Mechanisms of Rickettsia invasion, intracellular survival, and actin-based motility

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PROJECT SUMMARY/ABSTRACT The Rickettsiae are obligate intracellular bacterial pathogens that cause serious diseases, such as spotted fever and typhus. We study the model spotted fever group (SFG) species Rickettsia parkeri, which causes an eschar-associated human rickettsiosis, is experimentally tractable, and has emerging mouse models of pathogenesis, making it ideal for revealing molecular mechanisms of SFG Rickettsia infection and virulence. During infection, SFG Rickettsia invade host cells by mobilizing the actin cytoskeleton, escape from the phagosome into the cytosol, replicate while avoiding degradation by autophagy, and harness actin polymerization 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 components to promote their infection cycle. To bridge these gaps, in the current funding period we have pioneered an innovative combination of bacterial genetics and host cell biology to identify key Rickettsia factors that manipulate host cells. In unpublished work, we discovered that outer membrane protein OmpB is crucial for both invasion and avoidance of autophagy. We also observed that patatin-like phospholipase Pat1 plays a role in phagosome escape and/or autophagy evasion. Additionally, in published work, we demonstrated that Rickettsia use two actin-polymerizing surface proteins to direct sequential phases of motility – with RickA driving early motility and surface cell antigen Sca2 driving late motility. However, key outstanding questions remain, including: How do Rickettsia engage host receptors to promote invasion? How do Rickettsia degrade membranes during phagosome escape or inhibit membrane engulfment to avoid autophagy? And how do Rickettsia coordinate and use two actin assembly factors in distinct phases of motility? Our preliminary and published findings suggest the overall hypothesis that OmpB, Pat1, RickA, and Sca2 are multifunctional proteins that mobilize or disrupt host cell components and play a crucial role in infection in vivo. This hypothesis will be tested in three Aims focused on uncovering the mechanisms through which OmpB, Pat1, RickA, and Sca2 influence invasion, intracellular survival, and motility. The Aims are to: (1) define the role of Rickettsia surface protein OmpB in invasion and intracellular survival; (2) investigate the role of Pat1 phospholipase in phagosome escape and intracellular survival; and (3) determine how and why Rickettsia use two distinct actin-based motility mechanisms. The proposed studies will advance the field by revealing 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 diagnostics and treatments for rickettsial and other infections.