Bacterial Targets of T3SS Effector Proteases

项目摘要。 许多革兰氏阴性细菌病原体通过使用分泌系统注射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。其中一些注射的蛋白质“效应物”是酶， 通过催化添加不寻常的后- 翻译修饰III型分泌系统（T3 SS）效应子在毒力和它们的毒性中起重要作用。 免疫机制的研究为先天免疫系统的功能和组成部分提供了深刻的见解。 T3 SS效应物被认为是无活性的，直到它们被注射到宿主细胞中，在那里它们然后折叠成它们的细胞骨架。 活性构象然而，最近的工作与NleB和SseK糖基转移酶从E。大肠杆菌， 啮齿类柠檬酸杆菌和肠道沙门氏菌挑战了这一教条。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， 改良C。啮齿动物在氧化应激条件下的生存。SseK 1在肠道沙门氏菌中具有活性， 其中它糖基化并增强几种酶（GloA、GloB、GloC和YajL）的活性，所述酶是 对沙门氏菌抵抗甲基乙二醛应激的能力至关重要。本文提出的研究旨在扩大 以前的研究结果，并确定在何种程度上，其他T3 SS效应与定义的酶活性， 在细菌中活跃。为此，E。大肠杆菌T3 SS效应蛋白酶NleC、NleD和EspL将被 其特征在于它们的细菌内活性。这些蛋白酶的天然细菌底物将是 鉴定，并定量蛋白水解活性对病原体蛋白丰度的影响。 将开发重组系统以监测C中NleC、NleD和EspL的活性。啮齿动物。的 这些效应蛋白酶的内源性细菌底物将通过使用无偏的， 称为“底物的末端胺同位素标记（TAILS）”的最先进的蛋白质组学方法。拟议 研究代表了细菌内T3 SS效应蛋白活性的首次全面分析， 细胞，因此，很可能通过证明效应子 功能并不简单地限于它们在修饰宿主细胞蛋白中的公知活性。这些概念可以 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.