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Francisella tularensis can cause severe disease in humans via the respiratory or cutaneous routes and a case fatality ratio of up to 10 % is reported due to lack of proper antibiotic treatment, while F. novicida causes disease in severely immunocompromised individuals. Efforts are needed to develop effective vaccine candidates against Francisella species. Thus, in this study, a systematic computational work frame was used to deeply investigate the whole proteome of Francisella novicida containing 1728 proteins to develop vaccine against F. tularensis and related species. Whole-proteome analysis revealed that four proteins including (A0Q492) (A0Q7Y4), (A0Q4N4), and (A0Q5D9) are the suitable vaccine targets after the removal of homologous, paralogous and prediction of subcellular localization. These proteins were used to predict the T cell, B cell, and HTL epitopes which were joined together through suitable linkers to construct a multi-epitopes vaccine (MEVC). The MEVC was found to be highly immunogenic and non-allergenic while the physiochemical properties revealed the feasible expression and purification. Moreover, the molecular interaction of MEVC with TLR2, molecular simulation, and binding free energy analyses further validated the immune potential of the construct. According to Jcat analysis, the refined sequence demonstrates GC contents of 41.48 % and a CAI value of 1. The in-silico cloning and optimization process ensured compatibility with host codon usage, thereby facilitating efficient expression. Computational immune simulation studies underscored the capacity of MEVC to induce both primary and secondary immune responses. The conservation analysis further revealed that the selected epitopes exhibit 100 % conservation across different species and thus provides wider protection against Francisella. • Francisella novicida can cause severe disease in humans via the respiratory or cutaneous routes. • Investigation of the whole-proteome revealed four proteins as suitable vaccine targets. • We designed a multi-epitopes vaccine (MEVC) from T cell, B cell, and HTL epitopes. • Molecular docking, simulation, in silico cloning and immune simulation confirmed the potential of our MEVC. • Our MEVC could also provide cross-protection against the other species of Francisella. [ABSTRACT FROM AUTHOR]

Two component system bacterial response regulators are typically DNA-binding proteins which enable the genetic regulation of many adaptive bacterial behaviors. Despite structural similarity across response regulator families, there is a diverse array of DNA-binding mechanisms. Bacteria usually encode several dozen two-component system response regulators, but Francisella tularensis only encodes three. Due to their simplified response regulatory network, Francisella species are a model for studying the role of response regulator proteins in virulence. Here, we show that Francisella response regulators QseB, KdpE, and BfpR all utilize different DNA-binding mechanisms. Our evidence suggests that QseB follows a simple mechanism whereby it binds a single inverted repeat sequence with a higher affinity upon phosphorylation. This behavior is independent of whether QseB is a positive or negative regulator of the gene as demonstrated by qseB and priM promoter sequences, respectively. Similarly, KdpE binds DNA more tightly upon phosphorylation, but also exhibits a cooperative binding isotherm. While we propose a KdpE binding site, it is possible that KdpE has a complex DNA-binding mechanism potentially involving multiple copies of KdpE being recruited to a promoter region. Finally, we show that BfpR appears to bind a region of its own promoter sequence with a lower affinity upon phosphorylation. Further structural and enzymatic work will need to be performed to deconvolute the KdpE and BfpR binding mechanisms. • Identification of putative QseB and KdpE binding sequences. • QseB and KdpE have increased DNA-binding affinity upon beryllofluoridation. • KdpE exhibits a cooperative binding isotherm upon beryllofluoridation. • BfpR treated with a small molecule phosphodonor shows decreased affinity for DNA. • KdpE REC structure has an altered dimer interface compared to other response regulators. [ABSTRACT FROM AUTHOR]

Utilization of fluorescent proteins is widespread for the study of microbial pathogenesis and host-pathogen interactions. Here, we discovered that linkage of the 36 N-terminal amino acids of FTL_0580 (a hypothetical protein of Francisella tularensis) to fluorescent proteins increases the fluorescence emission of bacteria that express these recombinant fusions. This N-terminal peptide will be referred to as 580N. Western blotting revealed that the linkage of 580N to Emerald Green Fluorescent Protein (EmGFP) in F. tularensis markedly improved detection of this protein. We therefore hypothesized that transcripts containing 580N may be translated more efficiently than those lacking the coding sequence for this leader peptide. In support, expression of emGFP Ft that had been codon-optimized for F. tularensis , yielded significantly enhanced fluorescence than its non-optimized counterpart. Furthermore, fusing emGFP with coding sequence for a small N-terminal peptide (Serine-Lysine-Isoleucine-Lysine), which had previously been shown to inhibit ribosomal stalling, produced robust fluorescence when expressed in F. tularensis. These findings support the interpretation that 580N enhances the translation efficiency of fluorescent proteins in F. tularensis. Interestingly, expression of non-optimized 580N-emGFP produced greater fluorescence intensity than any other construct. Structural predictions suggested that RNA secondary structure also may be influencing translation efficiency. When expressed in Escherichia coli and Klebsiella pneumoniae bacteria, 580N-emGFP produced increased green fluorescence compared to untagged emGFP (neither allele was codon optimized for these bacteria). In conclusion, fusing the coding sequence for the 580N leader peptide to recombinant genes might serve as an economical alternative to codon optimization for enhancing protein expression in bacteria. • Bacteria expressing a fusion of a small peptide (580N) to EmGFP & TdTomato increases their brightness. • 580N likely antagonizes ribosomal stalling to increase translation of the fused protein. • Fusion of 580N to other recombinant proteins might serve as an economical alternative to codon optimization. [ABSTRACT FROM AUTHOR]

Abstract Bacteria that are highly virulent, expressing high infectivity, and able to survive nebulization, pose great risk to the human population. One of these is Francisella tularensis , the etiological agent of tularemia. F. tularensis is a subject of intense scientific interest due to the fact that vaccines for its immunoprophylaxis in humans are not yet routinely available. One of the substantial obstacles in developing such vaccines is our insufficient knowledge of processes that initiate and regulate the expression of effective protective immunity against intracellular bacteria. Here, we present data documenting the different pattern of cellular behavior occurring in an environment unaffected by microbiota using the model of germ-free mice mono-associated with F. tularensis subsp. holarctica strain LVS in comparison with a classic specific-pathogen-free murine model during early stages of infection. Highlights • Different pattern of cellular behavior in the environment unaffected by microbiota. • Decisive role of primary interaction between the invading bacterium and the host cell. • Majority of changes in composition of cell subtypes occur during 12 h post infection. • Existence of a hierarchy of functional innate immune response modules. [ABSTRACT FROM AUTHOR]

Francisella tularensis is a highly infectious intracellular bacterium causing tularemia disease and is regarded as a potential biological weapon. The development of a vaccine, effective treatment, or prophylactic substances targeted against tularemia is in the forefront of interest and could help to prevent or mitigate possible malevolent acts by bioterrorism utilizing F. tularensis. The viability of F. tularensis , and thus of a tularemia disease outbreak, might potentially be suppressed by simple commonly available natural substances. Epigallocatechin gallate (EGCG) is contained in green tea and its antimicrobial effect has been described. Here, we show that EGCG can suppress F. tularensis growth and is able to reduce the bacterium's ability to replicate inside mouse bone marrow-derived macrophages (BMMs) without side effects on BMMs' own viability. We suggest one (but not the only) mechanism of EGCG action. We demonstrate that EGCG can block the main functions of HU protein, the important regulator of F. tularensis virulence, leading to overall attenuation of F. tularensis viability. EGCG can delay death of mice infected by F. tularensis and can be used as a prophylactic agent against tularemia disease. Postponing death by up to 2 days can provide sufficient opportunity to administer another treatment agent. • Francisella tularensis constitutes risk as a potential biological weapon. • Therefore, a vaccine, therapies, and prophylaxes are much needed. • Contained in green tea, epigallocatechin gallate (EGCG) has antimicrobial effects. • EGCG can suppress F. tularensis growth and reduce its replication in macrophages. • Functions of F. tularensis virulence regulator HU protein can be blocked by EGCG. [ABSTRACT FROM AUTHOR]

