars T, Takahashi Y. Molecular expression of a cysteine proteinase of Clonorchis sinensis and its application to an enzyme-linked immunosorbent assay for immunodiagnosis of clonorchiasis. Clin Diagn Lab Immunol 2004;11:411–416. 15013996.
71. Na BK, Kang JM, Sohn WM. CsCF-6, a novel cathepsin F-like cysteine protease for nutrient uptake of Clonorchis sinensis. Int J Parasitol 2008;38:493–502. 17945236.
72. Ju JW, Joo HN, Lee MR, Cho SH, Cheun HI, Kim JY, Lee YH, Lee KJ, Sohn WM, Kim DM, Kim IC, Park BC, Kim TS. Identification of a serodiagnostic antigen, legumain, by immunoproteomic analysis of excretory-secretory products of Clonorchis sinensis adult worms. Proteomics 2009;9:3066–3078. 19526557.
73. Sajid M, McKerrow JH. Cysteine proteases of parasitic organisms. Mol Biochem Parasitol 2002;120:1–21. 11849701.
74. Hong SJ, Seong KY, Sohn WM, Song KY. Molecular cloning and immunological characterization of phosphoglycerate kinase from Clonorchis sinensis. Mol Biochem Parasitol 2000;108:207–216. 10838223.
75. Hong SJ, Shin JK, Kang SY, Ryu JR. Ultrastructural localization of phosphoglycerate kinase in adult Clonorchis sinensis. Parasitol Res 2003;90:369–371. 12720089.
76. Song L, Xu Z, Yu X. Molecular cloning and characterization of a phosphoglycerate mutase gene from Clonorchis sinensis. Parasitol Res 2007;101:709–714. 17468884.
77. Yang G, Jing C, Zhu P, Hu X, Xu J, Wu Z, Yu X. Molecular cloning and characterization of a novel lactate dehydrogenase gene from Clonorchis sinensis. Parasitol Res 2006;99:55–64. 16479375.
78. Zheng N, Xu J, Wu Z, Chen J, Hu X, Song L, Yang G, Ji C, Chen S, Gu S, Ying K, Yu X. Clonorchis sinensis: molecular cloning and functional expression of novel cytosolic malate dehydrogenase. Exp Parasitol 2005;109:220–227. 15755419.
79. Zheng N, Huang B, Xu J, Huang S, Chen J, Hu X, Ying K, Yu X. Enzymatic and physico-chemical characteristics of recombinant cMDH and mMDH of Clonorchis sinensis. Parasitol Res 2006;99:174–180. 16541263.
80. Zheng N, Huang B, Xu J, Huang S, Chen J, Hu X, Ji C, Ying K, Yu X. Cloning and expression of mitochondrial malate dehydrogenase of Clonorchis sinensis. Parasitol Res 2008;102:989–995. 18311572.
81. Lippe M, Andra J, Nickel R, Tanich E, Muller-Eberhard HJ. Amoebapores, a family pore-forming peptides from cytoplasmic granules of Entamoeba hystolytica: isolation, primary structure, and pore formation in bacterial cytoplasmic membranes. Mol Microbiol 1994;14:895–904. 7715451.
82. Lee JY, Cho PY, Kim TY, Kang SY, Song KY, Hong SJ. Hemolytic activity and developmental expression of pore-forming peptide, clonorin. Biochem Biophys Res Commun 2002;296:1238–1244. 12207906.
83. Hwang SY, Han HJ, Kim SH, Park SG, Seog DH, Kim NR, Han J, Chung JY, Kho WG. Cloning of a pore-forming subunit of ATP-sensitive potassium channel from Clonorchis sinensis. Korean J Parasitol 2003;41:129–133. 12815327.
84. Chu BD, Rim HJ, Kim SJ. A study on the body fluid antigen of Clonorchis sinensis using immunogold labeling method. Korean J Parasitol 1990;28:11–23.
85. Kim J, Chai JY, Kho WG, Cho KH, Lee SH. Immunohistochemical study on the antigenicity of each organ structure of Clonorchis sinensis. Korean J Parasitol 1991;29:21–29.
86. Hong SJ, Kim TY, Song KY, Sohn WM, Kang SY. Antigenic profile and localization of Clonorchis sinensis proteins in the course of infection. Korean J Parasitol 2001;39:307–312. 11775331.
87. Yong TS, Yang HJ, Park SJ, Kim YK, Lee DH, Lee SM. Immunodiagnosis of clonorchiasis using a recombinant antigen. Korean J Parasitol 1998;36:183–190. 9755589.
