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The essential micronutrient Selenium (Se) is co-translationally incorporated as selenocysteine into proteins. Selenoproteins contain one or more selenocysteines and are vital for optimum immunity. Interestingly, many pathogenic bacteria utilize Se for various biological processes suggesting that Se may play a role in bacterial pathogenesis. A previous study had speculated that Francisella tularensis, a facultative intracellular bacterium and the causative agent of tularemia, sequesters Se by upregulating Se-metabolism genes in type II alveolar epithelial cells. Therefore, we investigated the contribution of host vs. pathogen-associated selenoproteins in bacterial disease using F. tularensis as a model organism. We found that F. tularensis was devoid of any Se utilization traits, neither incorporated elemental Se, nor exhibited Se-dependent growth. However, 100% of Se-deficient mice (0.01 ppm Se), which express low levels of selenoproteins, succumbed to F. tularensis-live vaccine strain pulmonary challenge, whereas 50% of mice on Se-supplemented (0.4 ppm Se) and 25% of mice on Se-adequate (0.1 ppm Se) diet succumbed to infection. Median survival time for Se-deficient mice was 8 days post-infection while Se-supplemented and -adequate mice was 11.5 and >14 days post-infection, respectively. Se-deficient macrophages permitted significantly higher intracellular bacterial replication than Se-supplemented macrophages ex vivo, corroborating in vivo observations. Since Francisella replicates in alveolar macrophages during the acute phase of pneumonic infection, we hypothesized that macrophage-specific host selenoproteins may restrict replication and systemic spread of bacteria. F. tularensis infection led to an increased expression of several macrophage selenoproteins, suggesting their key role in limiting bacterial replication. Upon challenge with F. tularensis, mice lacking selenoproteins in macrophages (TrspM) displayed lower survival and increased bacterial burden in the lung and systemic tissues in comparison to WT littermate controls. Furthermore, macrophages from TrspM mice were unable to restrict bacterial replication ex vivo in comparison to macrophages from littermate controls. We herein describe a novel function of host macrophage-specific selenoproteins in restriction of intracellular bacterial replication. These data suggest that host selenoproteins may be considered as novel targets for modulating immune response to control a bacterial infection.

Francisella tularensis (Ft) is a Gram-negative, facultative intracellular coccobacillus that is the etiological agent of tularemia. Interestingly, the disease tularemia has variable clinical presentations that are dependent upon the route of infection with Ft. Two of the most likely routes of Ft infection include intranasal and intradermal, which result in pneumonic and ulceroglandular tularemia, respectively. While there are several differences between these two forms of tularemia, the most notable disparity is between mortality rates: the mortality rate following pneumonic tularemia is over ten times that of the ulceroglandular disease. Understanding the differences between intradermal and intranasal Ft infections is important not only for clinical diagnoses and treatment but also for the development of a safe and effective vaccine. However, the immune correlates of protection against Ft, especially within the context of infection by disparate routes, are not yet fully understood. Recent advances in different animal models have revealed new insights in the complex interplay of innate and adaptive immune responses, indicating dissimilar patterns in both responses following infection with Ft via different routes. Further investigation of these differences will be crucial to predicting disease outcomes and inducing protective immunity via vaccination or natural infection.

