Surface molecules of extracellular vesicles secreted by the helminth pathogen Fasciola hepatica direct their internalisation by host cells

Helminth parasites secrete extracellular vesicles (EVs) that can be internalised by host immune cells resulting in modulation of host immunity. While the molecular cargo of EVs have been characterised in many parasites, little is known about the surface-exposed molecules that participate in ligand-receptor interactions with the host cell surface to initiate vesicle docking and subsequent internalisation. Using a membrane-impermeable biotin reagent to capture proteins displayed on the outer membrane surface of two EV sub-populations (termed 15k and 120k EVs) released by adult F. hepatica, we describe 380 surface proteins including an array of virulence factors, membrane transport proteins and molecules involved in EV biogenesis/trafficking. Proteomics and immunohistochemical analysis show that the 120k EVs have an endosomal origin and may be released from the parasite via the protonephridial (excretory) system whilst the larger 15k EVs are released from the gastrodermal epithelial cells that line the fluke gut. A parallel lectin microarray strategy was used to profile the topology of major surface oligosaccharides of intact fluorogenically-labelled EVs as they would be displayed to the host. Lectin profiles corresponding to glycoconjugates exposed on the surface of the 15 K and 120K EV sub-populations are practically identical but are distinct from those of the parasite surface tegument, although all are predominated by high mannose sugars. We found that while the F. hepatica EVs were resistant to exo- and endo-glycosidases, the glyco-amidase PNGase F drastically remodelled the surface oligosaccharides and blocked the uptake of EVs by host macrophages. In contrast, pre-treatment with antibodies obtained from infected hosts, or purified antibodies raised against the extracellular domains of specific EV surface proteins (DM9-containing protein, CD63 receptor and myoferlin), significantly enhanced their cellular internalisation. This work highlights the diversity of EV biogenesis and trafficking pathways used by F. hepatica and sheds light on the molecular interaction between parasite EVs and host cells. Author summary Over the last decade, it has become recognised that extracellular vesicles (EVs) are important mediators of communication by transferring molecular signals (including proteins, lipids, complex carbohydrates, mRNA, microRNA and other non-coding RNA species), between cells. Variously described as exosomes or microvesicles depending on their cellular origin and mode of biogenesis, EVs perform a variety of roles in the maintenance of normal physiology but also participate in pathological settings. EVs also play an important role in host-pathogen interactions, with recent work suggesting that they contribute to helminth immunomodulatory strategies. Here we have identified the proteins and sugars displayed on the outer surface of two sub-types of EVs released by the helminth pathogen Fasciola hepatica. We show that the proteins are antigenic and direct EV internalisation by host macrophages. Our study provides a better understanding of how parasite-derived EVs interact with host cells which is important for future development of therapeutics/vaccines that target this interface.