Project summary Active modulation of host function is essential for the success of bacterial pathogens. The ubiquitin network regulates virtually every cellular process in eukaryotes, particularly those involved in the detection, recognition and response to infection. Accordingly, 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 revealed that members of the SidE effector family feature enzymatic domains with unique activities for manipulating ubiquitin signals. First, these proteins contain a deubiquitinase (DUB) module that efficiently removes ubiquitin from ubiquitinated proteins. Second, these proteins catalyze ubiquitination by an unusual mechanism: the reaction does not require the E1, E2 enzymes or ATP, factors 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 L. pneumophila Dot/Icm type IV effectors. As an additional layer of complexity, we find that the biological activity of SidE effectors is regulated by the SidJ effector through its reversal of the SidE-catalyzed ubiquitination by a mechanism that appears to distinctly differ from classical DUBs. By biochemical and structural analyses, we will study the mechanism of action of these proteins. Different functional domains encoded in these effectors seem to work in concert to regulate each other for a balanced control of host functions. We will study biochemical basis of functional integration among distinct activities present within these effectors. 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.