Bacterial ribosomes frequently translate to the 3’ end of an mRNA without terminating at an in-frame stop codon. In all bacteria studied to date, these non-stop ribosomes are rescued usingtrans-translation. In some species, genes required fortrans-translation are essential, but other species can survive withouttrans-translation because they express an alternative ribosome rescue factor, ArfA or ArfB.Francisella tularensiscells lackingtrans-translation are viable, butF. tularensisdoes not encode ArfA or ArfB. Transposon mutagenesis followed by deep sequencing (Tn-seq) identified a new alternative ribosome rescue factor, now named ArfT.arfTcan be deleted in wild-type cells but not in cells that lacktrans-translation activity. Over-expression of ArfT suppresses the slow growth phenotype in cells lackingtrans-translation and counteracts growth arrest caused bytrans-translation inhibitors, indicating that ArfT rescues non-stop ribosomesin vivo. Ribosome rescue assaysin vitroshow that ArfT promotes hydrolysis of peptidyl-tRNA on non-stop ribosomes in conjunction withF. tularensisrelease factors. Unlike ArfA, which requires RF2 for activity, ArfT can function with either RF1 or RF2. Overall, these results indicate that ArfT is a new alternative ribosome rescue factor with a distinct mechanism from ArfA and ArfB.ImportanceFrancisella tularensisis a highly infectious intracellular pathogen that kills more than half of infected humans if left untreated.F. tularensishas also been classified as a potential bioterrorism agent with the greatest risk for deliberate misuse. Recently, compounds that inhibit ribosome rescue have been shown to have antibiotic activity againstF. tularensisand other important pathogens. Like all bacteria that have been studied,F. tularensisusestrans-translation as the main pathway to rescue stalled ribosomes. However, unlike most bacteria,F. tularensiscan survive without any of the known factors for ribosome rescue. Our work identifies aF. tularensisprotein, ArfT, that rescues stalled ribosomes in the absence oftrans-translation using a new mechanism. These results indicate that ribosome rescue activity is essential inF. tularensisand suggest that ribosome rescue activity might be essential in all bacteria.

Bacteria require at least one pathway to rescue ribosomes stalled at the ends of mRNAs. The primary pathway for ribosome rescue is trans -translation, which is conserved in >99% of sequenced bacterial genomes. Some species also have backup systems, such as ArfA or ArfB, which can rescue ribosomes in the absence of sufficient trans -translation activity. Small-molecule inhibitors of ribosome rescue have broad-spectrum antimicrobial activity against bacteria grown in liquid culture. These compounds were tested against the tier 1 select agent Francisella tularensis to determine if they can limit bacterial proliferation during infection of eukaryotic cells. The inhibitors KKL-10 and KKL-40 exhibited exceptional antimicrobial activity against both attenuated and fully virulent strains of F. tularensis in vitro and during ex vivo infection. Addition of KKL-10 or KKL-40 to macrophages or liver cells at any time after infection by F. tularensis prevented further bacterial proliferation. When macrophages were stimulated with the proinflammatory cytokine gamma interferon before being infected by F. tularensis , addition of KKL-10 or KKL-40 reduced intracellular bacteria by >99%, indicating that the combination of cytokine-induced stress and a nonfunctional ribosome rescue pathway is fatal to F. tularensis . Neither KKL-10 nor KKL-40 was cytotoxic to eukaryotic cells in culture. These results demonstrate that ribosome rescue is required for F. tularensis growth at all stages of its infection cycle and suggest that KKL-10 and KKL-40 are good lead compounds for antibiotic development.

Tularemia is an endemic zoonotic disease in many parts of the world including Asia. A cross-sectional study was conducted to determine genome-based prevalence of Francisella tularensis (Ft) in soil, assess an association between its occurrence in soil and likely predictors i.e., macro and micro-nutrients and several categorical variables, and determine seroconversion in small and large ruminants. The study included a total of 2,280 soil samples representing 456 villages in eight districts of the Punjab Province of Pakistan followed by an analysis of serum antibodies in 707 ruminants. The genome of Ft was detected in 3.25% (n = 74, 95% CI: 2.60–4.06) of soil samples. Soluble salts (OR: 1.276, 95% CI: 1.043–1.562, p = 0.015), Ni (OR: 2.910, 95%CI: 0.795–10.644, p = 0.106), Mn (OR:0.733, 95% CI:0.565–0.951, p = 0.019), Zn (OR: 4.922, 95% CI:0.929–26.064, p = 0.061) and nutrients clustered together as PC-1 (OR: 4.76, 95% CI: 2.37–9.54, p = 0.000) and PC-3 (OR: 0.357, 95% CI: 0.640, p = 0.001) were found to have a positive association for the presence of Ft in soil. The odds of occurrence of Ft DNA in soil were higher at locations close to a water source, including canals, streams or drains, [χ2 = 6.7, OR = 1.19, 95% CI:1.05–3.09, p = 0.004] as well as places where animals were present [χ2 = 4.09, OR = 2.06, 95% CI: 1.05–4.05, p = 0.02]. The seroconversion was detected in 6.22% (n = 44, 95% CI: 4.67–8.25) of domestic animals. An occurrence of Ft over a wide geographical region indicates its expansion to enzootic range, and demonstrates the need for further investigation among potential disease reservoirs and at-risk populations, such as farmers and veterinarians.

