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Tularemia is caused by subspecies of Francisella tularensis and can manifest in a variety of disease states, with the pneumonic presentation resulting in the greatest mortality. Despite decades of research, there are no approved vaccines against F. tularensis in the United States. Traditional vaccination strategies, such as live-attenuated or subunit vaccines, are not favorable due to inadequate protection or safety concerns. Because of this, novel vaccination strategies are needed to combat tularemia. Here we discuss the current state of and challenges to the tularemia vaccine field and suggest novel vaccine approaches going forward that might be better suited for protecting against F. tularensis infection. [ABSTRACT FROM AUTHOR]

CRISPR/Cas technology is a versatile tool for genome engineering in many organisms, including filamentous fungi. Cpf1 is a multi-domain protein of class 2 (type V) RNA-guided CRISPR/Cas endonuclease, and is an alternative platform with distinct features when compared to Cas9. However, application of this technology in filamentous fungi is limited. Here, we present a single CRISPR/Cpf1 plasmid system in Aspergillus aculeatus strain TBRC 277, an industrially relevant cell factory. We first evaluated the functionality of three Cpf1 orthologs from Acidaminococcus sp. BV3L6 (AsCpf1), Francisella tularensis subsp. novicida U112 (FnCpf1), and Lachnospiraceae bacterium (LbCpf1), in RNA-guided site-specific DNA cleavage at the pksP locus. FnCpf1 showed the highest editing efficiency (93 %) among the three Cpf1s. It was further investigated for its ability to delete a 1.7 kb and a 0.5 kb from pksP and pyr G genes, respectively, using two protospacers targeting these gene loci in a single crRNA array. Lastly, simultaneous editing of three sites within TBRC 277 genome was performed using three guide sequences targeting these two genes as well as an additional gene, kusA , which resulted in combined editing efficiency of 40 %. The editing of the NHEJ pathway by targeting kusA to generate a NHEJ-deficient strain of A. aculeatus TBRC 277 improved gene targeting efficiency and yielded more precise gene-editing than that of using wild-type strain. This promising genome-editing system can be used for strain improvement in industrial applications such as production of valuable bioproducts. [ABSTRACT FROM AUTHOR]

In Europe, tick-borne diseases (TBDs) cause significant morbidity and mortality, affecting both human and animal health. Ticks can transmit a wide variety of pathogens (bacteria, viruses, and parasites) and feed on many vertebrate hosts. The incidence and public health burden of TBDs are tending to intensify in Europe due to various factors, mainly anthropogenic and often combined. Early detection of tick-borne pathogens (TBPs), preventive measures and treatment are of great importance to control TBDs and their expansion. However, there are various limitations in terms of the sensitivity and/or specificity of detection and prevention methods, and even in terms of feasibility. Aptamers are single-stranded DNA or RNA that could address these issues as they are able to bind with high affinity and specificity to a wide range of targets (e.g., proteins, small compounds, and cells) due to their unique three-dimensional structure. To date, aptamers have been selected against TBPs such as tick-borne encephalitis virus, Francisella tularensis , and Rickettsia typhi. These studies have demonstrated the benefits of aptamer-based assays for pathogen detection and medical diagnosis. In this review, we address the applications of aptamers to TBDs and discuss their potential for improving prevention measures (use of chemical acaricides, vaccination), diagnosis and therapeutic strategies to control TBDs. • The incidence of tick-borne diseases is increasing in Europe, with a major impact on human and animal health. • Their control is hampered by the lack of sensitive and specific detection and prevention tools. • Aptamers are promising tools, as they can bind with high affinity and specificity to their target. • This review compares aptamers with the current approach to tick-borne disease prevention and control. [ABSTRACT FROM AUTHOR]

The siderophore rhizoferrin (N¹,N4-dicitrylputrescine) is produced in fungi and bacteria to scavenge iron. Putrescineproducing bacterium Ralstonia pickettii synthesizes rhizoferrin and encodes a single nonribosomal peptide synthetaseindependent siderophore (NIS) synthetase. From biosynthetic logic, we hypothesized that this single enzyme is sufficient for rhizoferrin biosynthesis. We confirmed this by expression of R. pickettii NIS synthetase in Escherichia coli, resulting in rhizoferrin production. This was further confirmed in vitro using the recombinant NIS synthetase, synthesizing rhizoferrin from putrescine and citrate. Heterologous expression of homologous lbtA from Legionella pneumophila, required for rhizoferrin biosynthesis in that species, produced siderophore activity in E. coli. Rhizoferrin is also synthesized by Francisella tularensis and Francisella novicida, but unlike R. pickettii or L. pneumophila, Francisella species lack putrescine biosynthetic pathways because of genomic decay. Francisella encodes a NIS synthetase FslA/FigA and an ornithine decarboxylase homolog FslC/FigC, required for rhizoferrin biosynthesis. Ornithine decarboxylase produces putrescine from ornithine, but we show here in vitro that FigA synthesizes N-citrylornithine, and FigC is an N-citrylornithine decarboxylase that together synthesize rhizoferrin without using putrescine. We co-expressed F. novicida figA and figC in E. coli and produced rhizoferrin. A 2.1 Å X-ray crystal structure of the FigC N-citrylornithine decarboxylase reveals how the larger substrate is accommodated and how active site residues have changed to recognize N-citrylornithine. FigC belongs to a new subfamily of alanine racemase-fold PLP-dependent decarboxylases that are not involved in polyamine biosynthesis. These data reveal a natural product biosynthetic workaround that evolved to bypass a missing precursor and re-establish it in the final structure. [ABSTRACT FROM AUTHOR]

