School of Biological Sciences, Queen's University Belfast, 19 Chlorine Gardens, Belfast, Northern Ireland, United Kingdom.
School of Biological Sciences, Queen's University Belfast, 19 Chlorine Gardens, Belfast, Northern Ireland, United Kingdom.
Int J Parasitol. 2020 Aug;50(9):671-683. doi: 10.1016/j.ijpara.2020.03.015. Epub 2020 Jun 20.
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 (120 K) 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 120 K EV release in vitro, suggesting that a ceramide-dependent mechanism could drive 120 K EV formation. In contrast, the release of the larger 15 K 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.
寄生虫的后生动物会分泌细胞外囊泡 (EVs)，这些囊泡具有很强的免疫调节作用。虽然已经对许多物种的 EV 货物进行了描述，但我们对控制其生物发生和释放的机制知之甚少。我们使用针对一组 Fasciola hepatica EV (FhEV) 标记蛋白的抗体，在寄生虫中鉴定出多个 EV 产生的部位。对于不同的标记蛋白，在胃皮层细胞和体被合胞体中观察到离散的免疫荧光模式，而原肾 (排泄) 系统和实质型 2 细胞被鉴定为 FhEV 产生 (或转运) 的另外部位。结扎用于机械性阻塞口吸盘、排泄孔或两者，以确定其对体外活成虫吸虫中 FhEV 释放的影响。结果表明，FhEV 主要来源于肠道，而体被的释放则较少。数据还表明，原肾系统有助于小 (120 K) EV 亚群的形成。神经鞘磷脂酶 (SMase) 活性是哺乳动物细胞中 EV 生物发生的关键驱动因素，我们之前通过质谱法在 FhEV 中鉴定出了 SMase。与分离的 FhEV 相关的 SMase 活性易受化学抑制剂 GW4869 的影响，GW4869 处理成虫可导致体外 120 K EV 的释放显著减少，表明 ceramide 依赖性机制可能驱动 120 K EV 的形成。相比之下，较大的 15 K EV 的释放仅受到适度影响，表明它们的形成独立于 SMase 活性。GW4869 处理的 F. hepatica 组织的超微结构观察显示，实质组织严重破坏，体被、胃皮层细胞和排泄管的上皮衬里出现空泡化。这项工作确立了靶向破坏寄生虫后生动物的 EV 生物发生和释放是可能的，并为未来研究 EV 分泌作为寄生虫控制的靶点提供了概念验证。
