Respiratory Tularemia Research Articles

Tularemia-associated hyperinflammation regulation by Natural Killer T cell subsets

Abstract Inflammation is critical for effective clearance of viral and bacterial challenges, while simultaneously a mechanism utilized by many highly infectious pathogens to cause disease. The bacterium Francisella tularensis (Ft), the causative agent of human tularemia, elicits a cytokine storm, in which the immune system over-responds to the presence of the bacterium causing a dysregulated release of proinflammatory and regulatory cytokines. Without treatment, tularemia mortality can be as high as 30%, with inhalation of as few as 10 CFUs inducing respiratory tularemia. Natural Killer T (NKT) cells are innate-like T lymphocytes with a diverse array of regulatory functions. NKT cells are activated very early following Ft infection suggesting a critical role in the innate immune response in tularemia. NKT cells are categorized into two distinct subsets, types I and II, with a yet to be defined understanding of their individual characteristics in regulating inflammation. Through a combination of in vitro and in vivo techniques, we have observed a cooperative suppression of inflammation when both subsets of NKT cells are present. This phenotypic suppression is disrupted when each subtype responds individually to Ft. The cellular mechanism controlling this interdependence of function will yield a better understanding of NKT-regulated inflammation in tularemia, and more broadly characterize their involvement and potential therapeutic application for a number of NIH priority pathogens.

Disparate natural killer T cell subsets exhibit opposing regulatory effects on the cytokine storm.

Abstract Inflammation is both a critical component of the immune system for effective clearance of microbial challenges and a mechanism by which some pathogens cause disease, e.g., tularemia, dengue fever, and COVID-19. The bacterium Francisella tularensis (Ft), the causative agent of tularemia, elicits a cytokine storm in which the immune system over-responds to the bacterium, releasing massive amounts of proinflammatory cytokines. Untreated, tularemia can reach a mortality rate as high as 30% in healthy adults with inhalation of as few as 10 colony forming units inducing respiratory tularemia. While Ftis spread from zoonotic sources in nature, the ease of aerosol spread and high mortality led to its weaponization and continued concern for bioterrorism applications. Natural Killer T (NKT) cells are important immune cells with the capability of modulating the inflammatory response. NKT cells are activated early following Ftinfection suggesting a role in the innate immune response in tularemia. NKT cells are categorized into two distinct subsets, type I and type II based on their antigen recognition, or not, of α-galactosylceramide. Literature suggests that these subsets have opposing regulatory phenotypes, however, a clear understanding of their roles in regulating inflammation remains unknown and an emerging frontier in NKT research. Our lab has observed opposing roles in suppressing the cytokine storm by NKT subtypes. Ongoing studies will elucidate the mechanism utilized to influence the cytokine storm. This project will yield a better understanding of NKT cell-mediated inflammation during Ftinfection and, more broadly, characterize their involvement and potential for therapeutic targeting for a number of NIH priority pathogens. Supported by grants from National Institute of General Medical Sciences (NIGMS) Research Initiative for Scientific Enhancement (RISE) grant R25 GM 06921-18

UNDERSTANDING THE NEED FOR A BROAD DIFFERENTIAL DIAGNOSIS: A CASE OF PNEUMONIC TULAREMIA

UNDERSTANDING THE NEED FOR A BROAD DIFFERENTIAL DIAGNOSIS: A CASE OF PNEUMONIC TULAREMIA

Transcription factor profiling reveals activation of Type II Natural Killer T cell effector functions in response to Francisella tularensis infection.

