Silencing of Opisthorchis viverrini Tetraspanin Gene Expression Results in Reduced Secretion of Extracellular Vesicles.

Sujittra Chaiyadet,Thewarach Laha,Michael Smout,Alex Loukas,Watchara Krueajampa,Paul J Brindley,Sophita Thongsen,Javier Sotillo

doi:10.3389/fcimb.2022.827521

Abstract

Inter-phylum transfer of molecular information is exquisitely exemplified in the uptake of parasite extracellular vesicles (EVs) by their target mammalian host tissues. The oriental liver fluke, Opisthorchis viverrini is the major cause of bile duct cancer in people in Southeast Asia. A major mechanism by which O. viverrini promotes cancer is through the secretion of excretory/secretory products which contain extracellular vesicles (OvEVs). OvEVs contain microRNAs that are predicted to impact various mammalian cell proliferation pathways, and are internalized by cholangiocytes that line the bile ducts. Upon uptake, OvEVs drive relentless proliferation of cholangiocytes and promote a tumorigenic environment, but the underlying mechanisms of this process are unknown. Moreover, purification and characterization methods for helminth EVs in general are ill defined. We therefore compared different purification methods for OvEVs and characterized the sub-vesicular compartment proteomes. Two CD63-like tetraspanins (Ov-TSP-2 and TSP-3) are abundant on the surface of OvEVs, and could serve as biomarkers for these parasite vesicles. Anti-TSP-2 and -TSP-3 IgG, as well as different endocytosis pathway inhibitors significantly reduced OvEV uptake and subsequent proliferation of cholangiocytes in vitro. Silencing of Ov-tsp-2 and tsp-3 gene expression in adult flukes using RNA interference resulted in substantial reductions in OvEV secretion, and those vesicles that were secreted were deficient in their respective TSP proteins. Our findings shed light on the importance of tetraspanins in fluke EV biogenesis and/or stability, and provide a conceivable mechanism for the efficacy of anti-tetraspanin subunit vaccines against a range of parasitic helminth infections.

Highlights

Perhaps the best example of inter-kingdom molecular communication is that between parasitic helminth-derived extracellular vesicles (EVs) and the cells that they target lining the epithelial/endothelial surfaces of their respective vertebrate hosts
To investigate the purity of EVs isolated following two standard methodologies -ultracentrifugation or ultracentrifugation coupled with iodixanol (Optiprep) density gradient, we compared the number of vesicles, purity and presence of traditional markers of mammalian cell EVs, such as tetraspanins, in all isolated fractions
The roles that EVs play in inter-phylum molecular communication is becoming readily apparent, notably for the promise they hold in developing next-generation therapeutics (Woith et al, 2019)

Summary

Introduction

Perhaps the best example of inter-kingdom molecular communication is that between parasitic helminth-derived extracellular vesicles (EVs) and the cells that they target lining the epithelial/endothelial surfaces of their respective vertebrate hosts. The carcinogenic liver fluke, Opisthorchis viverrini, resides in the bile ducts of its human host where it secretes EVs that are internalized by biliary epithelial cells, or cholangiocytes (Chaiyadet et al, 2015b). Multiple processes are involved in liver fluke-induced cancer, including mechanical damage caused by the parasite’s physical attachment to and grazing on the biliary epithelium, chronic immunopathological processes that induce pro-inflammatory cytokines such as IL-6, and the release of parasite-derived excretory/secretory (ES) products into the bile duct that drive unchecked cell proliferation (Sripa et al, 2018). Several mechanisms by which EVs deliver their content into target cells have been described (Mulcahy et al, 2014; Mathieu et al, 2019), including clathrin-dependent mechanisms in neurons (Fruhbeis et al, 2013) and clathrin-independent mechanisms in endothelial cells (Nanbo et al, 2013; Svensson et al, 2013), or even fusion of the EV membrane with the plasma membrane of its target cell (Parolini et al, 2009; Montecalvo et al, 2012; Andreu and Yanez-Mo, 2014)

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Conclusion