Effector-mediated ubiquitin manipulation in Legionella pneumophila pathogenesis

泛素网络调控着几乎所有的宿主过程，特别是膜运输和免疫。嗜肺军团菌通过其Dot/ICM转运蛋白将数百个效应物转运到宿主胞浆中，广泛调节宿主过程。使用基于活性的探针、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个军团菌效应器参与了泛素信号转导。这些蛋白质的功能要么是典型的E3泛素连接酶、脱泛素素连接酶，要么是泛素连接酶，它们无视标准泛素化的催化机制。为了探索参与增选宿主泛素网络的其他效应器，我们创造了缺乏一组或每一个参与泛素信号转导的特定效应器的突变体。利用生物素连接酶介导的邻近标记，我们已经确定了能够催化泛素化的额外的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。本项目的目标是通过生化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成分的合作。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小体的生物发生。最后，我们将设计实验来解决Dot/ICM效应器(具有或不具有E3泛素连接酶活性)在Sec22b重新招募到细菌吞噬小体中潜在的功能冗余问题。这些实验结果不仅揭示了细胞内病原体利用宿主功能的新机制，而且还将深入了解宿主泛素化途径的调节，这两者都有可能被用于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.