Abstract The bacterium Francisella tularensis (Ft) is the causative agent of human tularemia. When untreated, tularemia can reach a mortality rate as high as 30% in healthy adults with inhalation of as few as 10 colony forming units (CFU) inducing respiratory tularemia. While Ft is spread from zoonotic sources in nature, the ease of aerosol spread and high mortality in respiratory form led to its weaponization and concern for bioterrorism. Exposure to Ft elicits the cytokine storm, in which the immune system over-responds to the presence of the bacterium releasing massive amounts of proinflammatory and regulatory cytokines. Natural Killer T (NKT) cells are important immune cells that have both activating and inhibitory effector mechanisms. NKT cells are activated very early following Ft infection suggesting a critical role in the innate immune response to the bacterium. NKT cells are categorized into two distinct subsets based on their antigen recognition of CD1d-tetramers loaded with α-galactosylceramide (αGalCer-tetramer), though their individual characteristics in regulating inflammation are ill-defined. Type I NKT cells, similar to CD4+ αβ T helper cells, have been shown to express varied transcription factor (TF) profiles, e.g., T-bet, GATA3, and RORγT, contributing to either Th1, Th2 or Th17 phenotypes. It is not currently known whether this same TF profile classification exists within type II NKTs. The purpose of this research project is to investigate the activation of the type II NKT cell subset and characterize their TF profiles in response to Ft infection. This data will lead to a better understanding of the role of type II NKT cells in Francisella-mediated immunity. Supported by National Institute of General Medical Sciences (NIGMS) Research Initiative for Scientific Enhancement (RISE) grant R25 GM 06921-18

Pulmonary tularemia in a person taking immunosuppressive drugs: a literature review and a clinical case report

Ieva Radavičiūtė 1, Gintarė Žilinskaitė 1, Birutė Zablockienė 2, 3 1 Vilnius University Faculty of Medicine, Vilnius, Lithuania 2 Vilnius University Faculty of Medicine, Institute of Clinical Medicine, Clinic of Infectious diseases and Dermatovenerology, Vilnius, Lithuania 3 Vilnius University Hospital Santaros Klinikos, Center of Infectious Diseases, Vilnius, Lithuania Abstract Introduction: Aerobic Gram negative bacterium – Francisella tularensis may cause a rare zoonotic disease tularemia. The reservoir of infection are various animals and arthropods. The infection can be transmitted through the respiratory tract, skin, mucous membranes, gastrointestinal tract or by insect bites. Consequently the clinical forms of tularemia are ulcerative glands, lymphadenopathy, ocular hemorrhage, oral and pharyngeal tularemia, gastrointestinal, respiratory and typhoidal tularemia. One of the most severe forms of the disease is pulmonary tularemia, which has a high mortality rate and is particularly dangerous in patients taking immunosuppressive drugs. Clinical case: This article presents a clinical case of a patient treated for rheumatoid arthritis with a TNF-alpha inhibitor (Infliximab). He contacted a doctor in the spring of 2020 due to a dry cough. In September of 2020 a diagnosis of tularemia was confirmed after various diagnostic tests. Antibacterial treatment was effective, but the patient was recommended not to resume biologic therapy with TNF-alpha inhibitors in the setting of tularemia. Conclusion: Tularemia is a rare but easily contagious infectious disease that often takes time to diagnose. In this clinical case, tularemia was more severe in an immunosuppressed patient who developed severe lesions such as lung lesions and lymphadenopathy than in a normal tularemia case patient. For such patients it is particularly important to recognize tularemia, perform laboratory tests to diagnose the zoonosis, and decide on further treatment with immunosuppressive therapy. Keywords: Immunodeficiency; biological therapy; pneumonia, tularemia.

Nlrp3 Increases the Host's Susceptibility to Tularemia.

