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Previously, we identified a spontaneous, essentially avirulent mutant, FSC043, of the highly virulent strain SCHU S4 of Francisella tularensis subsp. tularensis. We have now characterized the phenotype of the mutant and the mechanisms of its attenuation in more detail. Genetic and proteomic analyses revealed that the pdpE gene and most of the pdpC gene were very markedly downregulated and, as previously demonstrated, that the strain expressed partially deleted and fused fupA and fupB genes. FSC043 showed minimal intracellular replication and induced no cell cytotoxicity. The mutant showed delayed phagosomal escape; at 18 h, colocalization with LAMP-1 was 80%, indicating phagosomal localization, whereas the corresponding percentages for SCHU S4 and the Delta fupA mutant were < 10%. However, a small subset of the FSC043-infected cells contained up to 100 bacteria with LAMP-1 colocalization of around 30%. The unusual intracellular phenotype was similar to that of the Delta pdpC and Delta pdpC Delta pdpE mutants. Complementation of FSC043 with the intact fupA and fupB genes did not affect the phenotype, whereas complementation with the pdpC and pdpE genes restored intracellular replication and led to marked virulence. Even higher virulence was observed after complementation with both double-gene constructs. After immunization with the FSC043 strain, moderate protection against respiratory challenge with the SCHU S4 strain was observed. In summary, FSC043 showed a highly unusual intracellular phenotype, and based on our findings, we hypothesize that the mutation in the pdpC gene makes an essential contribution to the phenotype.

Francisella tularensis is a highly virulent intracellular bacterium capable of rapid multiplication in phagocytic cells. Previous studies have revealed that activation of F. tularensis-infected macrophages leads to control of infection and reactive nitrogen and oxygen species make important contributions to the bacterial killing. We investigated the effects of adding S-nitroso-acetyl-penicillamine (SNAP), which generates nitric oxide or 3-morpholinosydnonimine hydrochloride (SIN-1), which indirectly leads to formation of peroxynitrite, to J774 murine macrophage-like cell cultures infected with F. tularensis LVS. Addition of SNAP led to significantly increased colocalization between LAMP-1 and bacteria, indicating containment of F. tularensis in the phagosome within 2 h, although no killing occurred within 4 h. A specific inhibitory effect on bacterial transcription was observed since the gene encoding the global regulator MglA was inhibited 50- to 100-fold. F. tularensis-infected J774 cells are incapable of secreting TNF-alpha in response to Escherichia coli LPS but addition of SNAP almost completely reversed the suppression. Similarly, infection with an MglA mutant did not inhibit LPS-induced TNF-alpha secretion of J774 cells. Strong staining for nitrotyrosine was observed in SNAP-treated bacteria and mass spectrometry identified nitration of two ribosomal 50S proteins, a CBS domain pair protein, and bacterioferritin. The results demonstrate that addition of SNAP initially did not affect the viability of intracellular F. tularensis LVS but led to containment of the bacteria in the phagosome. Moreover, the treatment resulted in nitration of several F. tularensis proteins.

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.

The Francisella tularensis live vaccine strain (LVS), in contrast to its iglC mutant, replicates in the cytoplasm of macrophages. We studied the outcome of infection of the murine macrophagelike cell line J774A.1 with LVS and with iglC, iglD, and mglA mutants, the latter of which is deficient in a global regulator. Compared to LVS, all of the mutants showed impaired intracellular replication up to 72 h, and the number of the mglA mutant bacteria even decreased. Colocalization with LAMP-1 was significantly increased for all mutants compared to LVS, indicating an impaired ability to escape into the cytoplasm. A lysosomal acidity-dependent dye accumulated in approximately 40% of the vacuoles containing mutant bacteria but not at all in vacuoles containing LVS. Preactivation of the macrophages with gamma interferon inhibited the intracellular growth of all strains and significantly increased acidification of phagosomes containing the mutants, but it only slightly increased the LAMP-1 colocalization. The intracellular replication and phagosomal escape of the iglC and iglD mutants were restored by complementation in trans. In conclusion, the IglC, IglD, and MglA proteins each directly or indirectly critically contribute to the virulence of F. tularensis LVS, including its intracellular replication, cytoplasmic escape, and inhibition of acidification of the phagosomes.