Francisella tularensis (Ft) is a facultative intracellular pathogen and the causative agent of tularemia. Although it is clear that inhalation of as few as ten Ft subsp. tularensis can be fatal, and that macrophages are a major reservoir of this organism in vivo, how Ft evades elimination is only beginning to be defined, and we know even less about the effects of this pathogen on other cell types, including dendritic cells (DC). Because DC play central role in antigen presentation, elucidating the extent to which human pathogenic strains of Ft alter DC function has important implications for the development of new vaccines, and in this regard it is noteworthy that the fate of many other pathogens in macrophage and DC are distinct. Our longterm goal is to dissect at the molecular level the mechanisms by which Ft disrupts the function of human phagocytes. During the previous funding period we identified protein kinase Ca as a macrophage factor targeted by this pathogen to disrupt phagosome maturation at an eariy stage, prior to Ft egress and replication in the cytosol. PKCa activity declines throughout infected macrophages, and because this kinase also controls a subset of INFy-regulated processes including MHC Class II antigen presentation, we hypothesize that Ft may use a similar strategy to disrupt the function of human DC. Nevertheless, the fate of Ft in this cell type has not been described, and the extent to which the innate immune response can be manipulated to favor Ft clearance is unclear. To address this knowledge gap, we will define Ft fate in human DC, including DC that were 'conditioned'by co-culture with alveolar epithelial cells, and quantify effects of Ft on antigen presentation. In previous work we also defined a role for the mannose receptor (MR) in Ft uptake. A distinguishing feature of Ft is a mannose-rich surface capsule-like material (CLM) that together with LPS protects the organism from complement-mediated lysis. Whether CLM contains or masks MR ligands is unclear, and effects of CLM on other aspects of the Ft lifecycle remain obscure. To date, 9 CLM mutants in Ft strain SchuS4 have been isolated in Project 1, and our preliminary characterization of mutants in FTT1238C, FTT1447C, FTT1512c, FTT1291, FTT0673C-0674 suggests for the first time that alterations in CLM structure or abundance can diflFerentially effect Ft uptake and intracellular growth. Thus, the Specific Aims of this study are: 1) to elucidate the fate of virulent Ft subsp. tularensis strain SchuS4 and Ft subsp. holarctica strain 1547-57 in human DC;2) to evaluate the potential for translational modulation of macrophage and DC function to increase bacterial killing and favor antigen presentation;and 3) to begin to define the role of capsule-like material in Ft fate in macrophages and DC in vivo and in vitro.
Inhalation of a type of bacteria called Francisella tularensis causes severe, sometime fatal pneumonia. In this study we will determine how Francisella avoids being killed after it gets inside a type of immune system cell called a dendritic cell (DC), determine the role of sugars on the bacterial surface in bacterial survival, and also determine whether the functions of DC and another immune cell called macrophages can be manipulated to favor Francisella eradication. The results obtained upon completion of this study may lead to new treatments to combat or prevent this severe and often fatal lung infection.
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