Lipidomic Analysis of Extracellular Vesicles from the Pathogenic Phase of Paracoccidioides brasiliensis

BackgroundFungal extracellular vesicles are able to cross the cell wall and transport molecules that help in nutrient acquisition, cell defense, and modulation of the host defense machinery. Methodology/Principal FindingsHere we present a detailed lipidomic analysis of extracellular vesicles released by Paracoccidioides brasiliensis at the yeast pathogenic phase. We compared data of two representative isolates, Pb3 and Pb18, which have distinct virulence profiles and phylogenetic background. Vesicle lipids were fractionated into different classes and analyzed by either electrospray ionization- or gas chromatography-mass spectrometry. We found two species of monohexosylceramide and 33 phospholipid species, including phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol. Among the phospholipid-bound fatty acids in extracellular vesicles, C181 predominated in Pb3, whereas C18:2 prevailed in Pb18. The prevalent sterol in Pb3 and Pb18 vesicles was brassicasterol, followed by ergosterol and lanosterol. Inter-isolate differences in sterol composition were observed, and also between extracellular vesicles and whole cells. Conclusions/SignificanceThe extensive lipidomic analysis of extracellular vesicles from two P. brasiliensis isolates will help to understand the composition of these fungal components/organelles and will hopefully be useful to study their biogenesis and role in host-pathogen interactions.背景 真菌胞外囊泡（extracellular vesicles）能够穿透细胞壁，转运参与营养获取、细胞防御以及调控宿主防御系统的分子。 方法/主要结果 本研究对致病酵母相的巴西副球孢子菌（Paracoccidioides brasiliensis）释放的胞外囊泡开展了全面的脂质组学分析。我们选取了两个具有不同毒力特征与系统发育背景的代表性分离株Pb3和Pb18进行数据对比。将囊泡脂质按不同类别分级分离后，分别通过电喷雾电离质谱（electrospray ionization-mass spectrometry）或气相色谱-质谱联用（gas chromatography-mass spectrometry）技术进行分析。本研究共鉴定出两种单己糖神经酰胺（monohexosylceramide）与33种磷脂物种，涵盖磷脂酰胆碱（phosphatidylcholine）、磷脂酰乙醇胺（phosphatidylethanolamine）、磷脂酸（phosphatidic acid）、磷脂酰丝氨酸（phosphatidylserine）、磷脂酰肌醇（phosphatidylinositol）以及磷脂酰甘油（phosphatidylglycerol）。在胞外囊泡的磷脂结合脂肪酸中，Pb3分离株以C18:1为优势脂肪酸，而Pb18分离株则以C18:2占主导。Pb3与Pb18分离株的囊泡中，优势甾醇为菜油甾醇（brassicasterol），其次为麦角固醇（ergosterol）与羊毛甾醇（lanosterol）。研究观察到不同分离株之间，以及胞外囊泡与全细胞之间的甾醇组成均存在差异。 结论/意义 本研究针对两株巴西副球孢子菌胞外囊泡开展的全面脂质组学分析，有助于解析这类真菌组分/细胞器的组成，有望为其生物发生过程以及宿主-病原体互作中的功能研究提供参考依据。