需钠弧菌外膜囊泡的蛋白质组分析与异源蛋白的递送

Proteome analysis and heterologous cargo delivery of Vibrio natriegens outer membrane vesicles

摘要:

[背景] 需钠弧菌（Vibrio natriegens）是一种快速生长的革兰氏阴性菌，作为一种新兴工具在生物技术领域有重要的应用潜力。此前的研究主要集中在开发利用V. natriegens成为体内外重组蛋白生产的工具。然而，许多支持细菌进行快速生长和蛋白质生产的生理活动大部分仍未确定。外膜囊泡（Outer Membrane Vesicle，OMV）是由革兰氏阴性细菌普遍产生的一种球形小泡，其不仅具有重要的功能，而且还可以作为一种应用于疫苗治疗的高效运载工具。[目的] 表征指数生长期OMV的蛋白质组并利用OMV进行异源蛋白的递送。[方法] 使用透射电镜、动态光散射和质谱学的方法，观察OMV的形态及粒径分布并鉴定蛋白组成。以超折叠绿色荧光蛋白（Superfolded Green Fluorescent Protein，sfGFP）作为货物蛋白来确定OMV蛋白载体。[结果] 从细菌培养的指数期中期和末期分别提取的OMV中鉴定到了288个和317个蛋白。这些蛋白分属不同的功能组，包括ABC转运蛋白、鞭毛、双组分系统。相比之下，同时鉴定了全细胞样品，其在指数期中期和末期分别含有1 480个和1 565个蛋白。我们筛选OMV的蛋白作为候选载体发现了一种属于OmpA家族的蛋白（命名为OmpA24），其能够将sfGFP以融合货物蛋白的形式运载到OMV中。[结论] 首次证实V. natriegens能够在指数生长期产生OMV，并展示了第一个不同生长时期OMV和全细胞的蛋白质组鉴定结果。OmpA24是将外源融合货物蛋白呈递到OMV中的有前景的载体。本研究有助于促进V. natriegens在蛋白表达和OMV介导的分泌中的应用。

Abstract:

[Background] The fast-growing Gram-negative bacterium Vibrio natriegens is a burgeoning tool in biotechnology. Previous research has mainly focused on developing tools for in vitro and in vivo recombinant protein production using V. natriegens. However, many physiological activities that support fast growth and protein production remain largely uncharacterized. Ubiquitously produced by bacteria, outer membrane vesicles (OMVs) not only carry out important functions but also can serve as a useful delivery tool for vaccine and therapeutics development. [Objective] Characterize the proteomes of OMVs during exponential phase growth and to employ OMVs for heterologous protein delivery. [Methods] Using transmission electron microscopy, dynamic light scattering, and mass spectrometry, we characterized the morphology and size distribution of extracted OMVs and their protein composition. We used the superfolded green fluorescent protein (sfGFP) as cargo to determine OMVs protein carriers. [Results] OMVs of mid- and late-exponential phases cultures contain 288 and 317 proteins, respectively. These proteins belong to multiple functional groups including ABC transporters, flagella and two-component systems. By contrast, we identified 1 480 and 1 565 proteins in whole cell samples under these two conditions, respectively. We screened OMV proteins for candidate carriers and found an OmpA-family protein that we name OmpA24 could enrich the sfGFP as a protein-fusion cargo in OMVs. [Conclusion] We demonstrate for the first time that V. natriegens can produce OMVs throughout exponential growth and present the first proteomic snapshot of OMVs and related whole cell samples under different growth phases. OmpA24 protein is a promising carrier for delivery of heterologous protein-fusion cargo into OMVs. This study will facilitate the application of V. natriegens in protein expression and OMV-mediated secretion.