Bacterial pathogens display an impressive arsenal of molecular mechanisms that allow survival in diverse host niches. Subversion of plasma membrane and cytoskeletal functions are common themes associated to infection by both extracellular and intracellular pathogens. Moreover, intracellular pathogens modify the structure/stability of their membrane-bound compartments and escape degradation from phagocytic or autophagic pathways. Here, we review the manipulation of host membranes by Listeria monocytogenes , Francisella tularensis , Shigella flexneri and Yersinia spp. These four bacterial model pathogens exemplify generalized strategies as well as specific features observed during bacterial infection processes. [ABSTRACT FROM AUTHOR]

The detection of bacterial pathogens from complex sample matrices by PCR requires efficient DNA extraction. In this study, a protocol for extraction and purification of DNA from swabs, air, and water samples using a microfluidic chip system was established. The optimized protocol includes a combination of thermal, chemical and enzymatic lysis followed by chip-based DNA purification using magnetic particles. The procedure was tested using Gram-positive Bacillus thuringiensis Berliner var. kurstaki as a model organism for Bacillus anthracis and the attenuated live vaccine strain of Francisella tularensis subsp. holarctica as Gram-negative bacterium. The detection limits corresponded to 10 3 genome equivalents per milliliter (GE/ml) for surface water samples spiked with F. tularensis and 10 2 GE/ml for B. thuringiensis . In air, 10 GE of F. tularensis per 10 L and 1 GE of B. thuringiensis per 10 L were detectable. For swab samples obtained from artificially contaminated surfaces the detection limits were 4 × 10 3 GE/cm 2 for F. tularensis and 4 × 10 2 GE/cm 2 for B. thuringiensis . Suitability of the chip-assisted procedure for DNA preparation of real samples was demonstrated using livestock samples. The presence of thermophilic Campylobacter spp. DNA could be confirmed in air samples collected on pig and broiler farms. [ABSTRACT FROM AUTHOR]

Autophagy is a conserved lysosomal recycling process, which maintains cellular homeostasis during stress and starvation conditions by degrading and recycling proteins, lipids, and carbohydrates, ultimately increasing nutrient availability in eukaryotes. An additional function of autophagy, termed xenophagy, is to detect, capture, and destroy invading microorganisms, such as viruses, bacteria, and protozoa, providing autophagy with a role in innate immunity. Many intracellular pathogens have, however, developed mechanisms to avoid xenophagy and have evolved strategies to take advantage of select autophagic processes to undergo their intracellular life cycle. This review article will discuss the molecular mechanisms used by the intracellular bacterial pathogens Francisella spp . and Brucella spp . to manipulate components of the autophagic pathway, promoting cytosolic growth in the case of Francisella spp . and facilitating cellular egress and cell-to-cell spread in the case of Brucella spp . These examples highlight how successful, highly infectious bacterial pathogens avoid or subvert host autophagy mechanisms normally employed to maintain eukaryotic homeostasis. [ABSTRACT FROM AUTHOR]

Extracellular form of Francisella is able to cross various cell barriers and invade multiple organs, such as skin, liver, lung and central nervous system. Transient adhesion of Francisella to endothelial cells may trigger the process of translocation. In this report, we showed that Francisella tularensis subsp. holarctica ( Fth ) is able to adhere to the endothelial cells, while ICAM-1 may serve as an adhesion molecule for Fth . Pull down and affinity ligand binding assays indicated that the PilE4 could be the probable ligand for ICAM-1. Further deciphering of this ligand:receptor interaction revealed that PilE4 interacts with Ig-like C2-type 1 domain of ICAM-1. To corroborate the role of PilE4 and ICAM-1 interaction in adhesion of extracellular form of Fth to endothelial cells, ICAM-1 was blocked with monoclonal anti-ICAM-1 antibody prior to the incubation with Fth and numbers of adherent bacteria were counted. Blocking of the ICAM-1 significantly reduced (500–fold, P < 0.05) number of adherent Fth compared to unblocked cells. PilE4:ICAM-1 interaction unfolded here may provide a new perspective on molecules involved in the adhesion of extracellular form of Francisella to endothelial cells and probably its translocation across endothelial barriers. [ABSTRACT FROM AUTHOR]

• ACOD KO animals experience prolonged Francisella tularensis LVS infection. • ACOD KO mice are less susceptible to virulent F. tularensis infection. • Survival following challenge correlates with improved polyfunctional T cell responses. • T cells take up itaconate but it does not alter cytokine production. • The impact of itaconate on the T cell response is indirect via infected APCs. The metabolite itaconate plays a critical role in modulating inflammatory responses among macrophages infected with intracellular pathogens. However, the ability of itaconate to influence developing T cells responses is poorly understood. To determine if itaconate contributes to the quality of T cell mediated immunity against intracellular infection, we used Francisella tularensis as a model of vaccine induced immunity. Following vaccination with F. tularensis live vaccine strain, itaconate deficient mice (ACOD KO) had a prolonged primary infection but were more resistant to secondary infection with virulent F. tularensis relative to wild type controls. Improved resistance to secondary challenge was associated with both increased numbers and effector function of CD4+ and CD8+ T cells in ACOD KO mice. However, additional data suggest that improved T cell responses was not T cell intrinsic. These data underscore the consequences of metabolic perturbations within antigen presenting cells on the development of vaccine-elicited immune responses. [ABSTRACT FROM AUTHOR]

Abstract: To fully understand the interactions of a pathogen with its host, it is necessary to analyze the RNA transcripts of both the host and pathogen throughout the course of an infection. Although this can be accomplished relatively easily on the host side, the analysis of pathogen transcripts is complicated by the overwhelming amount of host RNA isolated from an infected sample. Even with the read depth provided by second-generation sequencing, it is extremely difficult to get enough pathogen reads for an effective gene-level analysis. In this study, we describe a novel capture-based technique and device that considerably enriches for pathogen transcripts from infected samples. This versatile method can, in principle, enrich for any pathogen in any infected sample. To test the technique’s efficacy, we performed time course tissue culture infections using Rift Valley fever virus and Francisella tularensis. At each time point, RNA sequencing (RNA-Seq) was performed and the results of the treated samples were compared with untreated controls. The capture of pathogen transcripts, in all cases, led to more than an order of magnitude enrichment of pathogen reads, greatly increasing the number of genes hit, the coverage of those genes, and the depth at which each transcript was sequenced. [Copyright &y& Elsevier]

