Subproject 1: Horwitz Francisella tularensis, a facultative intracellular pathogen, causes serious and potentially life threatening illness, and because the bacterium grows readily in liquid culture, has high infectivity, is easily dispersed, and was previously stockpiled as a germ warfare agent, it is a CDC Category A potential agent of bioterrorism. Moreover, F. tularensis can be engineered to carry antibiotic resistance genes. For these reasons, new approaches to prevention and treatment of tularemia are needed. Devising such strategies requires an improved understanding of F. tularensis interaction with host macrophages. We have recently uncovered key and unique features of F. tularensis - macrophage interaction. We have found that virulent F. tularensis a) enters macrophages by a unique complement dependent process involving engulfment within a spacious pseudopod loop; b) enters a phagosomal compartment that exhibits arrested maturation and acquires a unique fibrillar coat; c) inhibits phagosome acidification; and d) contrary to previous reports, disrupts the phagosomal membrane and escapes into the cytoplasm. We propose to study the molecular basis for these phenomena and their role in pathogenesis. Specifically, we shall a) examine the roles of professional phagocyte receptors, serum ligands, complement pathways, and host cell processes in phagocytosis; b) determine the host cell and bacterial contributions to the composition of the F. tularensis phagosome and the identity of the components of the fibrillar coat using a mass spectrometry based proteomics analysis of isolated phagosomes, complemented by confocal immunofluorescence and cryoimmunoelectron microscopy; c) identify proteins secreted by F. tularensis growing intracellularly, which are likely to be important in altering phagosome maturation and in phagosome escape, using metabolic radiolabeling and 2-D gel electrophoresis combined with mass spectrometry based proteomics analysis; and d) study the role of these proteins in bacterial virulence by immunoelectron microscopy and by constructing F. tularensis deletion mutants. These studies will increase our understanding of how F. tularensis subverts the host cell membrane trafficking pathways and disrupts its phagosomal membrane, identify promising therapeutic targets, and guide new strategies for the prevention and treatment of tularemia.
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