Effector-mediated ubiquitin manipulation in Legionella pneumophila pathogenesis

泛素网络调节几乎所有的宿主过程，特别是膜运输和免疫。细菌病原体嗜肺军团菌广泛地调节宿主过程，使用数百个效应物通过其Dot/Icm转运蛋白转运到宿主细胞质中。使用基于活性的探针，生物信息学分析加上仔细的生化和结构分析，已确定在28个军团菌效应参与选择泛素信号转导。这些蛋白质作为典型的E3泛素连接酶、去泛素化酶或作为无视典型泛素化的催化机制的泛素连接酶起作用。为了探索参与选择宿主泛素网络的其他效应子，我们创建了缺乏特定组或参与泛素信号传导的每个已知效应子的突变体。使用生物素连接酶介导的邻近标记，我们已经确定了额外的点/Icm底物能够催化泛素化。我们还获得了证据，军团菌蛋白质的存在，直接修改泛素。本项目的目标是通过生物化学和结构分析来确定这些蛋白质的功能，它们的催化机制以及它们与宿主泛素化组分的合作。我们还将研究细菌因子对它们活性的调节，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，以解决潜在的功能冗余之间的Dot/Icm效应（有或没有E3泛素连接酶活性）的Sec 22 b的招聘细菌吞噬体的问题。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，还将揭示宿主泛素化途径的调节，这两者都有可能被利用来开发新的疾病诊断和治疗方法。

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.