Alteration of host protein stability by Legionella

描述（由申请方提供）：细胞内病原体嗜肺军团菌感染可导致称为军团病的严重肺炎。嗜肺军团菌使用专门的IV型分泌装置，也称为Dot/Icm系统，将超过150种效应蛋白直接分泌到宿主细胞中。移位的细菌效应子建立了一个支持L.真核细胞中的嗜肺菌。虽然有大量文献描述了这些效应物中的几种如何改变宿主细胞功能，但大多数效应物的靶点仍然难以捉摸。将特定效应物与特定功能联系起来的一个重要问题是许多效应物的冗余或重叠活性。这意味着L。缺乏任何一种效应子的嗜肺菌突变株通常没有明显的表型，从而妨碍了对其宿主靶标的鉴定。虽然很好地理解许多L.尽管嗜肺菌效应子通过诸如E3泛素连接酶活性的功能直接改变宿主蛋白质，但很少有开发出大规模监测病原体诱导的宿主蛋白质稳定性变化的方法。在这里，我们提出了一种新的筛选方法，称为“全球蛋白质稳定性”（GPS）系统，以确定宿主细胞的蛋白质，其稳定性被分泌的L。嗜肺效应器。一旦我们确定了当功能性IV型分泌系统存在时稳定或不稳定的宿主蛋白质，我们将测试减少或增加这些蛋白质的流行（试图逆转军团菌效应子的作用）是否会损害L.嗜肺菌在宿主细胞内复制和存活。一旦我们确定了哪些宿主蛋白质必须改变才能使L。pneumophila复制，我们将采取有针对性的方法，以确定哪些L。嗜肺菌效应子引起宿主蛋白质的这些基本变化。此外，GPS屏幕还可以识别仍然难以捉摸的效应器的特定“家族”的目标，例如L。嗜肺菌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，如效应子的冗余，并确定仍然神秘的效应子的功能。机体诱导的宿主改变是发病机制的关键，但以前不可能在GPS系统允许的范围内研究单个宿主蛋白质的改变。本提案中描述的实验允许首次解剖细菌感染如何在转录水平之外全面调节宿主细胞蛋白质和途径。

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.