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进行注射与哺乳动物细胞相互作用 病毒蛋白直接成被感染的宿主细胞。这些注入的蛋白质“效应子”中的一些是酶 修改结构并抑制哺乳动物蛋白质的功能，通过催化不寻常的后加入 翻译修改。 III型分泌系统（T3SS）生效器在病毒及其中起着重要的作用 机制为先天免疫系统的功能和组成部分提供了深入的了解。 据信T3SS效应是无效的，直到将它们注入宿主细胞为止，然后它们折叠成 主动构象。但是，最近与大肠杆菌的Nleb和Ssek糖基转移酶一起工作， 柠檬酸啮齿动物和沙门氏菌肠质挑战了这一教条。 NLEB糖基酸盐和激活 细菌谷胱甘肽合成酶（GSHB）酶，导致谷胱甘肽的产生和 在氧化应激条件下改善了啮齿动物的生存。 SSEK1活跃在肠沙门氏菌中， 它的糖基化并增强了几种酶（Gloa，Glob，Gloc和Yajl）的活性 沙门氏菌抵抗甲基甘氨酸应激的能力至关重要。这里提出的研究试图扩展 以前的发现并确定其他具有定义酶促活性的T3SS效应器的程度是 活跃于细菌中。为此，大肠杆菌T3SS效应蛋白酶NLEC，NLED和ESPL将是 以细菌性活动为特征。这些蛋白酶的天然细菌底物将是 将量化蛋白水解活性对病原体蛋白丰度的影响。 将开发重组系统以监测NLEC，NLED和ESPL在C. rodentium中的活性。这 这些效应蛋白酶的内源性细菌底物将通过使用无偏的， ART蛋白质组学方法称为“底物的终末胺同位素标记（Tails）”。提议 研究代表了细菌中T3SS效应蛋白活性的首次综合分析 细胞，因此很可能通过证明效应器来对现场产生持久的变革性影响 功能不仅限于其在修饰宿主细胞蛋白上的知名活动。这样的概念可以 很容易扩展到其他病原体，其他酶活性和其他分泌系统。