The impact of primary immunization route on the outcome of infection with SARS-CoV-2 in a hamster model of COVID-19.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has resulted in over 6.7 million deaths worldwide. COVID-19 vaccines administered parenterally via intramuscular or subcutaneous (SC) routes have reduced the severity of respiratory infections, hospitalization rates, and overall mortality. However, there is a growing interest in developing mucosally delivered vaccines to further enhance the ease and durability of vaccination. This study compared the immune response in hamsters immunized with live SARS-CoV-2 virus via SC or intranasal (IN) routes and assessed the outcome of a subsequent IN SARS-CoV-2 challenge. Results showed that SC-immunized hamsters elicited a dose-dependent neutralizing antibody response but of a significantly lower magnitude than that observed in IN-immunized hamsters. The IN challenge with SARS-CoV-2 in SC-immunized hamsters resulted in body weight loss, increased viral load, and lung pathology than that observed in IN-immunized and IN-challenged counterparts. These results demonstrate that while SC immunization renders some degree of protection, IN immunization induces a stronger immune response and better protection against respiratory SARS-CoV-2 infection. Overall, this study provides evidence that the route of primary immunization plays a critical role in determining the severity of a subsequent respiratory infection caused by SARS-CoV-2. Furthermore, the findings suggest that IN route of immunization may be a more effective option for COVID-19 vaccines than the currently used parenteral routes. Understanding the immune response to SARS-CoV-2 elicited via different immunization routes may help guide more effective and long-lasting vaccination strategies.

Comparative analysis of absent in melanoma 2-inflammasome activation in Francisella tularensis and Francisella novicida.

Francisella tularensis is a highly virulent Gram-negative bacterium that causes the fatal zoonotic disease tularemia. The mechanisms and signaling pathways leading to the absent in melanoma 2 (Aim2) inflammasome activation have been elegantly elucidated using Francisella novicida as a model. Although not pathogenic for humans, F. novicida can cause tularemia in mice, and the inflammatory response it triggers is the polar opposite to that observed in mice infected with F. tularensis strains. This study aimed to understand the mechanisms of Aim2 inflammasome activation in F. tularensis-infected macrophages. The results reveal that macrophages infected with the F. tularensis live vaccine strain (LVS) induce lower levels of Aim2-dependent IL-1ß than those infected with F. novicida. The suppression/weak activation of Aim2 in F. tularensis LVS-infected macrophages is due to the suppression of the cGAS-STING DNA-sensing pathway. Furthermore, the introduction of exogenous F. tularensis LVS DNA into the cytosol of the F. tularensis LVS-infected macrophages, alone or in conjunction with a priming signal, failed to restore IL-1ß levels similar to those observed for F. novicida-infected macrophages. These results indicated that, in addition to the bacterial DNA, DNA from some other sources, specifically from the damaged mitochondria, might contribute to the robust Aim2-dependent IL-1ß levels observed in F. novicida-infected macrophages. The results indicate that F. tularensis LVS induces mitophagy that may potentially prevent the leakage of mitochondrial DNA and the subsequent activation of the Aim2 inflammasome. Collectively, this study demonstrates that the mechanisms of Aim2 inflammasome activation established for F. novicida are not operative in F. tularensis.

Chronically hypertensive transgenic mice expressing human AT1R haplotype-I exhibit increased susceptibility to Francisella tularensis.

