Macrophage Selenoproteins Restrict Intracellular Replication of Francisella tularensis and Are Essential for Host Immunity.

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.

Case Report: Atypical Cutaneous Manifestations of Tularemia after Horsefly Bite.

Tularemia is an infectious zoonosis caused by Francisella tularensis, an aerobic, noncapsulated, Gram-negative coccobacillus. It is more common in the northern hemisphere, and there are sporadic reports in non-endemic areas. The bacterium is usually transmitted by the bite or feces of a tick or other arthropods such as mosquitoes and horseflies. We report a case of an Italian patient with tularemia after a horsefly bite.

Humanized Mice for the Evaluation of Francisella tularensis Vaccine Candidates.

Francisella tularensis (FT), a highly infectious pathogen, is considered to be a potential biological weapon owing to the current lack of a human vaccine against it. Tul4 and FopA, both outer membrane proteins of FT, play an important role in the bacterium's immunogenicity. In the present study, we evaluated the immune response of mice-humanized with human CD34+ cells (hu-mice)-to a cocktail of recombinant Tul4 and FopA (rTul4 and rFopA), which were codon-optimized and expressed in Escherichia coli. Not only did the cocktail-immunized hu-mice produce a significant human immunoglobulin response, they also exhibited prolonged survival against an attenuated live vaccine strain as well as human T cells in the spleen. These results suggest that the cocktail of rTul4 and rFopA had successfully induced an immune response in the hu-mice, demonstrating the potential of this mouse model for use in the evaluation of FT vaccine candidates.

Characterization of a Francisella tularensis-Caenorhabditis elegans Pathosystem for the Evaluation of Therapeutic Compounds.

Francisella tularensis is a highly infectious Gram-negative intracellular pathogen that causes tularemia. Because of its potential as a bioterrorism agent, there is a need for new therapeutic agents. We therefore developed a whole-animal Caenorhabditis elegans-F. tularensis pathosystem for high-throughput screening to identify and characterize potential therapeutic compounds. We found that the C. elegans p38 mitogen-activate protein (MAP) kinase cascade is involved in the immune response to F. tularensis, and we developed a robust F. tularensis-mediated C. elegans killing assay with a Z' factor consistently of >0.5, which was then utilized to screen a library of FDA-approved compounds that included 1,760 small molecules. In addition to clinically used antibiotics, five FDA-approved drugs were also identified as potential hits, including the anti-inflammatory drug diflunisal that showed anti-F. tularensis activity in vitro Moreover, the nonsteroidal anti-inflammatory drug (NSAID) diflunisal, at 4× MIC, blocked the replication of an F. tularensis live vaccine strain (LVS) in primary human macrophages and nonphagocytic cells. Diflunisal was nontoxic to human erythrocytes and HepG2 human liver cells at concentrations of ≥32 µg/ml. Finally, diflunisal exhibited synergetic activity with the antibiotic ciprofloxacin in both a checkerboard assay and a macrophage infection assay. In conclusion, the liquid C. elegans-F. tularensis LVS assay described here allows screening for anti-F. tularensis compounds and suggests that diflunisal could potentially be repurposed for the management of tularemia.

Nasal Acai polysaccharides potentiate innate immunity to protect against pulmonary Francisella tularensis and Burkholderia pseudomallei Infections.

Pulmonary Francisella tularensis and Burkholderia pseudomallei infections are highly lethal in untreated patients, and current antibiotic regimens are not always effective. Activating the innate immune system provides an alternative means of treating infection and can also complement antibiotic therapies. Several natural agonists were screened for their ability to enhance host resistance to infection, and polysaccharides derived from the Acai berry (Acai PS) were found to have potent abilities as an immunotherapeutic to treat F. tularensis and B. pseudomallei infections. In vitro, Acai PS impaired replication of Francisella in primary human macrophages co-cultured with autologous NK cells via augmentation of NK cell IFN-γ. Furthermore, Acai PS administered nasally before or after infection protected mice against type A F. tularensis aerosol challenge with survival rates up to 80%, and protection was still observed, albeit reduced, when mice were treated two days post-infection. Nasal Acai PS administration augmented intracellular expression of IFN-γ by NK cells in the lungs of F. tularensis-infected mice, and neutralization of IFN-γ ablated the protective effect of Acai PS. Likewise, nasal Acai PS treatment conferred protection against pulmonary infection with B. pseudomallei strain 1026b. Acai PS dramatically reduced the replication of B. pseudomallei in the lung and blocked bacterial dissemination to the spleen and liver. Nasal administration of Acai PS enhanced IFN-γ responses by NK and γδ T cells in the lungs, while neutralization of IFN-γ totally abrogated the protective effect of Acai PS against pulmonary B. pseudomallei infection. Collectively, these results demonstrate Acai PS is a potent innate immune agonist that can resolve F. tularensis and B. pseudomallei infections, suggesting this innate immune agonist has broad-spectrum activity against virulent intracellular pathogens.

