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

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.

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

“…2). Similar findings have been reported in previous studies on TSP family in other helminth species including adult worms of S. mansoni [33] and O. viverrini [27,42], as well as cuticle dissociation in the free-living nematode C. elegans [46]. The present findings indicate that the TSP Fig.…”

“…Tran et al [33] showed Sm-tsp1 and Sm-tsp2 were highly expressed in cercariae and eggs of S. mansoni, respectively. 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].…”

“…3). In O. viverrini, silencing of three TSP members resulted in thinner and largely vacuolated tegument [27,42]. The tegumental distal cytoplasm of tsp-2-specific dsRNA-treated schistosomula and adult worms of S. mansoni was highly vacuolated and much thinner than that of the luciferase dsRNA control [33].…”

Schistosoma haematobiumextracellular vesicle proteins confer protection in a heterologous model of schistosomiasis

“…TSPs are also found from the proteomic analysis of other helminth EVs (reviewed in [77]). In trematodes, TSPs are involved in tegument development [84-86]. TSP LELs have been tested as vaccine candidates in trematode models of infection [42,47,87] and antibodies produced against TSP vaccine candidates present in S. mansoni and O. viverrini EVs blocked the internalisation of EVs by host cells in both parasites, and decreased pathogenesis in the case of O. viverrine [29,42,88], suggesting a possible mechanism of vaccine efficacy.…”