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.

13The quite recent discovery that parasites release extracellular vesicles (EVs) that can transfer 14 a range of effector molecules to host cells has made us re-think our understanding of the host-15 parasite interface. In this opinion article we will consider how recent proteomics and 16 transcriptomics studies, together with ultrastructural observations, suggest that more than one 17 mechanism of EV biogenesis can occur in helminths. We propose that distinct EV sub-types 18 have roles in immune-modulation and repair of drug-induced damage, and put forward the 19 case for targeting EV biogenesis pathways to achieve parasite control. In doing so we raise a 20 number of outstanding research questions that must be addressed before this can happen. released by mammalian cells (Table 1) nematodes has yet to be documented although exosomes released by C. elegans epithelial The mechanism of bleb formation in helminths has yet to be determined, but it is 214 thought to be a calcium-dependent process [63,64]. MVs also bud directly from the plasma 215 membrane in response to raised intracellular Ca 2+ levels [65]. Given these biochemical and 216 structural similarities, it is conceivable that parasite blebs are formed using the same Concluding remarks 224The discovery of parasite-derived EVs has changed our view of the host-parasite interface. 225The formation of EVs enables the parasite to release a range of effector molecules (including

Parasitic helminths secrete extracellular vesicles (EVs) which have potent immunomodulatory effects. Whilst the cargo of EVs has been characterised for many species, we know little about the mechanisms that govern their biogenesis and release. Using antibodies raised against a panel of Fasciola hepatica EV (FhEV) marker proteins, we have identified multiple sites of EV production in the parasite. Discrete immunofluorescence patterns were observed within the gastrodermal cells and tegumental syncytium for different marker proteins whilst the protonephridial (excretory) system and parenchymal-type 2 cells were identified as additional sites of production (or transit) of FhEVs. Ligation was used to mechanically block the oral sucker, excretory pore, or both, to determine the effect on FhEV release from live adult flukes in vitro. This revealed that FhEVs are predominately derived from the gut, whilst the tegument releases EVs to a lesser extent. The data also suggest that the protonephridial system contributes to the small (120K) EV sub-population. Sphingomyelinase (SMase) activity is a key driver of EV biogenesis in mammalian cells and we have previously identified SMases in FhEVs by mass spectrometry. SMase activity associated with isolated FhEVs was susceptible to the chemical inhibitor GW4869 and treatment of adult flukes with GW4869 led to a significant reduction in 120K EV release in vitro suggesting that a ceramidedependent mechanism could drive 120K EV formation. In contrast, the release of the larger 15K EVs was only moderately impacted, indicating that they form independently of SMase activity.Ultrastructural observation of GW4869-treated F. hepatica tissue showed severe disruption to the parenchyma and vacuolation of the tegument, gastrodermal cells and epithelial lining of the excretory ducts. This work establishes that targeted disruption of EV biogenesis and release in helminths is possible and provides proof-of-concept for future studies investigating EV secretion as a target for parasite control.

A comparative genomics approach was used to determine whether established extracellular vesicle (EV) biogenesis pathways are conserved in helminths. This revealed conservation of membrane and cytoskeletal-organising proteins as well as the endosomal sorting complex required for transport (ESCRT) previously described in mammalian cells. Domain-level analysis of this complex in helminths, however, indicated that some species may rely on atypical proteins to support subunit interactions and cargo recruitment. Interestingly, helminths displayed phylum-level divergence of proteins associated with loading RNA into EVs. These findings provide a framework for functional studies of helminth EV biogenesis and cargo sorting.

. Extracellular vesicles (EVs) are nanoparticles (30 to 1000 nm in diameter) surrounded by a lipid-bilayer which carry bioactive molecules between local and distal cells and participate in intercellular communication. Because of their small size and heterogenous nature they are challenging to characterize. Here, we discuss commonly used techniques that have been employed to yield information about EV size, concentration, mechanical properties, and protein content. These include dynamic light scattering, nanoparticle tracking analysis, flow cytometry, transmission electron microscopy, atomic force microscopy, western blotting, and optical methods including super-resolution microscopy. We also introduce an innovative technique for EV characterization which involves immobilizing EVs on a microscope slide before staining them with antibodies targeting EV proteins, then using the reflectance mode on a confocal microscope to locate the EV plane. By then switching to the microscope’s fluorescence mode, immunostained EVs bearing specific proteins can be identified and the heterogeneity of an EV preparation can be determined. This approach does not require specialist equipment beyond the confocal microscopes that are available in many cell biology laboratories, and because of this, it could become a complementary approach alongside the aforementioned techniques to identify molecular heterogeneity in an EV preparation before subsequent analysis requiring specialist apparatus.