Francisella tularensis (F. tularensis) is a Gram-negative, intracellular bacterium and the causative agent of a fatal human disease known as tularemia. The CDC has classified F. tularensis as a Tier 1 Category A select agent based on its ease of aerosolization, low infectious dose, past use as a bioweapon, and the potential to be used as a bioterror agent. Francisella has a unique replication cycle. Upon its uptake, Francisella remains in the phagosomes for a short period and then escapes into the cytosol, where the replication occurs. Francisella is recognized by cytosolic pattern recognition receptors, Absent In Melanoma 2 (Aim2) and Nacht LRR and PYD domains containing Protein 3 (Nlrp3). The recognition of Francisella ligands by Aim2 and Nlrp3 triggers the assembly and activation of the inflammasome. The mechanism of activation of Aim2 is well established; however, how Nlrp3 inflammasome is activated in response to F. tularensis infection is not known. Unlike Aim2, the protective role of Nlrp3 against Francisella infection is not fully established. This study investigated the role of Nlrp3 and the potential mechanisms through which Nlrp3 exerts its detrimental effects on the host in response to F. tularensis infection. The results from in vitro studies demonstrate that Nlrp3 dampens NF-κB and MAPK signaling, and pro-inflammatory cytokine production, which allows replication of F. tularensis in infected macrophages. In vivo, Nlrp3 deficiency results in differential expression of several genes required to induce a protective immune response against respiratory tularemia. Nlrp3-deficient mice mount a stronger innate immune response, clear bacteria efficiently with minimal organ damage, and are more resistant to Francisella infection than their wild-type counterparts. Together, these results demonstrate that Nlrp3 enhances the host’s susceptibility to F. tularensis by modulating the protective innate immune responses. Collectively, this study advances our understanding of the detrimental role of Nlrp3 in tularemia pathogenesis.

Deletion Mutants of Francisella Phagosomal Transporters FptA and FptF Are Highly Attenuated for Virulence and Are Protective Against Lethal Intranasal Francisella LVS Challenge in a Murine Model of Respiratory Tularemia.

Francisella tularensis (Ft) is a Gram-negative, facultative intracellular bacterium that is a Tier 1 Select Agent of concern for biodefense for which there is no licensed vaccine. A subfamily of 9 Francisella phagosomal transporter (fpt) genes belonging to the Major Facilitator Superfamily of transporters was identified as critical to pathogenesis and potential targets for attenuation and vaccine development. We evaluated the attenuation and protective capacity of LVS derivatives with deletions of the fptA and fptF genes in the C57BL/6J mouse model of respiratory tularemia. LVSΔfptA and LVSΔfptF were highly attenuated with LD50 values of >20 times that of LVS when administered intranasally and conferred 100% protection against lethal challenge. Immune responses to the fpt mutant strains in mouse lungs on day 6 post-infection were substantially modified compared to LVS and were associated with reduced organ burdens and reduced pathology. The immune responses to LVSΔfptA and LVSΔfptF were characterized by decreased levels of IL-10 and IL-1β in the BALF versus LVS, and increased numbers of B cells, αβ and γδ T cells, NK cells, and DCs versus LVS. These results support a fundamental requirement for FptA and FptF in the pathogenesis of Ft and the modulation of the host immune response.

Structural and functional analysis of the Francisella lysine decarboxylase as a key actor in oxidative stress resistance

Francisella tularensis is one of the most virulent pathogenic bacteria causing the acute human respiratory disease tularemia. While the mechanisms underlying F. tularensis pathogenesis are largely unknown, previous studies have shown that a F. novicida transposon mutant with insertions in a gene coding for a putative lysine decarboxylase was attenuated in mouse spleen, suggesting a possible role of its protein product as a virulence factor. Therefore, we set out to structurally and functionally characterize the F. novicida lysine decarboxylase, which we termed LdcF. Here, we investigate the genetic environment of ldcF as well as its evolutionary relationships with other basic AAT-fold amino acid decarboxylase superfamily members, known as key actors in bacterial adaptative stress response and polyamine biosynthesis. We determine the crystal structure of LdcF and compare it with the most thoroughly studied lysine decarboxylase, E. coli LdcI. We analyze the influence of ldcF deletion on bacterial growth under different stress conditions in dedicated growth media, as well as in infected macrophages, and demonstrate its involvement in oxidative stress resistance. Finally, our mass spectrometry-based quantitative proteomic analysis enables identification of 80 proteins with expression levels significantly affected by ldcF deletion, including several DNA repair proteins potentially involved in the diminished capacity of the F. novicida mutant to deal with oxidative stress. Taken together, we uncover an important role of LdcF in F. novicida survival in host cells through participation in oxidative stress response, thereby singling out this previously uncharacterized protein as a potential drug target.