Age-related illnesses, including hypertension and accompanying metabolic disorders, compromise immunity and exacerbate infection-associated fatalities. Renin-angiotensin system (RAS) is the key mechanism that controls blood pressure. Upregulation of RAS through angiotensin receptor type 1 (AT1R), a G-protein coupled receptor, contributes to the pathophysiological consequences leading to vascular remodeling, hypertension, and end-organ damage. Genetic variations that increase the expression of human AT1R may cause the above pathological outcomes associated with hypertension. Previously we have shown that our chronically hypertensive transgenic (TG) mice containing the haplotype-I variant (Hap-I, hypertensive genotype) of human AT1R (hAT1R) gene are more prone to develop the metabolic syndrome-related disorders as compared to the TG mice containing the haplotype-II variant (Hap-II, normotensive genotype). Since aging and an increased risk of hypertension can impact multiple organ systems in a complex manner, including susceptibility to various infections, the current study investigated the susceptibility and potential effect of acute bacterial infection using a Gram-negative intracellular bacterial pathogen, Francisella tularensis in our hAT1R TG mice. Our results show that compared to Hap-II, F. tularensis-infected aged Hap-I TG mice have significantly higher mortality post-infection, higher bacterial load and lung pathology, elevated inflammatory cytokines and altered gene expression profile favoring hypertension and inflammation. Consistent with worsened phenotype in aged Hap-I mice post-Francisella infection, gene expression profiles from their lungs revealed significantly altered expression of more than 1,400 genes. Furthermore, bioinformatics analysis identified genes associated with RAS and IFN-γ pathways regulating blood pressure and inflammation. These studies demonstrate that haplotype-dependent over-expression of the hAT1R gene leads to enhanced susceptibility and lethality due to F. tularensis LVS infection, which gets aggravated in aged animals. Clinically, these findings will help in exploring the role of AT1R-induced hypertension and enhanced susceptibility to infection-related respiratory diseases.

The severity of SARS-CoV-2 infection in K18-hACE2 mice is attenuated by a novel steroid-derivative in a gender-specific manner.

BACKGROUND AND PURPOSE: COVID-19 infections caused by SARS-CoV-2 disseminated through human-to-human transmission can evoke severe inflammation. Treatments to reduce the SARS-CoV-2-associated inflammation are needed and are the focus of much research. In this study, we investigated the effect of N-ethyl-N'-[(3ß,5α)-17-oxoandrostan-3-yl] urea (NEOU), a novel 17α-ketosteroid derivative, on the severity of COVID-19 infections. EXPERIMENTAL APPROACH: Studies were conducted in SARS-CoV-2-infected K18-hACE2 mice. KEY RESULTS: SARS-CoV-2-infected K18-hACE2 mice developed severe inflammatory crises and immune responses along with up-regulation of genes in associated signalling pathways in male more than female mice. Notably, SARS-CoV-2 infection down-regulated genes encoding drug metabolizing cytochrome P450 enzymes in male but not female mice. Treatment with NEOU (1 mg·kg-1 ·day-1 ) 24 or 72 h post-viral infection alleviated lung injury by decreasing expression of genes encoding inflammatory cytokines and chemokines while increasing expression of genes encoding immunoglobins. In situ hybridization using RNA scope™ probes and immunohistochemical assays revealed that NEOU increased resident CD169+ immunoregulatory macrophages and IBA-1 immunoreactive macrophage-dendritic cells within alveolar spaces in the lungs of infected mice. Consequentially, NEOU reduced morbidity more prominently in male than female mice. However, NEOU increased median survival time and accelerated recovery from infection by 6 days in both males and females. CONCLUSIONS AND IMPLICATIONS: These findings demonstrate that SARS-CoV-2 exhibits gender bias by differentially regulating genes encoding inflammatory cytokines, immunogenic factors and drug-metabolizing enzymes, in male versus female mice. Most importantly, we identified a novel 17α-ketosteroid that reduces the severity of COVID-19 infection and could be beneficial for reducing impact of COVID-19.

ThioredoxinA1 Controls the Oxidative Stress Response of Francisella tularensis Live Vaccine Strain (LVS).

