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Abstract Absent in melanoma 2 (AIM2) is a sensor of cytosolic dsDNA and is responsible for the activation of inflammatory and host immune responses to DNA viruses and intracellular bacteria. AIM2 is a member of the hematopoietic interferon-inducible nuclear proteins with a 200 amino-acid repeat (HIN200) family, containing a pyrin domain (PYD) at the N-terminus. Several studies have demonstrated that AIM2 is responsible for host defense against intracellular bacteria such as Francisella tularensis , Listeria monocytogenes and Mycobacerium tuberculosis. However, the role of AIM2 in host defenses against Brucella is poorly understood. In this study, we have shown that AIM2 senses Brucella DNA in dendritic cells to induce pyroptosis and regulates type I IFN. Confocal microscopy of infected cells revealed co-localization between Brucella DNA and endogenous AIM2. Dendritic cells from AIM2 KO mice infected with B. abortus showed impaired secretion of IL-1β as well as compromised caspase-1 cleavage. AIM2 KO mice displayed increased susceptibility to B. abortus infection in comparison to wild-type mice, and this susceptibility was associated with defective IL-1β production together with reduced IFN-γ responses. In summary, the increased bacterial burden observed in vivo in AIM2 KO animals confirmed that AIM2 is essential for an effective innate immune response against Brucella infection. [ABSTRACT FROM AUTHOR]

Respiratory infection of mice with Francisella novicida has recently been used as a model for the highly virulent human pathogen Francisella tularensis . Similar to F. tularensis , even small doses of F. novicida administered by respiratory routes are lethal for inbred laboratory mice. This feature obviously limits study of infection-induced immunity. Parenteral sublethal infections of mice with F. novicida are feasible, but the resulting immune responses are incompletely characterized. Here we use parenteral intradermal (i.d.) and intraperitoneal (i.p.) F. novicida infections of C57BL/6J mice to determine the role of B cells in controlling primary and secondary F. novicida infections. Despite developing comparable levels of F. novicida -primed T cells, B cell knockout mice were much more susceptible to both primary i.d. infection and secondary i.p. challenge than wild type (normal) C57BL/6J mice. Transfer of F. novicida -immune sera to either wild type C57BL/6J mice or to B cell knockout mice did not appreciably impact survival of subsequent lethal F. novicida challenge. However, F. novicida -immune mice that were depleted of T cells after priming but just before challenge survived and cleared secondary i.p. F. novicida challenge. Collectively these results indicate that B cells, if not serum antibodies, play a major role in controlling F. novicida infections in mice. [ABSTRACT FROM AUTHOR]

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]

Francisella tularensis inhalation results in bacterial interaction with numerous lung cell types, including those of the epithelium. This work investigates a three-dimensional cell-culture system to characterise the epithelial response to F. tularensis . Immortalised human pneumocytes (A549s) grown in rotating-wall vessel (RWV) bioreactors display an in vivo-like phenotype, which has been confirmed to be driven by specific transcriptional events (8454 genes, p ≤ 0.05). These data support the RWV model as a more in vivo-like culture system to investigate the lung epithelium, compared to monolayer counterparts. RWV-cultured A549s were infected with F. tularensis SchuS4 and LVS and intracellular replication mapped over 22 h compared to monolayer cells. The RWV-cultured A549s were more resistant to SchuS4 and LVS infection ( p ≤ 0.05). Transcriptomics identified 2086 genes ( p ≤ 0.05) as candidates for host–pathogen interactions which result in the observed increase in resistance of the RWV-cultured A549 cells. Gene and pathway analysis identified processes involved in MMP modulation, endocytosis, mucin production and the complement pathway amongst others. The role of these pathways during infection was further characterised using chemical inhibitors. This work has revealed several new hypotheses worthy of further testing in order to understand the epithelial host response to F. tularensis infection. [ABSTRACT FROM AUTHOR]