Further Characterization of the Capsule-Like Complex (CLC) Produced by Francisella tularensis Subspecies tularensis: Protective Efficacy and Similarity to Outer Membrane Vesicles.

Francisella tularensis is the etiologic agent of tularemia, and subspecies tularensis (type A) is the most virulent subspecies. The live vaccine strain (LVS) of subspecies holarctica produces a capsule-like complex (CLC) that consists of a large variety of glycoproteins. Expression of the CLC is greatly enhanced when the bacteria are subcultured in and grown on chemically defined medium. Deletion of two genes responsible for CLC glycosylation in LVS results in an attenuated mutant that is protective against respiratory tularemia in a mouse model. We sought to further characterize the CLC composition and to determine if a type A CLC glycosylation mutant would be attenuated in mice. The CLCs isolated from LVS extracted with 0.5% phenol or 1 M urea were similar, as determined by gel electrophoresis and Western blotting, but the CLC extracted with urea was more water-soluble. The CLC extracted with either 0.5% phenol or 1 M urea from type A strains was also similar to the CLC of LVS in antigenic properties, electrophoretic profile, and by transmission electron microscopy (TEM). The solubility of the CLC could be further enhanced by fractionation with Triton X-114 followed by N-Lauroylsarcosine detergents; the largest (>250 kDa) molecular size component appeared to be an aggregate of smaller components. Outer membrane vesicles/tubules (OMV/T) isolated by differential centrifugation and micro-filtration appeared similar to the CLC by TEM, and many of the proteins present in the OMV/T were also identified in soluble and insoluble fractions of the CLC. Further investigation is warranted to assess the relationship between OMV/T and the CLC. The CLC conjugated to keyhole limpet hemocyanin or flagellin was highly protective against high-dose LVS intradermal challenge and partially protective against intranasal challenge. A protective response was associated with a significant rise in cytokines IL-12, IL-10, and IFN-γ. However, a type A CLC glycosylation mutant remained virulent in BALB/c mice, and immunization with the CLC did not protect mice against high dose respiratory challenge with type A strain SCHU S4.

An Improved Tobacco Mosaic Virus (TMV)-Conjugated Multiantigen Subunit Vaccine Against Respiratory Tularemia.

Francisella tularensis, the causative agent of the fatal human disease known as tularemia is classified as a Category A Select Agent by the Centers for Disease Control. No licensed vaccine is currently available for prevention of tularemia in the United States. Previously, we published that a tri-antigen tobacco mosaic virus (TMV) vaccine confers 50% protection in immunized mice against respiratory tularemia caused by F. tularensis. In this study, we refined the TMV-vaccine formulation to improve the level of protection in immunized C57BL/6 mice against respiratory tularemia. We developed a tetra-antigen vaccine by conjugating OmpA, DnaK, Tul4, and SucB proteins of Francisella to TMV. CpG was also included in the vaccine formulation as an adjuvant. Primary intranasal (i.n.) immunization followed by two booster immunizations with the tetra-antigen TMV vaccine protected 100% mice against i.n. 10LD100 challenges dose of F. tularensis live vaccine strain (LVS). Mice receiving three immunization doses of tetra-antigen TMV vaccine showed only transient body weight loss, cleared the infection rapidly, and showed minimal histopathological lesions in lungs, liver, and spleen following a lethal respiratory challenge with F. tularensis LVS. Mice immunized with the tetra-antigen TMV vaccine also induced strong ex vivo recall responses and were protected against a lethal challenge as late as 163 days post-primary immunization. Three immunization with the tetra-antigen TMV vaccine also induced a stronger humoral immune response predominated by IgG1, IgG2b, and IgG2c antibodies than mice receiving only a single or two immunizations. Remarkably, a single dose protected 40% of mice, while two doses protected 80% of mice from lethal pathogen challenge. Immunization of Interferon-gamma (IFN-γ)-deficient mice with the tetra-antigen TMV vaccine demonstrated an absolute requirement of IFN-γ for the generation of protective immune response against a lethal respiratory challenge with F. tularensis LVS. Collectively, this study further demonstrates the feasibility of TMV as an efficient platform for the delivery of multiple F. tularensis antigens and that tetra-antigen TMV vaccine formulation provides complete protection, and induces long-lasting protective and memory immune responses against respiratory tularemia caused by F. tularensis LVS.