[Evaluation of immunobiological activity of Francisella tularensis C-complex preparations as promising component of subunit vaccines].

Data on influence of Francisella tularensis C-complex preparations on formation of immunity against tularemia are presented. Study of cellular immunity characteristics as well as dynamics of antibody response was carried out on white mice and guinea pigs models. Absence of toxicity, pyrogenicity, and negative effects on immunocompetent cells in combination with protective activity points to possibility of use the C-complex as a component of a subunit vaccine.

[Immunization with cellulose-immobilized antigens. The development of A. E. Gurvitch concept].

A highly purified TUL4-CBD chimeric protein was obtained by one stage purification method. TUL4-CBD protein consists of TUL4 Francisella tularensis mature peptide sequence, Gly-Ser spacer and cellulose binding domain (CBD) of Anaerocellum thermophilum. The TUL4-CBD protein was shown to induce production of specific antibodies to TUL4 protein in laboratory animals.

[Experimental study on the possibility of using live tularemia vaccine to increase resistance to heterologous infection disease]. / Eksperimental'noe obosnovanie vozmozhnosti primeneniia tuliaremiinoi zhivoi vaktsiny glia povysheniia rezistentnosti k geterologichnym infektsionnym zabolevaniiam.

In experiments on guinea pigs immunized with Francisella tularensis 15, or live tularemia vaccine (LTV), the level of heterologous protective effect to dangerous infectious diseases caused by Yersinia pestis, Burkholderia pseudomallei, B. mallei, Mycobacterium tuberculosis was studied. The study revealed that during the first 4 weeks after the subcutaneous immunization with LTV the level of resistance of the immunized animals to heterologous infective agent reliably increased as indicated by the survival rate of the animals, as well as by the survival time of those killed by infection, in comparison with the controls. Later (on day 150 after immunization) differences in death rate between the groups perceptibly decreased. Nevertheless, the 1 1/2-fold increase of the survival time of the challenged immunized animals in comparison with the controls proved the possibility of using immunization with LTV for the urgent prophylaxis and treatment not only of tularemia, but also of plague, glanders, melioidosis and tuberculosis.

Do homeopathic nosodes protect against infection? An experimental test.

CONTEXT: For centuries, homeopathic practitioners have claimed that serially agitated dilutions of infectious agents (called "nosodes") are effective in the prevention of infectious disease. However, no rigorous tests of this claim have been performed. OBJECTIVE: To test whether a nosode of Francisella tularensis-infected tissue could protect from subsequent challenge with this pathogen in vivo. DESIGN: Experimental laboratory test. SETTING: A P3 containment laboratory at an infectious disease research facility. PARTICIPANTS: 142 male C3H/HeN specific, pathogen-free mice. INTERVENTION: Six levels of a nosode prepared from tularemia-infected tissue were produced. All exposures were below the lowest level at which a classical vaccination response was expected. The nosode and dilutent control solutions were administered orally (.03 mL, 3 times per week) for 1 month before and after challenge. Animals were challenged with a potentially lethal dose (LD50 or LD75) of F tularensis, then evaluated for time of death and total mortality. MAIN OUTCOME MEASURES: Mortality and time to death. RESULTS: In a series of 15 trials (n = 142), the tularemia nosode consistently produced increased mean times to death. All but 2 of 15 trials showed reduced time to death in the nosode group and decreased mortality compared with controls. Protection rates averaged 22% over controls compared to 100% protection by standard vaccination. CONCLUSIONS: This study found partial protection from a nosode of tularemia in dilutions below those expected to have protective effects, but not as great as those produced by standard vaccination. If homeopathic nosodes can induce protection from infectious agents for which vaccination is currently unavailable, they may provide an interim method of reducing morbidity or mortality from such agents.

[The efficacy and outlook for the study of live vaccines for the prevention of melioidosis]. / Effektivnost' i perspektivy issledovaniia zhivykh vaktsin dlia profilaktiki melioidoza.

The effect of immunization with Burkholderia pseudomallei, (Pur- and Ts), heterologous vaccines and the recombinant culture of Francisella tularensis RM2, carrying a plasmid with fragments of B. pseudomallei chromosome, was studied on four species of experimental animals, essentially differing by their sensitivity to melioidosis. B. pseudomallei mutants formed the statistically significant level of protection in subcutaneously challenged animals, moderately sensitive to melioidosis, but were not effective when tested, under the same conditions, in animals, highly sensitive to melioidosis. The effect produced by the experimental vaccines under study in animals of all species, subjected to aerogenic challenge, was leveled. The study showed good prospects for the use of tularemia vaccine with a view to create heterologous immunity to melioidosis and the possibility of its use as the basis of bivalent gene engineering vaccine.

[The outlook for the development of live vaccines for the prevention of melioidosis]. / Perspektivy razrabotki zhivykh vaktsin dlia profilaktiki melioidoza.