Progress towards a safe and effective vaccine for the prevention of tularemia has been hindered by a lack of knowledge regarding the correlates of protective adaptive immunity and a lack of tools to generate this knowledge. CD8+ T cells are essential for protective immunity against virulent strains of Francisella tularensis, but to-date, it has not been possible to study these cells in an antigen-specific manner. Here, we report the development of a tool for expression of the model antigen ovalbumin (OVA) in F. tularensis, which allows for the study of CD8+ T cell responses to the bacterium. We demonstrate that in response to intranasal infection with the F. tularensis Live Vaccine Strain, adoptively transferred OVA-specific CD8+ T cells expand after the first week and produce IFN-γ but not IL-17. Effector and central memory subsets develop with disparate kinetics in the lungs, draining lymph node and spleen. Notably, OVA-specific cells are poorly retained in the lungs after clearance of infection. We also show that intranasal vaccination leads to more antigen-specific CD8+ T cells in the lung-draining lymph node compared to scarification vaccination, but that an intranasal booster overcomes this difference. Together, our data show that this novel tool can be used to study multiple aspects of the CD8+ T cell response to F. tularensis. Use of this tool will enhance our understanding of immunity to this deadly pathogen.

Progress towards a safe and effective vaccine for the prevention of tularemia has been hindered by a lack of knowledge regarding the correlates of protective adaptive immunity and a lack of tools to generate this knowledge. CD8+T cells are essential for protective immunity against virulent strains ofFrancisella tularensis, but to-date, it has not been possible to study these cells in a pathogen-specific manner. Here, we report the development of a tool for expression of the model antigen ovalbumin (OVA) inF. tularensis, which allows for the study of CD8+T cell responses to the bacterium. We demonstrate that in response to intranasal infection with theF. tularensisLive Vaccine Strain, pathogen-specific CD8+T cells expand after the first week and produce IFN-γ but not IL-17. Effector and central memory subsets develop with disparate kinetics in the lungs, draining lymph node and spleen. Notably,F. tularensis-specific cells are poorly retained in the lungs after clearance of infection. We also show that intranasal vaccination leads to more pathogen-specific CD8+T cells in the lung-draining lymph node compared to scarification vaccination, but that an intranasal booster overcomes this difference. Together, our data show that this novel tool can be used to study multiple aspects of the CD8+T cell response toF. tularensis. Use of this tool will enhance our understanding of immunity to this deadly pathogen.

Francisella tularensis subsp. holarctica is found in North America and much of Europe and causes the disease tularemia in humans and animals. An aquatic cycle has been described for this subspecies, which has caused waterborne outbreaks of tularemia in at least 10 countries. In this study, we sought to identify the mechanosensitive channel(s) required for the bacterium to survive the transition from mammalian hosts to freshwater, which is likely essential for the transmission of the bacterium between susceptible hosts. A single 165-amino-acid MscS-type mechanosensitive channel ( Ft MscS) was found to protect F. tularensis subsp. holarctica from hypoosmotic shock, despite lacking much of the cytoplasmic vestibule domain found in well-characterized MscS proteins from other organisms. The deletion of this channel did not affect virulence within the mammalian host; however, Ft MscS was required to survive the transition from the host niche to freshwater. The deletion of Ft MscS did not alter the sensitivity of F. tularensis subsp. holarctica to detergents, H 2 O 2 , or antibiotics, suggesting that the role of Ft MscS is specific to protection from hypoosmotic shock. The deletion of Ft MscS also led to a reduced average cell size without altering gross cell morphology. The mechanosensitive channel identified and characterized in this study likely contributes to the transmission of tularemia between hosts by allowing the bacterium to survive the transition from mammalian hosts to freshwater. IMPORTANCE The contamination of freshwater by Francisella tularensis subsp. holarctica has resulted in a number of outbreaks of tularemia. Invariably, the contamination originates from the carcasses or excreta of infected animals and thus involves an abrupt osmotic downshock as the bacteria enter freshwater. How F. tularensis survives this drastic change in osmolarity has not been clear, but here we report that a single mechanosensitive channel protects the bacterium from osmotic downshock. This channel is functional despite lacking much of the cytoplasmic vestibule domain that is present in better-studied organisms such as Escherichia coli ; this report builds on previous studies that have suggested that parts of this domain are dispensable for downshock protection. These findings extend our understanding of the aquatic cycle and ecological persistence of F. tularensis , with further implications for mechanosensitive channel biology.

