Effector-mediated ubiquitin manipulation in Legionella pneumophila pathogenesis

泛素网络几乎调节了宿主的每一个过程，特别是膜运输和免疫。细菌病原体嗜肺军团菌通过其Dot/Icm转运体转运到宿主细胞质中，通过数百种效应物广泛调节宿主过程。利用基于活性的探针、生物信息学分析以及仔细的生化和结构分析，已经确定了28种军团菌效应物参与了泛素信号转导。这些蛋白要么作为典型的E3泛素连接酶，去泛素酶，要么作为泛素连接酶，无视典型泛素化的催化机制。为了探索参与吸收宿主泛素网络的其他效应物，我们创建了缺乏一组特定或每个已知的泛素信号传导效应物的突变体。使用生物素连接酶介导的接近标记，我们已经确定了能够催化泛素化的额外Dot/Icm底物。我们还获得了直接修饰泛素的军团菌蛋白存在的证据。本项目的目的是通过生化和结构分析来确定这些蛋白的功能、催化机制以及它们与宿主泛素化成分的合作关系。我们还将研究来自细菌的因子对其活性的调节，并确定这种活性如何有助于支持细胞内细菌复制的吞噬体的生物发生。最后，我们将设计实验来解决Dot/Icm效应物（具有或不具有E3泛素连接酶活性）在将Sec22b招募到细菌吞噬体中的潜在功能冗余问题。这些实验的结果不仅将揭示细胞内病原体利用宿主功能的新机制，而且还将揭示宿主泛素化途径的调控，这两者都有可能被用于开发疾病诊断和治疗的新方法。

The ubiquitin network regulates virtually every host processes, particularly membrane trafficking and immunity. The bacterial pathogen Legionella pneumophila extensively modulates host processes using hundreds of effectors translocated into the host cytosol by its Dot/Icm transporter. The use of activity-based probes, bioinformatics analysis coupled with careful biochemical and structural analyses has identified at 28 Legionella effectors involved in co-opting ubiquitin signaling. These proteins function either as canonical E3 ubiquitin ligases, deubiquitinases or as ubiquitin ligases that defy the catalytic mechanism of canonical ubiquitination. To explore additional effectors involved in co-opting the host ubiquitin network, we have created mutants that lack a specific set or each of the known effectors involved in ubiquitin signaling. Using biotin ligase-mediated proximity labeling, we have identified additional Dot/Icm substrates capable of catalyzing ubiquitination. We have also obtained evidence for the existence of Legionella proteins that directly modify ubiquitin. The goal of this project is to determine the function of these proteins, their catalytic mechanism and their cooperation with host ubiquitination components by biochemical and structural analyses. We will also study the regulation of their activity by factors from the bacterium and determine how such activity contributes to the biogenesis of the phagosome supportive of intracellular bacterial replication. Finally, we will design experiments to address the issue of the potential functional redundancy among Dot/Icm effectors (with or without E3 ubiquitin ligase activity) in the recruitment of Sec22b to the bacterial phagosome. Results from these experiments will reveal not only novel mechanisms of host function exploitation by intracellular pathogens, but also insights into the regulation of host ubiquitination pathways, both of which have the potential to be capitalized to develop novel methods for diagnosis and treatment of diseases.