88. Yang HJ, Park SJ, Im KI, Yong TS. Identification of a Clonorchis sinensis gene encoding a vitellaria antigenic protein containing repetitive sequences. Mol Biochem Parasitol 2000;111:213–216. 11087931.
89. Kim TY, Kang SY, Ahn IY, Cho SY, Hong SJ. Molecular cloning and characterization of antigenic protein with a repeating region from Clonorchis sinensis. Korean J Parasitol 2001;39:57–66. 11301591.
90. Lee HJ, Lee CS, Kim BS, Joo KH, Lee JS, Kim TS, Kim HR. Purification and charicterization of a 7-kDa protein from Clonorchis sinensis adult worms. J Parasitol 2002;88:499–504. 12099418.
91. Lee M, Chung YB, Lee SK, Chung BS, Li S, Choi MH, Hong ST. The identification of a Clonorchis sinensis gene encoding an antigenic egg protein. Parasitol Res 2005;95:224–226. 15616856.
92. Chung YB, Yang HJ, Hong SJ, Kang SY, Lee M, Kim TY, Choi MH, Chai JY, Hong ST. Molecular cloning and immunolocalization of the 17 kDa myoglobin of Clonorchis sinensis. Parasitol Res 2003;90:365–368. 12720088.
93. Sim S, Park GM, Yong TS. Cloning and charicterization of Clonorchis sinensis myoglobin using immune sera against excretory-secretory antigenes. Parasitol Res 2003;91:338–343. 14574567.
94. Lee JY, Kim TY, Gan XX, Kang SY, Hong SJ. Use of a recombinant Clonorchis sinensis pore-forming peptide, clonorin, for serological diagnosis of clorchiasis. Parasitol Int 2003;52:175–178. 12798930.
95. Hu F, Yu X, Ma C, Zhou H, Zhou Z, Li Y, Lu F, Xu J, Wu Z, Hu X. Clonorchis sinensis: expression, characterization, immunolocalization and serological reactivity of one excretory/secretory antigen-LPAP homologue. Exp Parasitol 2007;117:157–164. 17507009.
96. Lee JS, Yong TS. Expression and cross-species reactivity of fatty acid-binding protein of Clonorchis sinensis. Parasitol Res 2004;93:339–343. 15197581.
97. Ma C, Hu X, Hu F, Li Y, Chen X, Zhou Z, Lu F, Xu J, Wu Z, Yu X. Molecular characterization and serodiagnosis analysis of a novel lysophospholipase from Clonorchis sinensis. Parasitol Res 2007;101:419–425. 17318582.
98. Shen C, Lee JA, Allam SR, Bae YM, Han ET, Takeo S, Tsuboi T, Hong ST, Choi MH. Serodiagnostic applicability of recombinant antigens of Clonorchis sinensis expressed by wheat germ cell-free protein synthesis system. Diagn Microbiol Infect Dis 2009;64:334–339. 19376673.
99. Kim TY, Kang SY, Park SH, Sukontason K, Sukontason K, Hong SJ. Cystatin capture enzyme-linked immunosorbent assay for serodiagnosis of human clonorchiasis and profile of captured antigenic protein of Clonorchis sinensis. Clin Diagn Lab Immunol 2001;8:1076–1080. 11687443.
100. Willadsen P. Antigen cocktails: valid hypothesis or unsubstantiated hope? Trends Parasitol 2008;24:164–167. 18342573.
101. Cardoso FC, Pacifico RNA, Mortara RA, Oliveira SC. Human antibody responses of patients living in endemic areas for schistosomiasis to the tegumental protein Sm29 identified through genomic studies. Clin Exp Immunol 2006;144:382–391. 16734606.
102. Loukas A, Tran M, Pearson MS. Schistosome membrane proteins as vaccines. Int J Parasitol 2007;37:257–263. 17222846.
103. Zhou Z, Hu X, Huang Y, Hu H, Ma C, Chen X, Hu F, Xu J, Lu F, Wu Z, Yu X. Molecular cloning and identification of a novel Clonorchis sinensis gene encoding a tegumental protein. Parasitol Res 2007;101:737–742. 17476530.
104. Zhou Z, Xia H, Hu X, Huang Y, Ma C, Chen X, Hu F, Xu J, Lu F, Wu Z, Yu X. Immunogenicity of recombinant Bacillus subtilis spores expressing Clonorchis sinensis tegumental protein. Parasitol Res 2008;102:293–297. 17924143.
105. Huang Y, Zhou Z, Hu X, Wei Q, Xu J, Wu Z, Yu X. A novel tegumental protein 31.8 kDa of Clonorchis sinensis: sequence analysis, expression, and immunolocalization. Parasitol Res 2007;102:77–81. 17768637.

Article information Continued