Heparin-Peptide Nanogranules for Thrombosis-Actuated Anticoagulation.

Despite nearly a century of clinical use as a blood thinner, heparin's rapid serum clearance and potential to induce severe bleeding events continue to urge the development of more effective controlled delivery strategies. Subcutaneous depots that steadily release the anticoagulant into circulation represent a promising approach to reducing overdose frequency, sustaining therapeutic concentrations of heparin in plasma, and prolonging anticoagulant activity in a safe and effective manner. Subcutaneously deliverable heparin-peptide nanogranules that allow for long-lasting heparin bioavailability in the circulatory system, while enabling on-demand activation of heparin's anticoagulant effects in the thrombus microenvironment, are reported. Biophysical studies demonstrate this responsive behavior is due to the sequestration of heparin within self-assembling peptide nanofibrils and its mechanically actuated decoupling to elicit antithrombotic effects at the clotting site. In vivo studies show these unique properties converge to allow subcutaneous nanogranule depots to extend heparin serum concentrations for an order of magnitude longer than standard dosing regimens while enabling prolonged and controlled anticoagulant activity. This biohybrid delivery system demonstrates a potentially scalable platform for the development of safer, easier to administer, and more effective antithrombotic nanotechnologies.

The role of selenoproteins in neutrophils during inflammation.

Polymorphonuclear neutrophils (PMNs)-derived ROS are involved in the regulation of multiple functions of PMNs critical in both inflammation and its timely resolution. Selenium is an essential trace element that functions as a gatekeeper of cellular redox homeostasis in the form of selenoproteins. Despite their well-studied involvement in regulating functions of various immune cells, limited studies have focused on the regulation of selenoproteins in PMN and their associated functions. Ex-vivo treatment of murine primary bone marrow derived PMNs with bacterial endotoxin lipopolysaccharide (LPS) indicated temporal regulation of several selenoprotein genes at the mRNA level. However, only glutathione peroxidase 4 (Gpx4) was significantly upregulated, while Selenof, Selenow, and Gpx1 were significantly downregulated in a temporal manner at the protein level. Exposure of PMNs isolated from tRNASec (Trsp)fl/fl S100A8Cre (TrspN) PMN-specific selenoprotein knockout mice, to the Gram-negative bacterium, Citrobacter rodentium, showed decreased bacterial growth, reduced phagocytosis, as well as impaired neutrophil extracellular trap (NET) formation ability, when compared to the wild-type PMNs. Increased extracellular ROS production upon LPS stimulation was also observed in TrspN PMNs that was associated with upregulation of Alox12, Cox2, and iNOS, as well as proinflammatory cytokines such as TNFα and IL-1ß. Our data indicate that the inhibition of selenoproteome expression results in alteration of PMN proinflammatory functions, suggesting a potential role of selenoproteins in the continuum of inflammation and resolution.

Inhalable SARS-CoV-2 Mimetic Particles Induce Pleiotropic Antigen Presentation.

Coronavirus disease 2019 (Covid-19) has caused over 5.5 million deaths worldwide, and viral mutants continue to ravage communities with limited access to injectable vaccines or high rates of vaccine hesitancy. Inhalable vaccines have the potential to address these distribution and compliance issues as they are less likely to require cold storage, avoid the use of needles, and can elicit localized immune responses with only a single dose. Alveolar macrophages represent attractive targets for inhalable vaccines as they are abundant within the lung mucosa (up to 95% of all immune cells) and are important mediators of mucosal immunity, and evidence suggests that they may be key cellular players in early Covid-19 pathogenesis. Here, we report inhalable coronavirus mimetic particles (CoMiP) designed to rapidly bind to, and be internalized by, alveolar macrophages to deliver nucleic acid-encoded viral antigens. Inspired by the SARS-CoV-2 virion structure, CoMiP carriers package nucleic acid cargo within an endosomolytic peptide envelope that is wrapped in a macrophage-targeting glycosaminoglycan coating. Through this design, CoMiP mimic several important features of the SARS-CoV-2 virion, particularly surface topography and macromolecular chemistry. As a result, CoMiP effect pleiotropic transfection of macrophages and lung epithelial cells in vitro with multiple antigen-encoding plasmids. In vivo immunization yields increased mucosal IgA levels within the respiratory tract of CoMiP vaccinated mice.