Francisella tularensis is an intracellular, Gram-negative bacterium known for causing a disease known as tularemia in the Northern Hemisphere. F. tularensis is classified as a category A select agent by the CDC based on its possible use as a bioterror agent. F. tularensis overcomes oxidative stress encountered during its growth in the environment or host macrophages by encoding antioxidant enzymes such as superoxide dismutases, catalase, and alkylhydroperoxy reductase. These antioxidant enzymes are regulated by the oxidative stress response regulator, OxyR. In addition to these antioxidant enzymes, F. tularensis also encodes two thioredoxins, TrxA1 (FTL_0611) and TrxA2 (FTL_1224); however, their role in the oxidative stress response of F. tularensis is not known. This study investigated the role of thioredoxins of F. tularensis in the oxidative stress response and intracellular survival. Our results demonstrate that TrxA1 but not TrxA2 plays a major role in the oxidative stress response of F. tularensis. Most importantly, this study elucidates a novel mechanism through which the TrxA1 of F. tularensis controls the oxidative stress response by regulating the expression of the master regulator, oxyR. Further, TrxA1 is required for the intramacrophage survival and growth of Francisella. Overall, this study describes a novel role of thioredoxin, TrxA1, in regulating the oxidative stress response of F. tularensis. IMPORTANCE The role of thioredoxins in the oxidative stress response of F. tularensis is not known. This study demonstrates that of the two thioredoxins, TrxA1 is vital to counter the oxidative stress in F. tularensis live vaccine strain (LVS). Furthermore, this study shows differences in the well-studied thioredoxins of Escherichia coli. First, the expression of TrxA1 of F. tularensis is independent of the oxidative stress response regulator, OxyR. Second and most importantly, TrxA1 regulates the expression of oxyR and, therefore, the OxyR-dependent oxidative stress response of F. tularensis. Overall, this study reports a novel regulatory role of TrxA1 of F. tularensis in the oxidative stress response.

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.

An AraC/XylS Family Transcriptional Regulator Modulates the Oxidative Stress Response of Francisella tularensis.

Francisella tularensis is a Gram-negative bacterium that causes a fatal human disease known as tularemia. The Centers for Disease Control and Prevention have classified F. tularensis as a category A tier 1 select agent. The virulence mechanisms of Francisella are not entirely understood. Francisella possesses very few transcription regulators, and most of these regulate the expression of genes involved in intracellular survival and virulence. The F. tularensis genome sequence analysis reveals an AraC (FTL_0689) transcriptional regulator homologous to the AraC/XylS family of transcriptional regulators. In Gram-negative bacteria, AraC activates genes required for l-arabinose utilization and catabolism. The role of the FTL_0689 regulator in F. tularensis is not known. In this study, we characterized the role of FTL_0689 in the gene regulation of F. tularensis and investigated its contribution to intracellular survival and virulence. The results demonstrate that FTL_0689 in Francisella is not required for l-arabinose utilization. Instead, FTL_0689 specifically regulates the expression of the oxidative and global stress response, virulence, metabolism, and other key pathways genes required by Francisella when exposed to oxidative stress. The FTL_0689 mutant is attenuated for intramacrophage growth and virulence in mice. Based on the deletion mutant phenotype, FTL_0689 was termed osrR (oxidative stress response regulator). Altogether, this study elucidates the role of the osrR transcriptional regulator in tularemia pathogenesis. IMPORTANCE The virulence mechanisms of category A select agent Francisella tularensis, the causative agent of a fatal human disease known as tularemia, remain largely undefined. The present study investigated the role of a transcriptional regulator and its overall contribution to the oxidative stress resistance of F. tularensis. The results provide an insight into a novel gene regulatory mechanism, especially when Francisella is exposed to oxidative stress conditions. Understanding such Francisella- specific regulatory mechanisms will help identify potential targets for developing effective therapies and vaccines to prevent tularemia.

Aim2 and Nlrp3 Are Dispensable for Vaccine-Induced Immunity against Francisella tularensis Live Vaccine Strain.

Francisella tularensis is a facultative, intracellular, Gram-negative bacterium that causes a fatal disease known as tularemia. Due to its extremely high virulence, ease of spread by aerosolization, and potential to be used as a bioterror agent, F. tularensis is classified by the CDC as a tier 1 category A select agent. Previous studies have demonstrated the roles of the inflammasome sensors absent in melanoma 2 (AIM2) and NLRP3 in the generation of innate immune responses to F. tularensis infection. However, contributions of both the AIM2 and NLRP3 to the development of vaccine-induced adaptive immune responses against F. tularensis are not known. This study determined the contributions of Aim2 and Nlrp3 inflammasome sensors to vaccine-induced immune responses in a mouse model of respiratory tularemia. We developed a model to vaccinate Aim2- and Nlrp3-deficient (Aim2-/- and Nlrp3-/-) mice using the emrA1 mutant of the F. tularensis live vaccine strain (LVS). The results demonstrate that the innate immune responses in Aim2-/- and Nlrp3-/- mice vaccinated with the emrA1 mutant differ from those of their wild-type counterparts. However, despite these differences in the innate immune responses, both Aim2-/- and Nlrp3-/- mice are fully protected against an intranasal lethal challenge dose of F. tularensis LVS. Moreover, the lack of both Aim2 and Nlrp3 inflammasome sensors does not affect the production of vaccination-induced antibody and cell-mediated responses. Overall, this study reports a novel finding that both Aim2 and Nlrp3 are dispensable for vaccination-induced immunity against respiratory tularemia caused by F. tularensis.

