Manipulation of host immunity by a bacterial E3 ubiquitin ligase

通过细菌 E3 泛素连接酶操纵宿主免疫

• 批准号: 8214698
• 负责人: Gregory B Martin
• 金额: $ 23.05万
• 依托单位: BOYCE THOMPSON INST FOR PLANT RESEARCH
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8214698
• 关键词: Affect; Agriculture; Amino Acid Sequence; Animals; BAK1 gene; Bacterial Infections; Bacterial Proteins; Binding; Biological Models; Boxing; C-terminal; Cells; Collaborations; Complex; Development; Disease; Disease susceptibility; Enzymes; Escherichia coli; Family; Flagellin; Goals; Health; Host Defense; Human; Immune response; Immunity; In Vitro; Individual; Infection; Institutes; Knowledge; Learning; Legionella pneumophila; Mediating; Methods; Modification; Monitor; N-terminal; Natural Immunity; Pathogenesis; Phosphorylation; Phosphotransferases; Plants; Play; Positioning Attribute; Post-Translational Protein Processing; Process; Protein Kinase; Proteins; Protocols documentation; Pseudomonas; Pseudomonas syringae; Reporting; Research; Resources; Role; Salmonella; Serine; Shigella; Structure; Surface; System; Testing; Time; Tomatoes; Type III Secretion System Pathway; Ubiquitin; Ubiquitination; United States National Institutes of Health; Universities; Virulence; base; fluorophore; in vivo; member; mimicry; novel; novel strategies; pathogen; public health relevance; receptor; ubiquitin ligase; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): Bacterial pathogens of both plants and humans use a type III secretion system to inject effector proteins into the cells of their hosts. As many as 30 or more different 'type III effectors' can be delivered by an individual pathogen and once inside the host cell they manipulate various processes to promote bacterial virulence. The great diversity of type III effectors, and the fact that their amino acid sequences offer few clues to their function, has been an impediment to understanding how they manipulate the host. One host process, the attachment of ubiquitin to a protein in order to regulate its function or to cause its degradation, plays an important role in host immunity and offers a vulnerable target for type III effectors. This application concerns the characterization of a type III effector, AvrPtoB, from the plant pathogen Pseudomonas syringae. The crystal structure of a C-terminal domain of AvrPtoB revealed that it is a structural mimic of the U-box class of E3 ubiquitin ligases - a type of host protein that facilitates the attachment of ubiquitin to other proteins. The AvrPtoB E3 ligase is able to use host E2 conjugating enzymes to cause ubiquitination and degradation of a host protein kinase required for immunity. Despite these advances, much remains to be learned about how this bacterial E3 ligase binds and ubiquitinates its host substrates in order to subvert their normal functions. Recently virulence proteins from human pathogens such as Legionella pneumophila and Escherichia coli have also been found to express proteins of the U-box class of E3 ligases. Structural mimicry of this type of host protein therefore appears to be a common strategy for undermining host immunity. The long term objective of this project is to increase our understanding of the underlying mechanisms that bacterial E3 ligases use to manipulate host ubiquitination. This knowledge will be useful for the development of novel strategies for interfering with these virulence proteins in order to lessen the impact of pathogen infection in both plants and humans. The goals of this application are to: 1) Identify the host E2 conjugating enzyme used by AvrPtoB and characterize the role of a ubiquitin-interaction motif that lies close to the U-box; 2) Exploit the recently solved crystal structure of AvrPtoB in complex with one of its host targets to understand how AvrPtoB recognizes and binds its substrates; 3) Perform a screen to identify additional host proteins that AvrPtoB ubiquitinates and which are candidates for playing a role in host immunity; 4) Investigate the way in which certain host-mediated modifications of AvrPtoB (e.g. phosphorylation) influence its E3 ligase activity; and 5) Develop a method to visually monitor the delivery of AvrPtoB into the host cell. The method will be used to test the hypothesis that type III effectors that act early in the infection process are delivered before later-acting effectors. PUBLIC HEALTH RELEVANCE: Manipulation of host immunity by a bacterial E3 ubiquitin ligase Gregory B. Martin, Boyce Thompson Institute for Plant Research at Cornell University Project Narrative This research will elucidate how a bacterial virulence protein, which is injected into host cells, interferes with the host immune response. The knowledge gained will contribute to strategies to lessen the impact of bacterial diseases of importance to both agriculture and human health.

描述（由申请人提供）：植物和人类的细菌病原体都使用 III 型分泌系统将效应蛋白注射到宿主细胞中。单个病原体可以传递多达 30 种或更多不同的“III 型效应子”，一旦进入宿主细胞，它们就会操纵各种过程来增强细菌毒力。 III 型效应子的巨大多样性，以及它们的氨基酸序列几乎无法提供其功能的线索，一直是理解它们如何操纵宿主的障碍。一种宿主过程，即泛素与蛋白质的附着，以调节其功能或导致其降解，在宿主免疫中发挥着重要作用，并为 III 型效应子提供了一个脆弱的目标。本申请涉及来自植物病原体丁香假单胞菌的 III 型效应子 AvrPtoB 的表征。 AvrPtoB C 端结构域的晶体结构表明，它是 E3 泛素连接酶 U 盒类的结构模拟物，E3 泛素连接酶是一种促进泛素与其他蛋白质附着的宿主蛋白。 AvrPtoB E3 连接酶能够使用宿主 E2 缀合酶来引起免疫所需的宿主蛋白激酶的泛素化和降解。尽管取得了这些进展，但关于这种细菌 E3 连接酶如何结合并泛素化其宿主底物以破坏其正常功能，仍有许多待了解。最近还发现来自人类病原体（例如嗜肺军团菌和大肠杆菌）的毒力蛋白表达 E3 连接酶的 U-box 类蛋白。因此，这种类型宿主蛋白的结构模拟似乎是破坏宿主免疫的常见策略。该项目的长期目标是加深我们对细菌 E3 连接酶用于操纵宿主泛素化的基本机制的理解。这些知识将有助于开发干扰这些毒力蛋白的新策略，以减轻病原体感染对植物和人类的影响。本应用的目标是： 1) 鉴定 AvrPtoB 使用的宿主 E2 缀合酶，并表征靠近 U 盒的泛素相互作用基序的作用； 2) 利用最近解析的 AvrPtoB 与其宿主靶标之一复合物的晶体结构来了解 AvrPtoB 如何识别和结合其底物； 3) 进行筛选，以确定 AvrPtoB 泛素化的其他宿主蛋白，以及哪些蛋白是在宿主免疫中发挥作用的候选蛋白； 4) 研究 AvrPtoB 的某些宿主介导的修饰（例如磷酸化）如何影响其 E3 连接酶活性； 5) 开发一种视觉监测 AvrPtoB 递送至宿主细胞的方法。该方法将用于检验以下假设：在感染过程早期起作用的 III 型效应器先于后期起作用的效应器传递。 公共健康相关性：通过细菌 E3 泛素连接酶操纵宿主免疫康奈尔大学博伊斯·汤普森植物研究所的格雷戈里·B·马丁 (Gregory B. Martin) 项目叙述这项研究将阐明注射到宿主细胞中的细菌毒力蛋白如何干扰宿主免疫系统。宿主免疫反应。所获得的知识将有助于制定减轻细菌性疾病对农业和人类健康影响的战略。