烟曲霉的细胞外囊泡蛋白质及RNA分析

Protein and RNA analysis of extracellular vesicles of Aspergillus fumigatus

目的分析烟曲霉的细胞外囊泡(extracellular vesicles,EVs)中的重要成分,以进一步明确曲霉致病机制。方法通过离心法分离烟曲霉的囊泡,用电镜观察形态。采用马尔文纳米颗粒跟踪分析仪分析溶液中EVs的大小分布。通过质谱仪对囊泡内处理后的肽段进行分析。二级质谱数据使用Maxquant(v1.5.2.8)进行检索。检测RNA分布,通过数据库分析烟曲霉EVs中miRNA可能参与的一些通路。结果电镜下烟曲霉的EVs可见明显的双层脂质结构。NTA发现烟曲霉分析的EVs大小主要集中在130 nm左右。EVs蛋白中不稳定蛋白为9种(15%),其余为稳定蛋白,而等电点(isoelectric point,PI)>7的为6种蛋白。EVs中大部分是胞浆蛋白,其余比较多的是细胞外分泌蛋白,但仍有25%的蛋白不能定位。通过跨膜蛋白预测(transmembrane prediction,TMPred)和糖基磷脂酰肌醇(glycosylphosphatidylinositol,GPI)预测,有5种蛋白存在于双层脂质膜上的蛋白。检测到RNA中rRNA和tRNA分别占59.7%和29.4%,而miRNA占0.25%,发现EVs-miRNA主要可能影响代谢通路。结论我们证实了烟曲霉也能分泌EVs,其中位直径为130 nm,含有较多种蛋白,以烟曲霉胞浆蛋白为主,RNA以rRNA和tRNA为主,而其中miRNA可能涉及多种信号通路。

Objective To analyze the important components in the extracellular vesicles(EVs)of Aspergillus fumigatus to further clarify the pathogenesis of Aspergillus fumigatus.Methods The vesicles of Aspergillus fumigatus were separated by centrifugal method and the morphology was observed by electron microscopy.The size of EVs in solution was analyzed by Marvin nanoparticle tracking analyzer.The processed peptides in the vesicle were analyzed by mass spectrometer.Secondary mass spectrometry data was retrieved using Maxquant(v1.5.2.8).Results The EVs of Aspergillus fumigatus showed obvious bilayer lipid structure under electron microscope.NTA found that the EVs size of Aspergillus fumigatus analysis was mainly concentrated around 130nm.EVs protein contains 9 unstable proteins(15%),the rest was stable proteins,while PI>7 contains 6 proteins.In EVs cytoplasmic proteins was the most,followed by exocrine proteins.However,25%of EVs could not be located.As predicted by TMPred and GPI,there were 5 proteins located on the bilayer lipid membrane.In RNA,rRNA and tRNA accounted for 59.7%and 29.4%respectively,while miRNA accounted for 0.25%.EVS-miRNA might mainly affect metabolic pathways.Conclusions Aspergillus fumigatus could secrete EVs with a diameter of 130 nm,which containing a variety of proteins,mainly cytoplasmic proteins.EVS-RNA was dominated by rRNA and tRNA,while miRNAs might involve a variety of signaling pathways.