Stringent response governs the oxidative stress resistance and virulence of Francisella tularensis.

Francisella tularensis is a Gram-negative bacterium responsible for causing tularemia in the northern hemisphere. F. tularensis has long been developed as a biological weapon due to its ability to cause severe illness upon inhalation of as few as ten organisms and, based on its potential to be used as a bioterror agent is now classified as a Tier 1 Category A select agent by the CDC. The stringent response facilitates bacterial survival under nutritionally challenging starvation conditions. The hallmark of stringent response is the accumulation of the effector molecules ppGpp and (p)ppGpp known as stress alarmones. The relA and spoT gene products generate alarmones in several Gram-negative bacterial pathogens. RelA is a ribosome-associated ppGpp synthetase that gets activated under amino acid starvation conditions whereas, SpoT is a bifunctional enzyme with both ppGpp synthetase and ppGpp hydrolase activities. Francisella encodes a monofunctional RelA and a bifunctional SpoT enzyme. Previous studies have demonstrated that stringent response under nutritional stresses increases expression of virulence-associated genes encoded on Francisella Pathogenicity Island. This study investigated how stringent response governs the oxidative stress response of F. tularensis. We demonstrate that RelA/SpoT-mediated ppGpp production alters global gene transcriptional profile of F. tularensis in the presence of oxidative stress. The lack of stringent response in relA/spoT gene deletion mutants of F. tularensis makes bacteria more susceptible to oxidants, attenuates survival in macrophages, and virulence in mice. This work is an important step forward towards understanding the complex regulatory network underlying the oxidative stress response of F. tularensis.

Role of peroxiredoxin of the AhpC/TSA family in antioxidant defense mechanisms of Francisella tularensis.

Francisella tularensis is a Gram-negative, facultative intracellular pathogen and the causative agent of a lethal human disease known as tularemia. Due to its extremely high virulence and potential to be used as a bioterror agent, F. tularensis is classified by the CDC as a Category A Select Agent. As an intracellular pathogen, F. tularensis during its intracellular residence encounters a number of oxidative and nitrosative stresses. The roles of the primary antioxidant enzymes SodB, SodC and KatG in oxidative stress resistance and virulence of F. tularensis live vaccine strain (LVS) have been characterized in previous studies. However, very fragmentary information is available regarding the role of peroxiredoxin of the AhpC/TSA family (annotated as AhpC) of F. tularensis SchuS4; whereas the role of AhpC of F. tularensis LVS in tularemia pathogenesis is not known. This study was undertaken to exhaustively investigate the role of AhpC in oxidative stress resistance of F. tularensis LVS and SchuS4. We report that AhpC of F. tularensis LVS confers resistance against a wide range of reactive oxygen and nitrogen species, and serves as a virulence factor. In highly virulent F. tularensis SchuS4 strain, AhpC serves as a key antioxidant enzyme and contributes to its robust oxidative and nitrosative stress resistance, and intramacrophage survival. We also demonstrate that there is functional redundancy among primary antioxidant enzymes AhpC, SodC, and KatG of F. tularensis SchuS4. Collectively, this study highlights the differences in antioxidant defense mechanisms of F. tularensis LVS and SchuS4.

Intranasal administration of a two-dose adjuvanted multi-antigen TMV-subunit conjugate vaccine fully protects mice against Francisella tularensis LVS challenge.