Protection of vaccinated mice against pneumonic tularemia is associated with an early memory sentinel-response in the lung.

Francisella tularensis is the intracellular bacterial pathogen causing the respiratory life-threatening disease tularemia. Development of tularemia vaccines has been hampered by an incomplete understanding of the correlates of immunity. Moreover, the importance of lung cellular immunity in vaccine-mediated protection against tularemia is a controversial matter. Live attenuated vaccine strains of F. tularensis such as LVS (Live Vaccine Strain), elicit an immune response protecting mice against subsequent challenge with the virulent SchuS4 strain, yet the protective immunity against pulmonary challenge is limited in its efficacy and longevity. We established a murine intra-nasal immunization model which distinguishes between animals fully protected, challenged at 4 weeks post double-vaccination (200 inhalation Lethal Dose 50%, LD50, of SchuS4), and those which do not survive the lethal SchuS4 infection, challenged at 8 weeks post double vaccination. Early in the recall immune response in the lung (before day 3), disease progression and bacterial dissemination differed considerably between protected and non-protected immunized mice. Pre-challenge analysis, revealed that protected mice, exhibited significantly higher numbers of lung Ft-specific memory T cells compared to non-protected mice. Quantitative PCR analysis established that a higher magnitude, lung T cells response was activated in the lungs of the protected mice already at 24 h post-challenge. The data imply that an early memory response within the lung is strongly associated with protection against the lethal SchuS4 bacteria presumably by restricting the dissemination of the bacteria to internal organs. Thus, future prophylactic strategies to countermeasure F. tularensis infection may require modulation of the immune response within the lung.

Development of a Multivalent Subunit Vaccine against Respiratory Tularemia

Abstract Francisella tularensis (Ft); the causative agent of a fatal human disease tularemia is classified as a Category A Select Agent. No licensed vaccine is available for prevention of tularemia in the U.S.A. In this study, we used a novel Tobacco Mosaic Virus (TMV) based delivery platform for development of a fully protective multi-antigen subunit tularemia vaccine. Previously we have published that a trivalent TMV-conjugate vaccine confers 50% protection in immunized mice against respiratory Ft LVS challenge. In this study, we refined TMV-conjugate vaccine formulation to improve the level of protection in immunized C57BL/6 mice against respiratory tularemia. We developed a tetravalent vaccine by conjugating OmpA, DnaK, Tul4 and SucB proteins of Francisella to TMV. CpG adjuvant was also included in the vaccine formulation. C57BL/6 mice were immunized intranasally (i.n.) on days 0, 14 and 28 and challenged with 10LD100 of Ft LVS on day 49 post-primary immunization (PPI). Mice were monitored for survival, weight loss, antibody and recall responses, and duration of immune protection. 100% of immunized mice were protected against a 10LD100 i.n. challenge dose of Ft LVS. Mice vaccinated with TMV-tetravalent vaccine showed high levels of TH1 antibodies than those with trivalent vaccine formulation. The challenged mice exhibited significantly reduced bacterial burden in lungs, liver and spleen. Strong ex-vivo recall responses were observed in immunized mice as late as day 84 PPI and 80% of mice survived when challenged 163 days PPI. Collectively, this study demonstrates that tetravalent vaccine formulation provides complete protection, induces strong protective and memory immune responses against respiratory challenge with F. tularensis.

Respiratory Tularemia: Francisella Tularensis and Microarray Probe Designing.