A Single Mechanosensitive Channel Protects Francisella tularensis subsp. holarctica from Hypoosmotic Shock and Promotes Survival in the Aquatic Environment.

Francisella tularensis subsp. holarctica is found in North America and much of Europe and causes the disease tularemia in humans and animals. An aquatic cycle has been described for this subspecies, which has caused waterborne outbreaks of tularemia in at least 10 countries. In this study, we sought to identify the mechanosensitive channel(s) required for the bacterium to survive the transition from mammalian hosts to freshwater, which is likely essential for the transmission of the bacterium between susceptible hosts. A single 165-amino-acid MscS-type mechanosensitive channel (FtMscS) was found to protect F. tularensis subsp. holarctica from hypoosmotic shock, despite lacking much of the cytoplasmic vestibule domain found in well-characterized MscS proteins from other organisms. The deletion of this channel did not affect virulence within the mammalian host; however, FtMscS was required to survive the transition from the host niche to freshwater. The deletion of FtMscS did not alter the sensitivity of F. tularensis subsp. holarctica to detergents, H2O2, or antibiotics, suggesting that the role of FtMscS is specific to protection from hypoosmotic shock. The deletion of FtMscS also led to a reduced average cell size without altering gross cell morphology. The mechanosensitive channel identified and characterized in this study likely contributes to the transmission of tularemia between hosts by allowing the bacterium to survive the transition from mammalian hosts to freshwater.IMPORTANCE The contamination of freshwater by Francisella tularensis subsp. holarctica has resulted in a number of outbreaks of tularemia. Invariably, the contamination originates from the carcasses or excreta of infected animals and thus involves an abrupt osmotic downshock as the bacteria enter freshwater. How F. tularensis survives this drastic change in osmolarity has not been clear, but here we report that a single mechanosensitive channel protects the bacterium from osmotic downshock. This channel is functional despite lacking much of the cytoplasmic vestibule domain that is present in better-studied organisms such as Escherichia coli; this report builds on previous studies that have suggested that parts of this domain are dispensable for downshock protection. These findings extend our understanding of the aquatic cycle and ecological persistence of F. tularensis, with further implications for mechanosensitive channel biology.

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.

Increased susceptibility of IgA-deficient mice to pulmonary Francisella tularensis live vaccine strain infection.

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

Type I IFN signaling constrains IL-17A/F secretion by gammadelta T cells during bacterial infections.

Recognition of intracellular bacteria by macrophages leads to secretion of type I IFNs. However, the role of type I IFN during bacterial infection is still poorly understood. Francisella tularensis, the causative agent of tularemia, is a pathogenic bacterium that replicates in the cytosol of macrophages leading to secretion of type I IFN. In this study, we investigated the role of type I IFNs in a mouse model of tularemia. Mice deficient for type I IFN receptor (IFNAR1(-/-)) are more resistant to intradermal infection with F. tularensis subspecies novicida (F. novicida). Increased resistance to infection was associated with a specific increase in IL-17A/F and a corresponding expansion of an IL-17A(+) gammadelta T cell population, indicating that type I IFNs negatively regulate the number of IL-17A(+) gammadelta T cells during infection. Furthermore, IL-17A-deficient mice contained fewer neutrophils compared with wild-type mice during infection, indicating that IL-17A contributes to neutrophil expansion during F. novicida infection. Accordingly, an increase in IL-17A in IFNAR1(-/-) mice correlated with an increase in splenic neutrophil numbers. Similar results were obtained in a mouse model of pneumonic tularemia using the highly virulent F. tularensis subspecies tularensis SchuS4 strain and in a mouse model of systemic Listeria monocytogenes infection. Our results indicate that the type I IFN-mediated negative regulation of IL-17A(+) gammadelta T cell expansion is conserved during bacterial infections. We propose that this newly described activity of type I IFN signaling might participate in the resistance of the IFNAR1(-/-) mice to infection with F. novicida and other intracellular bacteria.

Humoral and cell-mediated immunity to the intracellular pathogen Francisella tularensis.

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

Vitamin D and the Ability to Produce 1,25(OH)2D Are Critical for Protection from Viral Infection of the Lungs.

