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

描述(申请人提供)：为了突破人类的免疫防御，细胞内细菌可以进入、隐藏并在宿主免疫细胞内复制，并颠覆其免疫功能。这项工作将表征导致这一现象的分子机制，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被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，对免疫功能受抑制的人来说，这可能是致命的。军团菌利用注入宿主细胞的大量蛋白质来控制各种宿主过程，并建立细胞内复制的生态位。本研究的目的是阐明嗜肺军团菌蛋白家族的功能(S)，这些蛋白被预测通过颠覆宿主泛素化调节网络来改变宿主细胞的反应。这些效应蛋白含有真核细胞的F-box结构域，该结构域针对泛素化蛋白。初步数据显示，这些F-box效应物在感染过程中被转移到宿主细胞的胞浆中，并与泛素化蛋白相结合。最初，这项研究的目标是利用免疫荧光和免疫沉淀分析来定位控制F-box效应器与哺乳动物细胞中泛素化蛋白和SCF E3泛素连接酶复合体相互作用的分子决定因素。体外泛素化实验将被用来确定这些细菌F-box效应器利用宿主泛素连接酶招募和泛素化目标蛋白的能力。串联标签亲和纯化和泛素化蛋白的蛋白质组学分析将识别军团菌在感染过程中靶向的潜在宿主调控元件。本研究将以临床分离株LP1为材料，建立缺失F-box效应子的缺失突变体，并研究这些蛋白对军团菌感染和复制宿主细胞能力的影响。这些突变菌株泛素化蛋白质组筛选确定的目标蛋白的能力将利用感染的细胞模型进行评估。这项工作将阐明人类病原体嗜肺军团菌通过控制宿主泛素化机制来颠覆宿主细胞过程的分子机制，潜在地识别其他能够施加类似控制的病原体利用的颠覆的共同特征，这些特征可以被药物靶向控制或预防感染。

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.