Alteration of host protein stability by Legionella

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。嗜肺军团菌使用一种特殊的IV型分泌装置，也称为Dot/Icm系统，直接向宿主细胞分泌150多种效应蛋白。易位的细菌效应物建立了一个液泡生态位，支持嗜肺乳杆菌在真核细胞中的复制。虽然有大量的文献描述了这些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。这意味着嗜肺乳杆菌突变株缺乏任何一种效应物通常没有明显的表型，阻碍了其宿主目标的识别。虽然人们很清楚许多嗜肺乳杆菌效应物通过E3泛素连接酶活性等功能直接改变宿主蛋白，但很少有方法可以大规模监测病原体诱导的宿主蛋白稳定性变化。在这里，我们提出了一种新的筛选方法，称为“全局蛋白稳定性”（GPS）系统，以鉴定宿主细胞蛋白的稳定性被分泌的嗜肺乳杆菌效应物改变。一旦我们确定了在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（试图逆转军团菌效应物的影响）是否会损害嗜肺乳杆菌在宿主细胞内复制和存活的能力。一旦我们确定了嗜肺乳杆菌复制必须改变哪些宿主蛋白，我们将采取有针对性的方法来确定哪些嗜肺乳杆菌效应物导致宿主蛋白发生这些基本变化。此外，GPS屏幕还可以识别仍然难以捉摸的特定效应物“家族”的目标，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，例如效应体的冗余，并识别仍然神秘的效应体的功能。生物体诱导的宿主改变是发病机制的关键，但以前不可能在GPS系统允许的规模上研究单个宿主蛋白质的改变。本提案中描述的实验首次允许解剖细菌感染如何全局调节宿主细胞蛋白和转录水平以外的途径。

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.