Intracellular bacteria have developed various mechanisms to enter and persist in host cells and, at the same time, to evade the host immune response. One such pathogen is Francisella tularensis, the etiological agent of tularemia. After phagocytosis, this Gram-negative bacterium quickly escapes from the phagocytic compartment and replicates in the host cell cytosol. For this mode of infection, several components of the Francisella pathogenicity island (FPI) are critical. Interestingly, some FPI proteins share homology to components of Type VI Secretion Systems (T6SSs), but their assembly and functionality remains to be shown in Francisella.The thesis focused on the characterization of several of these FPI components; more specifically, how they contribute to the infection cycle as well as their possible role in the putative T6SS. We identified three unique mutants, ΔiglG, ΔiglI and ΔpdpE, which to various degrees were able to escape the phagosomal compartment, replicate in the host cytosol and cause host cell cytotoxicity. In contrast, ΔiglE as well as mutants within the conserved core components of T6SSs, VgrG and DotU, were defective for all of these processes. In the case of IglE, which is a lipoprotein and localized to the outer membrane of the bacterial cell wall, residues within its N-terminus were identified to be important for IglE function. Consistent with a suggested role as a trimeric membrane puncturing device, VgrG was found to form multimers. DotU stabilized the inner membrane protein IcmF, in agreement with its function as a core T6SS component. The functionality of the secretion system was shown by the translocation of several FPI proteins into the cytosol of infected macrophages, among them IglE, IglC and VgrG, of which IglE was the most prominently secreted protein. At the same time, the secretion was dependent on the core components VgrG, DotU but also on IglG. Although we and others have shown the importance of FPI proteins for the escape of F. tularensis, it has been difficult to assess their role in the subsequent replication, since mutants that fail to escape never reach the growth-permissive cytosol. For this reason, selected FPI mutants were microinjected into the cytosol of different cell types and their growth compared to their replication upon normal uptake. Our data suggest that not only the metabolic adaptation to the cytosolic compartment is important for the replication of intracytosolic bacteria, but also the mechanism of their uptake as well as the permissiveness of the cytosolic compartment per se.

Francisella tularensis is a gram-negative, highly virulent, intracellular bacterium which causes the zoonotic disease tularemia. The subspecies tularensis and holarctica are clinically important, and the former is the more virulent. The intracellular lifestyle of F. tularensis is not completely understood, but after uptake in monocytes, the bacterium escapes from the phagosome within hours and replicates massively in the cytosol. The escape is dependent on factors encoded by the Intracellular Growth Locus (igl) operon, located in the Francisella Pathogenicity Island, FPI. The thesis was aimed to clarify and understand the interaction of F. tularensis strains with the endosomal pathway of monocytic cells in general and the roles of the Igl proteins and the global regulator MglA for this interaction in particular. A focus has also been to elucidate the roles of reactive oxygen and nitrogen species for the intracellular host-parasite interaction. We show that mutants in the IglB, IglC, or IglD proteins or their regulator MglA of the live vaccine strain, LVS (subspecies holarctica), all demonstrated reduced replication rates and lowered cytopathogenicity compared to the wild type in a J774 mouse macrophage cell model. Colocalization with LAMP-1 was significantly increased for the IglC, IglD and MglA mutants compared to LVS. This indicated an impaired ability to escape into the cytoplasm, while at the same time they, like LVS, partly prevented fusion with lysosomes. IFN-γ activation of the J774 host cells prior to infection had a bactericidal effect on LVS and all of the mutants, though the cidal effect was significantly more pronounced for the mutants. Following IFN-γ activation, a majority of the mutant-containing phagosomesfused with lysosomeswhile LVS remained localized in the cytosol without significantly increased interactions with the endosomal pathway. Previous studies have revealed that IFN-γ activation of F. tularensis-infected macrophages leads to control of infection but conclusions about the importance of reactive nitrogen and oxygen species on bacterial killing are inconsistent. We found that the growth inhibition resulting from IFN-γ activation could not be attributed to an increased oxidative burst since PMA-induced superoxide production was still inhibited by LVS to the same extent as in non-activated macrophages. On the other hand, reactive nitrogen species may in part have contributed to the cidal effect. To further assess the role of reactive nitrogen species to the killing of F. tularensis, nitric oxide was administrated exogenously to J774 cells infected with LVS. This led to significant killing of intracellular LVS with a concomitant increased phagosomal localization and downregulation of the virulence gene regulator mglA. These effects were reversed by addition of a peroxynitrite decomposition catalyst. A spontaneous avirulent mutant of subspecies tularensis, strain FSC043, was previously demonstrated to provide protective immunity in mice. Here, microscopic analyses of the strain revealed an unusual intracellular localization with a delayed phagosomal escape. This may account for the low virulence, while at the same time FSC043 remains immunogenic and thereby confers protection. The igl operon is intact in strain FCS043 and we hypothesize that a defect in the FPI gene pdpC contributed to the observed phenotype. Altogether, this thesis work demonstrates the importance of the mglA and igl genes for the virulence of F. tularensis and specifically their important roles for a functional phagosomal escape and inhibition of the host cell oxidative burst. Also, addition of exogenous nitric oxide likely leads to formation of peroxynitrite intracellularly, a reactive molecule which confines the bacterium to the phagosome and confers a significant bactericidal effect on intracellular F. tularensis.