Adenopathies are a frequent cause of recourse in internal medicine. When histological analysis reveals the presence of granuloma, multiple infectious or non-infectious etiologies are considered. If diagnoses of lymphoma, sarcoidosis or tuberculosis are easily mentioned, tularemia should also be considered in the differential diagnosis. A 54-year-old patient had a fever at the evening with night sweats and a cough resistant to two lines of antibiotics. A thoraco-abdomino-pelvic CT scan revealed hilar and mediastinal adenopathies that appeared hypermetabolic with PET-TDM, as well as pulmonary nodules. A PCR performed on lymph node biopsy and serology allowed the diagnosis of tularemia. The evolution was favourable after antibiotic treatment. The association of fever, night sweats, altered general state and mediastinal adenopathies should be considered as a diagnosis of tularemia. Ganglionic biopsy, combined with molecular biology techniques and serology, can confirm the diagnosis. [ABSTRACT FROM AUTHOR]

Disulfiram (DSF) can help treat alcohol dependency by inhibiting aldehyde dehydrogenase (ALDH). Genomic analysis revealed that Francisella tularensis , the causative agent of tularemia, has lost all but one ALDH-like domain and that this domain retains the target of DSF. In this study, minimum inhibitory concentration (MIC) assays demonstrated that both DSF and its primary metabolite diethyldithiocarbamate (DDC) have strong antimicrobial activity against F. tularensis strain SCHU S4, with the MIC of DSF determined as 2 µg/mL in comparison with 8 µg/mL for DDC. The activity of DSF was further confirmed using an in vitro human macrophage infection assay. Francisella tularensis bacteria in DSF-treated cells were reduced in comparison with untreated and DDC-treated cells, comparable with that observed in doxycycline-treated cells. This suggests that DSF may be suitable for further investigation as an in vivo therapy for tularemia. [ABSTRACT FROM AUTHOR]

• Tularemia can be transmitted through the ingestion of contaminated grape products. • F. tularensis can be introduced into the food chain via mechanical harvesting of grapes and presumably infected rodents. • The clinical course of tularemia can be unexpectedly long and require long antibiotic treatment durations. Tularemia is caused by Francisella tularensis and can occasionally establish foodborne transmission. Patients were identified by active case detection through contact with the treating physicians and consent for publication was obtained. Clinical data were accumulated through a review of the patient charts. Serology, culture, and PCR methods were performed for confirmation of the diagnosis. A 46-year-old patient was hospitalised in the University Hospital Frankfurt (a tertiary care hospital) for pharyngitis and cervical lymphadenitis with abscess. A diagnosis of tularemia was made serologically, but treatment with ciprofloxacin initially failed. F. tularensis was detected in pus from the lymph node using a specific real-time PCR. The use of RD1 PCR led to the identification of the subspecies holarctica. Antibiotic therapy with high-dose ciprofloxacin and gentamicin was administered and was subsequently changed to ciprofloxacin and rifampicin. During a must-tasting, five other individuals became infected with tularemia by ingestion of contaminated must. All patients required treatment durations of more than 14 days. Mechanically harvested agricultural products, such as wine must, can be a source of infection, probably due to contamination with animal carcasses. The clinical course of tularemia can be complicated and prolonged and requires differentiated antibiotic treatment. [ABSTRACT FROM AUTHOR]

The host is a nutrient-rich niche for microbial pathogens, but one that comes with obstacles and challenges. Many intracellular pathogens like Legionella pneumophila, Coxiella burnetii, Listeria monocytogenes, and Chlamydia trachomatis have developed bipartite metabolism within their hosts. This style of metabolic regulation enables pathogen sensing of specific nutrients to engage them into catabolic and anabolic processes, and contributes to temporal and spatial pathogen phenotypic modulation. Not only have intracellular pathogens adapted their metabolism to the host, they have also acquired idiosyncratic strategies to exploit host nutritional supplies and intercept metabolites. Francisella tularensis and Anaplasma phagocytophilum alter host autophagy, Shigella flexneri intercepts all host pyruvate, while L. pneumophila induces host protein degradation and blocks protein translation. Strategies of pathogen manipulation of host nutrients could serve as therapeutic targets. There is a general common theme of bipartite metabolism by intracellular pathogens. Pathogens have evolved with mechanisms to override host-limited availability of nutrients. Host nutrient supplies govern pathogen virulence properties. Import of host nutrients occurs across the pathogen-containing vacuoles using host SLCs. [ABSTRACT FROM AUTHOR]

