Proteomic analysis of bacterial extracellular vesicles enriched by ultracentrifugation and an ε-Poly- L-lysine-based method

Both Gram-negative and Gram-positive bacteria can release vesicle-like structures referred as bacterial extracellular vesicles (BEVs), which contain various bioactive compounds. BEVs play important roles in the microbial community interactions and host-microbe interactions. Markedly, BEVs can be delivered to host cell, thus modulating the development and function of the innate immune system. To clarify the compositions and biological functions of BEVs, these vesicles need to be collected with high purity and bioactivity. Here we propose an isolation strategy based on a broad-spectrum antimicrobial epsilon-poly-L–lysine (ε-PL) to precipitate BEVs at a relatively low centrifugal speed (10,000 × g). Compared to the standard ultracentrifugation strategy, our method can enrich BEVs from large volumes of media inexpensively and rapidly. The precipitated BEVs can be recovered by adjusting pH and ionic strength of the media, followed by ultrafiltration step to remove ε-PL and achieve buffer exchange. Protein compositions of the BEVs enriched by ultracentrifugation and ε-PL-based method are measured using LC-MS/MS. The resultant data indicate that protein compositions of the ε-PL-precipitated BEVs are comparable to those purified by ultracentrifugation.

革兰氏阴性菌与革兰氏阳性菌均可释放囊泡状结构，这类结构被称为细菌胞外囊泡（bacterial extracellular vesicles，BEVs），其内含多种生物活性物质。BEVs在微生物群落互作以及宿主-微生物互作中发挥关键作用。尤为关键的是，BEVs可被递送至宿主细胞，进而调控先天免疫系统的发育与功能。为阐明BEVs的组成与生物学功能，需以高纯度、高生物活性的标准获取这类囊泡。本研究提出一种基于广谱抗菌剂ε-聚-L-赖氨酸（epsilon-poly-L–lysine，ε-PL）的分离策略，可在相对较低的离心转速（10,000 × g）下沉淀BEVs。相较于标准超速离心策略，本方法可低成本、快速地从大体积培养基中富集BEVs。沉淀得到的BEVs可通过调节培养基的pH与离子强度进行回收，随后通过超滤步骤去除ε-PL并完成缓冲液置换。采用液相色谱-串联质谱（LC-MS/MS）对超速离心法与ε-PL沉淀法富集得到的BEVs的蛋白质组成进行检测。所得数据表明，经ε-PL沉淀的BEVs的蛋白质组成与超速离心纯化所得BEVs的组成相当。