T3SS Effector Regulation of Bacterial Metabolism

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缀来改变哺乳动物蛋白质的结构并抑制其功能 翻译修饰。 III 型分泌系统 (T3SS) 效应子在毒力及其毒力中发挥重要作用 机制提供了对先天免疫系统的功能和组成部分的深入了解。 T3SS 效应子被认为是不活跃的，直到它们被注射到宿主细胞中，然后它们折叠成活性的 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 啮齿类动物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并改善啮齿类 C. 啮齿类动物 在氧化应激条件下生存。 SseK1 在肠沙门氏菌中具有活性，它可以糖基化并 增强多种酶的活性，这些酶对于沙门氏菌抵抗甲基乙二醛的能力至关重要 压力。支持识别和理解细菌蛋白质功能意义的长期目标 NleB 和 SseK1 的糖基化，SseK1 的两个新细菌靶点，即 NagC 和 CRP 已被 发现了。 NagC 是一种双重激活剂-阻遏剂，控制 GlcNAc 的摄取和代谢。 NagC也 调节肠出血性大肠杆菌 (EHEC) 中肠细胞消失位点 (LEE) 基因的表达。李 是一个重要的致病岛，编码 T3SS 和许多效应蛋白。分解代谢阻遏物 蛋白质[(CRP);也称为分解代谢物激活蛋白（CAP）]，是介导 约 150 个基因的表达，包括那些对 GlcNAc 代谢重要的基因和一些 T3SS 成分 沙门氏菌。要测试的中心假设是 SseK1 对 NagC 和 CRP 的 Arg 糖基化影响 沙门氏菌毒力基因调控和代谢。具体目标是： 1）量化 NagC 和 CRP 的精氨酸糖基化影响这些转录因子结合 DNA 的能力； 2）表征 NagC 和 CRP 的精氨酸糖基化对沙门氏菌转录组的影响。拟议的工作高度 研究前提和假设新颖，适合R21资助机制 待测试。这些数据将为未来研究机械方面和 T3SS效应子调节细菌转录因子的功能意义。

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.