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Proteomic characterization of the internalization of Opisthorchis viverrini excretory/secretory products in human cells.

ABSTRACT: The association between liver fluke infection caused by Opisthorchis viverrini and cholangiocarcinoma (CCA - hepatic cancer of the bile duct epithelium) has been well established. Multiple mechanisms play a role in the development of CCA, but the excretory/secretory products released by O. viverrini (OvES) represent the major interface between the parasite and its host, and their uptake by biliary epithelial cells has been suggested to be responsible for proliferation of cholangiocytes, the cells that line the biliary epithelium. Despite recent progress in the study of the molecular basis of O. viverrini-host interactions, little is known about the effects that OvES induces upon internalization by host cells. In the present study we incubated non-cancerous human cholangiocytes (H69) and human colon cancer (CaCo-2) cells with OvES and performed a time-course quantitative proteomic analysis on the cells to determine the early changes induced by the parasite. Different KEGG pathways were altered in H69 cells compared to Caco-2 cells: glycolysis/gluconeogenesis and protein processing in the endoplasmic reticulum. In addition, the Reactome pathway analysis showed a predominance of proteins involved in cellular pathways related to apoptosis and apoptotic execution phase in H69 cells after incubation with OvES. The present study provides the first proteomic analysis to address the molecular mechanisms by which OvES products interact with host cells, and Sheds light on the cellular processes involved in O. viverrini-induced CCA.

SUBMITTER: Chaiyadet S

PROVIDER: S-EPMC5149449 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Proteomic characterization of the internalization of Opisthorchis viverrini excretory/secretory products in human cells.

Chaiyadet Sujittra S Smout Michael M Laha Thewarach T Sripa Banchob B Loukas Alex A Sotillo Javier J

Parasitology international 20160209 4

The association between liver fluke infection caused by Opisthorchis viverrini and cholangiocarcinoma (CCA - hepatic cancer of the bile duct epithelium) has been well established. Multiple mechanisms play a role in the development of CCA, but the excretory/secretory products released by O. viverrini (OvES) represent the major interface between the parasite and its host, and their uptake by biliary epithelial cells has been suggested to be responsible for proliferation of cholangiocytes, the cell ...[more]

PMID: 26873540

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Project description:AimTo investigate the mechanism of fibroblast cell proliferation stimulated by the Opisthorchis viverrini excretory/secretory (ES) product.MethodsNIH-3T3, mouse fibroblast cells were treated with O. viverrini ES product by non-contact co-cultured with the adult parasites. Total RNA from NIH-3T3 treated and untreated with O. viverrini was extracted, reverse transcribed and hybridized with the mouse 15K complementary DNA (cDNA) array. The result was analyzed by ArrayVision version 5 and GeneSpring version 5 softwares. After normalization, the ratios of gene expression of parasite treated to untreated NIH-3T3 cells of 2-and more-fold upregulated was defined as the differentially expressed genes. The expression levels of the signal transduction genes were validated by semi-quantitative SYBR-based real-time RT-PCR.ResultsAmong a total of 15,000 genes/ESTs, 239 genes with established cell proliferation-related function were 2 fold-and more-up-regulated by O. viverrini ES product compared to those in cells without exposure to the parasitic product. These genes were classified into groups including energy and metabolism, signal transduction, protein synthesis and translation, matrix and structural protein, transcription control, cell cycle and DNA replication. Moreover, the expressions of serine-threonine kinase receptor, receptor tyrosine kinase and collagen production-related genes were up-regulated by O. viverrini ES product. The expression level of signal transduction genes; pkC, pdgfr alpha, jak 1, eps 8, tgf beta 1i4, strap and h ras measured by real-time RT-PCR confirmed their expression levels to those obtained from cDNA array. However, only the up-regulated expression of pkC, eps 8 and tgfbeta 1i4 which are the downstream signaling molecules of either epidermal growth factor (EGF) or transforming growth factor-beta (TGF-beta) showed statistical significance (P < 0.05).ConclusionO. viverrini ES product stimulates the significant changes of gene expression in several functional categories and these mainly include transcripts related to cell proliferation. The TGF-beta and EGF signal transduction pathways are indicated as the possible pathways of O. viverrini-driven cell proliferation.

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.

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Proteomic characterization of the internalization of Opisthorchis viverrini excretory/secretory products in human cells.

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Proteomic characterization of the internalization of Opisthorchis viverrini excretory/secretory products in human cells.

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