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 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。T3SS 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，柠檬酸杆菌 而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），导致谷胱甘肽产生增强和C. rodentium 在氧化应激条件下存活。SseK 1在肠道沙门氏菌中具有活性，在那里它糖基化， 增强对沙门氏菌抵抗甲基乙二醛的能力至关重要的几种酶的活性 应力支持识别和理解细菌蛋白质的功能意义的长期目标 通过NleB和SseK 1的糖基化，SseK 1的两个新的细菌靶标，即NagC和CRP已经被发现。 发现了NagC是控制GlcNAc摄取和代谢的双重激活-阻遏物。NagC也 调节肠出血性大肠杆菌中肠上皮细胞消失位点（LEE）基因表达。大肠杆菌（EHEC）。李 是一个重要的致病岛，编码T3 SS和许多效应蛋白。分解代谢物阻遏物 蛋白[（CRP）;也被称为分解代谢物激活蛋白（CAP）]，是介导代谢产物的全局调节剂。 约150个基因的表达，包括对GlcNAc代谢和几个T3 SS组分重要的基因， 沙门氏菌。待检验的中心假设是SseK 1对NagC和CRP的Arg-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 NagC和CRP的Arg-糖基化影响这些转录因子结合DNA的能力; NagC和CRP的Arg-糖基化对沙门氏菌转录组的影响。建议的工作高度 由于研究前提的创新性和假设的新颖性， 接受测试这些数据将为今后的机制方面的调查建立框架， T3 SS效应子调节细菌转录因子的功能意义。