Bacterial Targets of T3SS Effector Proteases

T3SS 效应蛋白酶的细菌靶标

• 批准号: 10612861
• 负责人: Samir Elqaidi
• 金额: $ 7.25万
• 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/10612861
• 关键词: Amines; Amino Acids; Area; Arginine; Bacteria; Bacterial Physiology; Bacterial Proteins; Cells; Citrobacter rodentium; DNA Damage; Data; Data Set; Enzymes; Escherichia coli; Family; Foundations; Funding Mechanisms; Future; Glucosamine; Glutathione; Goals; Gram-Negative Bacteria; Infection; Inflammatory; Innate Immune System; Investigation; Isotope Labeling; Mammalian Cell; Mass Spectrum Analysis; Mitogen-Activated Protein Kinases; Molecular Conformation; Monitor; NF-kappa B; Names; Oxidative Stress; Paper; Pathway interactions; Peptide Hydrolases; Phenotype; Play; Post-Translational Protein Processing; Production; Proteins; Proteomics; Publishing; Pyruvaldehyde; Recombinants; Role; Salmonella; Salmonella enterica; Stress; Structure; System; Testing; Type III Secretion System Pathway; Virulence; Work; cytokine; enzyme activity; experimental study; glutathione synthase; glycation; glycosylation; glycosyltransferase; improved; insight; novel; p65; pathogen; pathogenic bacteria; response

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.

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部分的深入了解。 T3SS 效应器被认为是不活跃的，直到它们被注射到宿主细胞中，然后它们折叠成它们的 然而，最近对大肠杆菌 NleB 和 SseK 糖基转移酶的研究， 啮齿类柠檬酸杆菌和肠沙门氏菌挑战了 NleB 糖基化和激活的教条。 细菌谷胱甘肽合成酶（GshB），导致谷胱甘肽产量增加 提高啮齿类动物在氧化应激条件下的存活率 SseK1 在肠道沙门氏菌中具有活性， 它可以糖基化并增强多种酶（GloA、GloB、GloC 和 YajL）的活性，这些酶是 对于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展。 先前的发现并确定具有确定酶活性的其他 T3SS 效应子的程度 为此，大肠杆菌 T3SS 效应蛋白酶 NleC、NleD 和 EspL 将被激活。 这些蛋白酶的天然细菌底物将被表征为它们的细菌内活性。 鉴定并量化蛋白水解活性对病原体蛋白质丰度的影响。 将开发重组系统来监测啮齿类动物中 NleC、NleD 和 EspL 的活性。 这些效应蛋白酶的内源性细菌底物将通过使用公正的状态来识别 最先进的蛋白质组学方法称为“底物末端胺同位素标记（TAILS）”。 研究首次对细菌内 T3SS 效应蛋白的活性进行全面分析 细胞，因此，通过证明效应器可能对该领域产生持久的、变革性的影响 功能不仅仅限于其修饰宿主细胞蛋白质的众所周知的活动。 很容易扩展到其他病原体、其他酶活性和其他分泌系统。