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: 7576736
• 负责人: Stanimir Stefanov Ivanov
• 金额: $ 5.01万
• 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 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7576736
• 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; 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; Type IV Secretion System Pathway; Ubiquitination; Vacuole; Vesicle; Virulence; Water; Work; aerosolized; antimicrobial drug; bactericide; cell type; 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）：为了违反人类免疫防御，细胞内细菌可以进入，隐藏和复制在宿主免疫细胞中，并颠覆其免疫功能。这项工作将表征负责这种现象的分子机制，这可能导致新型抗微生物药物靶标的发展。细胞内人病原体肺炎肺癌是严重的细菌性肺炎的病因，称为军团症氏病。吸入雾化细菌会导致疾病爆发，这对于抑制免疫功能的人可能致命。 Legionella利用注入宿主细胞中注入的蛋白质的武器库来控制各种宿主过程并建立细胞内复制生态位。这项研究的目的是阐明肺炎军团蛋白质家族的功能，这些功能被预测通过颠覆宿主的泛素化调节网络来改变宿主细胞反应。这些效应蛋白包含真核F-box结构域，该结构域靶向蛋白质用于泛素化。初步数据表明，在感染过程中，这些F-box效应子被转移到宿主细胞的细胞质中，并与泛素化蛋白相关。最初，这项研究的目的是绘制使用免疫荧光和免疫沉淀分析的哺乳动物细胞中控制F-box效应子与泛素化蛋白相互作用的分子决定因素和SCF E3泛素连接酶复合物。体外泛素化测定法将用于确定这些细菌F-box效应子使用宿主泛素连接酶募集和泛素化靶蛋白的能力。串联标签亲和力纯化和蛋白质组学分析对F-盒效应器进行修饰的泛素化蛋白质的蛋白质组学分析将识别出感染过程中军团菌针对的潜在宿主调节元件。这项研究将使用临床分离株LP1来创建缺乏F-box效应子的缺失突变体，并研究这些蛋白质对军团菌在宿主细胞中感染和复制的能力的贡献。这些突变菌株通过蛋白质组学筛查确定的泛素化靶蛋白的能力将通过感染的细胞模型来评估。这项工作将阐明分子机制，使人类病原体肺炎肺癌通过控制宿主的泛素化机制来颠覆宿主的细胞过程，从而有可能识别其他能够施加相似控制的病原体所利用的颠覆性特征，可以由药物控制或预防感染。