Proteomic identification of novel secreted antibacterial toxins of the Serratia marcescens type VI secretion system.
Proteomic identification of novel secreted antibacterial toxins of the Serratia marcescens type VI secretion system.
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DOI：
10.1074/mcp.m113.030502
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发表时间：
2013-10
期刊：
影响因子：
--
通讯作者：
Coulthurst SJ
中科院分区：
文献类型：
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作者：
Fritsch MJ;Trunk K;Diniz JA;Guo M;Trost M;Coulthurst SJ
最近变得明显的是，VI型分泌系统（T6 SS）是许多细菌物种用于将效应蛋白注入真核或细菌细胞的复杂大分子机器，其对毒力和细菌间竞争具有重要意义。“抗菌”T6 SS，例如由机会性人类病原体粘质沙雷氏菌（Serratia marcescens）产生的T6 SS，赋予分泌细菌快速有效地杀死竞争细菌的能力。T6 SS的分泌底物的鉴定对于理解其作用和杀死其他细胞的能力至关重要，但迄今为止仅报道了有限数量的效应物。在这里，我们报告成功地使用无标记定量质谱法，以确定至少十一个基板的S。marcescens T6 SS，包括四个新的效应蛋白，这是不同于其他的T6 SS分泌蛋白的报告日期。这些新的效应物被确认为抗菌毒素，并且鉴定了能够中和其同源毒素的自我保护免疫蛋白。全球分泌组学研究还意外地揭示了S. marcescens T6 SS不同于铜绿假单胞菌的H1-T6 SS。结合磷酸化蛋白质组学和遗传分析表明，保守的PPKA依赖的T6 SS结构成分Fha的苏氨酸磷酸化是必需的T6 SS在S。而磷酸酶PppA可以逆转这种修饰。然而，PpkA激活的信号和机制与先前观察到的不同，并且似乎不需要细胞-细胞接触。因此，这项研究不仅证明了新的和物种特异性的抗菌效应物组合是由T6 SS分泌的，而且还首次表明T6 SS的PpkA依赖性翻译后调节是定制的，以适应不同细菌物种的需要。
It has recently become apparent that the Type VI secretion system (T6SS) is a complex macromolecular machine used by many bacterial species to inject effector proteins into eukaryotic or bacterial cells, with significant implications for virulence and interbacterial competition. “Antibacterial” T6SSs, such as the one elaborated by the opportunistic human pathogen, Serratia marcescens, confer on the secreting bacterium the ability to rapidly and efficiently kill rival bacteria. Identification of secreted substrates of the T6SS is critical to understanding its role and ability to kill other cells, but only a limited number of effectors have been reported so far. Here we report the successful use of label-free quantitative mass spectrometry to identify at least eleven substrates of the S. marcescens T6SS, including four novel effector proteins which are distinct from other T6SS-secreted proteins reported to date. These new effectors were confirmed as antibacterial toxins and self-protecting immunity proteins able to neutralize their cognate toxins were identified. The global secretomic study also unexpectedly revealed that protein phosphorylation-based post-translational regulation of the S. marcescens T6SS differs from that of the paradigm, H1-T6SS of Pseudomonas aeruginosa. Combined phosphoproteomic and genetic analyses demonstrated that conserved PpkA-dependent threonine phosphorylation of the T6SS structural component Fha is required for T6SS activation in S. marcescens and that the phosphatase PppA can reverse this modification. However, the signal and mechanism of PpkA activation is distinct from that observed previously and does not appear to require cell–cell contact. Hence this study has not only demonstrated that new and species-specific portfolios of antibacterial effectors are secreted by the T6SS, but also shown for the first time that PpkA-dependent post-translational regulation of the T6SS is tailored to fit the needs of different bacterial species.