Analysis of F-box domain containing effector proteins from Legionella pneumophila

含 F-box 结构域的嗜肺军团菌效应蛋白分析

• 批准号: 7333882
• 负责人: Stanimir Stefanov Ivanov
• 金额: $ 4.68万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-29 至 2010-02-27
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7333882
• 关键词: Address; Affinity Chromatography; Air; Amino Acid Motifs; Amoeba genus; Animal Disease Models; Bacteria; Bacterial Pneumonia; Biological Assay; Biological Models; Breathing; Cell model; Cell physiology; Cells; Clinical; Communities; Complex; Cytosol; Data; Development; Disease Outbreaks; Drug Delivery Systems; Endoplasmic Reticulum; Endosomes; Environment; Enzymes; F Box Domain; Family; Fresh Water; Genes; Genome; Goals; Gram-Negative Bacteria; Human; Immune; Immunofluorescence Immunologic; Immunoprecipitation; In Vitro; Incidence; Individual; Infection; Infection prevention; Laboratories; Legionella; Legionella pneumophila; Legionellosis; Legionnaires' Disease; Life; Lysosomes; Mammalian Cell; Maps; Microbial Biofilms; Modification; Molecular; Nature; Numbers; Paris, France; Pathogenicity; Pathway interactions; Phagocytes; Phagocytosis; Pharmacologic Substance; Philadelphia; Play; Pneumonia; Process; Proliferating; Proteins; Proteomics; Protozoa; Recruitment Activity; Regulatory Element; Role; Screening procedure; Site-Directed Mutagenesis; Sterilization for infection control; Swimming; Symptoms; System; Thinking; Type IV Secretion System Pathway; Ubiquitination; Vacuole; Vesicle; Virulence; Water; Work; aerosolized; antimicrobial drug; bactericide; cell type; day; immune function; lens; macrophage; multicatalytic endopeptidase complex; mutant; novel; pathogen; protein function; research study; response; ubiquitin ligase; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): To breach the human immune defenses, intracellular bacteria can enter, hide and replicate within the host immune cells as well as subvert their immunological functions. This work will characterize the molecular mechanisms that are responsible for this phenomenon, potentially leading to development of novel anti-microbial drug targets. The intracellular human pathogen Legionella pneumophila is the causative agent of the severe bacterial pneumonia known as Legionnaire's disease. Inhalation of aerosolized bacteria results in outbreaks of disease, which can be fatal in people with suppressed immune function. Legionella utilizes an arsenal of proteins injected in the host cell to assume control over various host processes and establish an intracellular replicative niche. The goal of this study is to elucidate the function(s) of a family of Legionella pneumophila proteins that are predicted to alter host cellular responses via subversion of the host ubiquitination regulatory networks. These effector proteins contain the eukaryotic F-box domain, which targets proteins for ubiquitination. Preliminary data showed that these F-box effectors are translocated into the cytosol of host cells during infection and associate with ubiquitinated proteins. Initially the goal of this study is to map the molecular determinant governing the interaction of the F-box effectors with ubiquitinated proteins and the SCF E3 ubiquitin ligase complex in mammalian cells utilizing immunofluorescence and immunoprecipitation analyses. In vitro ubiquitination assay will be used to determine the capacity of these bacterial F-box effectors to recruit and ubiquitinate target proteins utilizing host ubiquitin ligases. Tandem tag affinity purification and proteomics analysis of ubiquitinated proteins targeted for modification by the F- box effectors would identify potential host regulatory elements that are targeted by Legionella during infection. This study will use the clinical isolate strain Lp1 to create deletion mutant lacking the F-box effectors and investigate the contribution of these proteins to the capacity of Legionella to infect and replicate in host cells. The ability of these mutant strains to ubiquitinate target proteins identified by the proteomics screen will be assessed utilizing a cellular model of infection. This work will elucidate the molecular mechanism enabling the human pathogen Legionella pneumophila to subvert host cellular processes by taking control of the host ubiquitination machinery, potentially identifying common features of subversion utilized by other pathogens capable of exerting similar control, which can be targeted by pharmaceuticals to control or prevent infection.

描述（由申请人提供）：为了破坏人体免疫防御，细胞内细菌可以进入宿主免疫细胞内，隐藏和复制，并破坏其免疫功能。这项工作将表征负责这种现象的分子机制，可能导致新的抗微生物药物靶点的开发。细胞内的人类病原体嗜肺军团菌是称为军团病的严重细菌性肺炎的病原体。吸入雾化细菌会导致疾病爆发，这对免疫功能受抑制的人来说可能是致命的。军团菌利用注入宿主细胞的蛋白质库来控制各种宿主过程并建立细胞内复制生态位。本研究的目的是阐明嗜肺军团菌蛋白家族的功能，这些蛋白被预测通过颠覆宿主泛素化调控网络来改变宿主细胞反应。这些效应蛋白含有真核生物的F-box结构域，其靶向蛋白进行泛素化。初步数据显示，这些F-box效应物在感染过程中易位到宿主细胞的胞质溶胶中，并与泛素化蛋白质结合。最初，本研究的目标是利用免疫荧光和免疫沉淀分析，映射的分子决定因素，在哺乳动物细胞中的F-box效应与泛素化蛋白和SCF E3泛素连接酶复合物的相互作用。体外泛素化测定将用于确定这些细菌F盒效应物利用宿主泛素连接酶募集和泛素化靶蛋白的能力。通过串联标签亲和纯化和蛋白质组学分析，靶向被F盒效应物修饰的泛素化蛋白质将鉴定在感染期间被军团菌靶向的潜在宿主调节元件。本研究将使用临床分离菌株Lp 1创建缺失F-box效应子的缺失突变体，并研究这些蛋白质对军团菌感染宿主细胞和在宿主细胞中复制的能力的贡献。将利用感染的细胞模型来评估这些突变株使通过蛋白质组学筛选鉴定的靶蛋白泛素化的能力。这项工作将阐明的分子机制，使人类病原体嗜肺军团菌通过控制宿主泛素化机制，颠覆宿主细胞过程，潜在地识别颠覆的共同特征，利用其他病原体能够发挥类似的控制，这可以有针对性的药物，以控制或预防感染。