Abstract: The role of antibodies in the course of Francisella tularensis (F. tularensis) infection is still a subject of debate. The understanding of the poorly described role of humoral immunity is more than important for the effort to develop effective prophylactic procedure against the infection with Francisella virulent strains. We utilized the model of gamma-irradiated mice for the studies of the protective role of anti-F. tularensis antibodies in order to partially eliminate cellular responses. The model of gamma-irradiated mice can also demonstrate the responses of immunocompromised host to intracellular bacterial infection. The gamma-irradiation by doses greater than 3 Gy completely impairs the resistance to infection and causes a disbalance of cytokine production in mice. In this study, we demonstrate that passive transfer of immune sera protected irradiated mice against subsequent infection with strains of F. tularensis subsp. holarctica. Naïve mice of BALB/c or C3H/CBi strains were subjected to passive transfer of sera obtained from immunized mice with live vaccine strain (LVS) F. tularensis LVS, F. tularensis subsp. holarctica strain 15, heat-killed F. tularensis LVS, or heat-killed strain 15 two hours before infection with lethal doses of LVS or strain 15. The passive transfer of sera obtained from immunized mice conferred full protection of naïve unirradiated as well as sublethally irradiated mice against low lethal doses of infection with F. tularensis LVS or strain 15, in all variants of the experiments. In addition, the passively protected mice that survived the primary infection with F. tularensis LVS were protected also against further secondary challenge with a highly virulent strain of F. tularensis subsp. tularensis SchuS4. Moreover, the first evidence of combination of successful passive transfer of immunity by specific antisera and subsequent active immunization of immunocompromised animals is demonstrated. In summary, we demonstrate that B cell-mediated effector responses together with the induction of T cell-mediated immunity both play an important role in naïve and also in immunocompromised mice and this fact it would be appropriate to take into the account in the design of new vaccines. [Copyright &y& Elsevier]

Abstract: Francisella tularensis is a facultative intracellular bacterium and the causative agent of tularemia. Virulence factors for this bacterium, particularly those that facilitate host cell interaction, remain largely uncharacterized. However, genes homologous to those involved in type IV pilus structure and assembly, including six genes encoding putative major pilin subunit proteins, are present in the genome of the highly virulent Schu S4 strain. To analyze the roles of three putative pilin genes in pili structure and function we constructed individual pilE4, pilE5, and pilE6 deletion mutants in both the F. tularensis tularensis strain Schu S4 and the Live Vaccine Strain (LVS), an attenuated derivative strain of F. tularensis holarctica. Transmission electron microscopy (TEM) of Schu S4 and LVS wild-type and deletion strains confirmed that pilE4 was essential for the expression of type IV pilus-like fibers by both subspecies. By the same method, pilE5 and pilE6 were dispensable for pilus production. In vitro adherence assays with J774A.1 cells revealed that LVS pilE4, pilE5, and pilE6 deletion mutants displayed increased attachment compared to wild-type LVS. However, in the Schu S4 background, similar deletion mutants displayed adherence levels similar to wild-type. In vivo, LVS pilE5 and pilE6 deletion mutants were significantly attenuated compared to wild-type LVS by intradermal and subcutaneous murine infection, while no Schu S4 deletion mutant was significantly attenuated compared to wild-type Schu S4. While pilE4 was essential for fiber expression on both Schu S4 and LVS, neither its protein product nor the assembled fibers contributed significantly to virulence in mice. Absent a role in pilus formation, we speculate PilE5 and PilE6 are pseudopilin homologs that comprise, or are associated with, a novel type II-related secretion system in Schu S4 and LVS. [Copyright &y& Elsevier]

Abstract: Three cytokines use the IL-12p40 cytokine subunit namely: IL-12p70 (IL-12-comprised of IL-12p40 and IL-12p35), IL-23 (comprised of the IL-12p40 and IL-23p19 subunits) and homodimeric IL-12p40 (IL-12(p40)2). Following activation, immature dendritic cells (DCs) upregulate the chemokine receptor Chemokine-C-Receptor 7 (CCR7), and migrate in response to homeostatic chemokines such as chemokine (C–C motif) ligand 19 (CCL19). Induction of the cytokine IL-12p40 in response to pathogen-exposure, likely in its homodimeric form, is one of the primary events that mediates migration of DCs in response to CCL19. Here we show that following exposure to Francisella tularensis Live Vaccine Strain (LVS), DCs produce IL-12p40 and promote the migration of DCs to the chemokine CCL19 in an IL-12Rβ1- and IL-12p(40)2-dependent manner. Induction of IL-12p40 and resulting chemokine responsiveness in DCs is TLR2-dependent and coincides with the uptake of F. tularensis LVS and activation of DCs. Importantly, we show that IL-12Rβ1 signaling is required for DC migration from the lung to the draining lymph node following F. tularensis LVS exposure and coincides with accumulation of IL-12p40 expressing DCs in the draining lymph nodes. Together, these findings illustrate that IL-12p40 is induced rapidly in response to F. tularensis LVS and is required for DC migration through an IL-12Rβ1-IL-12(p40)2 dependent mechanism. [Copyright &y& Elsevier]

Abstract: Mast cells have classically been implicated in the triggering of allergic and anaphylactic reactions. However, recent findings have elucidated the ability of these cells to selectively release a variety of cytokines leading to bacterial clearance through neutrophil and dendritic cell mobilization, and suggest an important role in innate host defenses. Our laboratory has established a primary bone marrow derived mast cell–macrophage co-culture system and found that mast cells mediated a significant inhibition of Francisella tularensis live vaccine strain (LVS) uptake and replication within macrophages through contact and the secreted product interleukin-4 (IL-4). In this study, we utilized P815 mast cells and J774 macrophages to further investigate whether mast cell activation by non-FcεR driven signals could produce IL-4 and control intramacrophage LVS replication. P815 supernatants collected upon activation by the mast cell activating peptide MP7, as well as P815 cells co-cultured with J774 macrophages, exhibited marked inhibition of bacterial uptake and replication, which correlated with the production of IL-4. The inhibition noted in vitro was titratable and preserved at ratios relevant to cellular infiltration events following pulmonary challenge. Collectively, our data suggest that both primary mast cell and P815 mast cell (lacking FcεR) secreted IL-4 can control intramacrophage Francisella replication. [Copyright &y& Elsevier]

Abstract: The virulence mechanisms of Francisella tularensis, the causative agent of severe pneumonia in humans and a CDC category A bioterrorism agent, are not fully defined. As sepsis is the leading cause of mortality associated with respiratory infections, we determined whether, in the absence of any known bacterial toxins, a deregulated host response resulting in sepsis syndrome is associated with lethality of respiratory infection with the virulent human Type A strain SchuS4 of F. tularensis. The C57BL/6 mice infected intranasally with a lethal dose of SchuS4 exhibited high bacterial burden in systemic organs and blood indicative of bacteremia. In correlation, infected mice displayed severe tissue pathology and associated cell death in lungs, liver and spleen. Consistent with our studies with murine model strain Francisella novicida, infection with SchuS4 caused an initial delay in upregulation of inflammatory mediators followed by development of severe sepsis characterized by exaggerated cytokine release, upregulation of cardiovascular injury markers and sepsis mediator alarmins S100A9 and HMGB1. This study shows that pulmonary tularemia caused by the Type A strain of F. tularensis results in a deregulated host response leading to severe sepsis and likely represents the major cause of mortality associated with this virulent pathogen. [Copyright &y& Elsevier]

Abstract: A Francisella tularensis shuttle vector that constitutively expresses the Photorhabdus luminescens lux operon in type A and type B strains of F. tularensis was constructed. The bioluminescence reporter plasmid was introduced into the live vaccine strain of F. tularensis and used to follow F. tularensis growth in a murine intranasal challenge model in real-time by bioluminescence imaging. The results show that the new bioluminescence reporter plasmid represents a useful tool for tularemia research that is suitable for following F. tularensis growth in both in vitro and in vivo model systems. [ABSTRACT FROM AUTHOR]

Abstract: Francisella tularensis is a facultative intracellular, gram-negative bacterium that induces apoptosis in macrophages and B cells. Here we show apoptotic pathways that are activated in the Ramos human B cell line in the course of F. tularensis infection. Live bacteria F. tularensis FSC200 activate caspases 8, 9 and 3, as well as Bid; release cytochrome c and apoptosis-inducing factor from mitochondria; and induce depolarization of mitochondrial membrane potential in the Ramos cell line, thus leading these cells to apoptosis. Unlike live bacteria, killed F. tularensis FSC200 bacteria activated only caspase 3, and did not cause apoptosis of Ramos cells as measured by annexin V. Killed bacteria also caused accumulation of anti-apoptotic protein BclxL in mitochondrial membranes. Thus, live F. tularensis activates both caspase pathways (receptor-mediated and intrinsic) as well as caspase-independent mitochondrial death. [ABSTRACT FROM AUTHOR]