Francisella tularensis , the causative agent of tularemia, is most deadly in the pneumonic form; therefore, mucosal immunity is an important first line of defense against this pathogen. We have now evaluated the lethality of primary F. tularensis live vaccine strain (LVS) pulmonary infection in mice that are defective in IgA (IgA −/− mice), the predominant mucosal Ig isotype. The results showed that IgA −/− mice were more susceptible than IgA +/+ mice to intranasal F. tularensis LVS infection, despite developing higher levels of LVS-specific total, IgG, and IgM antibodies in the bronchoalveolar lavage specimens following infection. In addition, the absence of IgA resulted in a significant increase in bacterial loads and reduced survival. Interestingly, IgA −/− mice had lower pulmonary gamma interferon (IFN-γ) levels and decreased numbers of IFN-γ-secreting CD4 + and CD8 + T cells in the lung on day 9 postinfection compared to IgA +/+ mice. Furthermore, IgA −/− mice displayed reduced interleukin 12 (IL-12) levels at early time points, and supplementing IgA −/− mice with IL-12 prior to LVS challenge induced IFN-γ production by NK cells and rescued them from mortality. Thus, IgA −/− mice are highly susceptible to primary pulmonary LVS infections not only because of IgA deficiency but also because of reduced IFN-γ responses.

Francisella tularensis is a highly pathogenic gram negative bacterium that infects multiple sites in a host, including the skin and the respiratory tract, which can lead to the onset of a deadly disease with a 50% mortality rate. The live vaccine strain (LVS) of F. tularensis, while attenuated in humans but still virulent in mice, is not an option for vaccine use in the United States due to safety concerns, and currently no FDA approved vaccine exists. The purpose of the present work was to assess the ability of recombinant Francisella outer membrane protein A (FopA) to induce a protective response in mice. The gene encoding FopA from F. tularensis LVS was cloned and expressed in Escherichia coli. The resulting recombinant protein was affinity-purified from the E. coli outer membrane, incorporated into liposomes and administered to mice via multiple routes. FopA-immunized mice produced FopA-specific antibodies and were protected against both lethal intradermal and intranasal challenges with F. tularensis LVS. The vaccinated mice had reduced bacterial numbers in their lungs, livers and spleens during infection, and complete bacterial clearance was observed by day 28 post infection. Passive transfer of FopA-immune serum protected naïve mice against lethal F. tularensis LVS challenge, showing that humoral immunity played an important role in vaccine efficacy. FopA-immunization was unable to protect against challenge with the fully virulent SchuS4 strain of F. tularensis; however, the findings demonstrate proof of principle that an immune response generated against a component of a subunit vaccine is protective against lethal respiratory and intradermal tularemia.

Vaccination of mice with Francisella tularensis live vaccine strain (LVS) mutants described so far have failed to induce protection in C57BL/6 mice against challenge with the virulent strain F. tularensis SchuS4. We have previously reported that a mutant of F. tularensis LVS deficient in iron superoxide dismutase (sodB(Ft)) is hypersensitive to oxidative stress and attenuated for virulence in mice. Herein, we evaluated the efficacy of this mutant as a vaccine candidate against respiratory tularemia caused by F. tularensis SchuS4. C57BL/6 mice were vaccinated intranasally (i.n.) with the sodB(Ft) mutant and challenged i.n. with lethal doses of F. tularensis SchuS4. The level of protection against SchuS4 challenge was higher in sodB(Ft) vaccinated group as compared to the LVS vaccinated mice. sodB(Ft) vaccinated mice following SchuS4 challenge exhibited significantly reduced bacterial burden in lungs, liver and spleen, regulated production of pro-inflammatory cytokines and less severe histopathological lesions compared to the LVS vaccinated mice. The sodB(Ft) vaccination induced a potent humoral immune response and protection against SchuS4 required both CD4 and CD8 T cells in the vaccinated mice. sodB(Ft) mutants revealed upregulated levels of chaperonine proteins DnaK, GroEL and Bfr that have been shown to be important for generation of a potent immune response against Francisella infection. Collectively, this study describes an improved live vaccine candidate against respiratory tularemia that has an attenuated virulence and enhanced protective efficacy than the LVS.

