UC Berkeley

Bacteria in the genus Rickettsia are arthropod-borne obligate intracellular microbes that can cause a spectrum of diseases in humans with manifestations ranging from mild to severe. Following invasion of host cells, Rickettsia must escape from the membrane-bound vacuole to gain access to the cytosol, where they reside. In the cytosol, bacteria must avoid detection and degradation by host pathways such as autophagy. They also undergo actin-based motility and initiate cell-cell spread to infect new cells. Although we have a mechanistic understanding of invasion, actin-based motility, and cell-cell spread, how Rickettsia interacts with and manipulates host membranes is poorly understood. In particular, Rickettsia genomes encode factors predicted to interact with and mediate rupture of host membranes, such as phospholipases and hemolysins, but very little is known about how these proteins function during infection. In this dissertation, I describe the characterization of a conserved Rickettsia phospholipase, Pat1, to address key unanswered questions about the role of this membrane targeting enzyme in the Rickettsia intracellular life cycle and in pathogenesis. I investigated the role of Rickettsia Pat1 by characterizing the phenotype of a Rickettsia parkeri mutant with a transposon insertion in the pat1 gene. I found that Pat1 is critical Rickettsia factor for efficient escape from the vacuole into the cytosol, both following invasion and during cell-cell spread. This provides genetic evidence to support a long-held hypothesis that phospholipases mediate Rickettsia vacuolar escape. Pat1 is also important for preventing association of the bacteria with damaged membranes marked by galectin-3 and for initial targeting by autophagy via the autophagy adapter NDP52. Pat1 is also important for avoiding autophagy that occurred on bacteria not associated with damaged membranes and involved targeting by host polyubiquitin and the autophagy cargo adaptor p62. Moreover, Pat1 is critical for actin-based motility and escape from the secondary vacuole, two processes related to cell-cell spread. Although Pat1 does not affect growth inside tissue culture cells, it is required for virulence in a mouse model of infection. Altogether, the data presented in this dissertation suggest Pat1 is important at multiple steps of the Rickettsia life cycle that involve manipulating host membranes. This work also contributes more generally to our understanding of the role of bacterial patatin-like phospholipases in the host-microbe interaction. Future work on Rickettsia Pat1 will further define the mechanistic details of Pat1 function during infection, as well as how Pat1 activity is regulated, how it cooperates with other bacterial and host proteins to allow bacteria to efficiently access the cytosol, and what role it plays in animal infection.