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Several bacterial pathogens interact with their host through protein secretion effectuated by a type VI secretion system (T6SS). Francisella tularensis is a highly pathogenic intracellular bacterium that causes the disease tularemia. Proteins encoded by the Francisella pathogenicity island (FPI), which constitute a type VI secretion system, are essential for the virulence of the bacterium and a key mechanism behind this is the escape from the phagosome followed by productive cytosolic replication. It has been shown that T6SS in Francisella is distinct since all putative substrates of F. tularensis T6SS, except for VgrG, are unique to the species. Many of the FPI proteins are secreted into the macrophage cytosol and this is dependent on the functional components of DotU, VgrG, IglC and IglG. In addition, PdpC seems to have a regulatory role for the expression of iglABCD. Since previous results showed peculiar phenotypes of the Δ pdp C and Δ igl G mutants in mouse macrophages, their unique behavior was characterized in human monocyte-derived macrophages (HMDM) in this study. Our results show that both Δ pdpC and Δ iglG mutants of the live vaccine strain (LVS) of F. tularensis did not replicate within HMDMs. The Δ pdpC mutant did not escape from the Francisella containing phagosome (FCP), neither caused cytopathogenicity in primary macrophages and was attenuated in a mouse model. Interestingly, the Δ iglG mutant escaped from the HMDMs FCP and also caused pathological changes in the spleen and liver tissues of intradermally infected C57BL/6 mice. The Δ iglG mutant, with its unique phenotype, is a potential vaccine candidate. [ABSTRACT FROM AUTHOR]

Abstract: Although survival of primary infection with the live vaccine strain (LVS) of Francisella tularensis depends on interferon gamma (IFN-γ), the relative importance of IFN-γ to secondary protective immunity in vivo has not been clearly established. Here we examine the role of IFN-γ in T cell priming and expression of vaccine-induced protection against lethal intraperitoneal challenge of mice. Large amounts of IFN-γ were detected between days 3 and 7 in the sera of LVS-immunized mice, while relatively small amounts were found transiently after secondary LVS challenge. Consistent with the production of this cytokine, mice lacking IFN-γ (gamma interferon knockout, GKO, mice) could not be successfully vaccinated with LVS or an attenuated mglA mutant of F. novicida to withstand secondary Francisella LVS challenge. Further, splenocytes from such primed mice did not adoptively transfer protection to naive GKO recipient mice in vivo, nor control the intramacrophage growth of LVS in vitro. Finally, LVS-immune WT mice depleted of IFN-γ prior to intraperitoneal challenge survived only the lowest doses of challenge. Thus successful priming of protective LVS-immune T cells, as well as complete expression of protection against Francisella during secondary challenge, depends heavily on IFN-γ. [Copyright &y& Elsevier]

Abstract: We demonstrated previously that mice treated with bacterial or oligonucleotide DNA containing unmethylated CpG motifs are transiently protected against lethal parenteral challenge with the intracellular bacterium Francisella tularensis Live Vaccine Strain (LVS). Here we explore the cellular basis of this protection. Wild-type mice that were treated with CpG oligonucleotide DNA and challenged with a lethal dose of LVS survived, while mice lacking TLR9 did not. In vitro, treatment of LVS-infected macrophages and/or naive splenocytes with oligo DNA had no impact on intracellular bacterial replication. In contrast, in vitro co-culture of LVS-infected macrophages with splenocytes obtained from mice treated with oligo DNA in vivo resulted in control of intracellular LVS growth. Control was reversed by antibodies to interferon-γ or to tumor necrosis factor-α and by inhibition of nitric oxide, and to a lesser degree by antibodies to Interleukin-12. Further, splenocytes from DNA-primed normal, T cell KO, B cell KO, lymphocyte-deficient scid, or perforin KO mice all controlled intra-macrophage LVS growth. Enriched DNA-primed natural killer cells, but not B cells, clearly controlled intracellular LVS growth. Thus, NK cells contribute to DNA-mediated protection by production of cytokines including IFN-γ and TNF-α, resulting in nitric oxide production and control of intracellular Francisella replication. [Copyright &y& Elsevier]

