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Molecular expression and enzymatic characterization of thioredoxin from the carcinogenic human liver fluke Opisthorchis viverrini.

ABSTRACT: The human liver fluke, Opisthorchis viverrini, induces inflammation of the hepatobiliary system. Despite being constantly exposed to inimical oxygen radicals released from inflammatory cells, the parasite survives for years. Defense against oxidative damage can be mediated through glutathione and/or thioredoxin utilizing systems. Here, we report the molecular expression and biochemical characterization of a thioredoxin (Trx) from O. viverrini. O. viverrini Trx cDNA encoded a polypeptide of 105 amino acid residues, of molecular mass 11.63 kDa. The predicted protein has similarity to previously characterized thioredoxins with 26-51% identity. Recombinant O. viverrini Trx (Ov-Trx-1) was expressed as soluble protein in E. coli. The recombinant protein showed insulin reduction activity and supported the enzymatic function of O. viverrini thioredoxin peroxidase. Expression of Ov-Trx-1 at mRNA and protein levels was observed in all obtainable developmental stages of the liver fluke. Ov-Trx-1 was also detected in excretory-secretory products released by adult O. viverrini. Immunohistochemistry, Ov-Trx-1 was expressed in nearly all parasite tissue excepted ovary and mature sperms. Interestingly, Ov-Trx-1 was observed in the infected biliary epithelium but not in normal bile ducts. These results suggest that Ov-Trx-1 is essential for the parasite throughout the life cycle. In the host-parasite interaction aspect, Ov-Trx-1 may support thioredoxin peroxidase in protecting the parasite against damage induced by reactive oxygen species from inflammation.

SUBMITTER: Suttiprapa S

PROVIDER: S-EPMC3725289 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Molecular expression and enzymatic characterization of thioredoxin from the carcinogenic human liver fluke Opisthorchis viverrini.

Suttiprapa Sutas S Matchimakul Pitchaya P Loukas Alex A Laha Thewarach T Wongkham Sopit S Kaewkes Sasithorn S Brindley Paul J PJ Sripa Banchob B

Parasitology international 20110629 1

The human liver fluke, Opisthorchis viverrini, induces inflammation of the hepatobiliary system. Despite being constantly exposed to inimical oxygen radicals released from inflammatory cells, the parasite survives for years. Defense against oxidative damage can be mediated through glutathione and/or thioredoxin utilizing systems. Here, we report the molecular expression and biochemical characterization of a thioredoxin (Trx) from O. viverrini. O. viverrini Trx cDNA encoded a polypeptide of 105 a ...[more]

PMID: 21740981

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Project description:Cholangiocarcinoma (CCA)--cancer of the bile ducts--is associated with chronic infection with the liver fluke, Opisthorchis viverrini. Despite being the only eukaryote that is designated as a 'class I carcinogen' by the International Agency for Research on Cancer, little is known about its genome.Approximately 5,000 randomly selected cDNAs from the adult stage of O. viverrini were characterized and accounted for 1,932 contigs, representing ~14% of the entire transcriptome, and, presently, the largest sequence dataset for any species of liver fluke. Twenty percent of contigs were assigned GO classifications. Abundantly represented protein families included those involved in physiological functions that are essential to parasitism, such as anaerobic respiration, reproduction, detoxification, surface maintenance and feeding. GO assignments were well conserved in relation to other parasitic flukes, however, some categories were over-represented in O. viverrini, such as structural and motor proteins. An assessment of evolutionary relationships showed that O. viverrini was more similar to other parasitic (Clonorchis sinensis and Schistosoma japonicum) than to free-living (Schmidtea mediterranea) flatworms, and 105 sequences had close homologues in both parasitic species but not in S. mediterranea. A total of 164 O. viverrini contigs contained ORFs with signal sequences, many of which were platyhelminth-specific. Examples of convergent evolution between host and parasite secreted/membrane proteins were identified as were homologues of vaccine antigens from other helminths. Finally, ORFs representing secreted proteins with known roles in tumorigenesis were identified, and these might play roles in the pathogenesis of O. viverrini-induced CCA.This gene discovery effort for O. viverrini should expedite molecular studies of cholangiocarcinogenesis and accelerate research focused on developing new interventions, drugs and vaccines, to control O. viverrini and related flukes.

