背景:已知蠕虫细胞外囊泡(EV)在寄生蠕虫之间具有三向通讯功能,它们的宿主和宿主相关的微生物群。它们被认为是可能携带毒力因子的生物容器,因此,作为治疗和预防目标候选人具有吸引力。这项研究旨在描述和表征Sparicotylechrysophrii(Polyopisthocootyla:Microcoticalae)分泌的电动汽车,金头seabream(Sparusaurata)的以血液为食的g寄生虫,对地中海水产养殖造成重大经济损失。
方法:为了鉴定参与细胞外囊泡生物发生的蛋白质,来自chrysophrii的基因组数据集是使用来自华支睾吸虫的已知蛋白质序列在计算机上进行挖掘的。,棘球蚴属。,Fasciolaspp.,筋膜。,Opisthorchisspp.,并吸虫属。和血吸虫。在通过高压冷冻和冷冻替代固定成年chrysophrii标本后,通过透射电子显微镜观察了EV的位置和超微结构。使用新开发的超离心-大小排阻色谱方案从成年沙棘S.chrashophrii(n=200)中分离和纯化EV,和电动汽车通过纳米粒子跟踪分析和串联质谱进行表征。
结果:在chrysophrii中鉴定出涉及EV生物发生的59种蛋白质,在夹钳衬里的胸壁区域的合胞层中观察到与外体相容的EV。分离和纯化的纳米颗粒的平均尺寸为251.8nm,得到1.71×108个颗粒·mL-1。蛋白质成分分析确定了与肽水解酶相关的蛋白质,GTPases,EF-手结构域蛋白,有氧能量代谢,抗凝剂/脂质结合,血红素解毒,铁运,EV生物发生相关,囊泡运输和其他细胞骨架相关蛋白。几种确定的蛋白质,如亮氨酰和丙氨酰氨基肽酶,钙蛋白酶,铁蛋白,动力蛋白轻链,14-3-3,热休克蛋白70,膜联蛋白,微管蛋白,谷胱甘肽S-转移酶,超氧化物歧化酶,烯醇化酶和果糖二磷酸醛缩酶,已经被提议作为用于治疗或预防目的的靶候选物。
结论:据我们所知,我们首次明确证明了外寄生虫扁虫分泌的电动汽车,在基因组和转录组水平上推断它们的生物发生机制,并通过确定它们的位置和蛋白质组成。在EVs蛋白质库中识别多个治疗靶标,首次为基于靶标的药物发现和疫苗开发提供了机会。在鱼-外寄生虫模型中。
BACKGROUND: Helminth extracellular vesicles (EVs) are known to have a three-way communication function among parasitic helminths, their host and the host-associated microbiota. They are considered biological containers that may carry virulence factors, being therefore appealing as therapeutic and prophylactic target candidates. This study aims to describe and characterise EVs secreted by Sparicotyle chrysophrii (Polyopisthocotyla: Microcotylidae), a blood-feeding gill parasite of gilthead seabream (Sparus aurata), causing significant economic losses in Mediterranean aquaculture.
METHODS: To identify proteins involved in extracellular vesicle biogenesis, genomic datasets from S. chrysophrii were mined in silico using known protein sequences from Clonorchis spp., Echinococcus spp., Fasciola spp., Fasciolopsis spp., Opisthorchis spp., Paragonimus spp. and Schistosoma spp. The location and ultrastructure of EVs were visualised by transmission electron microscopy after fixing adult S. chrysophrii specimens by high-pressure freezing and freeze substitution. EVs were isolated and purified from adult S. chrysophrii (n = 200) using a newly developed ultracentrifugation-size-exclusion chromatography protocol for Polyopisthocotyla, and EVs were characterised via nanoparticle tracking analysis and tandem mass spectrometry.
RESULTS: Fifty-nine proteins involved in EV biogenesis were identified in S. chrysophrii, and EVs compatible with
ectosomes were observed in the syncytial layer of the haptoral region lining the clamps. The isolated and purified nanoparticles had a mean size of 251.8 nm and yielded 1.71 × 108 particles · mL-1. The protein composition analysis identified proteins related to peptide hydrolases, GTPases, EF-hand domain proteins, aerobic energy metabolism, anticoagulant/lipid-binding, haem detoxification, iron transport, EV biogenesis-related, vesicle-trafficking and other cytoskeletal-related proteins. Several identified proteins, such as leucyl and alanyl aminopeptidases, calpain, ferritin, dynein light chain, 14-3-3, heat shock protein 70, annexin, tubulin, glutathione S-transferase, superoxide dismutase, enolase and fructose-bisphosphate aldolase, have already been proposed as target candidates for therapeutic or prophylactic purposes.
CONCLUSIONS: We have unambiguously demonstrated for the first time to our knowledge the secretion of EVs by an ectoparasitic flatworm, inferring their biogenesis machinery at a genomic and transcriptomic level, and by identifying their location and protein composition. The identification of multiple therapeutic targets among EVs\' protein repertoire provides opportunities for target-based drug discovery and vaccine development for the first time in Polyopisthocotyla (sensu Monogenea), and in a fish-ectoparasite model.