Carcinogenic liver fluke Opisthorchis viverrini oxysterols detected by LC-MS/MS survey of soluble fraction parasite extract.
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ABSTRACT:
SUBMITTER: Vale N
PROVIDER: S-EPMC3797210 | biostudies-literature | 2013 Dec
REPOSITORIES: biostudies-literature
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Carcinogenic liver fluke Opisthorchis viverrini oxysterols detected by LC-MS/MS survey of soluble fraction parasite extract.
Vale Nuno N Gouveia Maria João MJ Botelho Mónica M Sripa Banchob B Suttiprapa Sutas S Rinaldi Gabriel G Gomes Paula P Brindley Paul J PJ Correia da Costa José Manuel JM
Parasitology international 20130820 6
Liquid chromatography in tandem mass spectrometry (LC-MS/MS) has emerged as an informative tool to investigate oxysterols (oxidized derivatives of cholesterol) in helminth parasite associated cancers. Here, we used LC-MS/MS to investigate in soluble extracts of the adult developmental stage of Opisthorchis viverrini from experimentally infected hamsters. Using comparisons with known bile acids and the metabolites of estrogens, the LC-MS data indicated the existence of novel oxysterol derivatives ...[more]
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Project description:Opisthorchiasis caused by Opisthorchis viverrini remains a major public health problem in many parts of Southeast Asia, including Thailand, Lao PDR, Vietnam and Cambodia. The infection is associated with a number of hepatobiliary diseases, including cholangitis, obstructive jaundice, hepatomegaly, cholecystitis and cholelithiasis. Multi-factorial etiology of cholangiocarcinoma, mechanical damage, parasite secretions, and immunopathology may enhance cholangiocarcinogenesis. Moreover, both experimental and epidemiological evidences strongly implicate liver fluke infection as the major risk factor in cholangiocarcinoma, cancer of the bile ducts. The liver fluke infection is induced by eating raw or uncooked fish products that is the tradition and popular in the northeastern and northern region, particularly in rural areas, of Thailand. The health education programs to prevent and control opisthorchiasis are still required in the high-risk areas.
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Project description:BackgroundCholangiocarcinoma (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.ResultsApproximately 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.ConclusionThis 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.
| S-EPMC1913519 | biostudies-literature
Project description:Functional genomics have not been reported for Opisthorchis viverrini or the related fish-borne fluke, Clonorchis sinensis. Here we describe the introduction by square wave electroporation of Cy3-labeled small RNA into adult O. viverrini worms. Adult flukes were subjected to square wave electroporation employing a single pulse for 20 ms of 125V in the presence of 50 ?g/ml of Cy3-siRNA. The parasites tolerated this manipulation and, at 24 and 48 h after electroporation, fluorescence from the Cy3-siRNA was evident throughout the parenchyma of the worms, with strong fluorescence evident in the guts and reproductive organs of the adult worms. Second, other worms were treated using the same electroporation settings with double stranded RNA targeting an endogenous papain-like cysteine protease, cathepsin B. This manipulation resulted in a significant reduction in specific mRNA levels encoding cathepsin B, and a significant reduction in cathepsin B activity against the diagnostic peptide, Z-Arg-Arg-AMC. This appears to be the first report of introduction of reporter genes into O. viverrini and the first report of experimental RNA interference (RNAi) in this fluke. The findings indicated the presence of an intact RNAi pathway in these parasites which, in turn, provides an opportunity to probe gene functions in this neglected tropical disease pathogen.