Vitamin D supplementation is linked to improved outcomes from respiratory virus infection, and the COVID-19 pandemic renewed interest in understanding the potential role of vitamin D in protecting the lung from viral infections. Therefore, we evaluated the role of vitamin D using animal models of pandemic H1N1 influenza and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. In mice, dietary-induced vitamin D deficiency resulted in lung inflammation that was present prior to infection. Vitamin D sufficient (D+) and deficient (D-) wildtype (WT) and D+ and D- Cyp27B1 (Cyp) knockout (KO, cannot produce 1,25(OH)2D) mice were infected with pandemic H1N1. D- WT, D+ Cyp KO, and D- Cyp KO mice all exhibited significantly reduced survival compared to D+ WT mice. Importantly, survival was not the result of reduced viral replication, as influenza M gene expression in the lungs was similar for all animals. Based on these findings, additional experiments were performed using the mouse and hamster models of SARS-CoV-2 infection. In these studies, high dose vitamin D supplementation reduced lung inflammation in mice but not hamsters. A trend to faster weight recovery was observed in 1,25(OH)2D treated mice that survived SARS-CoV-2 infection. There was no effect of vitamin D on SARS-CoV-2 N gene expression in the lung of either mice or hamsters. Therefore, vitamin D deficiency enhanced disease severity, while vitamin D sufficiency/supplementation reduced inflammation following infections with H1N1 influenza and SARS-CoV-2.

Differential Immune Response Following Intranasal and Intradermal Infection with Francisella tularensis: Implications for Vaccine Development.

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

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.

Antibacterial effects of low-temperature plasma generated by atmospheric-pressure plasma jet are mediated by reactive oxygen species.

Emergence and spread of antibiotic resistance calls for development of non-chemical treatment options for bacterial infections. Plasma medicine applies low-temperature plasma (LTP) physics to address biomedical problems such as wound healing and tumor suppression. LTP has also been used for surface disinfection. However, there is still much to be learned regarding the effectiveness of LTP on bacteria in suspension in liquids, and especially on porous surfaces. We investigated the efficacy of LTP treatments against bacteria using an atmospheric-pressure plasma jet and show that LTP treatments have the ability to inhibit both gram-positive (S. aureus) and gram-negative (E. coli) bacteria on solid and porous surfaces. Additionally, both direct LTP treatment and plasma-activated media were effective against the bacteria suspended in liquid culture. Our data indicate that reactive oxygen species are the key mediators of the bactericidal effects of LTP and hydrogen peroxide is necessary but not sufficient for antibacterial effects. In addition, our data suggests that bacteria exposed to LTP do not develop resistance to further treatment with LTP. These findings suggest that this novel atmospheric-pressure plasma jet could be used as a potential alternative to antibiotic treatments in vivo.

Contribution of citrulline ureidase to Francisella tularensis strain Schu S4 pathogenesis.

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

Role of Selenoproteins in Bacterial Pathogenesis.

The trace element selenium is an essential micronutrient that plays an important role in maintaining homeostasis of several tissues including the immune system of mammals. The vast majority of the biological functions of selenium are mediated via selenoproteins, proteins which incorporate the selenium-containing amino acid selenocysteine. Several bacterial infections of humans and animals are associated with decreased levels of selenium in the blood and an adjunct therapy with selenium often leads to favorable outcomes. Many pathogenic bacteria are also capable of synthesizing selenocysteine suggesting that selenoproteins may have a role in bacterial physiology. Interestingly, the composition of host microbiota is also regulated by dietary selenium levels. Therefore, bacterial pathogens, microbiome, and host immune cells may be competing for a limited supply of selenium. Elucidating how selenium, in particular selenoproteins, may regulate pathogen virulence, microbiome diversity, and host immune response during a bacterial infection is critical for clinical management of infectious diseases.

Cross Sectional Study and Risk Factors Analysis of Francisella tularensis in Soil Samples in Punjab Province of Pakistan.

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

Adaptation of Francisella tularensis to the mammalian environment is governed by cues which can be mimicked in vitro.

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

Pertussis toxin inhibits neutrophil recruitment to delay antibody-mediated clearance of Bordetella pertussis.