Abstract: Biosynthesis and acquisition of nutrients during infection are integral to pathogenesis. Members of a metabolic pathway, the glycine cleavage system, have been identified in virulence screens of the intracellular bacterium Francisella tularensis but their role in pathogenesis remains unknown. This system generates 5,10-methylenetetrahydrofolate, a precursor of amino acid and DNA synthesis, from glycine degradation. To characterize this pathway, deletion of the gcvT homolog, an essential member of this system, was performed in attenuated and virulent F. tularensis strains. Deletion mutants were auxotrophic for serine but behaved similar to wild-type strains with respect to host cell invasion, intracellular replication, and stimulation of TNF-α. Unexpectedly, the glycine cleavage system was required for the pathogenesis of virulent F. tularensis in a murine model. Deletion of the gcvT homolog delayed mortality and lowered bacterial burden, particularly in the liver and bloodstream. To reconcile differences between the cell culture model and animal model, minimal tissue culture media was employed to mimic the nutritionally limiting environment of the host. This reevaluation demonstrated that the glycine cleavage system contributes to the intracellular replication of virulent F. tularensis in serine limiting environments. Thus, the glycine cleavage system is the serine biosynthetic pathway of F. tularensis and contributes to pathogenesis in vivo. [Copyright &y& Elsevier]

Abstract: Francisella tularensis is a highly infectious facultative intracellular bacterium causing the zoonotic disease tularemia. Numerous attributes required for F. tularensis intracellular multiplication have been identified recently. However, the mechanisms by which the majority of them interfere with the infected host are still poorly understood. The following review summarizes our current knowledge on the different steps of Francisella intramacrophagic life cycle and expands on the importance of nutrient acquisition. [Copyright &y& Elsevier]

Abstract: Pneumonic tularemia is a potentially fatal disease caused by the Category A bioterrorism agent Francisella tularensis. Understanding the pulmonary immune response to this bacterium is necessary for developing effective vaccines and therapeutics. In this study, characterization of immune cell populations in the lungs of mice infected with the type A strain Schu S4 revealed a significant loss in natural killer (NK) cells over time. Since this decline in NK cells correlated with morbidity and mortality, we hypothesized these cells contribute to host defense against Schu S4 infection. Depletion of NK cells prior to Schu S4 challenge significantly reduced IFN-γ and granzyme B in the lung but had no effect on bacterial burden or disease progression. Conversely, increasing NK cell numbers with the anti-apoptotic cytokine IL-15 and soluble receptor IL-15Rα had no significant impact on Schu S4 growth in vivo. A modest decrease in median time to death, however, was observed in live vaccine strain (LVS)-vaccinated mice depleted of NK1.1+ cells and challenged with Schu S4. Therefore, NK cells do not appear to contribute to host defense against acute respiratory infection with type A F. tularensis in vivo, but they play a minor role in protection elicited by LVS vaccination. [Copyright &y& Elsevier]

Abstract: In this paper we evaluate the role of human γδ T cells in control of Francisella tularensis infection. Using an in vitro model of infection, a reduction in bacterial numbers was detected in the presence of human γδ T cells for both attenuated LVS and virulent SCHU S4 strains of F. tularensis. Antibody neutralisation of IFN-γ caused an increase in survival of F. tularensis LVS suggesting that γδ T cell-mediated control of F. tularensis infection is partially mediated by IFN-γ. [Copyright &y& Elsevier]

