Effector-mediated ubiquitin manipulation in Legionella pneumophila pathogenesis

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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.