Abstract Francisellosis is a disease produced by Francisella spp. which affects farmed fish. Eleginops maclovinus specimens can be caught close to salmon farming centers, feeding on un-consumed pellet, making the transmission of pathogens such as Francisella noatunensis possible. The aim of this study was to evaluate the effect of F. noatunensis on liver intermediary metabolism in E. maclovinus. 144 fish were injected intraperitoneally with F. noatunensis at a low dose LD (1 × 101 cells/μL), medium dose MD (1 × 105 cells/μL), high dose HD (1 × 1010 cells/μL), or with culture medium C (control), and sampled at 1, 3, 7, 14, 21 and 28 days post injection (dpi). No mortality was recorded during the experimental period, but there was a marked metabolic response in fish injected with high doses. Metabolites in plasma were lowest in the high bacterial dose. Cortisol levels were highest at day 7 in the high dose and then decreased from day 14 until the end of the study. Liver enzymes showed a similar pattern to plasma metabolites, with decreased enzymatic activity, mostly with the high bacteria dose. PK was the exception, with increased enzymatic activity in a dose-dependent manner over time. Liver metabolites were highly variable, except in the high bacterial dose where variability and total levels decreased significantly. Our results show that fish infection with F. noatunensis induces a clear stress response, especially with at the highest dose, shifting intermediary metabolism towards mobilization of energy and suggesting that E. maclovinus detects experimental infection of F. noatunensis as a stressor, which it is dependent on the bacterial dose. Graphical abstract Unlabelled Image Highlights • Francisella noatunesis subsp. noatunensis is an emerging pathogen of salmonids that can infect the native fauna • Eleginops maclovinus is a native euryhaline species with the potential to spread pathogens to other ecological niches. • The aim of this study was to evaluate the metabolic response of E. maclovinus challenged with the pathogen F. noatunesis in a time course experiment. • Our results show that fish infection with F. noatunensis induces a clear stress response, especially with the highest dose. • This study described the rearrangement of energy and enzyme reserves of the hepatic intermediate metabolism in E. maclovinus [ABSTRACT FROM AUTHOR]

Abstract Absent in melanoma 2 (AIM2) is a sensor of cytosolic dsDNA and is responsible for the activation of inflammatory and host immune responses to DNA viruses and intracellular bacteria. AIM2 is a member of the hematopoietic interferon-inducible nuclear proteins with a 200 amino-acid repeat (HIN200) family, containing a pyrin domain (PYD) at the N-terminus. Several studies have demonstrated that AIM2 is responsible for host defense against intracellular bacteria such as Francisella tularensis , Listeria monocytogenes and Mycobacerium tuberculosis. However, the role of AIM2 in host defenses against Brucella is poorly understood. In this study, we have shown that AIM2 senses Brucella DNA in dendritic cells to induce pyroptosis and regulates type I IFN. Confocal microscopy of infected cells revealed co-localization between Brucella DNA and endogenous AIM2. Dendritic cells from AIM2 KO mice infected with B. abortus showed impaired secretion of IL-1β as well as compromised caspase-1 cleavage. AIM2 KO mice displayed increased susceptibility to B. abortus infection in comparison to wild-type mice, and this susceptibility was associated with defective IL-1β production together with reduced IFN-γ responses. In summary, the increased bacterial burden observed in vivo in AIM2 KO animals confirmed that AIM2 is essential for an effective innate immune response against Brucella infection. [ABSTRACT FROM AUTHOR]

Abstract Alveolar macrophages resident in the lung are prominent phagocytic effector cells of the pulmonary innate immune response, and paradoxically, are attractive harbors for pathogens. Consequently, facultative intracellular bacteria, such as Francisella tularensis , can cause severe systemic disease and sepsis, with high morbidity and mortality associated with pulmonary infection. Current clinical treatment, which involves exhaustive oral or intravenous antibiotic therapy, has limitations such as systemic toxicity and off-target effects. Pulmonary administration represents a promising alternative to systemic dosing for delivering antibiotics directly to the lung. Here, we present synthesized mannosylated ciprofloxacin polymeric prodrugs for efficient pulmonary delivery, targeting, and subsequent internalization by alveolar macrophages. We demonstrate significant improvement in efficacy against intracellular infections in an otherwise uniformly lethal airborne Francisella murine model (F. novicida). When administered to the lungs of mice in a prophylactic regimen, the mannosylated ciprofloxacin polymeric prodrugs led to 50% survival. In a treatment regimen that was concurrent with infection, the survival of mice increased to 87.5%. Free ciprofloxacin antibiotic was ineffective in both cases. This significant difference in antibacterial efficacy demonstrates the impact of this delivery platform based on improved physiochemical, pharmacokinetic, and pharmacodynamic properties of ciprofloxacin administered via our glycan polymeric prodrug. This modular platform provides a route for overcoming the limitations of free drug and increasing efficacy in treatment of intracellular infection. Graphical abstract Image 1 [ABSTRACT FROM AUTHOR]

