Division of Molecular Microbiology and.
Mol Cell Proteomics. 2013 Oct;12(10):2735-49. doi: 10.1074/mcp.M113.030502. Epub 2013 Jul 9.
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.
最近，人们已经清楚地认识到，VI 型分泌系统（T6SS）是一种复杂的大分子机器，许多细菌物种都利用它将效应蛋白注入真核或细菌细胞，这对毒力和细菌间竞争具有重要意义。“抗菌”T6SS，如机会性病原体粘质沙雷氏菌（Serratia marcescens）产生的 T6SS，赋予了分泌细菌快速而有效地杀死竞争细菌的能力。鉴定 T6SS 的分泌底物对于理解其杀死其他细胞的作用和能力至关重要，但到目前为止，只有有限数量的效应物被报道。在这里，我们报告了使用无标记定量质谱法成功鉴定了至少 11 种粘质沙雷氏菌 T6SS 的底物，其中包括 4 种新型效应蛋白，它们与迄今为止报道的其他 T6SS 分泌蛋白不同。这些新的效应物被证实为抗菌毒素，并且鉴定了能够中和其同源毒素的自我保护免疫蛋白。这项全面的分泌组学研究还意外地揭示了粘质沙雷氏菌 T6SS 的蛋白质磷酸化基于的翻译后调控与模式菌铜绿假单胞菌的 H1-T6SS 不同。结合磷酸蛋白质组学和遗传分析表明，T6SS 结构成分 Fha 的保守 PpkA 依赖性苏氨酸磷酸化对于粘质沙雷氏菌 T6SS 的激活是必需的，而磷酸酶 PppA 可以逆转这种修饰。然而，PpkA 的激活信号和机制与以前观察到的不同，似乎不需要细胞间接触。因此，这项研究不仅证明了 T6SS 分泌了新的和物种特异性的抗菌效应物组合，而且还首次表明，T6SS 的 PpkA 依赖性翻译后调控是根据不同细菌物种的需求定制的。
