Molecular Characterization of a Tetraspanin from the Human Liver Fluke, Opisthorchis viverrini

Abstract

BackgroundThe 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 FindingsThe 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/SignificanceThis 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.

Biological and morphological consequences of dsRNA-induced suppression of tetraspanin mRNA in developmental stages of Echinococcus granulosus

“…In Opisthorchis viverrini the highest expression of Ov-tsp-2 and Ov-tsp-3 was observed in the eggs [42]. In contrast, Piratae et al [27] found highly expressed Ov-tsp-1 in the metacercariae of this species.…”

“…No other data are available on the effects of TSP1 suppression in the strobilated forms of E. granulosus. In two studies on adult worms of O. viverrini, treatment by specific dsRNAs, resulted in expression suppression of Ov-tsp-1, Ov-tsp2 and Ov-tsp-3 [27,42].…”

“…TSPs participate in a broad spectrum of cellular activities, including cell proliferation, cell fusion, motility, adhesion, migration, sperm-egg fusion and signal transduction pathways [25,26]. TSPs in trematodes have an important role in the development, maturation and stability of the tegument and are involved in the immune evasion of schistosomes [27,28]. Recently, many studies have focused on some members of the TSPs family as a vaccine candidate against schistosomiasis [20,28], alveolar echinococcosis [29,30], filariasis [31] and clonorchiasis [32].…”

Suppression of Ov-grn-1 encoding granulin of Opisthorchis viverrini inhibits proliferation of biliary epithelial cells

“…Ov-grn-1 cDNA was amplified using specific primers as follows: forward primer, Ov -GRN-EXF: 5'-GGGATCGGTTAGTCTAATCTCC-3', and reverse primer, Ov -GRN EXR: 5'-GATCATGGGGGTTCACTGTC) (spanning Ov - grn-1 coding DNA position 6-365) amplifying a 359 bp product which is larger than the dsRNA used for electroporation and therefore cannot be amplified from exogenous dsRNA. The endogenous actin gene (GenBank AY005475) was used as a housekeeping control (Piratae et al, 2012). Real time PCR was carried out using an ABI7500 thermal cycler and the SYBR Green assay.…”

Opisthorchis viverrini: Analysis of the sperm-specific rhophilin associated tail protein 1-like

“…In Thailand, O. viverrini has attracted intense scientific interest due to the connection of opisthorchiasis and cholangiocarcinoma, a bile duct cancer with a uniquely high prevalence rate in this country (Sripa et al, 2007). Among others, released antigens, tegumental surface antigens and egg antigens of the parasite have been analyzed for their application as diagnostic markers, targets for intervention or their role in tumorigenesis (Ruangsittichai et al, 2006;Pinlaor et al, 2009;Smout et al, 2009;Eursitthichai et al, 2010;Piratae et al, 2012;Sripa et al, 2012) and yet, as this research will demonstrate, antigens of the parasite sperm could also be involved in the host-parasite interaction and might warrant further attention.…”