| Francisella tularensis is a facultative intracellular pathogen of macrophages and the causative agent of tularemia. Inhalation of as few as 10 organisms can cause a severe pneumonic infection that is potentially fatal in the absence of antibiotics. A central aspect of Francisella pathogenesis is its ability to survive and replicate inside host cells, including macrophages. However, how this bacterium resists killing by macrophages is unclear. In this thesis, Francisella-macrophage interactions are explored at the molecular level. The overall goal of this work is to better understand the mechanisms by which Francisella evades innate host defenses with a particular focus on bacterial entry into macrophages and dissection of the mechanisms by which antimicrobial systems are inhibited. We identify the first receptor mediating the uptake of unopsonized Francisella, the macrophage mannose receptor, and define a model in which this receptor and complement receptors play distinct roles during phagocytosis. We also find that Francisella uses two distinct strategies to prevent activation of the macrophage NADPH oxidase, the first via receptors used for initial uptake and the second mediated by potential secreted virulence factors. We show that Francisella initially resides within macrophages in an unusual phagosome that accumulates lamp1 but excludes most other late endosome markers such as the mannose 6-phosphage receptor, lysobisphosphatidic acid and Rab7. We also identify protein kinase C alpha as the first host molecule targeted by this bacterium to block phagosome maturation. We find that phagosome acidification is necessary for vacuole escape, but that replication in the macrophage cytosol is dependent on specific bacterial genes. Finally, show that Francisella phagosomes accumulate robust amounts of the GTPase Rab5 immediately preceding compartment disruption, which may provide a clue to the mechanism of phagosome disassembly. Taken together, we find that Francisella specifically targets oxidative and non-oxidative host defenses to resist killing and create an intracellular niche optimized for bacterial survival. The findings presented here significantly advance our understanding of Francisella-macrophage interactions and the pathogenesis of tularemia. |
