Suppression of mRNAs encoding CD63 family tetraspanins from the carcinogenic liver fluke Opisthorchis viverrini results in distinct tegument phenotypes

Sujittra Chaiyadet,Yada Plosan,Thewarach Laha,Michael Smout,Alex Loukas,Supawadee Piratae,Banchob Sripa,Wiphawi Hipkaeo,Watchara Krueajampa,Javier Sotillo,Paul J Brindley

doi:10.1038/s41598-017-13527-5

Abstract

The liver fluke Opisthorchis viverrini infects 10 million people in Southeast Asia and causes cholangiocarcinoma (CCA). Fluke secreted and tegumental proteins contribute to the generation of a tumorigenic environment and are targets for drug and vaccine-based control measures. Herein, we identified two tetraspanins belonging to the CD63 family (Ov-TSP-2 and Ov-TSP-3) that are abundantly expressed in the tegument proteome of O. viverrini. Ov-tsp-2 and tsp-3 transcripts were detected in all developmental stages of O. viverrini. Protein fragments corresponding to the large extracellular loop (LEL) of each TSP were produced in recombinant form and antibodies were raised in rabbits. Ov-TSP-2 and TSP-3 were detected in whole worm extracts and excretory/secretory products of O. viverrini and reacted with sera from infected hamsters and humans. Antibodies confirmed localization of Ov-TSP-2 and TSP-3 to the adult fluke tegument. Using RNA interference, Ov-tsp-2 and tsp-3 mRNA expression was significantly suppressed for up to 21 days in vitro. Ultrastructural observation of tsp-2 and tsp-3 dsRNA-treated flukes resulted in phenotypes with increased tegument thickness, increased vacuolation (tsp-2) and reduced electron density (tsp-3). These studies confirm the importance of CD63 family tegument tetraspanins in parasitic flukes and support efforts to target these proteins for vaccine development.

Highlights

Liver fluke infection caused by Opisthorchis viverrini remains a serious public health problem in Southeast Asia[1]
Ov-tsp-2 contained four transmembrane domains consisting of a small extracellular loop (SEL; residues 34–52), a larger extracellular loop (LEL; residues 109–186), a small inner loop (IL; residues 79–84), and short cytoplasmic tail
Using transmission electron microscopy (TEM) we showed that the tegument of O. viverrini exposed to Ov-tsp-2 double-stranded RNAs (dsRNAs) (Fig. 7C) was 4-fold thicker than the tegument of flukes treated with irrelevant dsRNAs (P < 0.0001; Fig. 7E), measuring on average 3.2306 ± 0.040 μm compared with 0.8770 ± 0.033 μm for luciferase controls (Fig. 7B)

Summary

Introduction

Liver fluke infection caused by Opisthorchis viverrini remains a serious public health problem in Southeast Asia[1]. The adult stage of O. viverrini resides in the bile ducts of the host where it secretes protein antigens that stimulate immunopathology[3]. Tetraspanins is a superfamily of membrane proteins characterized by the presence of four transmembrane domains and a large extracellular loop (LEL). They contribute to the maintenance of tegument membrane integrity[6] and play a key role in tegument formation in parasitic flukes[7,8]. We identified and characterized two tetraspanin-encoding cDNAs designated Ov-tsp-2 and Ov-tsp-3 and investigated the impact of silencing their expression on tegument architecture in adult flukes. Understanding the functions of tetraspanins in O. viverrini will facilitate efforts to develop a vaccine against this carcinogenic fluke

Methods

Results

Conclusion