Bacterial pathogens such as Yersinia pestis, Francisella tularensis, and Bacillus anthracis are classified into the highest rank of potential bioterrorism agents. Colorimetric lateral flow assay (LFA) strips are commercially available but these conventional strips have drawbacks in terms of low sensitivity and limit of quantitative analysis. Therefore, there is an urgent need for a new sensing platform to detect these pathogens in the early contamination stage. In this study, a novel surface-enhanced Raman scattering (SERS)-based LFA strip was developed for sensitive detection of bacterial pathogens. Target-specific SERS nanotags (Raman reporter-labeled gold nanoparticles) were used as an alternative to the gold nanoparticles in conventional LFA strips. Using these SERS nanotags the presence of bacteria could be identified through a simple color change in the test line. Additionally, highly sensitive and accurate quantitative analysis could be performed by monitoring the characteristic Raman peak intensity of SERS nanotags that were captured in the test line. This highly sensitive method required a short assay time (15 min) and a tiny volume of pathogen sample (40 μL). We believe that the proposed SERS-based LFA technique has great potential as a valuable tool in the early detection of specific bacterial pathogens in the field due to its excellent analytical sensitivity. [ABSTRACT FROM AUTHOR]

The bacterial acyl protein thioesterase (APT) homologue FTT258 from the gram-negative pathogen Francisella tularensis exists in equilibrium between a closed and open state. Interconversion between these two states is dependent on structural rearrangement of a dynamic loop overlapping its active site. The dynamics and structural properties of this loop provide a simple model for how the catalytic activity of FTT258 could be spatiotemporally regulated within the cell. Herein, we characterized the dual roles of this dynamic loop in controlling its catalytic and membrane binding activity. Using a comprehensive library of loop variants, we determined the relative importance of each residue in the loop to these two biological functions. For the catalytic activity, a centrally located tryptophan residue (Trp66) was essential, with the resulting alanine variant showing complete ablation of enzyme activity. Detailed analysis of Trp66 showed that its hydrophobicity in combination with spatial arrangement defined its essential role in catalysis. Substitution of other loop residues congregated along the N-terminal side of the loop also significantly impacted catalytic activity, indicating a critical role for this loop in controlling catalytic activity. For membrane binding, the centrally located hydrophobic residues played a surprising minor role in membrane binding. Instead general electrostatic interactions regulated membrane binding with positively charged residues bracketing the dynamic loop controlling membrane binding. Overall for FTT258, this dynamic loop dually controlled its biological activities through distinct residues within the loop and this regulation provides a new model for the spatiotemporal control over FTT258 and potentially homologous APT function. [ABSTRACT FROM AUTHOR]

Rift Valley fever virus is a mosquito-borne virus which is associated with acute hemorrhagic fever leading to large outbreaks among ruminants and humans in Africa and the Arabian Peninsula. RVFV circulates between mosquitoes, ruminants, camels and humans, which requires divergent amplification and maintenance strategies that have not been fully explored on the cellular and molecular level. We therefore assessed monoclonal antibodies for their applicability to monitor the expression pattern and kinetics of viral proteins in different RVFV infected cell species. Sequences of RVFV vaccine strain MP-12 were used in a bacterial expression system to produce recombinant non-structural proteins directed to NSs and NSm. After immunization of balb/c mice a set of monoclonal antibodies were generated and extensively characterized. The kinetics of RVFV proteins in vertebrate (Vero76) and mosquito-derived (C6/36) cells were evaluated with monoclonal antibodies against the nucleocapsid protein (NP) and the glycoproteins (Gn and Gc) as well as with the newly generated NSs and NSm derived monoclonal antibodies. Significant differences of viral protein distribution and accumulation in vertebrate compared to mosquito-derived cells could be demonstrated. Differences were observed for the nonstructural NSm and most intriguingly for the NSs protein indicating significant divergency of replication strategies of RVFV in Vero 76 cells and C6/36 cells. The described monoclonal antibodies are therefore powerful tools to elucidate the discrepancies of virus replication and interaction within the mammalian host compared to the mosquito vector. [ABSTRACT FROM AUTHOR]

This study presents the engineering of a less endotoxic Salmonella Typhimurium strain by manipulating the lipid-A structure of the lipopolysaccharide (LPS) component. Salmonella lipid A was dephosphorylated by using lpxE from Francisella tularensis. The 1-phosphate group from lipid-A was removed selectively , resulting in a close analog of monophosphoryl lipid A. We observed a significant impact of ∆ pagL on major virulence factors such as biofilm formation, motility, persistency, and immune evasion. In correlation with biofilm and motility retardation, adhesion and invasion were elevated but with reduced intracellular survival, a favorable phenotype prospect of a vaccine strain. Western blotting and silver staining confirmed the absence of the O-antigen and truncated lipid-A core in the detoxified Salmonella mutant. In vitro and in vivo studies demonstrated that the dephosphorylated Salmonella mutant mediated lower pro-inflammatory cytokine secretion than the wild-type strain. The vaccine strains were present in the spleen and liver for five days and were cleared from the organs by day seven. However, the wild-type strain persisted in the spleen, liver, and brain, leading to sepsis-induced death. Histological evaluations of tissue samples further confirmed the reduced endotoxic activity of the detoxified Salmonella mutant. The detoxification strategy did not compromise the level of protective immunity, as the vaccine strain could enhance humoral and cellular immune responses and protect against the wild-type challenge in immunized mice. • Engineering a live attenuated Salmonella Typhimurium (SG) strain with reduced endotoxicity. • Knocking out of pagL hindered the motility and biofilm formation ability of Salmonella. • Lipid A modified Salmonella strain generated significantly lower levels of pro-inflammatory cytokines. • Monophosphoryl lipid A created by insertion of lpxE showed improved adjuvanticity. • Detoxified Salmonella mutant strain showed enhanced protective immune responses against a virulent wild-type challenge. [ABSTRACT FROM AUTHOR]

