Francisella tularensis infection induces macrophage cell death

Francisella tularensis, the causative agent of tularemia, is a potent human and animal pathogen. Its principal survival mechanism is rapid intracellular multiplication. The mechanisms that enables it to multiply intracellularly have been ill-defined and the thesis focused on characterizing the outcome of the macrophage-Francisella interaction and also if the interactions differ between the various subspecies of F tularensis. The nature of host cell death was examined and the correlation of macrophage killing with intramacrophage Francisella growth was investigated by in vitro infection of J774A.1 macrophages with either the live vaccine (LVS) strain of F. tularensis, belonging to subspecies holarctica, or the subspecies novicida strain U112 Macrophage entry was in both cases cytochalasin D-sensitive but the intramacrophage growth of the two Francisella strains led to distinct types of host cell death, i.e., apoptosis vs. necrosis. The macrophage apoptosis induced by infection with the LVS strain was mediated via the intrinsic pathway with critical involvement of caspase-3 and the mitochondria. The infected and apoptotic macrophages were shrunken, their chromatin was specifically degraded and revealed a typical DNA ladder pattern upon electrophoresis. Moreover, they were TUNEL positive, indicating the occurrence of apoptosis-dependent DNA fragmentation. The necessity of intracellular growth for the apoptosis was shown by the use of an isogenic mutant, denoted iglC, which lacked the ability to multiply intracellularly and this infection did not result in apoptosis. The F. novicida strain U112, on the other hand, inhibited NF- B activity and ultimately induced macrophage necrosis. The infected and necrotic macrophages were enlarged, their chromatin was randomly degraded which gave a diffuse DNA pattern typical of necrosis. There was no apoptosis-specific caspase activation. By the use of an isogenic mutant, denoted mglA, it was shown that intracellular replication was necessary for the induction of necrosis. A hemolytic protein, novilysin A, was found in the F. novicida strain U112 but lacking in other subspecies of F. tularensis. The protein is a putative virulence factor but most likely not involved in the induction of necrosis. Its significance for the pathogenesis of F. novicida remains to be determined. The findings of the thesis provide a detailed picture of the interaction between the host cells and various subspecies of F. tularensis. It also shows that the outcome of the interaction is critically dependent on the type of F. tularensis subspecies. The findings also question the use of F. novicida as a model organism for understanding pathogenicity mechanisms of the species in general. The induction of the host cell death is presumably an important mechanism for the survival of F. tularensis since it allows the bacterium to escape from cells deplete of nutrients and subsequently to invade cells with an intact supply of nutrients necessary for its continuous multiplication