A novel ex vivo immunoproteomic approach characterising Fasciola hepatica tegumental antigens identified using immune antibody from resistant sheep
A novel ex vivo immunoproteomic approach characterising Fasciola hepatica tegumental antigens identified using immune antibody from resistant sheep
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DOI：
10.1016/j.ijpara.2017.02.004
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发表时间：
2017-08-01
影响因子：
4
通讯作者：
Spithill, Terry W.
中科院分区：
文献类型：
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作者：
Cameron, Timothy C.;Cooke, Ira;Spithill, Terry W.
如果我们要开发新的免疫疗法来控制片形吸虫病，就需要更彻底地了解片形吸虫及其宿主之间的免疫相互作用。对最终宿主对肝片形吸虫和巨型片形吸虫获得性免疫反应的目标抗原的深入了解，将有可能确定候选疫苗抗原。印度尼西亚细尾羊在感染巨型飞虫4周内表现出高水平的获得性免疫，印度尼西亚细尾羊血清中的抗体可促进体外对巨型飞虫幼体表面被的抗体依赖细胞介导的细胞毒性。鉴于F. hepatca和F. gigantica蛋白序列高度相似，我们假设F. gigantica感染羊的抗体可以用来鉴定F. hepatca被膜中的同源蛋白。从感染前和感染后4周的印度尼西亚细尾羊的血清中提取纯化的IgG，与活的离体肝炎成虫孵育，分离形成的免疫沉淀（免疫沉淀），通过液相色谱-电喷雾电离-串联质谱分析，鉴定参与免疫应答的蛋白质。使用第4周的IgG，共鉴定出38种蛋白，其中包括8种预测的膜蛋白，20种分泌蛋白，9种预测与溶酶体、细胞质或细胞骨架相关的蛋白和1种未知的细胞定位蛋白。三种膜蛋白是转运蛋白，包括一种多药耐药蛋白、一种氨基酸渗透酶和一种葡萄糖转运蛋白。有趣的是，38种蛋白质中有21种与最近报道的成年肝梭菌的小外泌体样细胞外囊泡相关的蛋白质相匹配，这表明印度尼西亚细尾第4周IgG要么识别从细胞外囊泡释放的单个蛋白质，要么是免疫沉淀完整的外泌体样细胞外囊泡。共鉴定出5种细胞外囊泡膜蛋白，包括2种与囊泡运输/胞吐有关的蛋白（VPS4、液泡蛋白分选相关蛋白4b和Niemann-Pick Cl蛋白）。对38个免疫溶酶蛋白发育转录的RNAseq分析显示，这些序列的表达丰度范围很广，其中21/38个转录本从囊蚴到成虫生命周期阶段都有较高的表达水平。免疫脱液的一个显著特征是缺乏胞质蛋白，而胞质蛋白是体外培养的成年吸虫损伤的分泌标记物，这表明观察到的蛋白质不是从体内受损的吸虫泄漏的无意污染物。鉴定巨型F.和肝F.之间共有的被膜蛋白抗原，对于开发一种对这两种吸虫都有效的双用途疫苗是有益的。(C) 2017澳大利亚寄生虫学学会。Elsevier Ltd.出版。版权所有。
A more thorough understanding of the immunological interactions between Fasciola spp. and their hosts is required if we are to develop new immunotherapies to control fasciolosis. Deeper knowledge of the antigens that are the target of the acquired immune responses of definitive hosts against both Fasciola hepatica and Fasciola gigantica will potentially identify candidate vaccine antigens. Indonesian Thin Tail sheep express a high level of acquired immunity to infection by F. gigantica within 4 weeks of infection and antibodies in Indonesian Thin Tail sera can promote antibody-dependent cell-mediated cytotoxicity against the surface tegument of juvenile F. gigantica in vitro. Given the high protein sequence similarity between F. hepatica and F. gigantica, we hypothesised that antibody from F. gigantica-infected sheep could be used to identify the orthologous proteins in the tegument of F. hepatica. Purified IgG from the sera of E gigantica-infected Indonesian Thin Tail sheep collected pre-infection and 4 weeks p.i. were incubated with live adult F. hepatica ex vivo and the immunosloughate (immunoprecipitate) formed was isolated and analysed via liquid chromatography-electrospray ionisation-tandem mass spectrometry to identify proteins involved in the immune response. A total of 38 proteins were identified at a significantly higher abundance in the immunosloughate using week 4 IgG, including eight predicted membrane proteins, 20 secreted proteins, nine proteins predicted to be associated with either the lysosomes, the cytoplasm or the cytoskeleton and one protein with an unknown cellular localization. Three of the membrane proteins are transporters including a multidrug resistance protein, an amino acid permease and a glucose transporter. Interestingly, a total of 21 of the 38 proteins matched with proteins recently reported to be associated with the proposed small exosome-like extracellular vesicles of adult F. hepatica, suggesting that the Indonesian Thin Tail week 4 IgG is either recognising individual proteins released from extracellular vesicles or is immunoprecipitating intact exosome-like extracellular vesicles. Five extracellular vesicle membrane proteins were identified including two proteins predicted to be associated with vesicle transport/exocytosis (VPS4, vacuolar protein sorting-associated protein 4b and the Niemann-Pick Cl protein). RNAseq analysis of the developmental transcription of the 38 immunosloughate proteins showed that the sequences are expressed over a wide abundance range with 21/38 transcripts expressed at a relatively high level from metacercariae to the adult life cycle stage. A notable feature of the immunosloughates was the absence of cytosolic proteins which have been reported to be secreted markers for damage to adult flukes incubated in vitro, suggesting that the proteins observed are not inadvertent contaminants leaking from damaged flukes ex vivo. The identification of tegument protein antigens shared between F. gigantica and F. hepatica is beneficial in terms of the possible development of a dual purpose vaccine effective against both fluke species. (C) 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.