In this study, we tested farm vegetables and agricultural water for the presence of foodborne pathogens, and evaluated farming practices of vegetable farms in Rwanda. Farm vegetable samples were found to be contaminated with foodborne pathogens at considerably high rate (overall 15/99 = 15%). Specifically, the prevalence of pathogens in farm vegetables varied from 1.0% (1/99) for Listeria monocytogenes , 3.0% (3/99) for thermo-tolerant Campylobacter spp., 5.1% (5/99) for Salmonella spp. to 6.1% (6/99) pathogenic Escherichia coli. In agricultural water from rivers, lakes, lagoons, ground and marshlands, prevalence of DNA from pathogens varied from 3% (1/30) for Enteroinvasive E. coli (EIEC); 7% (2/30) for Enteroaggregative E. coli (EAEC); 13% (4/30) for Enterotoxigenic E. coli. (ETEC) and Vibrio cholera ; 20% (6/30) for Yersinia pestis ; 27% (8/30) for Francisella tularensis ; 40% (12/30) for Cyclospora to 87% (26/30) for thermo-tolerant Campylobacter spp. DNA of the following pathogens was not detected (0/30) in water: entero pathogenic E. coli (EPEC), shiga toxin producing E. coli (STEC), Salmonella spp., L. monocytogenes , Burkholderia , Rickettsia , Toxoplasma gondii , Giardia lamblia , Cryptosporidium , Entamoeba histolytica and Hepatitis E. About farming practices, 60% of the visited vegetable farms practiced irrigation and all the water used was from un-protected sources (from marshlands [70% ], rivers [18% ], lakes [7% ], runoff lagoons [5% ]). Over 80% of the farms applied overhead irrigation methods and none of the farms had implemented measures to restrict the access of domestic and wild animals, while 50% of the farms used untreated manure. The reported high detection rate of foodborne pathogens DNA in agricultural water and the observed risky farming practices, provides a likely explanation of the reported prevalence of pathogens in farm vegetables and presents an important public health concern if these vegetables are to be consumed raw. [ABSTRACT FROM AUTHOR]

We have previously reported benzimidazole-based compounds to be potent inhibitors of FabI for Francisella tularensis ( Ft FabI), making them promising antimicrobial hits. Optically active enantiomers exhibit markedly differing affinities toward Ft FabI. The IC 50 of benzimidazole (−)- 1 is ∼100× lower than the (+)-enantiomer, with similar results for the 2 enantiomers. Determining the absolute configuration for these optical compounds and elucidating their binding modes is important for further design. Electronic circular dichroism (ECD) quantum calculations have become important in determining absolute configurations of optical compounds. We determined the absolute configuration of (−)/(+)- 1 and (−)/(+)- 2 by comparing experimental spectra and theoretical density functional theory (DFT) simulations of ECD spectra at the B3LYP/6-311+G(2d, p) level using Gaussian09. Comparison of experimental and calculated ECD spectra indicates that the S configuration corresponds to the (−)-rotation for both compounds 1 and 2 , while the R configuration corresponds to the (+)-rotation. Further, molecular dynamics simulations and MM-GBSA binding energy calculations for these two pairs of enantiomers with Ft FabI show much tighter binding MM-GBSA free energies for S - 1 and S - 2 than for their enantiomers, R - 1 and R - 2 , consistent with the S configuration being the more active one, and with the ECD determination of the S configuration corresponding to (−) and the R configuration corresponding to (+). Thus, our computational studies allow us to assign (−) to ( S )- and (+) to ( R )- for compounds 1 and 2 , and to further evaluate structural changes to improve efficacy. [ABSTRACT FROM AUTHOR]

A decrease in mass of isografts and a decline in islet function are major challenges in islet transplantations. Despite this, transplantation of 84 h harvested pancreatic duct ligation (PDL) tissues have been shown to have the same functional ability to foetal pancreata, but there was only 40% success in reverting hyperglycaemia. We tested the potential of early islets with mesenchymal stromal cells (MSCs) to promote isogeneic grafts survival and to restore normoglycemia in diabetic rats, in comparison with late islets. Islets were isolated from injured adult pancreata of donor rats at 24 h post ligation either with MSCs (24 h islet/MSC+) or without MSCs (24 h islet/MSC-), and at 84 h without MSCs (84 h islet/MSC-). These cells were transplanted under the renal capsule of syngeneic STZ-diabetic recipient rats. The islet grafts were monitored using the BGLs of recipients and the immunohistomorphology of the grafts were analysed using anti-insulin and anti-Ki67 antibodies. The mean BGL in 24 h islet/MSC+ recipients was reduced over time toward the control value. The curves of the mean BGLs in the control islet/MSC- and the 24 h islet/MSC- recipients dropped significantly below the control normal glucose group’s levels to reach their nadirs on weeks 4 and 6, respectively. Both curves had a peak overshoot on week 9, with no statistical significant difference between them. Engrafted islets were evident in these recipients, lasted for 5 and 6 weeks and correspondingly survived failure. However, insulin+ cells were present in the isografts of all recipients; but, only isografts in the 24 h islet/MSC+ presented with a homogenous subcapsular beta cell mass. In addition, the tendency of 24 h islet/MSC- to restore normoglycaemia with its survival capacity was statistically highly significant compared to the 84 islet/MSC- recipients (80%; 20%; p = 0.001). Transplantation of early islets with MSCs from injured adult pancreata prolongs islet graft survival and improves isograft function in diabetic rats. This novel observation requires much further exploration for its clinical application, but this model already provides hope for new sources of donor islets for transplantation. [ABSTRACT FROM AUTHOR]