The effectiveness of immunization with Burkholderia pseudomallei attenuated strains (Pur and Ts), heterologous vaccines and the recombinant culture of Francisella tularensis RM2 carrying a plasmid with fragments of B. pseudomallei chromosome was studied in four species of experimental animals, essentially differing in their sensitivity to melioidosis. The most immunogenic B. pseudomallei mutants, introduced subcutaneously, created a statistically significant level of protection in animals, moderately sensitive to melioidosis, but proved to be ineffective in highly sensitive animal models when tested under the same conditions. In aerogenic infection the effectiveness of the experimental vaccines under study in all species of the animals was on the same level. The study showed good prospects of using tularemia vaccine for inducing heterologous immunity to melioidosis, as well as the possibility of its use as the basis of a bivalent gene-engineering vaccine.

[The immunological efficacy of Francisella tularensis outer membranes for hamadryas baboons]. / Immunologicheskaia éffektivnost' vneshnikh membran Francisella tularensis dlia obez'ian-gamadril.

The protective properties of the preparation of F. tularensis outer membranes (OM), obtained from F. tularensis vaccine strain 15, were studied in experiments on hamadryas baboons challenged subcutaneously with F. tularensis virulent strain Schu (nonarctic subspecies). The subcutaneous immunization with the OM preparation prevented the development of clinically pronounced infection in more than 70% of the monkeys challenged with F. tularensis strain Schu in a dose of 787 live microbial cells 30 days after immunization. Antibody titers determined in the immunized monkeys with the use of the agglutination test (AT) and the passive hemagglutination test (PHAT) were usual in minimal diagnostic limits (1:80 for AT and 1:320 for PHAT) and did not significantly rise by day 20 after immunization. In all intact animals infected with F. tularensis strain Schu the development of the infectious process was registered, which was accompanied by a rise in temperature exceeding 39.5 degrees C and a rise in the titer of specific antibodies.

[The protective properties of the outer membranes of Francisella tularensis in an experimental infection in guinea pigs]. / Zashchitnye svoistva vneshnykh membran Francisella tularensis pri éksperimental'noi infektsii morskikh svinok.

Subcutaneous immunization, made in a single injection, with outer membrane preparations obtained from F.tularensis vaccine strain 15 and virulent strain A'Cole results in intensive immunity to tularemia in guinea pigs, ensuring the protection of 60-100% of the animals within a month after challenge with F.tularensis virulent strain 503 in a dose of 1,000 DCL. The development of protective effect induced by F.tularensis outer membranes can be observed during the first 24 hours and reaches its maximum by days 15-21 after immunization.

Growth inhibition of Francisella tularensis live vaccine strain by IFN-gamma-activated macrophages is mediated by reactive nitrogen intermediates derived from L-arginine metabolism.

We have examined the abilities of the recombinant murine lymphokines IFN-gamma, granulocyte-macrophage (GM)-CSF, and IL-4 to stimulate the in vitro antimicrobial activity of macrophages against the live vaccine strain (LVS) of Francisella tularensis. Resident peritoneal macrophages from C57BL/6 strain mice were cultured overnight with IFN-gamma, GM-CSF, or IL-4, and then infected with LVS. In macrophages treated with IFN-gamma, the growth of LVS was suppressed by a factor of 100- to 1000-fold in comparison with untreated cells. This effect was dose-dependent and was enhanced by the addition of LPS. In contrast, macrophages treated with either GM-CSF or IL-4 exhibited no such enhanced antitularemic activity, even in the presence of LPS. Because reactive nitrogen intermediates derived from L-arginine metabolism have been implicated in the killing of various infectious organisms, we evaluated the possibility that such a mechanism might contribute to the antitularemic activity of IFN-gamma-stimulated macrophages. Macrophages were treated with NG-monomethyl-L-arginine (NMMA), an inhibitor of L-arginine metabolism in mammalian cells, during the activation procedure and throughout the course of infection. NMMA had no effect on the growth of LVS in unstimulated macrophages. In macrophages activated with IFN-gamma, however, NMMA suppressed their capacity to inhibit LVS growth. This effect was proportional to the dose of NMMA added and reversible by supplementing the medium with additional L-arginine, and there was a direct correlation between the production of nitrite by activated macrophages and their ability to inhibit LVS growth. Furthermore, the growth of LVS was inhibited by nitrogen metabolites in a cellfree system. The results of this study indicate that the mechanism of action of IFN-gamma on the resistance of macrophages to LVS growth is related, at least in part, to the production of reactive nitrogen metabolites.

[The preventive activity of monoclonal antibodies specific to the lipopolysaccharide of Francisella tularensis]. / Preventivnaia aktivnost' monoklonal'nykh antitel, spetsifichnykh k lipopolisakharidu Francisella tularensis.

The preventive activity of five monoclonal antibodies (McAb) in experimental tularemia was evaluated. McAb produced by hybridoma FB11-k (IgG2a), specific to F. tularensis lipopolysaccharide, prevented the death of mice and guinea pigs infected with F. tularensis virulent strain 503 of the holarctic subspecies.