| S-EPMC3682765 | biostudies-literature
Project description:Opisthorchis viverrini is a fish-borne trematode endemic in East Asia. Following ingestion, the flukes locate to the biliary tre where chronic infection frequently leads to cholangiocarcinoma (CCA). The mechanisms by which O. viverrini infection culminates in CCA remain unknown. An unexplored aspect is its influence on the host microbiome. In the hamster, infection with this pathogen reliably leads to CCA. Genomic DNAs of microbiota from colorectal contents and bile of hamsters and from whole O. viverrini were examined in this model of fluke-induced CCA. Microbial communities were characterized by high-throughput sequencing of variable regions 7-9 of prokaryotic 16S ribosomal DNA. Of ?1 million sequences, 536,009 with useable reads were assignable to 29,776 operational taxonomy units (OTUs) and, in turn, to 20 phyla and 273 genera of Bacteria or Archaea. Microbial community analyses revealed that fluke infection perturbed the gastrointestinal tract microbiome, increasing Lachnospiraceae, Ruminococcaceae, and Lactobacillaceae, while decreasing Porphyromonadaceae, Erysipelotrichaceae, and Eubacteriaceae (P?0.05). More than 60 OTUs were detected in the biliary system, which confirmed bacteriobilia and a noteworthy community of microbes associated with the parasites. The fluke-associated microorganisms included potential pathogens from the Enterobacteriaceae and Listeriaceae and others, including Cyanobacteria and Deinococci, usually found in external environments. Given that opisthorchiasis is distinguished from other helminth infections by a robust inflammatory phenotype with conspicuously elevated IL-6, and that inflammation of the biliary system leads to periductal fibrosis, which is a precursor of CCA, the flukes and their microbiota may together drive this distinctive immune response.
| S-EPMC3804743 | biostudies-literature
Project description:ObjectivesTo 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.MethodsThe 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.ResultsOv-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.ConclusionsThe 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.
| S-EPMC2670890 | biostudies-literature
Project description: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.
| S-EPMC3725289 | biostudies-literature
Project description: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 many years. The mechanisms by which it avoids oxidative damage are unknown. In this study, thioredoxin peroxidase (TPx), a member of the peroxiredoxin superfamily, was cloned from an O. viverrini cDNA library. O. viverrini TPx cDNA encoded a polypeptide of 212 amino acid residues, of molecular mass 23.57kDa. The putative amino acid sequence shared 60-70% identity with TPXs from other helminths and from mammals, and phylogenetic analysis revealed a close relationship between TPxs from O. viverrini and other trematodes. Recombinant O. viverrini TPx was expressed as soluble protein in Escherichia coli. The recombinant protein dimerized, and its antioxidant activity was deduced by observing protection of nicking of supercoiled plasmid DNA by hydroxyl radicals. Antiserum raised against O. viverrini TPx recognized native proteins from egg, metacercaria and adult developmental stages of the liver fluke and excretory-secretory products released by adult O. viverrini. Immunolocalization studies revealed ubiquitous expression of TPx in O. viverrini organs and tissues. TPx was also detected in bile fluid and bile duct epithelial cells surrounding the flukes 2 weeks after infection of hamsters with O. viverrini. In addition, TPx was observed in the secondary (small) bile ducts where flukes cannot reach due to their large size. These results suggested that O. viverrini TPx plays a significant role in protecting the parasite against damage induced by reactive oxygen species from inflammation.
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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.
| S-EPMC4283030 | biostudies-literature
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.
| S-EPMC4033688 | biostudies-literature
Project description:Infection with the human liver fluke, Opisthorchis viverrini, is a serious public health problem in Thailand, Laos and nearby locations in Southeast Asia. Both experimental and epidemiological evidence strongly implicate liver fluke infection in the etiology of one of the liver cancer subtypes, cholangiocarcinoma (CCA). To identify parasite proteins critical for liver fluke survival and the etiology of CCA, OFFGEL electrophoresis and multiple reaction monitoring were employed to characterize 300 parasite proteins from the O. viverrini excretory/secretory products and, utilizing selective labeling and sequential solubilization, from the host-exposed tegument. The excretory/secretory included a complex mixture of proteins that have been associated with cancers, including proteases of different mechanistic classes and orthologues of mammalian growth factors and anti-apoptotic proteins. Also identified was a cysteine protease inhibitor which, in other helminth pathogens, induces nitric oxide production by macrophages, and, hence may contribute to malignant transformation of inflamed cells. More than 160 tegumental proteins were identified using sequential solubilization of isolated teguments, and a subset of these was localized to the surface membrane of the tegument by labeling living flukes with biotin and confirming surface localization with fluorescence microscopy. These included annexins, which are potential immuno-modulators, and orthologues of the schistosomiasis vaccine antigens Sm29 and tetraspanin-2. Novel roles in pathogenesis were suggested for the tegument-host interface since more than ten surface proteins had no homologues in the public databases. The O. viverrini proteins identified here provide an extensive catalogue of novel leads for research on the pathogenesis of opisthorchiasis and the development of novel interventions for this disease and CCA, as well as providing a scaffold for sequencing the genome of this fluke.
| S-EPMC3038172 | biostudies-literature