Abstract: Francisella tularensis causes the zoonosis tularemia in humans, and inhaled F. tularensis ssp. novicida induces lethal murine tularemia. Transcription of virulence factors in F. novicida is regulated by macrophage growth locus A (mglA), a global regulator required for bacterial replication in macrophages in vitro. We examined the infectivity and immunogenicity of attenuated F. novicida ΔmglA in the lung in vivo. Aerosolized ΔmglA caused replicative pulmonary infection that peaked at 7 days and was cleared thereafter, without clinical evidence of disease. In contrast, inhalation of wild type F. novicida resulted in more rapid bacterial replication and dissemination leading to death within 96h. Early containment of ΔmglA infection was partially dependent on myeloid differentiation factor 88 and interferon-γ but did not require B or T cells. However, lymphocytes were necessary for subsequent bacterial clearance. Infection with ΔmglA elicited specific IgG1-predominant antibodies and variable interferon-γ recall responses to wild type F. novicida. Inoculation of mice with aerosolized ΔmglA afforded no protection against a subsequent low-dose aerosol challenge with wild type F. novicida. These findings establish that inhalation of F. novicida ΔmglA results in replicative infection that elicits innate and adaptive immune responses but not protective immunity against invasive pneumonic tularemia. [Copyright &y& Elsevier]

Abstract: Francisella tularensis is a highly virulent intracellular pathogen responsible for tularemia. This bacterium is capable of infecting many mammalian species and various cell types, but little is known about the mechanisms of survival and interactions with host cells. We examined the number of infected host cells, cytotoxicity and the role of apoptosis or necrosis in infection-induced cell death. Our results demonstrate that F. tularensis LVS induces apoptosis of infected macrophages within 10 h. At later time points we were also able to detect a dramatic increase in the proportion of necrotic macrophages. We investigated the signalling pathways involved in infection-induced cell death by analysing three mitogen-activated protein kinase (MAPK) pathways that are known to be activated by LPS stimulation; p42/p44 MAPK (Erk1/2), transcription factor c-Jun and p38 MAPK. We identified post-translational activation of both p42 MAPK and p44 MAPK by phosphorylation at threonine and tyrosine residues after infection. Furthermore, treatment of infected cells with MEK1/2 inhibitors abrogated phosphorylation of p42/p44 MAPK and inhibited macrophage apoptosis and necrosis after infection. In contrast, phosphorylation and kinase activity of p38 MAPK was significantly lower in F. tularensis-infected cells, and inhibition of p38 MAPK activity induced apoptosis in uninfected cells. When we monitored JNK-dependent phosphorylation of the transcription factor c-Jun, we did not observe any reactivity with either SAPK/JNK or phospho-SAPK/JNK antibodies at any time point. In conclusion, we demonstrate that F. tularensis LVS infection induces macrophage apoptosis. This process requires activation of the p42/p44 MAPK pathway and is associated with reduced p38 MAPK activity, indicating that infection-induced cell death can be caused by perturbation of these two signalling pathways. [Copyright &y& Elsevier]

To further understand the role of LPS in the pathogenesis of Francisella infection, we characterized murine infection with F. novicida, and compared immunobiological activities of F. novicida LPS and the LPS from F. tularensis live vaccine strain (LVS). F. novicida had a lower intradermal LD50 in BALB/cByJ mice than F. tularensis LVS, and mice given a lethal F. novicida dose intraperitoneally died faster than those given the same lethal F. tularensis LVS dose. However, the pattern of in vivo dissemination was similar, and in vitro growth of both bacteria in bone marrow-derived macrophages was comparable. F. novicida LPS stimulated very modest in vitro proliferation of mouse splenocytes at high doses, but F. tularensis LVS LPS did not. Murine bone marrow macrophages treated in vitro with F. novicida LPS produced IL12 and TNF-α, but did not produce detectable interferon-γ, IL10, or nitric oxide; in contrast, murine macrophages treated with F. tularensis LVS LPS produced none of these mediators. In contrast to clear differences in stimulation of proliferation and especially cytokines, both types of purified LPS stimulated early protection against lethal challenge of mice with F. tularensis LVS, but not against lethal challenge with F. novicida. Thus, although LPS recognition may not be a major factor in engendering protection, the ability of F. novicida LPS to stimulate the production of proinflammatory cytokines including TNF-α likely contributes to the increased virulence for mice of F. novicida compared to F. tularensis LVS. [Copyright &y& Elsevier]