Abstract: The potential for genetic modification of biological warfare agents makes rapid identification of antibiotic resistant strains critical for the implementation of suitable infection control measures. The fluorinated quinolone, ciprofloxacin, is an antibiotic effective for treating bacterial infections by inhibiting the enzyme activity of the DNA type II topoisomerases DNA gyrase and topoisomerase IV. The genes that encode the subunits of DNA gyrase (gyrA and gyrB) and topo IV (par C and parE) contain hotspots within an area known as the quinolone resistance-determining region (QRDR). Base pair changes within this region give rise to mutations that cause resistance to the antibiotic by altering amino acids within the enzymes. Ciprofloxacin-resistant (cipror) strains of Bacillus anthracis, Yersinia pestis, and Francisella tularensis with one or more known mutations within the QRDR of gyrA, gyrB, parC, and parE genes were tested with SimpleProbe™ and High Resolution Melt (HRM) dye chemistries and Pyrosequencing™ genetic analysis to evaluate the ability to rapidly detect ciprofloxacin-induced mutations. While SimpleProbe™ and Pyrosequencing™ successfully identified all known mutants, the HRM assay identified all but those resulting from G↔C or A↔T substitutions. [Copyright &y& Elsevier]

Abstract: Francisella infects the lungs causing pneumonic tularemia. Focusing on the lung’s host defense, we have examined antimicrobial peptides as part of the innate immune response to Francisella infection. Interest in antimicrobial peptides, such as the cathelicidins, has grown due their potential therapeutic applications and the increasing problem of bacterial resistance to commonly used antibiotics. Only one human cathelicidin, LL-37, has been characterized. Helical cathelicidins have also been discovered in snakes including the Chinese King Cobra, Naja atra (NA-CATH). Four synthetic 11-residue peptides (ATRA-1, -2, -1A and -1P) containing variations of a repeated motif within NA-CATH were designed. We hypothesized that these smaller synthetic peptides could have excellent antimicrobial effectiveness with shorter length (and less cost), making them strong potential candidates for development into broad-spectrum antimicrobial compounds. We tested the susceptibility of F. novicida to four ATRA peptides, LL-37, and NA-CATH. Two of the ATRA peptides had high antimicrobial activity (μM), while the two proline-containing ATRA peptides had low activity. The ATRA peptides did not show significant hemolytic activity even at high peptide concentration, indicating low cytotoxicity against host cells. NA-CATH killed Francisella bacteria more quickly than LL-37. However, LL-37 was the most effective peptide against F. novicida (EC50=50nM). LL-37 mRNA was induced in A549 cells by Francisella infection. We recently demonstrated that F. novicida forms in vitro biofilms. LL-37 inhibited F. novicida biofilm formation at sub-antimicrobial concentrations. Understanding the properties of these peptides, and their endogenous expression in the lung could lead to potential future therapeutic interventions for this lung infection. [Copyright &y& Elsevier]

Abstract: Real-time polymerase chain reaction (PCR) assays were developed to detect Francisella tularensis (Ft), the causative agent of tularaemia in humans. Two real-time PCRs (FTT0376 and FTT0523) were designed in genetic sequences identified by the Insignia genome comparison tool (http://insignia.cbcb.umd.edu/) as being unique to pathogenic subspecies of F. tularensis. Both PCRs identified all pathogenic F. tularensis subspecies but did not cross react with avirulent Francisella philomiragia or F. tularensis ssp. novicida or other environmental bacteria. Limits of detection from DNA purified from pure culture (FTT0376 ∼ 80 Ft genome equivalents (GEs) per PCR; FTT0523 ∼ 20 Ft GEs per PCR;) and DNA purified from spiked blood samples (4 × 104 to 4 × 103 cfu ml−1, both assays) were determined. [Copyright &y& Elsevier]

Summary: Histidine acid phosphatases catalyze the transfer of a phosphoryl group from phosphomonoesters to water at acidic pH using an active-site histidine. The histidine acid phosphatase from the category A pathogen Francisella tularensis (FtHAP) has been implicated in intramacrophage survival and virulence, motivating interest in understanding the structure and mechanism of this enzyme. Here, we report a structure-based study of ligand recognition by FtHAP. The 1.70-Å-resolution structure of FtHAP complexed with the competitive inhibitor l(+)-tartrate was solved using single-wavelength anomalous diffraction phasing. Structures of the ligand-free enzyme and the complex with inorganic phosphate were determined at resolutions of 1.85 and 1.70 Å, respectively. The structure of the Asp261Ala mutant enzyme complexed with the substrate 3′-AMP was determined at 1.50 Å resolution to gain insight into substrate recognition. FtHAP exhibits a two-domain fold similar to that of human prostatic acid phosphatase, consisting of an α/β core domain and a smaller domain that caps the core domain. The structures show that the core domain supplies the phosphoryl binding site, catalytic histidine (His17), and an aspartic acid residue (Asp261) that protonates the leaving group, while the cap domain contributes residues that enforce substrate preference. FtHAP and human prostatic acid phosphatase differ in the orientation of the crucial first helix of the cap domain, implying differences in the substrate preferences of the two enzymes. 3′-AMP binds in one end of a 15-Å-long tunnel, with the adenine clamped between Phe23 and Tyr135, and the ribose 2′-hydroxyl interacting with Gln132. The importance of the clamp is confirmed with site-directed mutagenesis; mutation of Phe23 and Tyr135 individually to Ala increases K m by factors of 7 and 10, respectively. The structural data are consistent with a role for FtHAP in scavenging phosphate from small molecules present in host macrophage cells. [Copyright &y& Elsevier]

Abstract: Francisella tularensis is a Gram-negative bacterium that can cause gastrointestinal or oropharyngeal tularemia in humans from ingestion of contaminated food or water. Despite the potential for accidental or intentional contamination of foods with F. tularensis, there are few studies on the long-term survivability of this organism in food matrices. Infant formula has previously been implicated as a vehicle for the transmission of a variety of bacterial pathogens in infants. In this study, we investigated the survival of F. tularensis in dehydrated infant formula under various storage conditions. F. tularensis was stored for up to 12 weeks in dehydrated infant formula in an ambient air, dry or nitrogen atmosphere. Viable counts of fresh F. tularensis at 12 weeks in infant formula revealed a 4.15, 3.37 and 3.72-log decrease in ambient air, dry and nitrogen atmosphere, respectively. D-values were calculated (in weeks) as 3.99, 4.68 and 4.47 in air, dry and nitrogen atmosphere, respectively. [Copyright &y& Elsevier]

Abstract: Francisella tularensis is the causative agent of tularemia, a zoonotic disease often transmitted to humans by infected animals. The lack of useful specific genetic tools has long hampered the study of F. tularensis subspecies. We identified and characterized two new plasmids, pF242 and pF243, isolated from Francisella philomiragia strains ATCC 25016 and ATCC 25017, respectively. Sequence analysis revealed that pF242 and pF243 are closely related to pC194 and pFNL10 plasmids, respectively. Two generations of pF242- and pF243-based shuttle vectors, harboring several antibiotic resistance markers, were developed. We used the first generation to compare transformation efficiencies in two virulent F. tularensis subspecies. We found that electroporation was more efficient than cryotransformation: almost all vectors tested were successfully introduced by electroporation into Francisella strains with a high level of efficiency. The second generation of shuttle vectors, containing a multiple cloning site and/or gfp gene downstream of Francisella groES promotor, was used for GFP production in F. tularensis. The development of new shuttle vectors offers new perspectives in the genetic manipulation of F. tularensis, helping to elucidate the mechanisms underlying its virulence. [Copyright &y& Elsevier]