Five new fawcettimine-type alkaloids ( 1 – 5 ) and three new natural products ( 6 – 8 ), along with four known analogues, fawcettimine ( 9 ), fewcettidine ( 10 ), lycopoclavamine B ( 11 ), and lycopladine B ( 12 ), were isolated from the whole plant of Lycopodium complanatum var. glaucum Ching. The structures of lycogladines A-H ( 1 – 8 ) were determined based on HRESIMS, 1D and 2D NMR spectroscopic analysis, as well as single-crystal X-ray diffraction. Compound 1 possesses a new ring system that is formed through the linkage of C-13- O C-2, which is rarely present in Lycopodium alkaloids (LAs). Compounds 1 – 8 were tested for their β -site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) and acetylcholinesterase (AChE) inhibitory activities. [ABSTRACT FROM AUTHOR]

Interval exercise delays critical mechanical-ventilatory constraints with positive consequences on Dyspnoea and exercise tolerance in COPD. We hypothesized that those advantages of interval exercise would be partially off-set in patients showing excessive ventilation ( V ˙ E) to metabolic demand ( V ˙ CO 2 ). Sixteen men (FEV 1  = 42.3 ± 8.9%) performed, on different days, 30 s and 60 s bouts at 100% peak ( on ) interspersed by moderate exercise at 40% ( off ). Nine patients did not sustain exercise for 30 min irrespective of on duration. They presented with higher V ˙ E/ V ˙ CO 2 nadir (35 ± 3 vs . 30 ± 5) and dead space/tidal volume (0.39 ± 0.05 vs . 0.34 ± 0.06) compared to their counterparts (p < 0.05). [Lactate], operating lung volumes and symptom burden (dyspnoea and leg effort) were also higher (p < 0.05). Unloading off decreased the metabolic-ventilatory demands, thereby allowing 7/9 patients to exercise for 30 min. Increased wasted ventilation accelerates the rate at which critical mechanical constraints and limiting dyspnoea are reached during interval exercise in patients with COPD. [ABSTRACT FROM AUTHOR]

Rhodococcus equi is an opportunistic, intracellular pathogen that causes pyogranulomatous pneumonia in foals and immunocompromised people. Currently, there is no experimental model of R. equi pneumonia other than intra-bronchial experimental infection of foals with R. equi , which is labor-intensive and costly. This study’s objective was to develop a guinea pig (GP) model of R. equi pneumonia that would facilitate development of novel approaches for controlling and preventing this disease. Guinea pigs were infected with either 10 1 , 10 2 , 10 3 , or 10 4 colony forming units (CFUs) of a virulent strain of R. equi using a Madison aerosol chamber, or 10 6 or 10 7 CFUs of this strain intratracheally. Animals were monitored daily for clinical signs of pneumonia, and were euthanized and necropsied on days 1, 3, 7, or 35 post-infection (PI). Lung homogenates were plated onto selective agar to determine bacterial load. No clinical signs of disease were observed regardless of the inoculum dose or infection method. No bacteria were recovered from GPs euthanized at 35 days PI. Histology and immunostaining of T-cells, B-cells, and macrophages in lungs showed that inflammatory responses in infected GPs were similarly unremarkable irrespective of dose or route of infection. Guinea pigs appear to be resistant to pulmonary infection with virulent R. equi even at doses that reliably produce clinical pneumonia in foals. [ABSTRACT FROM AUTHOR]

Varicella-zoster virus (VZV) is a causative agent for chickenpox and shingles. Comparative genomic sequence analysis of clinical and vaccine strains suggested potential sites responsible for attenuation. In this study, low and high passages of two VZV clinical strains cultured in human fibroblast cells were compared for genomic DNA sequences and growth characteristics. Mutations were detected at 187 and 162 sites in the strain YC01 and YC02, respectively. More than 86% of mutations were found in open reading frames, and ORF62 exhibited highest frequency of mutations. T to C and A to G transitions accounted for more 90% of all possible substitutions. Forty mutations were common to two strains, including 27 in ORF62. Mutations found in attenuated vaccine strains were also detected at 7 positions. Both high and low passage strains were infectious and grew similarly in human fibroblast cells. In guinea pig cells, however, high passage strain remained infectious while low passage strain lost infectivity. This study may provide new insight into the attenuating mutations associated with in vitro passaging of VZV. [ABSTRACT FROM AUTHOR]

