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The carcinogenic liver fluke, Clonorchis sinensis: new assembly, reannotation and analysis of the genome and characterization of tissue transcriptomes.

ABSTRACT: Clonorchis sinensis (C. sinensis), an important food-borne parasite that inhabits the intrahepatic bile duct and causes clonorchiasis, is of interest to both the public health field and the scientific research community. To learn more about the migration, parasitism and pathogenesis of C. sinensis at the molecular level, the present study developed an upgraded genomic assembly and annotation by sequencing paired-end and mate-paired libraries. We also performed transcriptome sequence analyses on multiple C. sinensis tissues (sucker, muscle, ovary and testis). Genes encoding molecules involved in responses to stimuli and muscle-related development were abundantly expressed in the oral sucker. Compared with other species, genes encoding molecules that facilitate the recognition and transport of cholesterol were observed in high copy numbers in the genome and were highly expressed in the oral sucker. Genes encoding transporters for fatty acids, glucose, amino acids and oxygen were also highly expressed, along with other molecules involved in metabolizing these substrates. All genes involved in energy metabolism pathways, including the ?-oxidation of fatty acids, the citrate cycle, oxidative phosphorylation, and fumarate reduction, were expressed in the adults. Finally, we also provide valuable insights into the mechanism underlying the process of pathogenesis by characterizing the secretome of C. sinensis. The characterization and elaborate analysis of the upgraded genome and the tissue transcriptomes not only form a detailed and fundamental C. sinensis resource but also provide novel insights into the physiology and pathogenesis of C. sinensis. We anticipate that this work will aid the development of innovative strategies for the prevention and control of clonorchiasis.

SUBMITTER: Huang Y

PROVIDER: S-EPMC3559784 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Project description:Clonorchis sinensis is the causative agent of the life-threatening disease endemic to China, Korea, and Vietnam. It is estimated that about 15 million people are infected with this fluke. C. sinensis provokes inflammation, epithelial hyperplasia, and periductal fibrosis in bile ducts, and may cause cholangiocarcinoma in chronically infected individuals. Accumulation of a large amount of biological information about the adult stage of this liver fluke in recent years has advanced our understanding of the pathological interplay between this parasite and its hosts. However, no developmental gene expression profiles of C. sinensis have been published. In this study, we generated gene expression profiles of three developmental stages of C. sinensis by analyzing expressed sequence tags (ESTs). Complementary DNA libraries were constructed from the adult, metacercaria, and egg developmental stages of C. sinensis. A total of 52,745 ESTs were generated and assembled into 12,830 C. sinensis assembled EST sequences, and then these assemblies were further categorized into groups according to biological functions and developmental stages. Most of the genes that were differentially expressed in the different stages were consistent with the biological and physical features of the particular developmental stage; high energy metabolism, motility and reproduction genes were differentially expressed in adults, minimal metabolism and final host adaptation genes were differentially expressed in metacercariae, and embryonic genes were differentially expressed in eggs. The higher expression of glucose transporters, proteases, and antioxidant enzymes in the adults accounts for active uptake of nutrients and defense against host immune attacks. The types of ion channels present in C. sinensis are consistent with its parasitic nature and phylogenetic placement in the tree of life. We anticipate that the transcriptomic information on essential regulators of development, bile chemotaxis, and physico-metabolic pathways in C. sinensis that presented in this study will guide further studies to identify novel drug targets and diagnostic antigens.

Project description:The small liver fluke Clonorchis sinensis causes hepatobiliary ductal infections in humans. Clonorchiasis is characterized histopathologically by ductal dysplasia, hyperplasia and metaplasia, which closely resembles cholangiocarcinoma (CCA). The disruption of programmed cell death is critical for malignant transformation, while molecular events underlying these phenomena have poorly been understood in clonorchiasis-related CCA tumorigenesis. We incorporated recombinant C. sinensis omega-class glutathione transferase (rCsGSTo) 1 or 2 into human intrahepatic biliary epithelial cells (HIBECs) and analyzed pathophysiological alterations of HIBECs upon the application of oxidative stress. rCsGSTos partially but significantly rescued HIBECs from cell death by inhibiting oxidative stress-induced apoptosis (p < 0.01). rCsGSTos modulated transcriptional levels of numerous genes. We analyzed 13 genes involved in programmed cell death (the upregulation of five antiapoptotic and two apoptotic genes, and the downregulation of one antiapoptotic and five apoptotic genes) and 11 genes associated with cell differentiation (the increase in seven and decrease in four genes) that showed significant modifications (p < 0.05). The induction profiles of the mRNA and proteins of these differentially regulated genes correlated well with each other, and mostly favored apoptotic suppression and/or cell differentiation. We detected increased active, phosphorylated forms of Src, PI3K/Akt, NF-κB p65, MKK3/6 and p38 MAPK, but not JNK and ERK1/2. CsGSTos were localized in the C. sinensis-infected rat cholangiocytes, where cytokeratin 19 was distributed. Our results demonstrated that CsGSTos excreted to the biliary lumen are internalized and accumulated in the host cholangiocytes. When cholangiocytes underwent oxidative stressful condition, CsGSTos appeared to be critically involved in both antiapoptotic process and the differentiation of host cholangiocytes through the regulation of target genes following the activation of responsible signal molecules.

Project description:Liver fluke proteins, including excretory-secretory products (ESPs) and tegument proteins, are critical for the pathogenesis, nutrient metabolism, etiology and immune response of liver cancer. To understand the functions of various proteins in Clonorchis sinensis physiology and human clonorchiasis, the ESPs and tegument proteins of C. sinensis were identified. Supernatants containing ESPs from adult C. sinensis after culture for 6 h were harvested and concentrated. The tegument was detached using a freeze/thaw method and successively extracted using various extraction buffers. The outer surface proteins of C. sinensis were labeled with biotin, and the biotinylated proteins were purified. The ESP, tegument and labeled outer surface proteins were identified and analyzed by high-resolution LC-MS/MS. The identified proteins were compared with those of other flukes, and the protein functions associated with pathogenesis, carcinogenesis and potential vaccine antigens and drug targets were predicted and analyzed. A total of 175 proteins were identified after the 6-h culture of C. sinensis ESPs. A total of 352 tegument proteins were identified through sequential solubilization of the isolated teguments, and a subset of these proteins were localized to the surface membrane of the tegument by labeling with biotin. Thirty identified proteins, including annexins, actin and tetraspanins, were identified as potential immunomodulators and promising vaccine antigens. Interestingly, among the 352 tegument proteins, as many as 155 were enzymes, and most were oxidoreductases, hydrolases or transferases. A comparison of the outer surface proteins of C. sinensis with those of other flukes indicated that flukes have some common outer surface proteins, such as actin, tetraspanin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and annexin. Granulin, thioredoxin peroxiredoxin, carbonyl reductase 1 and cystatin were identified in the C. sinensis proteome and predicted to be related to liver disease and cancer. The analysis of the C. sinensis proteome could contribute to a more in-depth understanding of complex parasite-host relationships, improve the diagnosis of clonorchiasis and benefit research on the pathogenesis and development of novel interventions, drugs and vaccines to control C. sinensis infection.

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The carcinogenic liver fluke, Clonorchis sinensis: new assembly, reannotation and analysis of the genome and characterization of tissue transcriptomes.

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