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Infection with the food-borne liver fluke Opisthorchis viverrini is the principal risk factor for cholangiocarcinoma (CCA) in the Mekong Basin countries of Thailand, Lao PDR, Vietnam, Myanmar and Cambodia. Using a novel model of CCA, involving infection with gene-edited liver flukes in the hamster during concurrent exposure to dietary nitrosamine, we explored the role of the fluke granulin-like growth factor Ov-GRN-1 in malignancy. We derived RNA-guided gene knockout flukes (ΔOv-grn-1) using CRISPR/Cas9/gRNA materials delivered by electroporation. Genome sequencing confirmed programmed Cas9-catalyzed mutations of the targeted genes, which was accompanied by rapid depletion of transcripts and the proteins they encode. Gene-edited parasites colonized the biliary tract of hamsters and developed into adult flukes. However, less hepatobiliary tract disease manifested during chronic infection with ΔOv-grn-1 worms in comparison to hamsters infected with control gene-edited and mock-edited parasites. Specifically, immuno- and colorimetric-histochemical analysis of livers revealed markedly less periductal fibrosis surrounding the flukes and less fibrosis globally within the hepatobiliary tract during infection with ΔOv-grn-1 genotype worms, minimal biliary epithelial cell proliferation, and significantly fewer mutations of TP53 in biliary epithelial cells. Moreover, fewer hamsters developed high-grade CCA compared to controls. The clinically relevant, pathophysiological phenotype of the hepatobiliary tract confirmed a role for this secreted growth factor in malignancy and morbidity during opisthorchiasis. Author summary: Infection with the human liver flukes, Opisthorchis viverrini, O. felineus and Clonorchis sinensis remains a public health concern in regions where these parasites are endemic. O. viverrini is endemic in the Mekong River drainage countries including Thailand and the Lao People's Democratic Republic. Infection follows the consumption of undercooked freshwater fish harboring the parasite. Liver fluke infection, opisthorchiasis, is associated with diseases of the liver and bile ducts including cancer of the biliary tract, cholangiocarcinoma, a cancer with a poor prognosis. This report characterizes, for the first time, experimental infection with gene-edited O. viverrini liver flukes during concurrent exposure to a dietary nitrosamine in a rodent model of liver fluke infection-associated cancer. Cancer development was slowed in hamsters infected with the parasite following CRISPR-based knock-out mutation and loss of a parasite gene known to stimulate growth of cells lining the bile ducts. These findings definitely link a parasite factor to cancer etiology, and present a new laboratory model to investigate risk factors for infection-associated cholangiocarcinoma and to assess efficacy of anti-infection/anti-cancer vaccines. [ABSTRACT FROM AUTHOR]

Mucin plays a crucial role in safeguarding mucosal tissues by obstructing the translocation of microorganisms. Mucosal tissue-dwelling parasites must devise a strategy to surmount this mucin barrier in order to establish colonization. In a recent discovery, it was observed that the liver fluke Opisthorchis viverrini secretes two mucinases, namely Ov -M60-like-1 and Ov -M60-like-2. Ov -M60-like-1 was previously characterized. Here, we study the Ov -M60-like-2 by utilizing the wheat germ expression system to produce recombinant proteins and conducted a functional analysis of its enzymatic activity on bovine submaxillary mucin (BSM). Subsequently, we delved deeper into understanding the role of this enzyme in host-parasite interactions by evaluating its mucinase activity on mucins from the bile duct of O. viverrini -infected hamsters. Through successful production of recombinant proteins using the wheat germ expression system, we observed that this enzyme displayed mucinase activity over a wide pH range (pH 2 to pH 10) against BSM. Our investigations revealed it ability to digest mucin from the bile duct. These findings suggest that Ov -M60-like-2 possess a mucinase activity, together with Ov -M60-like-1, enabling the liver fluke to successful colonization of the host's bile duct. [Display omitted] • The carcinogenic liver fluke Opisthorchis viverrini releases Ov -M60-like-2 metallopeptidases. • Ov -M60-like-2 displays mucinase activity over a wide pH range (pH 2 to pH 10) against mucin substrate. • Ov -M60-like-2 digest mucin from the bile duct, allows the liver fluke colonize the bile duct of the host. [ABSTRACT FROM AUTHOR]