A β-class carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic bacterium Francisella tularensis (FtuβCA) was cloned and purified, and the anion inhibition profile was investigated. Based on the measured kinetic parameters for the enzyme catalyzed CO 2 hydration reaction (k cat of 9.8 × 10 5 s −1 and a k cat /K M of 8.9 × 10 7 M −1 s −1 ), FtuβCA is a highly effective enzyme. The activity of FtuβCA was not inhibited by a range of anions that do not typically coordinate Zn(II) effectively, including perchlorate, tetrafluoroborate, and hexafluorophosphate. Surprisingly, some anions which generally complex well with many cations, including Zn(II), also did not effectively inhibit FtuβCA, e.g., fluoride, cyanide, azide, nitrite, bisulphite, sulfate, tellurate, perrhenate, perrhuthenate, and peroxydisulfate. However, the most effective inhibitors were in the range of 90–94 µM (sulfamide, sulfamic acid, phenylarsonic and phenylboronic acid). N,N -Diethyldithiocarbamate (K I of 0.31 mM) was a moderately potent inhibitor. As Francisella tularensis is the causative agent of tularemia, the discovery of compounds that can interfere with the life cycle of this pathogen may result in novel opportunities to fight antibiotic drug resistance. [ABSTRACT FROM AUTHOR]

Fluoroquinolones (FQs) are highly effective for treating tularaemia, a zoonosis caused by Francisella tularensis , but failures and relapses remain common in patients with treatment delay or immunocompromised status. FQ-resistant strains of F. tularensis harboring mutations in the quinolone–resistance determining region (QRDR) of gyrA and gyrB, the genes encoding subunits A and B of DNA gyrase, have been selected in vitro. Such mutants have never been isolated from humans as this microorganism is difficult to culture. In this study, the presence of FQ-resistant mutants of F. tularensis was assessed in tularaemia patients using combined culture- and PCR-based approaches. We analyzed 42 F. tularensis strains and 82 tissue samples collected from 104 tularaemia cases, including 32 (30.7%) with FQ treatment failure or relapse. Forty F. tularensis strains and 55 clinical samples were obtained before any FQ treatment, while 2 strains and 15 tissue samples were collected after treatment. FQ resistance was evaluated by the minimum inhibitory concentration (MIC) for the bacterial strains, and by newly developed PCR-based methods targeting the gyrA and g yrB QRDRs for both the bacterial strains and the clinical samples. None of the F. tularensis strains displayed an increased MIC compared with FQ–susceptible controls. Neither gyrA nor gyrB QRDR mutation was found in bacterial strains and tissue samples tested, including those from patients with FQ treatment failure or relapse. Further phenotypic and genetic resistance traits should be explored to explain the poor clinical response to FQ treatment in such tularaemia patients. [ABSTRACT FROM AUTHOR]

Tularaemia is a highly contagious infectious zoonosis caused by the bacterial agent Francisella tularensis . The aim of this study was to investigate the presence of antibodies to F. tularensis in febrile patients in northeastern Kenya. During 2014–2015, 730 patients were screened for anti- F. tularensis antibodies using a combination of ELISA and Western blot. Twenty-seven (3.7%) individuals were positive for F. tularensis . Tularaemia was not suspected by the treating clinicians in any of them. Our results suggest that tularaemia may be present in Kenya but remain unreported, and emphasizes the need for local clinicians to broaden their diagnostic repertoire when evaluating patients with undifferentiated febrile illness. [ABSTRACT FROM AUTHOR]

Background Tularemia is caused by Francisella tularensis , a gram-negative bacterium that has been weaponized as an aerosol. For protection of personnel conducting biodefense research, the United States Army required clinical evaluation of a new lot of tularemia live vaccine strain manufactured in accordance with Current Good Manufacturing Practices. Methods A phase 2 randomized clinical trial compared the new lot (DVC-LVS) to the existing vaccine that has been in use for decades (USAMRIID-LVS). The vaccines were delivered by scarification to 228 participants. Safety, reactogenicity, take and/or antibody levels were assessed on days 0, 1, 2, 8, 14, 28, 56, and 180. Principal Results Both vaccines were safe and had acceptable reactogenicity profiles during six months of follow-up. There were no serious or grade 3 and 4 laboratory adverse events. Moderate systemic reactogenicity (mostly headache or feeling tired) was reported by ∼23% of participants receiving either vaccine. Injection site reactogenicity was mostly mild itchiness and pain. The frequencies of vaccine take skin reactions were 73% (95% CI, 64, 81) for DVC-LVS and 80% (95% CI, 71, 87) for USAMRIID-LVS. The 90% CI for the difference in proportions was −6.9% (−16.4, 2.6). The rates of seroconversion measured by microagglutination assay on days 28 or 56 were 94% (95% CI, 88, 98; n = 98/104) for DVC-LVS and 94% (95% CI, 87, 97; n = 103/110) for USAMRIID-LVS (p = 1.00). Day 14 sera revealed more rapid seroconversion for DVC-LVS relative to USAMRIID-LVS: 82% (95% CI, 73, 89) versus 55% (95% CI, 45, 65), respectively (p < 0.0001). Major conclusions The DVC-LVS vaccine had similar safety, reactogenicity, take and antibody responses compared to the older USAMRIID vaccine, and was superior for early (day 14) antibody production. Vaccination take was not a sensitive surrogate for seroconversion in a multi-center study where personnel at five research clinics performed assessments. ClinicalTrials.gov identifier NCT01150695 [ABSTRACT FROM AUTHOR]

