Alteration of host protein stability by Legionella

军团菌改变宿主蛋白稳定性

• 批准号: 8176583
• 负责人: MICHAEL N STARNBACH
• 金额: $ 25.43万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8176583
• 关键词: Animal Model; Antibiotics; Bacterial Infections; Bacterial Proteins; Cell physiology; Cells; Critical Pathways; Development; Dissection; Eukaryotic Cell; Family; Genetic; Goals; Growth; Individual; Infection; Lead; Legionella; Legionella pneumophila; Legionnaires' Disease; Link; Literature; Mediating; Methods; Monitor; Organism; Pathogenesis; Pathway interactions; Phenotype; Pneumonia; Prevalence; Proteins; Research; Screening procedure; System; Testing; Time; Type IV Secretion System Pathway; Virulence Factors; Work; impaired capacity; innovation; interest; loss of function; mutant; novel; pathogen; prevent; research study; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): Infection with the intracellular pathogen Legionella pneumophila can lead to a severe pneumonia known as Legionnaires' disease. Legionella pneumophila uses a specialized type IV secretion apparatus, also known as the Dot/Icm system, to secrete over 150 effector proteins directly into the host cell. The translocated bacterial effectors establish a vacuolar niche that supports replication of L. pneumophila in eukaryotic cells. While there is an extensive literature describing how several of these effectors alter host cell functions, the targets of most have remained elusive. A significant problem in linking a particular effector to a particular function is the redundant or overlapping activity of many effectors. This means that L. pneumophila mutant strains deficient in any one effector often have no appreciable phenotype, preventing the identification of their host targets. While it is well appreciated that many L. pneumophila effectors directly alter host proteins through functions such as E3 ubiquitin ligase activity, there have been few methods developed to monitor pathogen-induced changes in host protein stability on a large scale. Here we propose to apply a novel screening method called the "Global Protein Stability" (GPS) system to identify host cell proteins whose stability is altered by the secreted L. pneumophila effectors. Once we have identified host proteins that are stabilized or destabilized when a functional type IV secretion system is present, we will test whether reducing or increasing the prevalence of these proteins (attempting to reverse the effects of the Legionella effectors) impairs the capacity of L. pneumophila to replicate and survive within host cells. Once we identify which host proteins must be altered in order for L. pneumophila to replicate, we will take a targeted approach to identify which of the L. pneumophila effectors are causing these essential changes to host proteins. In addition, the GPS screen may also identify the targets of specific "families" of effectors that have remained elusive, such as the L. pneumophila E3 ubiquitin ligases. The directed approach we propose allows us to overcome the difficulties inherent in target identification, such as the redundancy of effectors, and identify the functions of effectors that have remained cryptic. Organism-induced alterations of the host are key to pathogenesis, yet it has previously not been possible to study alterations to individual host proteins at the scale the GPS system permits. The experiments described in this proposal allow, for the first time, dissection of how bacterial infection globally regulates host cell proteins and pathways beyond the transcriptional level. PUBLIC HEALTH RELEVANCE: Legionella pneumophila, the causative agent of Legionnaires' disease replicates inside host cells. To manipulate the host cell and replicate intracellularly, the organism injects >150 of its proteins into host cells. The proposed research uses a large-scale approach to identify the targets of these injected bacterial proteins - identifying the host cell proteins that are destabilized or stabilized by the injected bacterial proteins. Once we identify which bacterial proteins are manipulating which host proteins, we can test methods to disrupt these interactions. This may lead to the development of new classes of antibiotics to treat bacterial infection.

描述（由申请人提供）： 细胞内病原体嗜肺军团菌感染可导致严重肺炎，称为军团病。嗜肺军团菌使用专门的 IV 型分泌装置（也称为 Dot/Icm 系统）将 150 多种效应蛋白直接分泌到宿主细胞中。易位的细菌效应子建立了一个液泡生态位，支持嗜肺军团菌在真核细胞中的复制。虽然有大量文献描述了其中几种效应器如何改变宿主细胞功能，但大多数效应器的目标仍然难以捉摸。将特定效应器与特定功能联系起来的一个重要问题是许多效应器的冗余或重叠活动。这意味着缺乏任何一种效应子的嗜肺军团菌突变株通常没有明显的表型，从而阻碍了对其宿主靶标的识别。虽然众所周知，许多嗜肺军团菌效应子通过 E3 泛素连接酶活性等功能直接改变宿主蛋白，但目前还没有开发出大规模监测病原体诱导的宿主蛋白稳定性变化的方法。在这里，我们建议应用一种称为“全局蛋白质稳定性”（GPS）系统的新型筛选方法来识别其稳定性被分泌的嗜肺军团菌效应子改变的宿主细胞蛋白质。一旦我们确定了在功能性 IV 型分泌系统存在时稳定或不稳定的宿主蛋白，我们将测试减少或增加这些蛋白的流行率（试图逆转军团菌效应子的影响）是否会损害嗜肺军团菌在宿主细胞内复制和生存的能力。一旦我们确定了哪些宿主蛋白必须被改变才能使嗜肺军团菌复制，我们将采取有针对性的方法来确定哪些嗜肺军团菌效应子正在导致宿主蛋白发生这些重要变化。此外，GPS屏幕还可以识别仍然难以捉摸的特定效应器“家族”的目标，例如嗜肺军团菌E3泛素连接酶。我们提出的定向方法使我们能够克服目标识别中固有的困难，例如效应器的冗余，并识别仍然神秘的效应器的功能。生物体引起的宿主改变是发病机制的关键，但以前不可能在 GPS 系统允许的范围内研究单个宿主蛋白质的改变。该提案中描述的实验首次允许剖析细菌感染如何在转录水平之外全面调节宿主细胞蛋白和途径。 公共卫生相关性：嗜肺军团菌是军团病的病原体，可在宿主细胞内复制。为了操纵宿主细胞并在细胞内复制，生物体将超过 150 种蛋白质注射到宿主细胞中。拟议的研究使用大规模方法来识别这些注射的细菌蛋白的靶标——识别被注射的细菌蛋白不稳定或稳定的宿主细胞蛋白。一旦我们确定了哪些细菌蛋白正在操纵哪些宿主蛋白，我们就可以测试破坏这些相互作用的方法。这可能会导致新型抗生素的开发来治疗细菌感染。