Tularemia is a potentially fatal zoonosis of humans caused by the facultative intracellular bacterium, Francisella tularensis (Ft). Indeed, inhalation of as few as ten organisms is sufficient to cause severe pneumonic disease, tissue necrosis and death. Because of its extreme virulence via the aerosol route, Ft is a potential bioweapon and has been classified as a Category A Select Agent. The results of a recent study demonstrate that matrix metalloprotease-9 is upregulated by Ft and that subsequent cleavage of extracellular matrix generates a chemoattractant that drives neutrophil migration into the lung. Although neutrophils are the dominant white blood cell in alveoli and bronchioles during acute infection (day 2-14), and engulf large numbers of bacteria in this locale, Ft are not killed and bacterial load increases markedly. These data suggest that disruption of neutrophil function by Ft may be an important aspect of virulence. Indeed, if PMN accumulation migration into the lung is compromised, mice survive an otherwise lethal dose of F. tularensis. Although relatively few pathogens resist elimination by PMN, almost nothing is known about neutrophil-Ft interactions at the molecular level. Our preliminary data now demonstrate that Ft has profound effects on human neutrophil function that include rapid and complete inhibition of the oxidative burst. In this regard it is noteworthy that cell activation by heterologous stimuli is also impaired, and as such our data suggest that effects of Ft extend beyond the confines of its own phagosome. Francisella also blocks non-oxidative killing arsenal of the neutrophil, and degranulation is inhibited or severely delayed. Later in infection, Ft breaches the phagosome membrane and resides in the nutrient-rich cytosol. Thus, we hypothesize that Ft disrupts both NADPH oxidase activity and phagosome-lysosome fusion as a means to evade intracellular killing. Our long term goal is to define at the molecular level the mechanisms by which Ft disrupts neutrophil function. To test this hypothesis we will: 1. Elucidate the effect of Ft on NADPH oxidase assembly and activity. 2. Define the composition of the phagosome and quantify granule mobilization; and 3. Discern the extent to which PMN function is impaired via effects of Ft virulence determinants on neutrophil gene expression and key intracellular signaling pathways. Toward this end we will utilize chemiluminescence fluorescence and other biochemical assays to quantify reactive oxygen species; confocal microscopy to localize NADPH oxidase components; immunofluorescence microscopy and immuno-electron microscopy to assess phagosome composition; transmission electron microscopy to quantify phagosome escape and PMN viability; in vitro kinase assays and immunoblotting to measure intracellular signaling; DNA microarrays to define changes in neutrophil gene expression; and both targeted allelic replacement strategies and transposon mutant library screening to begin to define Ft genes required for virulence in this system. ? ? Project Narrative: Inhalation of the bacterium called Francisella tularensis causes severe, sometime fatal pneumonia and no vaccine is available. In this study we will begin to determine how Francisella avoid being killed by a type of white blood cell called neutrophils (which accumulate in the lung during this infection). The results of this study may lead to new treatments to combat this severe and often fatal lung infection. ? ? ?
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