PROJECT SUMMARY Intracellular bacterial pathogens are a major cause of disease worldwide, and improvement of current therapeutic interventions as well as development of new ones relies on gaining a detailed perspective of the molecular strategies important for pathogen survival. A fundamental gap persists in the current understanding of how bacterial pathogens subvert host membrane transport processes and continued existence of this gap impedes our appreciation of the sophisticated mechanisms that bacterial pathogens use to coordinate virulence strategies and maintain a delicate balance between exploiting and preserving host cellular resources. Our long-term goal is to address this problem by systematically unveiling the host pathways critical for infection of human lung macrophages by the bacterial pathogen Legionella pneumophila, the causative agent of a severe pneumonia known as Legionnaires' disease. The pathogen evades host defenses by remaining enclosed in a plasma membrane-derived vacuole that it dramatically remodels to avoid fusion with lysosomes. This process relies on the bacterium's ability to translocate a vast number of (effector) proteins directly into the host cell cytosol. The evidence to date clearly demonstrates that bacterial effector proteins manipulate key regulators of membrane traffic to remodel the vacuole. Yet as far as which effector proteins manipulate membrane transport pathways and how bacteria manipulate host pathways to promote intracellular survival, a clear perspective has not yet emerged. The overall objective is to obtain a detailed understanding of which bacterial effectors manipulate host membrane transport to support survival of L. pneumophila within a vacuole derived from the plasma membrane. L. pneumophila effectors target host membrane compartments by interacting with host phosphoinositides, which are key lipids that define organelle identity in eukaryotic cells. We propose to use (1) phosphoinositide binding as a molecular handle to comprehensively identify L. pneumophila effectors target host membrane compartments using a robust biochemical approach followed by cellular validation, and (2) to begin characterizing novel phosphoinositide binding L. pneumophila effectors identified in our preliminary screen. The proposed research is significant because it is positioned to advance our understanding of how bacterial pathogens manipulate host membrane transport pathways to promote intracellular survival of bacteria. A significant collateral outcome is that these studies could suggest new molecular targets for intervention in L. pneumophila infections and related conditions.