DeADP-ribosylation of host targets mediated by a bacterial effector

致病菌嗜肺军团菌（Lp）是军团菌病的病原体， 利用其Dot/Icm蛋白分泌系统将近330个效应物注入宿主细胞质中， 产生和维持一种复制性细胞器的过程，这种细胞器被称为含军团菌的细胞器。 液泡（LCV）。过去几年的研究显示了一些关于泛素化的显著例子 由这些效应子催化的化学反应在真核生物中是无与伦比的。两个显著 已经确定了宿主靶的泛素化的例子，其中机制是独立的 属于真核细胞E1-E2-E3系统，与真核细胞E1-E2-E3系统截然不同。这增加了一个令人兴奋的新层， 复杂的泛素（Ub）操纵Lp，这是已知的具有几个经典的 Ub E3连接酶和去泛素化酶是其效应子。这就提出了一个问题， 军团菌对宿主遍在蛋白的操纵。是否有额外的新的Ub操作 蛋白质/酶在细菌的大型效应器武器库？ 我们已经进行了基于活性的富集分析，以确定新的Ub相互作用蛋白 为了解决这个问题。我们使用了Ub衍生的基于活性的探针（Ub-ABP） 从Lp的可溶性蛋白质组中富集蛋白质，并使用液体 通过柱色谱法与串联质谱法（LC-MS/MS）联用来分析样品。这让我们想到了Dot/ICM 底物MavL作为一种新型的Ub相互作用效应子，具有未知的生化和生物学功能。对 进一步分析，我们发现MavL是一种能够去除ADP-核糖的去ADP-核糖基糖水解酶 从ADP-核糖基化Ub（ADPR-Ub）的精氨酸侧链中分离ADPR（ADPR）。我们将使用邻近标记 在感染条件下实施生物素化策略以捕获MavL底物。以补充 这种策略，我们将工程酶的失活突变体成为蛋白质组范围的亲和工具， 从宿主细胞富集和鉴定MavL相互作用物。结合来自 蛋白质组学数据集，我们的目标是达到一个候选名单，将在基于细胞的测定验证 在感染的情况下。进一步，我们将试图通过这一点来提供Ub识别的结构基础。 新的致病效应物。综上所述，我们将能够描述一个独特的UB识别 deADP-核糖基化酶从病原性细菌，并探讨其宿主的目标，从而扩大 宿主-病原体相互作用中可逆ADP-核糖基化的范围。

The pathogenic bacteria Legionella pneumophila (Lp), the causative agent of Legionnaires’ disease, injects nearly 330 effectors using its Dot/Icm protein secretion system into host cytosol to remodel cellular processes toward creating and maintaining a replicative organelle called the legionella-containing vacuole (LCV). Research in the last few years has shown some remarkable examples of ubiquitination chemistry catalyzed by some of these effectors that are unparalleled in the eukaryotic world. Two notable examples of ubiquitination of host targets have been identified where the mechanisms are independent of and radically different from the eukaryotic E1-E2-E3 system. This has added an exciting new layer of complexity to ubiquitin (Ub) manipulation by Lp, which was already known to possess several classical Ub E3 ligases and deubiquitinases among its effectors. This raises a question as to how far and deep Legionella’s manipulation of the host ubiquitin goes. Are there additional new Ub-manipulating proteins/enzymes in the large effector arsenal of the bacteria? We have performed an activity-based enrichment analysis to identify novel Ub-interacting proteins among Lp effectors to address this question. We have used Ub-derived activity-based probes (Ub-ABPs) to enrich proteins from the soluble proteome of Lp and have identified candidates using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). This has led us to the Dot/Icm substrate MavL, with unknown biochemical and biological function, as a novel Ub-interacting effector. On further analysis, we find that MavL is a deADP-ribosyl glycohydrolase capable of removing ADP-ribose (ADPR) from an arginine side chain of ADP-ribosylated Ub (ADPR-Ub). We will use a proximity labeling biotinylation strategy implemented under infection conditions to capture MavL substrates. To complement this strategy, we will engineer an inactive mutant of the enzyme into an affinity tool for proteome-wide enrichment and identification of MavL interactors from host cells. Combining information from the proteomics datasets, we aim to arrive at a list of candidates which will be validated in cell-based assays under infection conditions. Further, we will try to provide the structural basis of Ub recognition by this novel pathogenic effector. Taken together, we will be able to characterize a unique Ub-recognizing deADP-ribosylating enzyme from a pathogenic bacterium and explore its host targets, thereby expanding the scope of reversible ADP-ribosylation in host-pathogen interaction.