Background: Francisella tularensis (F. tularensis) is the etiological microorganism for tularemia. There are different forms of tularemia such as respiratory tularemia. Respiratory tularemia is the most severe form of tularemia with a high rate of mortality; if not treated. Therefore, traditional microbiological tools and Polymerase Chain Reaction (PCR) are not useful for a rapid, reliable, accurate, sensitive and specific diagnosis. But, DNA microarray technology does. DNA microarray technology needs to appropriate microarray probe designing.Objective:The main goal of this original article was to design suitable long oligo microarray probes for detection and identification of F. tularensis.Method:For performing this research, the complete genomes of F. tularensis subsp. tularensis FSC198, F. tularensis subsp. holarctica LVS, F. tularensis subsp. mediasiatica, F. tularensis subsp. novicida (F. novicida U112), and F. philomiragia subsp. philomiragia ATCC 25017 were studied via NCBI BLAST tool, GView and PanSeq Servers and finally the microarray probes were produced and processed via AlleleID 7.7 software and Oligoanalyzer tool, respectively.Results:In this in silico investigation, a number of long oligo microarray probes were designed for detecting and identifying F. tularensis. Among these probes, 15 probes were recognized as the best candidates for microarray chip designing.Conclusion:Calibrated microarray probes reduce the biasis of DNA microarray technology as an advanced, rapid, accurate and cost-effective molecular diagnostic tool with high specificity and sensitivity. Professional microarray probe designing provides us with much more facility and flexibility regarding preparation of a microarray diagnostic chip.

Lipoxin A4, a 5-lipoxygenase pathway metabolite, modulates immune response during acute respiratory tularemia.

Respiratory infection with Francisella tularensis (Ft) is characterized by a muted, acute host response, followed by sepsis-like syndrome that results in death. Infection with Ft establishes a principally anti-inflammatory environment that subverts host-cell death programs to facilitate pathogen replication. Although the role of cytokines has been explored extensively, the role of eicosanoids in tularemia pathogenesis is not fully understood. Given that lipoxin A4 (LXA4) has anti-inflammatory properties, we investigated whether this lipid mediator affects host responses manifested early during infection. The addition of exogenous LXA4 inhibits PGE2 release by Ft-infected murine monocytes in vitro and diminishes apoptotic cell death. Tularemia pathogenesis was characterized in 5‑lipoxygenase-deficient (Alox5-/-) mice that are incapable of generating LXA4 Increased release of proinflammatory cytokines and chemokines, as well as increased apoptosis, was observed in Alox5-/- mice as compared with their wild-type counterparts. Alox5-/- mice also exhibited elevated recruitment of neutrophils during the early phase of infection and increased resistance to lethal challenge. Conversely, administration of exogenous LXA4 to Alox5-/- mice made them more susceptible to infection thus mimicking wild-type animals. Taken together, our results suggest that 5-LO activity is a critical regulator of immunopathology observed during the acute phase of respiratory tularemia, regulating bacterial burden and neutrophil recruitment and production of proinflammatory modulators and increasing morbidity and mortality. These studies identify a detrimental role for the 5-LO-derived lipid mediator LXA4 in Ft-induced immunopathology. Targeting this pathway may have therapeutic benefit as an adjunct to treatment with antibiotics and conventional antimicrobial peptides, which often have limited efficacy against intracellular bacteria.

Respiratory and oral vaccination improves protection conferred by the live vaccine strain against pneumonic tularemia in the rabbit model.

Tularemia is a severe, zoonotic disease caused by a gram-negative bacterium, Francisella tularensis We have previously shown that rabbits are a good model of human pneumonic tularemia when exposed to aerosols containing a virulent, type A strain, SCHU S4. We further demonstrated that the live vaccine strain (LVS), an attenuated type B strain, extended time to death when given by scarification. Oral or aerosol vaccination has been previously shown in humans to offer superior protection to parenteral vaccination against respiratory tularemia challenge. Both oral and aerosol vaccination with LVS were well tolerated in the rabbit with only minimal fever and no weight loss after inoculation. Plasma antibody titers against F. tularensis were higher in rabbits that were vaccinated by either oral or aerosol routes compared to scarification. Thirty days after vaccination, all rabbits were challenged with aerosolized SCHU S4. LVS given by scarification extended time to death compared to mock-vaccinated controls. One orally vaccinated rabbit did survive aerosol challenge, however, only aerosol vaccination extended time to death significantly compared to scarification. These results further demonstrate the utility of the rabbit model of pneumonic tularemia in replicating what has been reported in humans and macaques as well as demonstrating the utility of vaccination by oral and respiratory routes against an aerosol tularemia challenge.