Project description:BACKGROUND: The human liver fluke, Opisthorchis viverrini, is designated as a group 1 carcinogen, and is the major risk factor for cholangiocarcinoma in endemic countries throughout Southeast Asia. Proteins in the excretory-secretory products and tegumental surface membranes of the fluke have been proposed to play pivotal roles in parasite survival in the host, and subsequent pathogenesis. These macromolecules are therefore valid targets for the development of vaccines and new drugs to control the infection. Tetraspanins (TSP) are prominent components of the tegument of blood flukes where they are essential for tegument formation, are directly exposed to the immune system, and are major targets for a schistosomiasis vaccine. We propose that similar molecules in the surface membranes of O. viverrini are integral to tegument biogenesis and will be efficacious vaccine antigens. METHODOLOGY/PRINCIPAL FINDINGS: The cDNA sequence encoding O. viverrini tetraspanin-1 (Ov-TSP-1) was identified and cloned. The Ov-tsp-1gene was isolated from a cDNA library. Ov-tsp-1 mRNA was expressed most highly in metacercariae and eggs, and to a lesser extent in juvenile and adult worms. Immunolocalization with adult flukes confirmed that Ov-TSP-1 was expressed in the tegument and eggs in utero. Western blot analysis of rOv-TSP-1 probed with sera from O. viverrini-infected humans and hamsters indicated that both hosts raise antibody responses against the native TSP. Using RNA interference we silenced the expression level of Ov-tsp-1 mRNA in adult flukes by up to 72% by 10 days after delivery of dsRNA. Ultrastructural morphology of adult worms treated with Ov-tsp-1 dsRNA displayed a distinctly vacuolated and thinner tegument compared with controls. CONCLUSIONS/SIGNIFICANCE: This is the first report of a tetraspanin from the tegument of a liver fluke. Our data imply that tetraspanins play important structural roles in the development of the tegument in the adult fluke. Potential uses of O. viverrini tetraspanins as novel interventions are discussed.

Project description:To isolate and characterize an asparaginyl endopeptidase from the carcinogenic liver fluke, Opisthorchis viverrini, and evaluate its expression profile, biochemical activity, and potential as an immunodiagnostic antigen.The full length mRNA encoding an asparaginyl endopeptidase (family C13), Ov-aep-1, was isolated by immunoscreening of a cDNA bacteriophage library of adult O. viverrini using sera from patients infected with O. viverrini. Investigation of Ov-aep-1 transcripts in developmental stages of the parasite, and phylogenetic analysis, immunohistochemical localization, and recombinant protein expression and enzymology were employed to characterize the Ov-AEP-1 protein. Immunoblotting was used to assess the potential of this enzyme for immunodiagnosis of human opisthorchiasis.Ov-AEP-1 is characteristic of the C13 cysteine protease family. Ov-aep-1 transcripts were detected in adult and juvenile worms, eggs, and metacercariae. Phylogenetic analysis indicated that Ov-AEP-1 is closely related to homologous proteins in other trematodes. Recombinant Ov-AEP-1 was expressed in bacteria in inclusion bodies and refolded to a soluble form. Excretory-secretory (ES) products derived from adult O. viverrini and refolded recombinant Ov-AEP-1 both displayed catalytic activity against the diagnostic tripeptide substrate, Ala-Ala-Asn-aminomethylcoumarin. Rabbit antiserum raised to recombinant Ov-AEP-1 identified the native AEP-1 protease in both somatic extract and ES products of adult worms. Anti-Ov-AEP-1 IgG immunolocalized the anatomical site of expression to the gut of the fluke, implying a physiological role in digestion of food or activation of other digestive enzymes. Recombinant Ov-AEP-1 was recognized by serum antibodies from patients with opisthorchiasis but not other helminth infections, with a sensitivity and specificity of 85% and 100%, respectively. The positive and negative predictive values are 100% and 67%, respectively.The liver fluke, O. viverrini, has a gut-localized asparaginyl endopeptidase. Refolded recombinant Ov-AEP-1 is catalytically active and has potential for immunodiagnosis of human opisthorchiasis.