Whooping cough is considered a childhood disease, although there is growing evidence that children are infected by adult carriers. Additionally, increasing numbers of vaccinated adults are being diagnosed with Bordetella pertussis disease. Thus it is critical to understand how B. pertussis remains endemic even in highly vaccinated or immune populations. Here we used the mouse model to examine the nature of sterilizing immunity to B. pertussis. Antibodies were necessary to control infection but did not rapidly clear B. pertussis from the lungs. However, antibodies affected B. pertussis after a delay of at least a week by a mechanism that involved neutrophils and Fc receptors, suggesting that neutrophils phagocytose and clear antibody-opsonized bacteria via Fc receptors. B. pertussis blocked migration of neutrophils and inhibited their recruitment to the lungs during the first week of infection by a pertussis toxin-dependent (PTx-dependent) mechanism; a PTx mutant of B. pertussis induced rapid neutrophil recruitment and was rapidly cleared from the lungs by adoptively transferred antibodies. Depletion of neutrophils abrogated the defects of the PTx mutant. Together these results indicate that PTx inhibits neutrophil recruitment, which consequently allows B. pertussis to avoid rapid antibody-mediated clearance and therefore successfully infect immune hosts.

A New Mechanism for Ribosome Rescue Can Recruit RF1 or RF2 to Nonstop Ribosomes.

Bacterial ribosomes frequently translate to the 3' end of an mRNA without terminating at an in-frame stop codon. In all bacteria studied to date, these "nonstop" ribosomes are rescued using trans-translation. Genes required for trans-translation are essential in some species, but other species can survive without trans-translation because they express an alternative ribosome rescue factor, ArfA or ArfB. Francisella tularensis cells lacking trans-translation are viable, but F. tularensis does not encode ArfA or ArfB. Transposon mutagenesis followed by deep sequencing (Tn-seq) identified a new alternative ribosome rescue factor, now named ArfT. arfT can be deleted in wild-type (wt) cells but not in cells that lack trans-translation activity. Overexpression of ArfT suppresses the slow-growth phenotype in cells lacking trans-translation and counteracts growth arrest caused by trans-translation inhibitors, indicating that ArfT rescues nonstop ribosomes in vivo Ribosome rescue assays in vitro show that ArfT promotes hydrolysis of peptidyl-tRNA on nonstop ribosomes in conjunction with F. tularensis release factors. Unlike ArfA, which requires RF2 for activity, ArfT can function with either RF1 or RF2. Overall, these results indicate that ArfT is a new alternative ribosome rescue factor with a distinct mechanism from ArfA and ArfB.IMPORTANCEFrancisella tularensis is a highly infectious intracellular pathogen that kills more than half of infected humans if left untreated. F. tularensis has also been classified as a potential bioterrorism agent with a great risk for deliberate misuse. Recently, compounds that inhibit ribosome rescue have been shown to have antibiotic activity against F. tularensis and other important pathogens. Like all bacteria that have been studied, F. tularensis uses trans-translation as the main pathway to rescue stalled ribosomes. However, unlike most bacteria, F. tularensis can survive without any of the known factors for ribosome rescue. Our work identified a F. tularensis protein, ArfT, that rescues stalled ribosomes in the absence of trans-translation using a new mechanism. These results indicate that ribosome rescue activity is essential in F. tularensis and suggest that ribosome rescue activity might be essential in all bacteria.

Different mechanisms of vaccine-induced and infection-induced immunity to Bordetella bronchiseptica.

A recent resurgence in the number of cases of whooping cough, and other respiratory diseases caused by members of the bordetellae, in vaccinated populations has demonstrated the need for a thorough understanding of vaccine-induced immunity to facilitate more intelligent vaccine design. In this work, we use a murine model of respiratory infection using the highly successful animal pathogen, Bordetella bronchiseptica. Since previously infected animals have been shown to resist re-infection by B. bronchiseptica, we sought to examine the differences between vaccine-induced immunity and infection-induced immunity. Both prior infection and vaccination conferred nearly complete protection in the lungs, however, only prior infection resulted in significant protection in the upper respiratory tract. While immunity induced by prior infection offered significant protection even in the absence of complement or FcgammaRs, vaccination-induced protection required both complement and FcgammaRs. Although vaccination induced higher titers of B. bronchiseptica-specific antibodies, this serum was less effective than infection-induced serum in clearing bacteria from the lower respiratory tract. Together these findings highlight substantial differences between the mechanisms involved in vaccine- and infection-induced protective immunity.

Development of a novel Francisella tularensis Live Vaccine Strain expressing ovalbumin provides insight into antigen-specific CD8+ T cell responses.

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