Proteomic analysis of Vibrio cholerae outer membrane vesicles
Proteomic analysis of Vibrio cholerae outer membrane vesicles
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DOI：
10.1073/pnas.1403683111
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发表时间：
2014-04-15
影响因子：
11.1
通讯作者：
Mekalanos, John J.
中科院分区：
文献类型：
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作者：
Altindis, Emrah;Fu, Yang;Mekalanos, John J.
由革兰氏阴性菌产生的外膜囊泡(OMV)为在不破坏细胞的情况下确定细胞包膜蛋白提供了一种有趣的研究材料。OMVS也是很有前景的免疫原性平台，可能在细菌的生存和发病机制中发挥重要作用。我们使用溶液中胰酶消化与质谱仪相结合的方法，在激活ToxT毒力调节蛋白(ToxT)毒力调节子的条件下生长，鉴定了霍乱弧菌OMV中存在的90种蛋白质。使用ToxT和体内RNA-seq、Tn-seq和霍乱粪便蛋白质组和其他全基因组数据集评估ToxT表达谱和对毒力的潜在贡献。13个OMV相关蛋白似乎对细胞生长至关重要，因此可能是抗菌药物的靶点。另外12种非必需的OMV蛋白，包括DegP蛋白酶，是兔肠道定植所必需的。DegP突变体的比较蛋白质组学揭示了DegP在将9种蛋白质整合到OMV中的重要性，其中包括参与生物膜基质形成的蛋白质和II型分泌系统的各种底物。综上所述，这些结果表明，DegP在决定OMV含量方面发挥着重要作用，并影响肠道定植、II型分泌系统的正常功能和生物膜基质的形成等表型。
Outer membrane vesicles (OMVs) produced by Gram-negative bacteria provide an interesting research material for defining cell-envelope proteins without experimental cell disruption. OMVs are also promising immunogenic platforms and may play important roles in bacterial survival and pathogenesis. We used in-solution trypsin digestion coupled to mass spectrometry to identify 90 proteins present in OMVs of Vibrio cholerae when grown under conditions that activate the TCP pilus virulence regulatory protein (ToxT) virulence regulon. The ToxT expression profile and potential contribution to virulence of these proteins were assessed using ToxT and in vivo RNA-seq, Tn-seq, and cholera stool proteomic and other genome-wide data sets. Thirteen OMV-associated proteins appear to be essential for cell growth, and therefore may represent antibacterial drug targets. Another 12 nonessential OMV proteins, including DegP protease, were required for intestinal colonization in rabbits. Comparative proteomics of a degP mutant revealed the importance of DegP in the incorporation of nine proteins into OMVs, including ones involved in biofilm matrix formation and various substrates of the type II secretion system. Taken together, these results suggest that DegP plays an important role in determining the content of OMVs and also affects phenotypes such as intestinal colonization, proper function of the type II secretion system, and formation of biofilm matrix.