Tularemia is a fatal human disease caused by Francisella tularensis, a Gram-negative encapsulated coccobacillus bacterium. Due to its low infectious dose, ease of aerosolized transmission, and lethal effects, the CDC lists F. tularensis as a Category A pathogen, the highest level for a potential biothreat agent. Previous vaccine studies have been conducted with live attenuated, inactivated, and subunit vaccines, which have achieved partial or full protection from F. tularensis live vaccine strain (LVS) challenge, but no vaccine has been approved for human use. We demonstrate the improved efficacy of a multi-antigen subunit vaccine by using Tobacco Mosaic virus (TMV) as an antigen carrier for the F. tularensis SchuS4 proteins DnaK, OmpA, SucB and Tul4 (DOST). The magnitude and quality of immune responses were compared after mice were immunized by subcutaneous or intranasal routes of administration with a TMV-DOST mixture, with or without four different adjuvants. Immune responses varied in magnitude and isotype profile, by antigen, by route of administration, and by protection in an F. tularensis LVS challenge model of disease. Interestingly, our analysis demonstrates an overwhelming IgG2 response to SucB after intranasal dosing, as well as a robust cellular response, which may account for the improved two-dose survival imparted by the tetravalent vaccine, compared to a previous study that tested efficacy of TMV-DOT. Our study provides evidence that potent humoral, cellular and mucosal immunity can be achieved by optimal antigen combination, delivery, adjuvant and appropriate route of administration, to improve vaccine potency and provide protection from pathogen challenge.

Characterization of a Unique Outer Membrane Protein Required for Oxidative Stress Resistance and Virulence of Francisella tularensis.

Francisella tularensis, the causative agent of tularemia, lacks typical bacterial virulence factors and toxins but still exhibits extreme virulence. The bacterial multidrug efflux systems consist of an inner membrane, a transmembrane membrane fusion protein, and an outer membrane (OM) component that form a contiguous channel for the secretion of a multitude of bacterial products. Francisella contains three orthologs of the OM proteins; two of these, termed TolC and FtlC, are important for tularemia pathogenesis. The third OM protein, SilC, is homologous to the silver cation efflux protein of other bacterial pathogens. The silC gene (FTL_0686) is located on an operon encoding an Emr-type multidrug efflux pump of F. tularensis The role of SilC in tularemia pathogenesis is not known. In this study, we investigated the role of SilC in secretion and virulence of F. tularensis by generating a silC gene deletion (ΔsilC) mutant and its transcomplemented strain. Our results demonstrate that the ΔsilC mutant exhibits increased sensitivity to antibiotics, oxidants, silver, diminished intramacrophage growth, and attenuated virulence in mice compared to wild-type F. tularensis However, the secretion of antioxidant enzymes of F. tularensis is not impaired in the ΔsilC mutant. The virulence of the ΔsilC mutant is restored in NADPH oxidase-deficient mice, indicating that SilC resists oxidative stress in vivo Collectively, this study demonstrates that the OM component SilC serves a specialized role in virulence of F. tularensis by conferring resistance against oxidative stress and silver.IMPORTANCEFrancisella tularensis, the causative agent of a fatal human disease known as tularemia, is a category A select agent and a potential bioterror agent. The virulence mechanisms of Francisella are not completely understood. This study investigated the role of a unique outer membrane protein, SilC, of a multidrug efflux pump in the virulence of F. tularensis This is the first report demonstrating that the OM component SilC plays an important role in efflux of silver and contributes to the virulence of F. tularensis primarily by providing resistance against oxidative stress. Characterization of these unique virulence mechanisms will provide an understanding of the pathogenesis of tularemia and identification of potential targets for the development of effective therapeutics and prophylactics for protection from this lethal disease.

Necroptotic debris including damaged mitochondria elicits sepsis-like syndrome during late-phase tularemia.

Infection with Francisella tularensis ssp. tularensis (Ft) strain SchuS4 causes an often lethal disease known as tularemia in rodents, non-human primates, and humans. Ft subverts host cell death programs to facilitate their exponential replication within macrophages and other cell types during early respiratory infection (⩽72 h). The mechanism(s) by which cell death is triggered remains incompletely defined, as does the impact of Ft on mitochondria, the host cell's organellar 'canary in a coal mine'. Herein, we reveal that Ft infection of host cells, particularly macrophages and polymorphonuclear leukocytes, drives necroptosis via a receptor-interacting protein kinase 1/3-mediated mechanism. During necroptosis mitochondria and other organelles become damaged. Ft-induced mitochondrial damage is characterized by: (i) a decrease in membrane potential and consequent mitochondrial oncosis or swelling, (ii) increased generation of superoxide radicals, and (iii) release of intact or damaged mitochondria into the lung parenchyma. Host cell recognition of and response to released mitochondria and other damage-associated molecular patterns engenders a sepsis-like syndrome typified by production of TNF, IL-1ß, IL-6, IL-12p70, and IFN-γ during late-phase tularemia (⩾72 h), but are absent early during infection.