Abstract: Francisella tularensis is a highly infectious Gram-negative bacterium that is the causative agent of tularemia. Very little is known about the molecular mechanisms responsible for F. tularensis virulence, in part due to the paucity of genetic tools available for the study of F. tularensis. We have developed a gene knockout system for F. tularensis that utilizes retargeted mobile group II introns, or “targetrons”. These targetrons disrupt both single and duplicated target genes at high efficiency in three different F. tularensis subspecies. Here we describe in detail the targetron-based method for insertional mutagenesis of F. tularensis genes, which should facilitate a better understanding of F. tularensis pathogenesis. Group II introns can be adapted to inactivate genes in bacteria for which few genetic tools exist, thus providing a powerful tool to study the genetic basis of bacterial pathogenesis. [Copyright &y& Elsevier]

Abstract: Many receptors that are employed for the engulfment of apoptotic cells are also used for the recognition and phagocytosis of bacteria. Tyro3, Axl, and Mertk (TAM) are important in the phagocytosis of apoptotic cells by macrophages. Animals lacking these receptors are hypersensitive to bacterial products. In this report, we examine whether the TAM receptors are involved in the phagocytosis of bacteria. We found that macrophages lacking Mertk, Axl, Tyro3 or all three receptors were equally efficient in the phagocytosis of Gram-negative E. coli. Similarly, the phagocytosis of E. coli and Gram-positive S. aureus bioparticles by macrophages lacking TAM receptors was equal to wild-type. In addition, we found that Mertk did not play a role in killing of extracellular E. coli or the replication status of intracellular Francisella tularensis. Thus, while TAM receptors may regulate signal transduction to bacterial components, they are not essential for the phagocytosis and killing of bacteria. [Copyright &y& Elsevier]

Abstract: The analysis of large-scale gene expression profiles is still a demanding and extensive task. Modern machine learning and data mining techniques developed in linear algebra, like Independent Component Analysis (ICA), become increasingly popular as appropriate tools for analyzing microarray data. We applied ICA to analyze kinetic gene expression profiles of human monocyte derived macrophages (MDM) from three different donors infected with Francisella tularensis holartica and compared them to more classical methods like hierarchical clustering. Results were compared using a pathway analysis tool, based on the Gene Ontology and the MeSH database. We could show that both methods lead to time-dependent gene regulatory patterns which fit well to known TNFα induced immune responses. In comparison, the nonexclusive attribute of ICA results in a more detailed view and a higher resolution in time dependent behavior of the immune response genes. Additionally, we identified NFκB as one of the main regulatory genes during response to F. tularensis infection. [Copyright &y& Elsevier]

Abstract: Francisella tularensis is an extremely infectious airborne pathogen that has long been considered as a potential biological weapon. Enzymes of fatty acid synthesis (FAS) pathway are attractive targets for the development of new antibacterial agents because of differences between the biosynthesis pathways of bacteria and mammals. We report here the first expression of three functional enzymes in F. tularensis FAS-II pathway: FabH (3-oxoacyl-acyl carrier protein synthase III) which initiates elongation in FAS-II; FabD (Malonyl-CoA-acyl carrier protein transacylase) which catalyzes the transfer of a malonyl moiety from malonyl-CoA to ACP generating malonyl-ACP, and FabI (enoyl-ACP reductase) which catalyzes the reduction of enoyl-acyl-ACP derivatives. The genes encoding the FabD, FabH, and FabI were custom synthesized and cloned in pET15b expression vector. Each recombinant His-tagged fusion protein was overexpressed by IPTG induction, and then purified by affinity chromatography on a Ni-NTA column. The purified FabH and FabI have been used as targets for new drug development. Screening of a class of indole-2-carboxylic acid compounds has led to the discovery of several new compounds with promising activity against F. tularensis FabH or FabI enzymes. For example, indole derivative WIUAKP-001 inhibited 80% the FabH enzyme at 40μM with IC50 value of 2μM whereas WIUAKP-031 inhibited 98% the FabI enzyme at 37.5μM with IC50 value of 6μM. These compounds hold great promise for future development of new indole derivatives as inhibitors of type II FAS enzymes, and as potential new treatment for tularemia. [Copyright &y& Elsevier]

Abstract: The type A and B subspecies of Francisella tularensis cause severe disease, tularemia, in humans. However, only the former can be lethal especially if inhaled. It is likely that non-lethal infection is due at least in part to the ability of innate host defenses to control pathogen growth whilst acquired immunity develops. Most common small laboratory animals rapidly succumb to clinical strains of F. tularensis and are, therefore, poor models with which to study innate immunity. In an attempt to improve upon this situation in the present study, Sprague–Dawley and Fischer 344 rats were examined for their ability to combat challenge with type A and B strains of the pathogen. Sprague–Dawley rats were significantly more resistant than Fischer rats to infection with either subspecies. This correlated with the ability of Sprague–Dawley rats to arrest the growth of the pathogen at both the site of challenge and at sites of disseminated infection. The rapidity with which F. tularensis kills susceptible rats and the early onset of control of infection in resistant rats suggests that differences in innate immunity account for these disparate outcomes. Thus, the rat might be a more useful model for studying innate immunity to virulent F. tularensis than other small mammals. [Copyright &y& Elsevier]

Abstract: Immunity to Francisella tularensis is largely mediated by T lymphocytes but an important role of B lymphocytes in early stage of infection was previously uncovered. We wanted to find out if F. tularensis is able to infect B cells and/or influence them by direct contact. To investigate this possibility we infected B cell lines from mouse (A20) or humans (Ramos RA-1), or primary mouse spleen cells, with F. tularensis LVS and F. tularensis FSC200 in vitro. In all cases, we detected bacteria on the cell surface and inside the B cells using transmission electron microscopy. More than 20% cells were infected by microbes after 24h. The number of bacteria, determined by CFU, increased about 1log during 24h. Infection with live bacteria led to apoptosis of Ramos cells and mouse CD19+ spleen cells. Approximately 30% of cells were apoptotic after 24h and 70% after 48h, independently of the F. tularensis strain, while only 10% of non-infected cell were apoptotic at either time point. Apoptosis was confirmed by Western blot using anti-PARP antibodies. Thus, this study demonstrates unique phenomenon – namely, the ability of the intracellular pathogen F. tularensis to invade and induce apoptosis in B cells. [Copyright &y& Elsevier]

Abstract: The ability of human beta-defensins hBD-1, hBD-2, and hBD-3 to exert direct in vitro antimicrobial effects was evaluated using Francisella tularensis Live Vaccine Strain (LVS) and Francisella novicida. While hBD-2 showed some antimicrobial activity in these assays, only hBD-3 demonstrated significant potency against Francisella. Francisella tularensis LVS infection induced elevated levels of hBD-2 mRNA in human airway epithelial (A549) cells, while having no significant impact on the levels of hBD-3 and only a moderate effect on the level of hBD-1 mRNA. Francisella infection avoided stimulating the production of the most potent anti-Francisella host peptide, hBD-3, in A549 cells, although hBD-3 is stimulated by other treatments. The differential induction of beta-defensins in Francisella infected lung epithelial cells suggests a complex dynamic in the expression of antimicrobial peptides and the innate immune response. [Copyright &y& Elsevier]

