Bacterial Targets of T3SS Effector Proteases

项目摘要。 许多革兰氏阴性细菌通过分泌系统与哺乳动物细胞相互作用 毒力蛋白直接进入受感染的宿主细胞。这些被注入的蛋白质“效应器”中的一些是酶 通过催化不寻常的后蛋白的添加来改变哺乳动物蛋白质的结构和抑制其功能 翻译修饰语。III型分泌系统(T3SS)效应器在毒力和它们的 机制提供了对先天免疫系统的功能和组成部分的极大洞察力。 T3SS效应器被认为是不活跃的，直到它们被注射到宿主细胞中，然后它们在宿主细胞中折叠成 活跃的构象。然而，最近对来自大肠杆菌的NleB和SseK糖基转移酶的研究， 罗氏柠檬酸杆菌和肠道沙门氏菌对这一教条提出了挑战。NleB糖基化并激活 细菌谷胱甘肽合成酶(GshB)，导致谷胱甘肽产量增加和 改善了在氧化应激条件下轮齿藻的存活率。SseK1在肠沙门氏菌中很活跃， 在那里它糖基化并增强几种酶(GloA、Glob、Gloc和YajL)的活性 对沙门氏菌抵抗甲基乙二醛压力的能力至关重要。这里提出的研究试图扩展到 并确定其他具有确定的酶活性的T3SS效应器在多大程度上 在细菌内活跃的。要做到这一点，大肠杆菌T3SS效应蛋白NleC、NleD和Esp1将被 以其细菌内活性为特征的。这些蛋白酶的天然细菌底物将是 并将对蛋白分解活性对病原体蛋白丰度的影响进行量化。 将开发重组系统来监测轮齿芽孢杆菌中NleC、NleD和Espl的活性。这个 这些效应性蛋白酶的内源性细菌底物将通过使用无偏见的状态- 最先进的蛋白质组学方法被命名为底物(尾)的末端胺同位素标记。建议数 研究首次对细菌内T3SS效应蛋白的活性进行了全面分析 细胞，因此，很可能通过展示该效应器而对该领域产生持久的、变革性的影响 功能不仅仅局限于它们在修饰宿主细胞蛋白质方面众所周知的活动。这样的概念可以 很容易扩展到其他病原体、其他酶活性和其他分泌系统。

Project Summary. Many Gram-negative bacterial pathogens interact with mammalian cells by using secretion systems to inject virulence proteins directly into infected host cells. Some of these injected protein ‘effectors’ are enzymes that modify the structure and inhibit the function of mammalian proteins by catalyzing the addition of unusual post- translational modifications. Type III secretion system (T3SS) effectors play essential roles in virulence and their mechanisms have provided great insight into the functions and components of the innate immune system. T3SS effectors are believed to be inactive until they are injected into host cells, where they then fold into their active conformations. However, recent work with the NleB and SseK glycosyltransferases from E. coli, Citrobacter rodentium, and Salmonella enterica has challenged that dogma. NleB glycosylates and activates the bacterial glutathione synthetase (GshB) enzyme, resulting in enhanced glutathione production and improved C. rodentium survival in oxidative stress conditions. SseK1 is active within Salmonella enterica, where it glycosylates and enhances the activity of several enzymes (GloA, GloB, GloC, and YajL) that are critical to the ability of Salmonella to resist methylglyoxal stress. The studies proposed here seek to extend previous findings and determine the extent to which other T3SS effectors with defined enzymatic activities are active within the bacterium. To do this, the E. coli T3SS effector proteases NleC, NleD, and EspL will be characterized for their intra-bacterial activities. The natural bacterial substrates of these proteases will be identified and the impact of proteolytic activities on pathogen protein abundance will be quantified. Recombinant systems will be developed to monitor the activity of NleC, NleD, and EspL in C. rodentium. The endogenous bacterial substrates of these effector proteases will be identified by using an unbiased, state-of- the-art proteomic approach named ‘terminal amine isotopic labeling of substrates (TAILS)’. The proposed studies represent the first comprehensive analysis of the activities of T3SS effector proteins within the bacterial cell, and as such, are likely to have a lasting, transformative impact on the field by demonstrating that effector functions are not simply limited to their well-known activities in modifying host cell proteins. Such concepts can readily be extended to other pathogens, other enzyme activities, and other secretion systems.