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. 2012;6(12):e1939.
doi: 10.1371/journal.pntd.0001939. Epub 2012 Dec 6.

Molecular characterization of a tetraspanin from the human liver fluke, Opisthorchis viverrini

Supawadee Piratae  1 , Smarn Tesana, Malcolm K Jones, Paul J Brindley, Alex Loukas, Erica Lovas, Veerachai Eursitthichai, Banchob Sripa, Sirikanda Thanasuwan, Thewarach Laha
Affiliations

Molecular characterization of a tetraspanin from the human liver fluke, Opisthorchis viverrini

Supawadee Piratae et al. PLoS Negl Trop Dis. 2012.

Abstract

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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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Sequence and structure of Ov-TSP-1, a tetraspanin from Opisthorchis viverrini.
Panel A: Schematic illustration of the structural design of Ov-TSP-1. Panel B: Multiple sequence alignment of the deduced amino acid sequence of Ov-TSP-1 with other members of the tetraspanin superfamily. Large extracellular loops are enclosed in boxes. Identical residues of sequences are shown in black boxes, conserved substitutions in gray boxes.
Figure 2
Figure 2. Phylogenetic analysis of tetraspanins from Opisthorchis viverrini and homologs from related superfamilies.
O. viverrini tetraspanins can be grouped within extant two families, the CD family and CD63 family. CD family: Clonorchis sinensis (GAA49954.1), Schistosoma mansoni (XP_002580456.1), S. japonicum (CAX70118.1), Danio rerio (NP_001003735.1), Bos taurus (NP_776325.1), Mus musculus (NP_031683.1), Homo sapiens (NP_004347.1). CD63 family: C. sinensis (GAA50199.1), S. mansoni (AAN17276.1), S. japonicum (CAX70616.1), D. rerio (NP_955837.1), B. taurus (NP_991372.1), M. musculus (NP_031679.1), H. sapiens (NP_001771.1). Note that the mouse, bovine and human proteins presented on the CD clade are distinct proteins from those shown in the CD63 clade.
Figure 3
Figure 3. Expression of Ov-tsp-1 in different developmental stages of O. viverrini.
RNA levels relative to the gene encoding actin, Ov-actin, were analyzed by real-time qRT-PCR. Transcript levels were calculated from duplicate specimens from each treatment group and the data are presented as means ± SD.
Figure 4
Figure 4. Immunohistochemical detection of Ov-TSP-1 in tissue sections of adult O. viverrini in the bile ducts of an infected hamster.
(A–B) Mouse anti-Ov-TSP-1 IgG bound to the tegument (T) and parasite eggs (E). (C–D) Control serum from the same mouse prior to immunization did not bind to the same structures. Scale bars are shown.
Figure 5
Figure 5. Western blot analysis for recognition of Ov-TSP-1 by sera from O. viverrini infected humans and hamsters.
Lanes 1, immunoblot of rOv-TSP-1 probed with serum of O. viverrini infected human; lane 2, immunoblot of rOv-TSP-1 probed with serum of O. viverrini infected hamster; lane 3, immunoblot of rOv-TSP-1 probed with normal serum of hamster and lane 4, immunoblot of rOv-TSP-1 probed with sera of mice immunized with rOv-TSP-1.
Figure 6
Figure 6. Suppression of Ov-tsp-1 mRNA in adult O. viverrini by RNA interference (RNAi).
Real time qRT-PCR analysis of Ov-tsp-1 transcription levels relative to Ov-actin (mean± standard error) showing reduction in expression of Ov-tsp-1 mRNA in adult O. viverrini on days (D) 1–16 of ds-Ov-tsp-1 treatment by electroporation and soaking. Transcript levels were calculated in triplicate from three randomly-selected parasites from each treatment group and the data are presented as means ± SD. Student's t-tests confirmed significant differences as indicated.
Figure 7
Figure 7. Ultrastructure of the tegument of adult O. viverrini treated with Ov-tsp-1 dsRNA RNA observed using transmission electron microscopy.
Panel A = Tegument of non-treated adult O. viverrini in RPMI medium for 1 day. Panel B = Tegument of adult O. viverrini treated with firefly luciferase dsRNA for 1days. Panel C = Tegument of adult O. viverrini treated with Ov-tsp-1 dsRNA for 1days. Panel D = Tegument of non-treated adult O. viverrini in RPMI medium for 3 days. Panel E and F = Tegument of adult O. viverrini treated with Ov-tsp-1 dsRNA for 3 days. The tegument of knocked down tsp-1 worm is more highly vacuolated (indicated by arrows) and thinner compared with controls.
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This work was supported by the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, through the Health Cluster (SHeP-GMS), The National Research Project of Khon Kaen University. Supawadee was supported by a Postgraduate Study Support Grant of Faculty of Medicine, Khon Kaen University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.