The long-term goal of this research is to understand how bacterial pathogens interact with the host ubiquitination pathway during intracellular infection. The gram negative bacterial pathogen, Legionella pneumophila, establishes an intracellular replicative niche by modulating a number of host pathways, including endocytosis and secretory trafficking. We recently made a number of observations that suggest that the ubiquitin pathway is subject to modulation by L. pneumophila. Polyubiquitinated substrates rapidly associate with the Legionella containing vacuole (LCV) upon infection and a downstream component of the host ubiquitin-mediated proteolytic pathway, Cdc48/p97 is required for efficient intracellular growth of the bacteria. Additionally, the L. pneumophila genome contains putative translocated effectors which contain eukaryotic motifs typically associated with the ubiquitination pathway. Ubiquitination has been observed in association with a number of intracellular pathogens, where it likely plays a role in their detection by the host. Upon L. pneumophila infection, we hypothesize that both the host immune response and pathogenic bacteria interact with the ubiquitin system to achieve opposing goals. This application seeks to elucidate the manner in which L. pneumophila modulates this system to its benefit and the role this plays in pathogenesis. The aims of this research include: i. determination of the type(s) of polyubiquitin conjugated to substrates around the Legionella containing vacuole (LCV);ii. identification of Legionella proteins that modulate the host ubiquitin system;and iii. identification of LCV-associated ubiquitinated substrates. Ubiquitination interacts with a diverse set of biological pathways within the cell, with established roles in immunity, genetic disease, and cancer. There is growing evidence that host ubiquitination is involved in the detection of intracellular bacterial pathogens. By studying how Legionella pneumophila, the causative agent of Legionnaire's disease, co-opts this system to support its own growth, we will gain insight into both this pathogenesis and the ubiquitin system in general.