PROJECT SUMMARY/ABSTRACT Pathogenic Rickettsiae are obligate intracellular bacteria that cause diseases such as spotted fever and typhus. We study the spotted fever group (SFG) species Rickettsia parkeri, which causes an eschar-associated human rickettsiosis and is experimentally tractable, making it an ideal model for revealing molecular mechanisms of SFG Rickettsia infection and virulence. Following invasion of host cells, SFG Rickettsia escape from the phagosome into the cytosol, replicate while avoiding ubiquitylation and autophagy, and polymerize host actin to promote intracellular motility and cell-cell spread. However, there are fundamental gaps in our knowledge of the molecular mechanisms by which SFG Rickettsia exploit or disrupt host cell structures and pathways to promote their infection cycle. Towards bridging these gaps, in the current granting period we discovered that the patatin- like phospholipase enzyme Pat1 is important for virulence and for escaping from host membranes including phagosomes and autophagosomes. We further showed that outer membrane protein OmpB and lysine methylation are crucial for virulence and for shielding bacteria from ubiquitylation and autophagy. We also observed that two actin-based motility proteins, RickA and Sca2, function independently in cell-cell spread and virulence. Finally, we developed an interferon receptor-deficient mouse model of eschar-associated rickettsiosis that can be used to evaluate the role of bacterial factors in virulence. These findings support the overall scientific premise that rickettsial proteins manipulate host cell components to enable bacterial escape from host membranes, avoidance of host ubiquitylation pathways, and mobilization of the host cytoskeleton for movement. However, key outstanding questions remain. How does bacterial phospholipase activity contribute to infection? How does bacterial surface architecture prevent ubiquitylation by host machinery? How and why do Rickettsia use two mechanisms for intracellular movement? We will address these outstanding questions, testing the overall hypothesis that the structure, function, and regulation of bacterial secreted and surface proteins is critical for manipulating or avoiding host cell molecules and structures, enabling infection of cells and virulence in animals. This general hypothesis will be tested in three Aims focused on uncovering the roles of Pat1, OmpB, RickA, and Sca2 in escape from host membranes, intracellular survival, and motility. The Aims are to: (1) determine how Rickettsia phospholipase activity contributes to infection; (2) reveal how Rickettsia surface architecture enables avoidance of ubiquitylation; and (3) establish how and why Rickettsia use two mechanisms for intracellular movement. The impact will be to reveal crucial molecular mechanisms used by Rickettsia and other pathogens to manipulate host cells and the importance of these mechanisms to infectivity. Our studies may also lead to improved d...