Manipulation of the innate immune response and evasion of macrophage host defense mechanisms by Francisella tularensis Matthew Eugene Long , University of Iowa Follow

[摘要] Tularemia is a potentially fatally illness caused by the facultative intracellular Gram-negative bacterium Francisella tularensis. Virulent strains of F. tularensis can cause a fatal disease after inhalation of a few as ten organisms. Due to the highly pathogenic features of Francisella, it has been designated as a Tier 1 select agent, meaning that its possession and handling is highly restricted. Macrophages are phagocytes that play a central role in the innate immune response to infection that can be used by certain pathogens, including Francisella, as a niche for bacterial replication and dissemination during infection. After infection of macrophages Francisella escapes from the phagosome and replicates in the cytosol, however the bacterial factors required for these aspects of virulence are incompletely defined. Here we describe the isolation and characterization of F. tularensis subspecies tularensis strain Schu S4 mutants in iglI, iglJ, and pdpC, three genes located in the Francisella Pathogenicity Island. Our data demonstrate that these mutants were unable to replicate in macrophages due to a defect in phagosome escape. However, a small percentage of pdpC mutants were able to reach the cytosol and replicate moderately. Both iglJ and pdpC mutants were highly attenuated for virulence in a mouse intranasal infection model, however pdpC but not iglJ mutants, were able to disseminate from the lung before eventual clearance. These data demonstrated that the FPI genes tested were essential for F. tularensis Schu S4 virulence, but suggest that they may have different functions due to the unique phenotype observed for pdpC mutants. Our studies also characterized the role of F. tularensis O-antigen and capsule to facilitate interactions with components of the serum complement system; demonstrating that the O-antigen is required for binding of IgM to the bacteria in order to initiate complement opsonization. IgM dependent complement opsonization of both F. tularensis Schu S4 and LVS strains facilitated enhanced phagocytosis of the bacteria by complement receptors 3 and 4 of human macrophages. In addition, we examined the mechanisms of macrophage cytotoxicity and proinflammatory cytokine secretion that was induced after infection with a Schu S4 LPS O-antigen and capsule mutant. The response to the mutant was dependent on phagosome escapes, suggesting a cytosolic pattern recognition receptor was involved in recognition of the bacteria. We found that the cytotoxic and proinflammatory responses had both similar and distinct requirements between human and murine macrophages. Infection with the O-antigen mutant induced robust proinflammatory cytokine secretion that was dependent on caspase-1, cathepsin B, and ASC while cytotoxicity was partially dependent on these molecules. Importantly, we demonstrated that wild-type Schu S4 predominately activated apoptotic caspases, and not inflammatory caspases, during infection and had a blunted cytotoxic response. This was in contrast to the robust cytotoxicity and activation of inflammatory caspases after infection with the non-virulent strain LVS. Together, these studies demonstrated that the Schu S4 LPS O-antigen and capsule are required for evasion of macrophage cytosolic host defense mechanisms.