Project description:UNLABELLED:The snail Bithynia siamensis goniomphalos acts as the first intermediate host for the human liver fluke Opisthorchis viverrini, the major cause of cholangiocarcinoma (CCA) in Northeast Thailand. The undisputed link between CCA and O. viverrini infection has precipitated efforts to understand the molecular basis of host-parasite interactions with a view to ultimately developing new control strategies to combat this carcinogenic infection. To date most effort has focused on the interactions between the parasite and its human host, and little is known about the molecular relationships between the liver fluke and its snail intermediate host. In the present study we analyse the protein expression changes in different tissues of B. siamensis goniomphalos induced by infection with larval O. viverrini using iTRAQ labelling technology. We show that O. viverrini infection downregulates the expression of oxidoreductases and catalytic enzymes, while stress-related and motor proteins are upregulated. The present work could serve as a basis for future studies on the proteins implicated in the susceptibility/resistance of B. siamensis goniomphalos to O. viverrini, as well as studies on other pulmonate snail intermediate hosts of various parasitic flukes that infect humans. BIOLOGICAL SIGNIFICANCE:Despite the importance and high prevalence of opisthorchiasis in some regions of Southeast Asia and the direct relationship between infection by Opisthorchis viverrini and the incidence of cholangiocarcinoma, little is known of the modifications induced by this parasite in its snail intermediate hosts. This time-course study provides the first in-depth quantitative proteomic analysis of experimentally infected Bithynia siamensis goniomphalos. We show how motor and stress-related proteins are upregulated in infected snails, while O. viverrini infection downregulates the expression of oxidoreductases and catalytic enzymes. This work serves as a basis for the development of new strategies, focused on the invertebrate intermediate hosts, to control parasite transmission.

Project description:BackgroundOpisthorchiasis and Opisthorchis viverrini-associated bile duct cancer represent major public health threats in Thailand and Laos. The tegument of this food borne fluke plays pivotal roles in parasite metabolism, homeostasis and osmoregulation. Excretory/secretory products also pass from the fluke to the biliary environment, products that likely underlie pathogenesis of liver fluke infection. Aquaporins (AQPs), belong to the major intrinsic protein superfamily of integral plasma membrane channel proteins that selectively transport water across cell membranes. AQPs play key roles as water and ion transport channels through the tegument of helminth parasites.MethodsHere, two forms of AQP mRNAs from the adult developmental stage of O. viverrini, termed O. viverrini aquaporin-1 and -2 (Ov-aqp-1 and -2) were investigated. Roles of Ov-aqp-1 and -2 in the movement of water across the tegument of this carcinogenic liver fluke were investigated using RNA interference.ResultsOv-AQP-1 and Ov-AQP-2 contain unique characteristic asparagine-proline-alanine (NPA) motifs of AQP transmembrane proteins. Phylogenetic analysis indicated that Ov-AQPs belong to an expanding group of aquaglyceroporin-like water channel proteins characterized from helminth and protozoan parasites, which is pivotal to the specialized requirements of water and solute control during parasitism. Elevated transcription of Ov-aqp-1 was evident in the egg, cercaria, metacercaria and adult stages of O. viverrini, whereas Ov-aqp-2 transcripts were detected at higher level in egg, metacercaria, cercaria and adult stage, respectively. RNA interference using electroporated dsRNA suppressed transcript levels of Ov-aqp-1 and Ov-aqp-2 in adult worms by 58-99% over periods of up to 16 days in vitro. Suppression of Ov-aqp-1 and Ov-aqp-2 in vitro disabled water transport in adult flukes.ConclusionThe apparently pivotal roles of Ov-AQP in solute homeostasis at the fluke surface suggest that deeper investigation will be informative for the pathophysiology of O. viverrini, and may uncover intervention targets, particularly in view of the singularly notable predilection of this pathogen for residence within ducts of the biliary tree.

Dataset Information

Molecular expression and enzymatic characterization of thioredoxin from the carcinogenic human liver fluke Opisthorchis viverrini.

Publications

Molecular expression and enzymatic characterization of thioredoxin from the carcinogenic human liver fluke Opisthorchis viverrini.

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