Vaccination evokes gender-dependent protection against tularemia infection in C57BL/6Tac mice

Francisella tularensis (Ft) is a Category A biothreat agent for which there currently is no FDA-approved vaccine. Thus, there is a substantial effort underway to develop an effective tularemia vaccine. While it is well established that gender can significantly impact susceptibility to primary infection, the impact of gender on vaccine efficacy is not well established. Thus, development of a successful vaccine against tularemia will require an understanding of the impact gender has on vaccine-induced protection against this organism. In this study, a role for gender in vaccine-induced protection following Ft challenge is identified for the first time. In the present study, mucosal vaccination with inactivated Ft (iFt) LVS elicited gender-based protection in C57BL/6Tac mice against respiratory challenge with Ft LVS. Specifically, vaccinated male mice were more susceptible to subsequent Ft LVS challenge. This increased susceptibility in male mice correlated with increased bacterial burden, increased tissue inflammation, and increased proinflammatory cytokine production late in post-challenge infection. In contrast, improved survival of iFt-vaccinated female mice correlated with reduced bacterial burden and enhanced levels of Ft-specific Abs in serum and broncho-alveolar lavage (BAL) fluid post-challenge. Furthermore, vaccination with a live attenuated vaccine consisting of an Ft LVS superoxide dismutase (SodB) mutant, which has proven efficacious against the highly virulent Ft SchuS4 strain, demonstrated similar gender bias in protection post-Ft SchuS4 challenge. Of particular significance is the fact that these are the first studies to demonstrate that gender differences impact disease outcome in the case of lethal respiratory tularemia following mucosal vaccination. In addition, these studies further emphasize the fact that gender differences must be a serious consideration in any future tularemia vaccine development studies.

Induction of lipoxin A4 by Francisella tularensis: a mechanism for regulation of pro-inflammatory responses during early-phase tularemia (INM3P.401)

Abstract During respiratory tularemia, caused by inhalation of Francisella tularensis (Ft), we postulate that bacteria establish a principally anti-inflammatory environment and subvert host cell death programs to facilitate their unfettered replication within various cell types. Here we have explored the role of lipid mediators such as lipoxin A4 (LXA4) and prostaglandin E2 (PGE2) in host inflammatory responses manifested early during infection with Ft. An inverse relationship was observed between the production of LXA4 and PGE2 suggesting that Ft induces the generation of these eicosanoids to modulate cell death and enhance its own survival. This notion is supported by our findings that Ft triggers the release of LXA4, which inhibits apoptosis of macrophages. We also have elucidated the course of tularemia in mice deficient for 5-lipoxygenase (5-LO), leaving them incapable of generating LXA4, and observed elevated levels of PGE2, increased apoptosis, increased neutrophil recruitment and elevated release of pro-inflammatory cytokines and chemokines as compared to their wild-type counterparts. 5-LO deficient mice also were found to be less susceptible to lethal doses of Ft LVS as compared to wild-type mice. Our results suggests that 5-LO activity increases susceptibility to Ft infection, suggesting that targeting this pathway may have therapeutic benefit by limiting bacterial growth in tularemic individuals and perhaps those infected with other virulent respiratory pathogens.

Alarmin function of galectin-9 in murine respiratory tularemia.

Sepsis is a complex immune disorder that is characterized by systemic hyperinflammation. Alarmins, which are multifunctional endogenous factors, have been implicated in exacerbation of inflammation in many immune disorders including sepsis. Here we show that Galectin-9, a host endogenous β-galactoside binding lectin, functions as an alarmin capable of mediating inflammatory response during sepsis resulting from pulmonary infection with Francisella novicida, a Gram negative bacterial pathogen. Our results show that this galectin is upregulated and is likely released during tissue damage in the lungs of F. novicida infected septic mice. In vitro, purified recombinant galectin-9 exacerbated F. novicida-induced production of the inflammatory mediators by macrophages and neutrophils. Concomitantly, Galectin-9 deficient (Gal-9-/-) mice exhibited improved lung pathology, reduced cell death and reduced leukocyte infiltration, particularly neutrophils, in their lungs. This positively correlated with overall improved survival of F. novicida infected Gal-9-/- mice as compared to their wild-type counterparts. Collectively, these findings suggest that galectin-9 functions as a novel alarmin by augmenting the inflammatory response in sepsis development during pulmonary F. novicida infection.

