Project Summary Active modulation of host function is essential for the success of bacterial pathogens. The ubiquitin network regulates virtually every cellular process in eukaryote, particularly those involved in the detection, recognition and response to infection. It is thus not unexpected that many pathogens target host ubiquitination for their benefits. Earlier studies revealed that Legionella pneumophila, the causative agent of Legionnaires' disease, interferes with host ubiquitin signaling by using at least 9 of its Dot/Icm effectors. Our recent study has identified members of the SidE effector family as unique ubiquitin manipulation enzymes. First, these proteins contain a deubiquitinase motif that attacks ubiquitinated proteins. Second, these proteins catalyze ubiquitination by an unusual mechanism: the reaction does not require the E1, E2 enzymes or ATP, factor that are essential for all described ubiquitination events. Furthermore, these novel ubiquitin manipulating effectors are required for maximal intracellular bacterial replication, which differs sharply with the majority of Legionella type IV effectors. By biochemical and structural analyses, we will first study the mechanisms of action of these proteins. 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. These studies will not only reveal novel mechanisms of host function exploitation by intracellular pathogens, but also will have the potential to revise the current understanding of ubiquitination, an enormously important signaling mechanism.