Elucidation of a mechanism of oxidative stress regulation in Francisella tularensis live vaccine strain.

Francisella tularensis causes a lethal human disease known as tularemia. As an intracellular pathogen, Francisella survives and replicates in phagocytic cells, such as macrophages. However, to establish an intracellular niche, Francisella must overcome the oxidative stress posed by the reactive oxygen species (ROS) produced by the infected macrophages. OxyR and SoxR/S are two well-characterized transcriptional regulators of oxidative stress responses in several bacterial pathogens. Only the OxyR homolog is present in F. tularensis, while the SoxR homologs are absent. The functional role of OxyR has not been established in F. tularensis. We demonstrate that OxyR regulates oxidative stress responses and provides resistance against ROS, thereby contributing to the survival of the F. tularensis subsp. holarctica live vaccine strain (LVS) in macrophages and epithelial cells and contributing to virulence in mice. Proteomic analysis reveals the differential production of 128 proteins in the oxyR gene deletion mutant, indicating its global regulatory role in the oxidative stress response of F. tularensis. Moreover, OxyR regulates the transcription of the primary antioxidant enzyme genes by binding directly to their putative promoter regions. This study demonstrates that OxyR is an important virulence factor and transcriptional regulator of the oxidative stress response of the F. tularensis LVS.

Inhibitors of Ribosome Rescue Arrest Growth of Francisella tularensis at All Stages of Intracellular Replication.

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.

Antioxidant Defenses of Francisella tularensis Modulate Macrophage Function and Production of Proinflammatory Cytokines.

Francisella tularensis, the causative agent of a fatal human disease known as tularemia, has been used in the bioweapon programs of several countries in the past, and now it is considered a potential bioterror agent. Extreme infectivity and virulence of F. tularensis is due to its ability to evade immune detection and to suppress the host's innate immune responses. However, Francisella-encoded factors and mechanisms responsible for causing immune suppression are not completely understood. Macrophages and neutrophils generate reactive oxygen species (ROS)/reactive nitrogen species as a defense mechanism for the clearance of phagocytosed microorganisms. ROS serve a dual role; at high concentrations they act as microbicidal effector molecules that destroy intracellular pathogens, and at low concentrations they serve as secondary signaling messengers that regulate the expression of various inflammatory mediators. We hypothesized that the antioxidant defenses of F. tularensis maintain redox homeostasis in infected macrophages to prevent activation of redox-sensitive signaling components that ultimately result in suppression of pro-inflammatory cytokine production and macrophage microbicidal activity. We demonstrate that antioxidant enzymes of F. tularensis prevent the activation of redox-sensitive MAPK signaling components, NF-κB signaling, and the production of pro-inflammatory cytokines by inhibiting the accumulation of ROS in infected macrophages. We also report that F. tularensis inhibits ROS-dependent autophagy to promote its intramacrophage survival. Collectively, this study reveals novel pathogenic mechanisms adopted by F. tularensis to modulate macrophage innate immune functions to create an environment permissive for its intracellular survival and growth.

Development of a Multivalent Subunit Vaccine against Tularemia Using Tobacco Mosaic Virus (TMV) Based Delivery System.