Abstract: Interactions between Francisella tularensis and the host are slowly being elucidated. Microarray technology was used to further characterise the response of Balb/c mice after inhalation of the virulent F. tularensis, SchuS4. The validated array data revealed changes in expression of 476 genes across a 96 h time course following infection (p ≤ 0.05). These data confirm down-regulation of the toll-like receptor pathway (TLR3, 4, 5, 7 and 8), and the induction of IFN-γ inducible genes (T-cell specific GTPase, β2 microglobulin and interleukin 21). The overall response appears to be two staged with an initial up-regulation of genes involved in apoptosis, TNFα production and antigen presentation. This is followed by a large alteration of expression at 96 h as the host succumbs to infection. A key regulatory time-point has been identified at 24 h post challenge, where several transcriptional events may predicate the progression of infection; these include transcriptional regulators of inflammation and proteolytic pathways. Pathway analysis indicates a novel role for cell–cell adhesion and extracellular matrix modulation in infection. Transcripts representing cellular junctions, focal adhesion and adherens junctions changed following infection. Additionally, aspects of extracellular matrix remodelling have been confirmed at the protein level, suggesting an important role of the respiratory epithelium in host response to F. tularensis warranting further study. [Copyright &y& Elsevier]

Abstract: Francisella tularensis, the causative agent of tularemia, is a highly infectious intracellular pathogen with no licensed vaccine available today. The recent search for genome sequences involved in F. tularensis virulence mechanisms led to the identification of the 30-kb region defined as a Francisella pathogenicity island (FPI). In our previous iTRAQ study we described the concerted upregulation of some FPI proteins in different F. tularensis strains cultivated under stress conditions. Among them we identified the IglH protein whose role in Francisella virulence has not been characterized yet. In this work we deleted the iglH gene in a European clinical isolate of F. tularensis subsp. holarctica FSC200. We showed that the iglH gene is necessary for intracellular growth and escape of F. tularensis from phagosomes. We also showed that the iglH mutant is avirulent in a mouse model of infection and persists in the organs for about three weeks after infection. Importantly, mice vaccinated by infection with the iglH mutant were protected against subcutaneous challenge with the fully virulent parental FSC200 strain. This is the first report of a defined subsp. holarctica FPI deletion strain that provides protective immunity against subsequent subcutaneous challenge with a virulent isolate of F. tularensis subsp. holarctica. [Copyright &y& Elsevier]

Abstract: Francisella tularensis subspecies (subsp.) tularensis is a CDC Category A biological warfare agent and inhalation of as few as 15 bacilli can initiate severe disease. Relatively little is known about the cellular and molecular mechanisms of host defense against respiratory infection with subsp. tularensis. In this study, we examined the role of neutrophils and NADPH phagocyte oxidase in host resistance to pulmonary infection in a mouse intranasal infection model. We found that despite neutrophil recruitment to the lungs and increased concentrations of neutrophil-chemotactic chemokines (KC, MIP-2 and RANTES) in the bronchoalveolar lavage fluid following intranasal inoculation of the pathogen, neither depletion of neutrophils nor enhancement of their recruitment into the lungs had any impact on bacterial burdens or survival rate/time. Nevertheless, mice deficient in NADPH phagocyte oxidase (gp91phox–/– ) did exhibit higher tissue and blood bacterial burdens and succumbed to infection one day earlier than wild-type C57BL/6 mice. These results imply that although neutrophils are not a major effector cell in defense against subsp. tularensis infection, NADPH phagocyte oxidase does play a marginal role. [Copyright &y& Elsevier]

Abstract: Francisella tularensis is a category A select agent based on its infectivity and virulence but disease mechanisms in infection remain poorly understood. Murine pulmonary models of infection were therefore employed to assess and compare dissemination and pathology and to elucidate the host immune response to infection with the highly virulent Type A F. tularensis strain Schu4 versus the less virulent Type B live vaccine strain (LVS). We found that dissemination and pathology in the spleen was significantly greater in mice infected with F. tularensis Schu4 compared to mice infected with F. tularensis LVS. Using gene expression profiling to compare the response to infection with the two F. tularensis strains, we found that there were significant differences in the expression of genes involved in the apoptosis pathway, antigen processing and presentation pathways, and inflammatory response pathways in mice infected with Schu4 when compared to LVS. These transcriptional differences coincided with marked differences in dissemination and severity of organ lesions in mice infected with the Schu4 and LVS strains. Therefore, these findings indicate that altered apoptosis, antigen presentation and production of inflammatory mediators explain the differences in pathogenicity of F. tularensis Schu4 and LVS. [Copyright &y& Elsevier]