Background: The human liver fluke Opisthorchis viverrini is a food-borne trematode that causes hepatobiliary disease in humans throughout Southeast Asia. People become infected by consuming raw or undercooked fish containing metacercariae. Development of a vaccine to prevent or minimize pathology would decrease the risk of severe morbidity, including the development of bile duct cancer.Methods: We produced an oral vaccine based on recombinant Bacillus subtilis spores expressing the large extracellular loop (LEL) of O. viverrini tetraspanin-2 (Ov-TSP-2), a protein that is abundant on the surface of O. viverrini secreted extracellular vesicles (EVs). Recombinant spores expressing Ov-TSP-2-LEL were orally administered to hamsters prior to challenge infection with O. viverrini metacercariae.Results: Vaccinated hamsters generated serum IgG as well as bile IgG and IgA responses to Ov-TSP-2-LEL, and serum IgG from vaccinated hamsters blocked the uptake of fluke EVs by a human bile duct epithelial cell line. Vaccinated hamsters had 56% reductions in both adult flukes and fecal eggs compared to the control group.Conclusions: These findings indicate that oral vaccination of hamsters with recombinant B. subtilis spores expressing Ov-TSP-2-LEL is efficacious at reducing infection intensity and could form the basis of a vaccine for control of carcinogenic liver fluke infection in humans. [ABSTRACT FROM AUTHOR]

Background: The liver fluke Opisthorchis viverrini infects several million people in Southeast Asia. Adult flukes live in the bile ducts of humans, where they cause hepatobiliary pathology, including cholangiocarcinoma. Here, we investigated the potential of extracellular vesicles (EVs) secreted by the fluke and defined recombinant proteins derived from EVs to generate protective immunity in a hamster vaccination-challenge model. Methodology/Principal findings: EVs isolated from the excretory-secretory products of O. viverrini and two recombinant EV surface proteins encoding the large extracellular loops (LEL) of Ov-TSP-2 (rOv-TSP-2) and Ov-TSP-3 (rOv-TSP-3) were adjuvanted and used to vaccinate hamsters intraperitoneally followed by challenge infection with O. viverrini metacercariae. The number of adult flukes recovered from hamsters immunized with EVs, rOv-TSP-2, rOv-TSP-3 and rOv-TSP-2+rOv-TSP-3 were significantly reduced compared to control animals vaccinated with adjuvant alone. The number of eggs per gram feces was also significantly reduced in hamsters vaccinated with rOv-TSP-2 compared to controls, but no significant differences were found in the other groups. The average length of worms recovered from hamsters vaccinated with EVs, rOv-TSP-2 and rOv-TSP-3 was significantly shorter than that of worms recovered from the control group. Anti-EV IgG levels in serum and bile were significantly higher in hamsters vaccinated with EVs compared to control hamsters both pre- and post-challenge. In addition, levels of anti-rOv-TSP antibodies in the serum and bile were significantly higher than control hamsters both pre- and post-challenge. Finally, antibodies against rOv-TSP-2 and rOv-TSP-3 blocked uptake of EVs by human primary cholangiocyte in vitro, providing a plausible mechanism by which these vaccines exert partial efficacy and reduce the intensity of O. viverrini infection. Conclusion/Significance: Liver fluke EVs and recombinant tetraspanins derived from the EV surface when administered to hamsters induce antibody responses that block EV uptake by target bile duct cells and exert partial efficacy and against O. viverrini challenge. [ABSTRACT FROM AUTHOR]