Francisella tularensis is a facultative intracellular pathogen, and is the causative agent of a fatal human disease known as tularemia. F. tularensis is classified as a Category A Biothreat agent by the CDC based on its use in bioweapon programs by several countries in the past and its potential to be used as an agent of bioterrorism. No licensed vaccine is currently available for prevention of tularemia. In this study, we used a novel approach for development of a multivalent subunit vaccine against tularemia by using an efficient tobacco mosaic virus (TMV) based delivery platform. The multivalent subunit vaccine was formulated to contain a combination of F. tularensis protective antigens: OmpA-like protein (OmpA), chaperone protein DnaK and lipoprotein Tul4 from the highly virulent F. tularensis SchuS4 strain. Two different vaccine formulations and immunization schedules were used. The immunized mice were challenged with lethal (10xLD100) doses of F. tularensis LVS on day 28 of the primary immunization and observed daily for morbidity and mortality. Results from this study demonstrate that TMV can be used as a carrier for effective delivery of multiple F. tularensis antigens. TMV-conjugate vaccine formulations are safe and multiple doses can be administered without causing any adverse reactions in immunized mice. Immunization with TMV-conjugated F. tularensis proteins induced a strong humoral immune response and protected mice against respiratory challenges with very high doses of F. tularensis LVS. This study provides a proof-of-concept that TMV can serve as a suitable platform for simultaneous delivery of multiple protective antigens of F. tularensis. Refinement of vaccine formulations coupled with TMV-targeting strategies developed in this study will provide a platform for development of an effective tularemia subunit vaccine as well as a vaccination approach that may broadly be applicable to many other bacterial pathogens.

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.

EmrA1 membrane fusion protein of Francisella tularensis LVS is required for resistance to oxidative stress, intramacrophage survival and virulence in mice.

Francisella tularensis is a category A biodefence agent that causes a fatal human disease known as tularaemia. The pathogenicity of F. tularensis depends on its ability to persist inside host immune cells primarily by resisting an attack from host-generated reactive oxygen and nitrogen species (ROS/RNS). Based on the ability of F. tularensis to resist high ROS/RNS levels, we have hypothesized that additional unknown factors act in conjunction with known antioxidant defences to render ROS resistance. By screening a transposon insertion library of F. tularensis LVS in the presence of hydrogen peroxide, we have identified an oxidant-sensitive mutant in putative EmrA1 (FTL_0687) secretion protein. The results demonstrate that the emrA1 mutant is highly sensitive to oxidants and several antimicrobial agents, and exhibits diminished intramacrophage growth that can be restored to wild-type F. tularensis LVS levels by either transcomplementation, inhibition of ROS generation or infection in NADPH oxidase deficient (gp91Phox(-/-)) macrophages. The emrA1 mutant is attenuated for virulence, which is restored by infection in gp91Phox(-/-) mice. Further, EmrA1 contributes to oxidative stress resistance by affecting secretion of Francisella antioxidant enzymes SodB and KatG. This study exposes unique links between transporter activity and the antioxidant defence mechanisms of F. tularensis.

Repression of inflammasome by Francisella tularensis during early stages of infection.

Francisella tularensis is an important human pathogen responsible for causing tularemia. F. tularensis has long been developed as a biological weapon and is now classified as a category A agent by the Centers for Disease Control because of its possible use as a bioterror agent. F. tularensis represses inflammasome; a cytosolic multi-protein complex that activates caspase-1 to produce proinflammatory cytokines IL-1ß and IL-18. However, the Francisella factors and the mechanisms through which F. tularensis mediates these suppressive effects remain relatively unknown. Utilizing a mutant of F. tularensis in FTL_0325 gene, this study investigated the mechanisms of inflammasome repression by F. tularensis. We demonstrate that muted IL-1ß and IL-18 responses generated in macrophages infected with F. tularensis live vaccine strain (LVS) or the virulent SchuS4 strain are due to a predominant suppressive effect on TLR2-dependent signal 1. Our results also demonstrate that FTL_0325 of F. tularensis impacts proIL-1ß expression as early as 2 h post-infection and delays activation of AIM2 and NLRP3-inflammasomes in a TLR2-dependent fashion. An enhanced activation of caspase-1 and IL-1ß observed in FTL_0325 mutant-infected macrophages at 24 h post-infection was independent of both AIM2 and NLRP3. Furthermore, F. tularensis LVS delayed pyroptotic cell death of the infected macrophages in an FTL_0325-dependent manner during the early stages of infection. In vivo studies in mice revealed that suppression of IL-1ß by FTL_0325 early during infection facilitates the establishment of a fulminate infection by F. tularensis. Collectively, this study provides evidence that F. tularensis LVS represses inflammasome activation and that F. tularensis-encoded FTL_0325 mediates this effect.