Francisella tularensis induces apoptosis within macrophages but the temporal and spatial modulation through activation of caspase-1, caspase-3, and the anti-apoptosis nuclear transcription factor B (NF-κB) is not known. Whether escape of the bacteria into the cytosol is sufficient and/or essential for activation of NF-κB is not known. Our results show that F. tularensis subsp. novicida induces sustained nuclear translocation of NF-κB at early time points after infection of human monocytes derived macrophages (hMDMs). The sustained nuclear translocation of NF-κB is defective in the iglC mutant that fails to escape into the cytosol of macrophages. Nuclear translocation of NF-κB by the wild type strain is abolished upon treatment with the NF-κB inhibitor caffein acid phenyl ester. While the wild type strain triggers caspase-3 and caspase-1 activation by 6 h post-infection the iglC mutant is defective in triggering both caspases. In hMDMs treated with the apoptosis-inducing agent, staurosporin, there is an induction of cell death in the iglC mutant-infected macrophages despite reduced frequency of caspase-1 and caspase-3 activity. The wt-infected macrophages are resistant to cell death-induced agent. We conclude that although caspase-1 and capsase-3 are triggered within F. tularensis-infected hMDMs during early stages of infection, cell death is delayed, which is correlated with simultaneous activation of NF-κB. [Copyright &y& Elsevier]

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: Concerns over weaponizable bacteria have recently prompted considerable interest in Francisella tularensis (Ft). In addition to its potential illicit use, Ft occurs naturally in diverse ecological niches including mammals, arthropods, and fresh water protozoans. Here we review the current knowledge of Ft adaptation which has ramifications for both basic and applied research. [Copyright &y& Elsevier]

Abstract: The remarkable infectiousness of Francisella tularensis suggests that the bacterium efficiently evades innate immune responses that typically protect the host during its continuous exposure to environmental and commensal microbes. In our studies of the innate immune response to F. tularensis, we have observed that, unlike the live vaccine strain (LVS) of F. tularensis subsp. holarctica, F. tularensis subsp. novicida U112 opsonized in pooled human serum activated the NADPH oxidase when incubated with human neutrophils. Given previous observations that F. tularensis fixes relatively small quantities of complement component C3 during incubation in human serum and the importance of C3 to neutrophil phagocytosis, we hypothesized that F. tularensis subsp. novicida may fix C3 in human serum more readily than would LVS. We now report that F. tularensis subsp. novicida fixed approximately six-fold more C3 than did LVS when incubated in 50% pooled human serum and that this complement opsonization was antibody-mediated. Furthermore, antibody-mediated C3 deposition enhanced bacterial uptake and was indispensable for the neutrophil oxidative response to F. tularensis subsp. novicida. Taken together, our results reveal important differences between these two strains of F. tularensis and may, in part, explain the low virulence of F. tularensis subsp. novicida for humans. [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: We used the killing of Galleria mellonella (Lepidoptera: Pyralidae; the greater wax moth) caterpillar by the live vaccine strain (LVS) of Francisella tularensis to develop an invertebrate host system that can be used to study F. tularensis infection and the in vivo effects of antibacterial compounds on F. tularensis LVS. After injection into the insect hemocoel, F. tularensis LVS, killed caterpillars despite the association of LVS with hemocytes. The rate of killing depended on the number of bacteria injected. Antibiotic therapy with ciprofloxacin, levofloxacin or streptomycin administered before or after inoculation prolonged survival and decreased the tissue burden of F. tularensis in the hemocoel. Delayed drug treatment reduced the efficacy of antibacterials and especially streptomycin. The G. mellonella–F. tularensis LVS system may facilitate the in vivo study of F. tularensis, efficacy with antibacterial agents. [Copyright &y& Elsevier]

