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（由申请人提供）：细胞内病原体肺炎的感染可导致严重的肺炎被称为军团疾病。肺炎军团使用专门的IV型分泌设备，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，该泡酸盐支持真核细胞中肺炎乳杆菌的复制。尽管有广泛的文献描述了这些效应器中的几个如何改变宿主细胞功能，但大多数效应的目标仍然难以捉摸。将特定效应子与特定功能联系起来的一个重要问题是许多效应子的冗余或重叠活动。这意味着任何一种效应子缺乏的肺炎乳杆菌菌株通常没有明显的表型，从而阻止了其宿主靶标的识别。虽然许多肺炎乳杆菌效应子通过诸如E3泛素连接酶活性直接改变宿主蛋白的众所周知，但几乎没有开发用于监测病原体诱导的大规模宿主蛋白稳定性变化的方法。在这里，我们建议采用一种称为“全局蛋白质稳定性”（GPS）系统的新型筛选方法来识别宿主细胞蛋白，其稳定性被分泌的肺炎乳杆菌效应子改变。一旦我们确定了存在IV型功能性分泌系统时稳定或不稳定的宿主蛋白，我们将测试是否会降低或增加这些蛋白质的患病率（试图逆转军团菌效应子的影响）会损害肺炎乳杆菌的能力，以复制和在宿主细胞内生存。一旦我们确定了必须改变哪种宿主蛋白​​才能复制肺炎乳杆菌，我们将采用靶向方法来识别哪些肺炎乳杆菌效应子正在导致宿主蛋白的这些基本变化。此外，GPS屏幕还可以识别出仍然难以捉摸的特定效应子的靶标，例如肺炎乳杆菌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固有的困难，例如效应子的冗余，并确定效应子的功能仍然是隐秘的。生物体诱导的宿主改变是发病机理的关键，但以前无法在GPS系统允许的规模上研究对单个宿主蛋白的改变。该提案中描述的实验首次允许对全球细菌感染的解剖如何调节宿主细胞蛋白和转录水平的途径。