We investigated the role of interleukin-10 (IL-10) in cutaneous and pulmonary infection with Francisella tularensis . We found that after intradermal challenge of mice with the live vaccine strain (LVS) of F. tularensis , splenic IL-10 levels increased rapidly and reached a peak 5 days after infection. However, IL-10 expression after infection was detrimental, since IL-10 −/− mice showed increased bacterial clearance and were resistant to an infectious dose (>10 6 CFU/mouse) that was uniformly lethal for IL-10 +/+ mice. Furthermore, IL-10 +/+ mice treated with neutralizing anti-IL-10R monoclonal antibody were able to survive lethal cutaneous LVS challenge. The presence of IL-10 appeared to restrain the expression of IL-17, since high levels of splenic IL-17 were observed after intradermal LVS infection only in IL-10 −/− mice. Furthermore, treatment with neutralizing anti-IL-17R antibody ablated the enhanced survival observed in IL-10 −/− mice. However, neutralization of IL-10 activity in IL-17R −/− mice failed to provide protection. Thus, IL-10 suppresses a protective IL-17 response that is necessary for resistance to cutaneous LVS infection. Surprisingly, however, IL-10 −/− mice were significantly more susceptible to pulmonary infection with LVS. Finally, although IL-10 is a critical and novel regulator of immunity to F. tularensis LVS infection, its effects were masked during infection with the highly virulent SchuS4 strain. Taken together, these findings suggest that differentially regulating expression of the IL-10 pathway in various tissues could ultimately have prophylactic and therapeutic benefits for protection against tularemia.

The citrulline ureidase (CTU) activity has been shown to be associated with highly virulentFrancisella tularensisstrains, including Schu S4, while it is absent in avirulent or less virulent strains. A definitive role of thectugene in virulence and pathogenesis ofF. tularensisSchu S4 has not been assessed; thus, an understanding of the significance of this phenotype is long overdue. CTU is a carbon-nitrogen hydrolase encoded by the citrulline ureidase (ctu) gene (FTT0435) on theF. tularensisSchu S4 genome. In the present study, we evaluated the contribution of thectugene in the virulence of category A agentF. tularensisSchu S4 by generating a nonpolar deletion mutant, the Δctumutant. The deletion of thectugene resulted in loss of CTU activity, which was restored by transcomplementing thectugene. The Δctumutant did not exhibit any growth defect under acellular growth conditions; however, it was impaired for intramacrophage growth in resting as well as gamma interferon-stimulated macrophages. The Δctumutant was further tested for its virulence attributes in a mouse model of respiratory tularemia. Mice infected intranasally with the Δctumutant showed significantly reduced bacterial burden in the lungs, liver, and spleen compared to wild-type (WT) Schu S4-infected mice. The reduced bacterial burden in mice infected with the Δctumutant was also associated with significantly lower histopathological scores in the lungs. Mice infected with the Δctumutant succumbed to infection, but they survived longer and showed significantly extended median time to death compared to that shown by WT Schu S4-infected mice. To conclude, this study demonstrates thatctucontributes to intracellular survival, in vivo growth, and pathogenesis. However,ctuis not an absolute requirement for the virulence ofF. tularensisSchu S4 in mice.

Francisella tularensis can cause fatal respiratory tularemia in humans and animals and is increasingly being isolated in the United States and several European countries. The correlates of protective immunity against this intracellular bacterium are not known, and currently there are no licensed vaccines available for human use. Cell-mediated immunity has long been believed to be critical for protection, and the importance of humoral immunity is also now recognized. Furthermore, synergy between antibodies, T cell-derived cytokines, and phagocytes appears to be critical to achieve sterilizing immunity against F. tularensis. Thus, novel vaccine approaches should be designed to induce robust antibody and cell-mediated immune responses to this pathogen.