Abstract: The means by which Francisella tularensis, the causative agent of tularemia, are recognized by mammalian immune systems are poorly understood. Here we wished to explore the contribution of the MyD88/Toll-like receptor signaling pathway in initiating murine responses to F. tularensis Live Vaccine Strain (LVS). MyD88 knockout (KO) mice, but not TLR2-, TLR4- or TLR9-deficient mice, rapidly succumbed following in vivo bacterial infection via the intradermal route even with a very low dose of LVS (5×101) that was 100,000-fold less than the LD50 of normal wild-type (WT) mice. By day 5 after LVS infection, bacterial organ burdens were 5–6 logs higher in MyD88 knockout mice; further, unlike infected WT mice, levels of interferon-γ in the sera of LVS-infected MyD88 KO were undetectable. An in vitro culture system was used to assess the ability of bone marrow macrophages derived from either KO or WT mice to support bacterial growth, or to control intracellular bacterial replication when co-cultured with immune lymphocytes. In this assay, bacterial replication was similar in macrophages derived from either WT or any of the TLR KO mice. Bacterial growth was controlled in co-cultures containing macrophages from MyD88 KO mice or TLR KO mice as well as in co-cultures containing immune WT splenic lymphocytes and WT macrophages. Further, MyD88-deficient LVS-immune splenocytes controlled intracellular growth comparably to those from normal mice. Thus MyD88 is essential for innate host resistance to LVS infection, but is not required for macrophage control of intracellular bacterial growth. [Copyright &y& Elsevier]

Abstract: Research on the highly virulent and contagious, facultative intracellular bacterium Francisella tularensis has come into the limelight recently, but still little is known regarding its virulence mechanisms. This review summarizes recent studies on its intramacrophage survival mechanisms, some of which appear to be novel. [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]

Abstract: Intracellular killing of Francisella tularensis by macrophages depends on interferon-gamma (IFN-γ)-induced activation of the cells. The importance of inducible nitric oxide synthase (iNOS) or NADPH phagocyte oxidase (phox) for the cidal activity was studied. Murine IFN-γ-activated peritoneal exudate cells (PEC) produced nitric oxide (NO), measured as nitrite plus nitrate, and superoxide. When PEC were infected with the live vaccine strain, LVS, of F. tularensis, the number of viable bacteria was at least 1000-fold lower in the presence than in the absence of IFN-γ after 48 h of incubation. PEC from iNOS-gene-deficient (iNOS–/–) mice killed F. tularensis LVS less effectively than did PEC from wild-type mice. PEC from phox gene-deficient (p47 phox –/–) mice were capable of killing the bacteria, but killing was less efficient, although still significant, in the presence of NG-monomethyl-l-arginine (NMMLA), an inhibitor of iNOS. A decomposition catalyst of ONOO–, FeTPPS, completely reversed the IFN-γ-induced killing of F. tularensis LVS. Under host cell-free conditions, F. tularensis LVS was exposed to S-nitroso-acetyl-penicillamine (SNAP), which generates NO, or 3-morpholinosydnonimine hydrochloride (SIN-1), which generates NO and superoxide, leading to formation of ONOO–. During 6 h of incubation, SNAP caused no killing of F. tularensis LVS, whereas effective killing occurred in the presence of equimolar concentrations of SIN-1. The results suggest that mechanisms dependent on iNOS and to a minor degree, phox, contribute to the IFN-γ-induced macrophage killing of F. tularensis LVS. ONOO– is likely to be a major mediator of the killing. [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]

The immune response to intracellular bacterium, Francisella tularensis, which causes tularemia and is proposed to be a potential bioterrorism pathogen, has been studied in mice using the attenuated live vaccine strain (LVS). Here we review this infection model, which provides a convenient means of studying protective immune mechanisms not only for Francisella, but also for the large and important class of intracellular pathogens. [Copyright &y& Elsevier]