Liver fluke infection caused by Opisthorchis viverrini remains a major public health problem in many parts of Asia including Thailand, Lao PDR, Vietnam and Cambodia, where there is a strikingly high incidence of cholangiocarcinoma (CCA – hepatic cancer of the bile duct epithelium). Among other factors, uptake of O. viverrini excretory/secretory products ( Ov ES) by biliary epithelial cells has been postulated to be responsible for chronic inflammation and proliferation of cholangiocytes, but the mechanisms by which cells internalise O. viverrini excretory/secretory products are still unknown. Herein we incubated normal human cholangiocytes (H69), human cholangiocarcinoma cells (KKU-100, KKU-M156) and human colon cancer (Caco-2) cells with O. viverrini excretory/secretory products and analysed the effects of different endocytic inhibitors to address the mechanism of cellular uptake of ES proteins. Opisthorchis viverrini excretory/secretory products was internalised preferentially by liver cell lines, and most efficiently/rapidly by H69 cells. There was no evidence for trafficking of ES proteins to cholangiocyte organelles, and most of the fluorescence was detected in the cytoplasm. Pretreatment with clathrin inhibitors significantly reduced the uptake of O. viverrini excretory/secretory products, particularly by H69 cells. Opisthorchis viverrini excretory/secretory products induced proliferation of liver cells (H69 and CCA lines) but not intestinal (Caco-2) cells, and proliferation was blocked using inhibitors of the classical endocytic pathways (clathrin and caveolae). Opisthorchis viverrini excretory/secretory products drove IL6 secretion by H69 cells but not Caco-2 cells, and cytokine secretion was significantly reduced by endocytosis inhibitors. This the first known study to address the endocytosis of helminth ES proteins by host epithelial cells and sheds light on the pathways by which this parasite causes one of the most devastating forms of cancer in south-eastern Asia. [ABSTRACT FROM AUTHOR]

• A selenoprotein thioredoxin glutathione reductase (TGR) was identified from the carcinogenic liver fluke Opisthorchis viverrini. • The TGR coding sequence was cloned into pABC2 plasmid and transformed into C321.ΔA Escherichia coli to facilitate selenocysteine incorporation to produce an active enzyme. • Enzymatic activities of TGR expressed by pABC2/ C321.ΔA system was a magnitude higher than expressed by a conventional pET32/B21(DE3) system. • The recombinant Ov-TGR was inhibited by auranofin. • Auranofin caused lethal effect on both juvenile and adult stages of O. viverrini in vitro. The carcinogenic liver fluke Opisthorchis viverrini causes several hepatobiliary diseases including a bile duct cancer-cholangiocarcinoma (CCA), which is a major public health problem in many countries in the Greater Mekong Sub-region. Praziquantel is the main drug against this parasite, however, reduced drug efficacy has been observed in some endemic areas. Therefore, alternative drugs are needed to prepare for praziquantel resistance in the future. The selenoprotein thioredoxin glutathione reductase (TGR) enzyme, which plays a crucial role in cellular redox balance of parasitic flatworms, has been shown as a potential drug target against these parasites. Hence, this study aimed to investigate the TGR of O. viverrini and assess its potential as a drug target. An open reading frame (ORF) that encodes O. viverrini TGR (Ov-TGR) was cloned from an O. viverrini cDNA library and the nucleotide were sequenced. The 1,812 nucleotides of the Ov-TGR full ORF encoded a polypeptide of 603 amino acid residues with a predicted molecular mass of 66 kDa. The putative amino acid sequence shared 55–96.8% similarities with TGRs from other helminths and mammals. Phylogenetic analysis revealed a close relationship of Ov-TGR with that of other trematodes. The ORF of Ov-TGR was inserted into pABC2 plasmid and transformed into Escherichia coli strain C321.ΔA to facilitate selenocysteine incorporation. The recombinant Ov-TGR (rOv-TGR-SEC) was expressed as a soluble protein and detected as a dimer form in the non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Its thioredoxin reductase (TrxR) and glutathione reductase (GR) activities were detected using DTNB, Trx and GSSG substrates with the Michaelis constant (Km) of 292.6 ± 52.3 µM, 8.09 ± 1.91 µM and 13.74 ± 1.2 µM, respectively. The TGR enzyme activities were effectively inhibited by a well-known inhibitor, auranofin in a dose-dependent manner. Moreover, auranofin expressed a lethal toxic effect on both newly excysted juveniles (NEJs) and adult worms of O. viverrini in vitro. Taken together, these results indicated that Ov-TGR is crucial for O. viverrini survival and maybe a potential target for the development of novel agents against opisthorschiasis. Image, graphical abstract [ABSTRACT FROM AUTHOR]