Preclinical testing of a vaccine candidate against tularemia.

Tularemia is caused by a gram-negative, intracellular bacterial pathogen, Francisella tularensis (Ft). The history weaponization of Ft in the past has elevated concerns that it could be used as a bioweapon or an agent of bioterrorism. Since the discovery of Ft, three broad approaches adopted for tularemia vaccine development have included inactivated, live attenuated, or subunit vaccines. Shortcomings in each of these approaches have hampered the development of a suitable vaccine for prevention of tularemia. Recently, we reported an oxidant sensitive mutant of Ft LVS in putative EmrA1 (FTL_0687) secretion protein. The emrA1 mutant is highly sensitive to oxidants, attenuated for intramacrophage growth and virulence in mice. We reported that EmrA1 contributes to oxidant resistance by affecting the secretion of antioxidant enzymes SodB and KatG. This study investigated the vaccine potential of the emrA1 mutant in prevention of respiratory tularemia caused by Ft LVS and the virulent SchuS4 strain in C57BL/6 mice. We report that emrA1 mutant is safe and can be used at an intranasal (i. n.) immunization dose as high as 1x106 CFU without causing any adverse effects in immunized mice. The emrA1 mutant is cleared by vaccinated mice by day 14–21 post-immunization, induces minimal histopathological lesions in lungs, liver and spleen and a strong humoral immune response. The emrA1 mutant vaccinated mice are protected against 1000–10,000LD100 doses of i.n. Ft LVS challenge. Such a high degree of protection has not been reported earlier against respiratory challenge with Ft LVS using a single immunization dose with an attenuated mutant generated on Ft LVS background. The emrA1 mutant also provides partial protection against i.n. challenge with virulent Ft SchuS4 strain in vaccinated C57BL/6 mice. Collectively, our results further support the notion that antioxidants of Ft may serve as potential targets for development of effective vaccines for prevention of tularemia.

An outbreak of respiratory tularemia caused by diverse clones of Francisella tularensis.

The bacterium Francisella tularensis is recognized for its virulence, infectivity, genetic homogeneity, and potential as a bioterrorism agent. Outbreaks of respiratory tularemia, caused by inhalation of this bacterium, are poorly understood. Such outbreaks are exceedingly rare, and F. tularensis is seldom recovered from clinical specimens. A localized outbreak of tularemia in Sweden was investigated. Sixty-seven humans contracted laboratory-verified respiratory tularemia. F. tularensis subspecies holarctica was isolated from the blood or pleural fluid of 10 individuals from July to September 2010. Using whole-genome sequencing and analysis of single-nucleotide polymorphisms (SNPs), outbreak isolates were compared with 110 archived global isolates. There were 757 SNPs among the genomes of the 10 outbreak isolates and the 25 most closely related archival isolates (all from Sweden/Finland). Whole genomes of outbreak isolates were >99.9% similar at the nucleotide level and clustered into 3 distinct genetic clades. Unexpectedly, high-sequence similarity grouped some outbreak and archival isolates that originated from patients from different geographic regions and up to 10 years apart. Outbreak and archival genomes frequently differed by only 1-3 of 1 585 229 examined nucleotides. The outbreak was caused by diverse clones of F. tularensis that occurred concomitantly, were widespread, and apparently persisted in the environment. Multiple independent acquisitions of F. tularensis from the environment over a short time period suggest that natural outbreaks of respiratory tularemia are triggered by environmental cues. The findings additionally caution against interpreting genome sequence identity for this pathogen as proof of a direct epidemiological link.