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.

蠕虫寄生虫可分泌细胞外囊泡（extracellular vesicles, EVs），此类囊泡可被宿主免疫细胞摄取并调控宿主免疫。尽管诸多寄生虫的EVs分子载荷已得到系统表征，但针对参与配体-受体相互作用、结合宿主细胞表面以启动囊泡锚定及后续摄取过程的EV表面暴露分子，目前所知仍十分有限。本研究使用膜不透性生物素试剂，捕获由成虫肝片吸虫（F. hepatica）释放的两种EV亚群（命名为15k与120k EVs）外膜表面展示的蛋白质，共鉴定得到380种表面蛋白，涵盖一系列毒力因子、膜转运蛋白以及参与EV生物发生与转运的相关分子。蛋白质组学与免疫组织化学分析显示，120k EVs起源于内体，可能通过原肾（排泄）系统从寄生虫体内释放；而体积更大的15k EVs则由吸虫肠道内衬的胃皮上皮细胞分泌释放。本研究采用平行凝集素微阵列策略，对完整荧光标记EVs的主要表面寡糖拓扑结构进行谱分析，以还原其向宿主暴露的真实状态。结果表明，15k与120k EV亚群表面暴露的糖缀合物对应的凝集素谱几乎完全一致，但与寄生虫体表被膜的凝集素谱存在显著差异；不过所有样本均以高甘露糖型糖基作为主要糖链类型。研究发现，肝片吸虫EVs可抵抗外切糖苷酶与内切糖苷酶的降解，但糖苷酰胺酶PNGase F可显著重塑其表面寡糖结构，并阻断EVs被宿主巨噬细胞摄取。与之相反，用感染宿主获取的抗体，或针对特定EV表面蛋白（含DM9结构域蛋白、CD63受体及肌铁蛋白（myoferlin））的胞外结构域制备的纯化抗体进行预处理，可显著增强EVs的细胞摄取效率。本研究揭示了肝片吸虫所利用的EV生物发生与转运通路的多样性，并阐明了寄生虫EVs与宿主细胞之间的分子相互作用机制。