Effects of Francisella tularensis infection on macrophage intracellular signaling

Microbes contain a number of structural components, also known as pathogen associated molecular patterns (PAMP), which can be recognized by the host defense system. The PAMP serve as ligands for Toll-like receptors (TLR) expressed on phagocytic cells. Binding of PAMP to TLR leads to activation of a number of intracellular transduction pathways including NF-�B and mitogen-activated protein kinase pathways (MAPK). Activation of those pathways in turn leads to activation of proinflammatory immune responses and antimicrobial defense mechanisms. In this study we show that the intracellular pathogen F. tularensis LVS can induce a proinflammatory response during internalization in both human and mouse macrophages. However, after internalization, F. tularensis LVS effectively blocks all these pathways resulting in no secretion of proinflammatory cytokines in mouse macrophages. In human macrophages, a downregulation was observed, however, there was not a complete block as in the murine cells. Furthermore, the infected cells do not become activated by stimulation with TLR agonists such as Escherichia coli LPS or bacterial lipoprotein. This phenomenon is observed only when the cells were infected with the LVS strain, but not with killed bacteria or specific mutants such as iglC, or mglA mutants, suggesting that not only their presence but also rapid regulation of the Igl proteins are needed. Thus, Igl/Mgl system plays a very important role in mediating these inhibitory effects. To further investigate the role of the Igl/Mgl proteins in the virulence of F. tularensis, we established a novel model for tularemia, infection of Drosophila melanogaster. Using the fly model as a way to mimic the F. tularensis infection in arthropod vectors, which are important for its life cycle, we observed that bacterial mutants in each of the IglB, IglC, IglD, or MglA proteins all showed less efficient killing of the flies than did the parental LVS strain. Nitric oxide, superoxide, and the reaction product of the two mediators, peroxynitrite, all are important for regulation of eukaryotic cell signaling and effectuating bactericidal mechanisms. To this end, we investigated the role of these mediators for the F. tularensismediated TLR-signaling inhibition. We observed that whereas peroxynitrite had little effect on the inhibition, addition of SNAP, a donor of nitric oxide, partially restored the ability of the cells to respond to LPS stimulation and secrete proinflammatory cytokine such as TNF-�. However, since FeTTPS reversed the SNAP-mediated effect, the results indicate that NO forms peroxynitrite intracellular and that the latter molecule is the effector. Together these results indicate that F. tularensis contains PAMP that can activate proinflammatory immune response while the bacteria are located outside of phagocytic cell and during internalization, however, the intracellular bacteria have means to inhibit the activation of anti-bacterial systems of macrophages and block inflammatory responses completely in mouse cells and partially in human cells. This suppression may lead to disruption of synthesis of bactericidal effector molecules, allowing F. tularensis to survive and rapidly proliferate inside phagocytic cells. The ability to suppress the inflammatory responses is dependent on expression of the Igl/Mgl proteins. The Igl/Mgl system is also critical for the ability of F. tularensis to replicate in mammalian cells and in D. melanogaster