T3SS Effector Regulation of Bacterial Metabolism

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 效应器被认为是非活性的，直到它们被注入宿主细胞，然后它们在宿主细胞中折叠成它们的活性 构象。然而，最近对来自大肠杆菌、柠檬酸杆菌的NleB和SseK糖基转移酶的研究 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，并改善轮状芽孢杆菌 在氧化应激条件下存活。SseK1在肠沙门氏菌中是活跃的，它在那里糖基化和 增强几种酶的活性，这些酶对沙门氏菌抵抗乙二醛的能力至关重要 压力。支持确定和理解细菌蛋白的功能意义的长期目标 SseK1的两个新的细菌靶标NAGC和CRP被NleB和SseK1糖基化 被发现了。NAGC是一个双重的激活-抑制因子，控制GlcNAc的摄取和代谢。NAGC也 调节肠出血性大肠杆菌(EHEC)中肠上皮细胞消失(Lee)基因的表达。李氏家族 是一个重要的致病岛，编码T3SS和许多效应蛋白。分解代谢抑制因子 蛋白质[(CRP)；也被称为分解代谢激活蛋白(CAP)]，是一种全球调节因子，介导 ~150个基因的表达，包括与GlcNAc代谢有关的基因和几个T3SS组分 沙门氏菌。有待检验的中心假设是，SseK1对NAGC和CRP的精氨酸糖基化影响 沙门氏菌毒力基因调控与代谢。具体目标是：1)量化以下方面的程度 NAGC和CRP的精氨酸糖基化影响这些转录因子与DNA的结合能力；2)表征 NAGC和CRP精氨酸糖基化对沙门氏菌转录组的影响。拟议中的工作具有很高的 适用于R21资助机制，因研究前提创新和假设新颖 接受测试。这些数据将为今后对机械方面的调查和 细菌转录因子T3SS效应调控的功能意义

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 that are critical to the ability of Salmonella to resist methylglyoxal stress. In support of long-term goals to identify and understand the functional significance of bacterial protein glycosylation by NleB and SseK1, two new bacterial targets of SseK1, namely NagC and CRP have been discovered. NagC is a dual activator-repressor that controls GlcNAc uptake and metabolism. NagC also regulates locus of enterocyte effacement (LEE) gene expression in enterohemorrhagic E. coli (EHEC). The LEE is an important pathogenicity island that encodes the T3SS and many effector proteins. The catabolite repressor protein [(CRP); also referred to as the catabolite activator protein (CAP)], is a global regulator that mediates the expression of ~150 genes, including those important to GlcNAc metabolism and several T3SS components in Salmonella. The central hypothesis to be tested is that Arg-glycosylation of NagC and CRP by SseK1 affects Salmonella virulence gene regulation and metabolism. The specific aims are: 1) Quantify the extent to which Arg-glycosylation of NagC and CRP affects the ability of these transcription factors to bind DNA; 2) Characterize the impact of Arg-glycosylation of NagC and CRP on the Salmonella transcriptome. The proposed work is highly suitable for the R21 funding mechanism because of the innovation of the research premise and novel hypothesis to be tested. Such data will establish the framework for future investigation of the mechanistic aspects and functional significance of T3SS effector regulation of bacterial transcription factors.