Abstract: Francisella tularensis is a category A bioterror pathogen which in some cases can cause a severe and fatal human infection. Very few virulence factors are known in this species due to the difficulty in working with it as well as the lack of tools for genetic manipulation. This work describes the construction of a shuttle vector that can replicate in Escherichia coli and F. tularensis as well as two distinct promoter trap constructs based on the shuttle vector backbone. Replication in F. tularensis is based on the promiscuous origin of replication from the Staphylococcus aureus plasmid pC194. We demonstrate the novel plasmids can coexist with established F. tularensis vectors based on the pFNL10 plasmid, the current workhorse of F. tularensis genetics. Our promoter trap can identify promoters that are activated during intracellular growth and survival. These new vectors provide additional tools for the genetic manipulation of F. tularensis. [Copyright &y& Elsevier]

Abstract: Francisella tularensis, the causative agent of tularemia and Category A biodefense agent, is known to replicate within host macrophages, though the pathogenesis of this organism is incompletely understood. We have isolated a variant of F. tularensis live vaccine strain (LVS) based on colony morphology and its effect on macrophages. Human monocyte-derived macrophages produced more tumor necrosis factor α (TNFα), interleukin (IL)-1β, IL-6, and IL-12 p40 following exposure to the variant, designated the activating variant (ACV). The immunoreactivity of the lipopolysaccharide (LPS) from both LVS and ACV was comparable to the previously described blue variant and was distinct from the gray variant of LVS. We found, however, the soluble protein fractions of LVS and ACV differed. Further investigation using two-dimensional gel electrophoresis demonstrated higher levels of several proteins in the parental LVS isolate. The differentially expressed proteins featured several associated with virulence in F. tularensis and other pathogens, including intracellular growth locus C (IglC), a σ 54-modulation protein family member (YhbH), and aconitase. ACV reverted to the LVS phenotype, indicated by low cytokine induction and high IglC expression, after growth in a chemically defined medium. These data provide evidence that the levels of virulence factors in F. tularensis are modulated based on culture conditions and that this modulation impacts host responses. This work provides a basis for investigation of Francisella virulence factor regulation and the identification of additional factors, co-regulated with IglC, that affect macrophage responses. [Copyright &y& Elsevier]

Abstract: Francisella tularensis is the etiologic agent of the zoonotic disease tularemia and is thought to be maintained in the environment principally by various terrestrial and aquatic vertebrate animals. The organism is known to persist in water or mud for long periods of time and Francisella-specific DNA has been identified from water and soil. To gain a better understanding of the ecology and epidemiology of F. tularensis, it will be important to further explore its distribution in the environment. Therefore, methods must be established to efficiently extract Francisella-specific DNA from the soil and be able to eliminate potential PCR inhibitors. Thus, we evaluated five commercial DNA extraction kits for their ability to recover F. tularensis-specific DNA from soil samples and eliminate potential PCR inhibitors. The kits evaluated included the Puregene DNA purification kit, QIAamp Stool Mini kit, Epicentre Biotech SoilMaster DNA extraction kit, and the UltraClean™ and PowerMax™ soil DNA isolation kits from MoBio. Soil samples were spiked with γ-irradiated F. tularensis SHU-4 strain (corresponding to a range from 10 to 105 CFU). Spiked samples were extracted with each kit and evaluated using a F. tularensis-specific real-time PCR assay and an internal positive control assay that measures the presence of potential PCR inhibitors. DNA extraction using the UltraClean™ and PowerMax™ kits resulted in the most consistently positive results at the lowest limit of detection (20 and 100CFU/g soil, respectively) for all soil types tested, suggesting that these kits can provide the most sensitive methods for extracting F. tularensis from environmental soil samples. Processing time and cost were also evaluated. [Copyright &y& Elsevier]

Abstract: The virulence of Francisella tularensis LVS is determined in part by its ability to invade and replicate within macrophages and stimulate the production of inflammatory cytokines. The present study determined the effects of growing F. tularensis in macrophages on its ability to stimulate cytokine secretion by macrophages. F. tularensis grown in Mueller–Hinton broth (FtB) stimulated the secretion of large amounts of TNF-α, IL-12p40, IL-6 and MCP-1/CCL2 when incubated with macrophages overnight. In contrast, F. tularensis released from infected macrophages (FtMac) stimulated very little secretion of these cytokines by primary cultures of murine peritoneal macrophages, human monocytes or macrophage cell lines. Stimulation of nitric oxide production by FtMac was also less than that elicited by FtB. FtMac killed with gentamicin or paraformaldehyde also stimulated low levels of cytokine secretion. FtMac recovered the ability to stimulate cytokine secretion after overnight culture in broth. Infection of macrophages with FtMac inhibited the cytokine response to subsequent stimulation with LPS from Escherichia coli but did not affect Fcγ receptor-mediated phagocytosis. FtMac were ingested by macrophages at about half the rate of FtB, however, this did not account for the lower cytokine secretion. FtMac and FtB replicated at similar rates within macrophages. Finally, Mice infected with FtMac had a higher mortality rate than those infected with FtB. These results reveal that growth in macrophages causes a reversible phenotypic change in F. tularensis that is associated with decreased stimulation of cytokine secretion, inhibition of LPS-stimulated secretion of inflammatory cytokines by macrophages and increased lethality in mice. [Copyright &y& Elsevier]

Abstract: Understanding the pathogenesis of infectious diseases requires comprehensive knowledge of the proteins expressed by the pathogen during in vivo growth in the host. Proteomics provides the tools for such analyses but the protocols required to purify sufficient quantities of the pathogen from the host organism are currently lacking. Here, we present a rapid immunomagnetic protocol for the separation of Francisella tularensis, a highly virulent bacterium and potential biowarfare agent, from the spleens of infected mice. In less than one hour, bacteria can be isolated in quantities sufficient to carry out meaningful proteomic comparisons with in vitro grown bacteria. Furthermore, the isolates are virtually free from contaminating host proteins. Two-dimensional gel analysis revealed a host induced proteome in which 78 proteins were differentially expressed in comparison to in vitro grown controls. The results obtained clearly demonstrate the complexity of the adaptive response of F. tularensis to the host environment, and the difficulty of mimicking such behavior in vitro. [Copyright &y& Elsevier]

Abstract: Francisella tularensis subspecies tularensis (type A F. tularensis) is considered to be one of the most virulent of all bacterial pathogens. Mice are extremely susceptible to infection with this subspecies (LD100 via various inoculation routes is <10cfu). However, it has not been established whether overt virulence differences exist amongst type A strains of F. tularensis. To this end, the present study compared the virulence of two distinct type A strains, FSC033 and SCHU S4, for naïve mice and mice immunized with the live vaccine strain of the pathogen, F. tularensis LVS. One nominal isolate of SCHU S4 was found to be completely avirulent. Another isolate was highly virulent, but all examined cases appeared somewhat less virulent than FSC033. The implication of these findings for future infection and immunity studies is discussed. [Copyright &y& Elsevier]

Abstract: Francisella tularensis is the etiologic agent of the potentially fatal human disease tularemia and is capable of survival and multiplication within professional phagocytes of the host. While the mechanisms that allow intracellular survival of the bacterium are only now beginning to be elucidated at the molecular level, previous work demonstrated that F. tularensis produces copious levels of an acid phosphatase which in crude and purified form affected the dose-dependent abrogation of the respiratory burst of stimulated neutrophils. The work presented here was undertaken to provide a source of recombinant F. tularensis acid phosphatase for detailed biochemical, biological, and structural studies. Results from this work are consistent with the ability to generate milligram amounts of recombinant enzyme whose attributes are demonstrably equivalent to those of the native enzyme. Such properties include molecular mass, broad substrate specificity, sensitivity and resistance to various inhibitors, pH optimum, and reactivity with rabbit polyclonal antibody to the native enzyme. [Copyright &y& Elsevier]

