Alteration of host protein stability by Legionella

军团菌改变宿主蛋白稳定性

• 批准号: 8268377
• 负责人: MICHAEL N STARNBACH
• 金额: $ 21.19万
• 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/8268377
• 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.

描述(申请人提供)：感染细胞内病原体嗜肺军团菌可导致一种被称为军团病的严重肺炎。嗜肺军团菌使用一种专门的IV型分泌器，也被称为Dot/ICM系统，将150多种效应蛋白直接分泌到宿主细胞中。转位的细菌效应器建立了一个空泡生态位，支持嗜肺乳杆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的功能，但大多数效应器的靶点仍然难以捉摸。将特定效应器链接到特定功能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个效应器的嗜肺乳杆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。虽然许多嗜肺乳杆菌的效应者通过E3泛素连接酶活性等功能直接改变宿主蛋白，但很少有人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，我们建议应用一种新的筛选方法，称为“全球蛋白稳定性”(GPS)系统，以确定其稳定性被分泌的嗜肺乳杆菌效应器改变的宿主细胞蛋白。一旦我们确定了当存在IV型功能分泌系统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器的影响)是否会损害嗜肺乳杆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改变才能使嗜肺乳杆菌复制，我们就会采取有针对性的方法来确定哪些嗜肺乳杆菌的效应子导致了宿主蛋白的这些基本变化。此外，GPS屏幕还可以识别仍然难以捉摸的特定效应器“家族”的靶标，例如嗜肺乳杆菌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，并识别仍然未知的效应器的功能。生物体诱导的宿主变化是发病机制的关键，然而以前还不可能在GPS系统允许的范围内研究单个宿主蛋白的变化。这项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对宿主细胞蛋白质和途径进行全球调节。