T3SS Effector Regulation of Bacterial Metabolism

T3SS 细菌代谢的效应器调节

• 批准号: 10612897
• 负责人: Derek Mosier
• 金额: $ 22.43万
• 依托单位: KANSAS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-22 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10612897
• 关键词: Affect; Area; Arginine; Bacterial Physiology; Bacterial Proteins; Behavior; Biochemical; Biological Assay; Cells; Citrobacter rodentium; Cyclic AMP Receptor Protein; DNA; DNA Binding; DNA Damage; Data; Electrophoretic Mobility Shift Assay; Enterocytes; Enzymes; Escherichia coli; Escherichia coli EHEC; Family; Funding Mechanisms; Future; Gene Expression; Gene Expression Regulation; Genes; Glucosamine; Glutathione; Goals; Gram-Negative Bacteria; Inflammatory; Innate Immune System; Investigation; Mammalian Cell; Manuscripts; Mass Spectrum Analysis; Measurement; Mediating; Metabolism; Molecular Conformation; NF-kappa B; Names; Orthologous Gene; Oxidative Stress; Pathogenicity Island; Pathway interactions; Play; Post-Translational Protein Processing; Production; Protein Glycosylation; Proteins; Publishing; Pyruvaldehyde; Regulation; Regulon; Reporter; Repressor Proteins; Research; Role; Salmonella; Salmonella enterica; Stress; Structure; Surface Plasmon Resonance; System; Testing; Type III Secretion System Pathway; Virulence; Work; bacterial metabolism; complex biological systems; experimental study; glutathione synthase; glycation; glycosylation; glycosyltransferase; improved; innovation; insight; novel; pathogenic bacteria; transcription factor; transcriptome; transcriptome sequencing; uptake; virulence gene

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.

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 将毒力蛋白注入直接感染的宿主细胞中，其中一些注射蛋白“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 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的深入了解。 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 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 NleB 糖基化并激活细菌的观念。 谷胱甘肽合成酶 (GshB) 酶，导致谷胱甘肽产量增加并改善啮齿类 C. 啮齿类动物 SseK1 在肠道沙门氏菌中具有活性，可在氧化应激条件下进行糖基化并发挥作用。 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 支持识别和理解细菌蛋白质的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 NagC 是一种双重激活剂-阻遏剂，也控制 GlcNAc 的摄取和代谢。 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。 是一个重要的致病岛，编码 T3SS 和许多效应蛋白。 蛋白质[(CRP)；也称为分解代谢激活蛋白(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 影响 NagC 和 CRP 的 Arg 糖基化。 沙门氏菌毒力基因调控和代谢的具体目标是： 1) 量化沙门氏菌毒力基因调控和代谢的程度。 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 Arg 糖基化对沙门氏菌转录组的影响非常重要。 研究前提和假设新颖，适合R21资助机制 这些数据将为未来的机械方面的研究建立框架。 T3SS效应子调节细菌转录因子的功能意义。