Abstract: Francisella tularensis is a gram-negative facultative intracellular bacterium and the causative agent of tularemia. Two subspecies (type A and B strains) of the pathogen exist, the former being much more virulent than the latter for humans and other higher mammals. In this study, we examined the effect of virulent strains of F. tularensis infection on the thymus and thymocytes and the potential mechanisms involved. Low-dose aerosol exposure of C57BL/6 mice with type A, but not type B, F. tularensis caused severe reduction in thymus weight and destruction of thymocytes, particularly CD4+CD8+ thymocytes, by day 4 after infection. The depletion of thymocytes was accompanied by a significant increase in circulating cortisone levels and could be partially prevented by adrenalectomy. Moreover, thymus atrophy and thymocyte depletion following infection were abolished in mice deficient in tumor necrosis factor receptors 1 and 2, but not in FasL-deficient mice. The severe destruction of the thymus and selective depletion of immature thymocytes during type A F. tularensis infection may represent a key pathogenic mechanism in tularemia and could hinder the development of an effective primary immune response against this highly virulent pathogen. [Copyright &y& Elsevier]

Abstract: Monocytic cells constitute an important defense mechanism against invading pathogens by recognizing conserved pathogens components. The recognition leads to activation of intracellular pathways involving nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPK), such as the c-Jun NH2-terminal kinase (JNK), and p38. We show that in vitro infection with Francisella tularensis results in activation of NF-κB, phosphorylation of p38 and c-Jun, and secretion of TNF-α in adherent mouse peritoneal cells, in the mouse macrophage-like cell line J774A.1, in the human macrophage cell line THP-1, and in human peripheral blood monocytic cells. This occurred after infection with the human live vaccine strain, F. tularensis LVS or a mutant strain denoted ΔiglC, which lacks expression of a 23-kDa protein, or after addition of killed F. tularensis LVS. Addition of purified F. tularensis LPS resulted in no discernible effects on the cells. When the effects were followed up to 5h, activation persisted in cultures with killed bacteria or infected with the ΔiglC strain. In contrast, the signal transduction activation and secretion of TNF-α were down-regulated within the 5h period in mouse peritoneal cells, J774 cells or human peripheral blood mononuclear cells infected with F. tularensis LVS. Together, the results suggest that infection with live F. tularensis LVS bacteria leads to a rapid induction of a proinflammatory response in mouse and human cells but after internalization of bacteria, this response is completely or partly down-regulated in most cell types. This down-regulation does not occur when cells are infected with the mutant ΔiglC. [Copyright &y& Elsevier]

Abstract: Francisella tularensis is a gram-negative intracellular bacterium that has been classified as a Category A biothreat because of its ability to induce deadly pneumonic tularemia when inhaled. In the present study, an experimental model of F. tularensis LVS intranasal infection was used to study the immune cells involved in cytokine secretion in the lungs after infection. Dramatic increases in the numbers of cells secreting IFN-γ were observed 72h after intranasal infection of BALB/c and C57BL/6 mice with sublethal (1000CFU) or lethal (10,000CFU) doses of F. tularensis LVS and the cells primarily responsible for this IFN-γ expression were identified as CD11b+ DX5+ NK cells. The findings were further confirmed in C57BL/6 mice showing that cells responsible for IFN-γ secretion in the lungs were CD11b+ DX5+ NK1.1+. NK cell depletion studies showed a decrease in the percentage of IFN-γ secreting cells, due not only to a diminished proportion of IFN-γ secreting NK cells, but also to a reduced percentage of T cells secreting IFN-γ. The results indicate that IFN-γ is secreted in response to respiratory infection with F. tularensis LVS, and that NK cells are the early responders responsible for IFN-γ secretion. [Copyright &y& Elsevier]

Francisella tularensis, the causative agent of tularemia, is a gram-negative facultative intracellular bacterium. Toll-like receptor (TLR) 4 is considered to be critical for inducing host innate immunity against many gram-negative bacteria including many respiratory pathogens. To determine the role of TLR4 in host defense against airborne F. tularensis infection, TLR4-defective C3H/HeJ (TLR4d) or wild-type C3H/HeOuJ (WT) mice were challenged by low-dose aerosol with type A F. tularensis, and the course of the infection and host responses were compared at day 2 and 4 post-inoculation (dpi). At dpi 2, bacterial burdens in the lungs were similar between TLR4d and WT mice, but TLR4d mice surprisingly harbored approximately 10-fold fewer bacteria in their spleens and livers. However, the bacterial burdens at dpi 4, the mortality and median time to irreversible moribundity were indistinguishable between the two mouse strains. In addition, the inflammatory responses to the infection, as reflected by the cytokine levels and leukocyte influx in the bronchoalveolar lavage fluid and histopathological analysis, were similar between both mouse strains. Additionally, as with C3H mice, we found no difference in either the median time to death or the survival rate between TLR4-deleted C57BL/10ScNJ mice and WT C57BL/10 mice. Combined, these data suggest that TLR4 does not contribute to resistance of mice to airborne type A F. tularensis infection. [Copyright &y& Elsevier]

Francisella tularensis, the causative agent of tularemia, is a highly infectious pathogen of humans and animals, yet little is known about the surface proteins of this organism that mediate mechanisms of pathogenicity. λTnphoA was used to generate random alkaline phosphatase gene fusions in a F. tularensis subsp. tularensis (strain Schu S4) genomic library to identify genes encoding exported extracytoplasmic proteins. Eleven genes encoding membrane-associated proteins were identified by this method and their respective signal peptides were characterized. Three of the genes encoded conserved ‘housekeeping’ enzymes, while the other eight genes were unique to F. tularensis, encoding proteins with molecular masses ranging from 11 to 78kDa as deduced from the amino acid sequences. Two genes putatively encoded lipoproteins based on the presence of characteristic signal peptidase II cleavage sites. Four selected proteins were found associated with outer membranes from Schu S4 and LVS strains by Western blotting. Indirect immunofluorescence of strain Schu S4 cells also showed evidence of protein localization to the outer membrane. Protein database searches produced significant alignments with proteins from other bacteria involved in carbohydrate transport, lipid metabolism, and cell envelope biogenesis, thereby providing clues for putative functions. These findings demonstrated that TnphoA mutagenesis can be used in conjunction with F. tularensis genome sequence data to provide a foundation for studies to identify and define cellular surface protein virulence factors of this pathogen. [Copyright &y& Elsevier]

Previous studies have shown that IFN-γ, TNF-α and NOS-2, but not B cells, are crucial for host defense against primary systemic infection with the attenuated live vaccine strain (LVS) of Francisella tularensis. In this study, we examined the importance of these and additional immune components in host resistance against infection with virulent strains of F. tularensis initiated by systemic and airborne routes. Wild-type (WT) mice and mice deficient in IFN-γ, TNFR1R2, NOS-2, or B cells were equally susceptible to low dose (∼10 colony forming units) aerosol or intradermal challenge with virulent type B F. tularensis, and succumbed to the infection between days 6 and 8 post-inoculation. Quantitative bacteriology showed that IFN-γ−/− and B cell−/− mice consistently harbored up to one log10 more bacteria in their lungs, spleens and livers than WT mice at day 5 post aerosol exposure. Surprisingly, however, compared to other strains of KO mice and WT control mice, IFN-γ−/− mice showed only mild liver damage as assessed by histopathology and liver function tests. Additional experiments established that even mice with broad immunodeficiency (SCID, neutropenic, splenectomized or thymectomized mice and mice treated with corticosteroid) were no more susceptible to aerosol-initiated infection with virulent type B or type A F. tularensis than immunosufficient control mice. Combined, our results indicate that, unlike LVS, normal type A and type B F. tularensis strains are so extremely virulent that even immunocompetent mice are virtually defenseless to low dose aerosol and intradermal challenges with them. [Copyright &y& Elsevier]