The intracellular bacterium Francisella tularensis survives in mammals, arthropods, and freshwater amoeba. It was previously established that the conventional media used for in vitro propagation of this microbe do not yield bacteria that mimic those harvested from infected mammals; whether these in vitro-cultivated bacteria resemble arthropod- or amoeba-adapted Francisella is unknown. As a foundation for our goal of identifying F. tularensis outer membrane proteins which are expressed during mammalian infection, we first sought to identify in vitro cultivation conditions that induce the bacterium's infection-derived phenotype. We compared Francisella LVS grown in brain heart infusion broth (BHI; a standard microbiological medium rarely used in Francisella research) to that grown in Mueller-Hinton broth (MHB; the most widely used F. tularensis medium, used here as a negative control) and macrophages (a natural host cell, used here as a positive control). BHI- and macrophage-grown F. tularensis cells showed similar expression of MglA-dependent and MglA-independent proteins; expression of the MglA-dependent proteins was repressed by the supraphysiological levels of free amino acids present in MHB. We observed that during macrophage infection, protein expression by intracellular bacteria differed from that by extracellular bacteria; BHI-grown bacteria mirrored the latter, while MHB-grown bacteria resembled neither. Naïve macrophages responding to BHI- and macrophage-grown bacteria produced markedly lower levels of proinflammatory mediators than those in cells exposed to MHB-grown bacteria. In contrast to MHB-grown bacteria, BHI-grown bacteria showed minimal delay during intracellular replication. Cumulatively, our findings provide compelling evidence that growth in BHI yields bacteria which recapitulate the phenotype of Francisella organisms that have emerged from macrophages.

The role of Abs in protection against respiratory infection with the intracellular bacterium Francisella tularensis is not clear. To investigate the ability of Abs to clear bacteria from the lungs and prevent systemic spread, immune serum was passively administered i.p. to naive mice before intranasal F. tularensis live vaccine strain infection. It was found that immune serum treatment provided 100% protection against lethal challenge while normal serum or Ig-depleted immune serum provided no protection. Protective efficacy was correlated with increased clearance of bacteria from the lung and required expression of FcγR on phagocytes, including macrophages and neutrophils. However, complement was not required for protection. In vitro experiments demonstrated that macrophages were more readily infected by Ab-opsonized bacteria but became highly efficient in killing upon activation by IFN-γ. Consistent with this finding, in vivo Ab-mediated protection was found to be dependent upon IFN-γ. SCID mice were not protected by passive Ab transfer, suggesting that T cells but not NK cells serve as the primary source for IFN-γ. These data suggest that a critical interaction of humoral and cellular immune responses is necessary to provide sterilizing immunity against F. tularensis. Of considerable interest was the finding that serum Abs were capable of conferring protection against lethal respiratory tularemia when given 24–48 h postexposure. Thus, this study provides the first evidence for the therapeutic use of Abs in Francisella-infected individuals.

Respiratory infection with Francisella tularensis is the deadliest form of disease and represents the most likely route to be used by bioterrorists. Although mucosal surfaces represent the first line of defense against respiratory tularemia, and in fact, against the great majority of human pathogens, little is known about protective immunity at these sites. The objective of this chapter is to review recent data examining the importance of various pulmonary immune mechanisms in defense against F. tularensis infection and to evaluate potential strategies for induction of protective lung immunity. Aerosol and intranasal mouse infection models have yielded essentially equivalent results and have implicated an important role for Th1-type immune responses in protection, including IFN-gamma, TNF-alpha, and IL-12. The cells responsible for protection in the lung are not well-characterized but NK cells are an early target for activation after infection although it appears that CD8 T cells might be most critical for host resistance. In addition, it is becoming increasingly clear that antibodies can provide prophylactic and therapeutic protection against pulmonary infection but only in the presence of active cell-mediated immunity. In fact, in vitro exposure of resting macrophages to antibody-coated bacteria in the absence of IFN-gamma can actually enhance infection. Although various immune mechanisms can be shown to be important for protection against attenuated strains such as LVS, the real challenge for the future is to design efficacious approaches to prevent disease by highly virulent strains such as SchuS4.