T cells are crucial to the control and eradication of the facultative intracellular bacterium Francisella tularensis. A contributory role of humoral antibodies in the host defence remains to be assessed. We used B-cell-deficient mice to study the possible contribution of antibodies to the defence against the live vaccine strain (LVS) or a clinical isolate of F. tularensis, both belonging to the subspecies holarctica (type B). When B-cell-deficient (Igμ−/−) mice of the C57BL/10 background were administered immune serum one day before intradermal injection of LVS, they developed lower bacterial numbers in skin, liver, and spleen than did mice receiving normal serum, and survived a challenge inoculum that was lethal for mice given normal serum. Administration of immune serum to C57BL/10 mice afforded protection also against infection with the clinical isolate of F. tularensis subsp. holarctica. Five days after intradermal inoculation of bacteria of the isolate, animals receiving immune serum showed 4 log10 lower bacterial counts in liver and spleen than mice administered normal serum. In mice primed by LVS infection, T-cell immunity and host protection were strong and only a marginal contribution of immune serum against a secondary intradermal infection was demonstrated. Together, these findings show that specific antibodies contribute to the host defence of mice against F. tularensis subsp. holarctica. [Copyright &y& Elsevier]

BALB/c and C57BL/6 mice were challenged by aerosol or intradermally with low doses (∼10–20 colony forming units) of virulent type A and type B strains of the facultative intracellular pathogen, Francisella tularensis, and the course of infection was monitored. Both mouse strains were equally susceptible to infection, but type A strains reached lethal numbers a few days earlier than type B strains regardless of challenge route. BALB/c mice showed overt signs of infection for several days, whereas C57BL/6 mice remained asymptomatic until a few hours before death. Histological changes were extensive and severe in the liver and spleen, but much more limited in the lungs, even in mice challenged by aerosol. Thus, it appears that regardless of the route of infection, systemic rather than pulmonary infection was the likely cause of death following low dose challenge with virulent F. tularensis. [Copyright &y& Elsevier]

The present study examines the relationship between the structure and important biological effects of the lipopolysaccharide (LPS) of the intracellular bacterial pathogen, Francisella tularensis LVS. It shows that treating mice with sub-immunogenic amounts of intact F. tularensis LPS rapidly induces an enhanced resistance to intradermal or aerogenic challenge with strains of the pathogen of varying virulence. However, neither the free Lipid A nor core-O-chain produced by mild acid hydrolysis of LPS appeared able to elicit this host defense mechanism. [Copyright &y& Elsevier]

Francisella tularensis is a zoonotic, facultative intracellular bacterial pathogen capable of initiating infection, tularemia, via multiple routes including dermal micro-abrasions and inhalation. Mouse models of systemically-initiated infection with F. tularensis LVS have been used extensively to reveal potential host defence mechanisms against the pathogen. Such studies have demonstrated the critical need for neutrophils and interferon-gamma (IFN-γ) to combat the early stages of primary experimental tularaemia initiated by systemic routes. Surprisingly, however, the present study shows that these defences appear not to combat early pulmonary tularaemia initiated by inhalation of the pathogen into the lower airways. The results imply that the effectiveness of particular anti-bacterial host defences vary with invasion site. Thus, it is impossible to predict effective host defence mechanisms against inhalation-initiated tularaemia from current knowledge of anti- Francisella defences that have been shown to combat systemically-initiated infection. [Copyright &y& Elsevier]

The microbial community's structure and functions determine the health, quality, and anthropogenic conditions of the river ecosystems. The presence of Bacteria such as Arcobacter spp , Escherichia spp , and Campylobacters spp, have been shown to reflect the poor water quality of rivers. Apprehension of the microbial community in polluted water bodies is significant because it affects human health and the environment. Culture-independent metagenomic and metatranscriptomic approaches employed in the current study of the Cooum river unraveled the taxonomic classification of diverse microbes, including archaea, bacteria, viruses, and phages. The presence of abundant Macellibacteroides fermentans, Arcobacter bivolvorium , Arcobacter butzleri , Methanothrix soenhngeii , and Bacteroides graminisolvens were noted. Viruses and phages like Caudovirales , Human mastadenovirus C, Siphoviridae, Escherichia phage, Erwinia phage, Synechoccus phage , and Vibrio phage were relatively predominant. Various metabolic pathways like methane, sulfur, and nitrogen metabolism adopted by the microbiome confer dangerous gases. Mechanisms such as secretory systems, signal transduction, Chemotaxis, quorum sensing, transportation of chemicals and ions were significantly enriched. The microbes expressed antimicrobial resistance mechanisms as identified from the genes encoding beta-lactamase enzymes and aminoglycoside phosphotransferase enzymes. Metal resistance mechanisms against copper, tellurium, chromium, and cadmium were plentiful. Presence of human pathogens interactions with Yersinia pestis, Campylobacter jejuni, Escherichia coli, Helicobacter pylori, and Francisella tularensis subsp. tularensis suggested the possibilities of transmission of pathogenesis to humans. The current study is the first to apprehend the detailed microbiome composition of one of the highly polluted rivers in South India. The study elaborated the microbiome's structure, functions, and metabolic potential at a specific site of the polluted river. [Display omitted] • The study focused on Structural, Functional, Resistome of the microbiomes in the Cooum river through metagenomics analysis. • Extreme antimicrobial resistance mechanisms were demonstrated. • The metabolic pathway of methane, sulfur, and nitrogen were determined. • Metal resistance mechanisms were elucidated against copper, tellurium, chromium, and cadmium. [ABSTRACT FROM AUTHOR]

Arabinose 5-phosphate isomerase (API) catalyzes the reversible isomerization of Ribulose 5-phosphate (Ru5P) to Arabinose 5-Phosphate (Ar5P) for the production of 3-deoxy-2-octulosonic acid 8-phosphate (KDO), a component of bacterial lipopolysaccharide (LPS) of gram-negative bacteria. API is an attractive target for therapeutic development against gram-negative bacterial pathogens. The current assay method of API activity utilizes a general reaction for keto sugar determination in a secondary, 3-h color development reaction with 25 N sulfuric acid which poses hazard to both personnel and instrumentation. We therefore aimed to develop a more user friendly assay of the enzyme. Since Ru5P absorbs in the UV region and contains at least 2 chiral centers, it can be expected to display circular dichroism (CD). A wavelength scan revealed indeed Ru5P displays a pronounced negative ellipticity of 30,560 mDeg M−1cm−1 at 279 nm in Tris buffer pH 9.1 but Ar5P does not have any CD. API enzymatic reactions were monitored directly and continuously in real time by following the disappearance of CD from the Ru5P substrate, or by the appearance of CD from Ar5P substrate. The CD signal at this wavelength was not affected by absorption of the enzyme protein or of small molecules, or turbidity of the solution. Common additives in protein and enzyme reaction mixtures such as detergents, metals, and 5% dimethylsulfoxide did not interfere with the CD signal. Assay reactions of 1–3 min consistently yielded reproducible results. Introduction of accessories in a spectropolarimeter will easily adapt this assay to high throughput format for screening thousands of small molecules as inhibitor candidates of API. • Arabinose 5-phosphate isomerase. • Circular Dichroism-based enzyme assay. • KdsD pathway enzyme assay. • Continuous KdsD assay. [ABSTRACT FROM AUTHOR]