Francisella tularensis is a highly infectious Gram-negative coccobacillus which causes the disease tularemia. The potential for its misuse as a biological weapon has led disease control and prevention centers to classify this bacterium as a category A agent. Bacterial outer membrane vesicles (OMVs) are spherical particles 20–250 nm in size produced by all Gram-negative bacteria and constitute one of the major secretory pathways. Bacteria use them in interacting with both other bacterial cells and eukaryotic (host) cells. OMVs of Francisella contain number of its so far described virulence factors and immunomodulatory proteins. Their role in host–pathogen interactions can therefore be presumed, and the possibility exists also for their potential use in a subunit vaccine. Moreover, Francisella microbes produce both usual spherical and unusual tubular OMVs. Because OMVs emerge from the outermost surface of the bacterial cell, we focused on the secretion of OMVs in several mutant Francisella strains with disrupted surface structures (namely the O-antigen). O-antigen in Francisella is not only the structural component of LPS but also forms another important virulence factor: the O-antigen polysaccharide capsule. Mutant strain phenotypes were evaluated by growth curves, vesiculation rates, their sensitivity to the complement contained in serum, and proliferation inside murine bone marrow macrophages. Morphologies of both OMVs and the bacteria were visualized by electron microscopy. The O-antigen mutant strains were considerably attenuated in serum resistance and intracellular proliferation. All the strains showed lower ability to form the tubular OMVs. Some strains formed tubular protrusions from their outer membrane but their stability was weak. Some hypervesiculating strains were revealed that will serve as source of OMVs for further studies of their protective potential. Our results suggest the presence of LPS and the O-antigen capsule on the surface of Francisella to be critical not only for its virulence but also for the exceptional tubular shape of its OMVs. • O-antigen is important for tubular shape of vesicles in F. tularensis. • Hypervesiculating strains were found among O-antigen mutants in F. tularensis. • O-antigen mutants in F. tularensis are attenuated in serum resistance. • O-antigen mutants in F. tularensis are attenuated in intracellular proliferation. [ABSTRACT FROM AUTHOR]

Tularemia or rabbit fever is a transmissible disease from animals, rodents, and insects to human populations that is caused by Francisella tularensis. Epidemiological studies showed that tularemia is endemic throughout most different regions of the world. Recent evidence documented the transmission of the F. tularensis in a different part of Asia. Because there is no updated review information for tularemia in Iran, we performed this systematic review. In this study, we systematically explored biomedical databases (Google Scholar, Scopus, PubMed, and Web of sciences) to identify epidemiology, reservoirs, and carriers of Francisella in animal and human clinical specimens from 2010 to 2020, either in English or in Persian. Different studies have shown the different frequencies of F. tularensis among human and animal resources in eighteen provinces of Iran. In total, 1242 human clinical specimens, 1565 animal samples, and 355 environmental water samples were investigated to find F. tularensis in different provinces of Iran. According to the collected documents, 94 human clinical samples, 69 water samples, and 26 animal specimens were introduced as positive samples for the F. tularensis. According to studies, thirteen species of rodent and hare presented as an inter-epizootic reservoir. Only one species of tick (D. marginatus) was introduced as a vector for Francisella in Iran. According to these results, it is essential for exclusive attention to the prevalence of F. tularensis in different provinces of Iran. Furthermore, special planning should be done for prevention, control of the outbreak, and proper treatment of the tularemia. [ABSTRACT FROM AUTHOR]

A new β-class carbonic anhydrase (CA, EC 4.2.1.1) has been cloned, purified and characterized in the genome of the pathogenic bacterium Francisella tularensis responsible of the febrile illness tularemia. This enzyme, FtuβCA, showed a k cat of 9.8 × 10 5 s −1 and a k cat /K M of 8.9 × 10 7 M −1 s −1 for the CO 2 hydration, physiological reaction, being one of the most effective β-CAs known to date, with a catalytic activity only 1.68-times lower than that of the human(h) isoform hCA II. A panel of 39 simple aromatic and heterocyclic sulfonamides, as well as clinically used drugs incorporating sulfonamide/sulfamate zinc-binding groups, was used to investigate the inhibition profile of FtuβCA with these classes of derivatives. The enzyme generally showed a weaker affinity for these inhibitors compared to other α- and β-CAs investigated earlier, with only acetazolamide and its deacetylated precursor having inhibition constant <1 µM. Indeed, the two compounds acetazolamide AAZ and its deacetylated precursor 13 (K I s of 655–770 nM), as well as metanilamide and methazolamide (K I s of 2.53–2.92 µM), were the best FtuβCA inhibitors detected so far. As the physiological role of bacterial β-CAs is poorly understood for the virulence/life cycle of these pathogens, the present study may constitute a starting point for the design of effective pathogenic bacteria CA inhibitors with potential use as antiinfectives. [ABSTRACT FROM AUTHOR]

Francisella tularensis subsp. tularensis strain SchuS4 (Ftt) is a highly virulent intracellular bacterium. Inhalation of 10 or fewer organisms results in an acute and potentially lethal disease called pneumonic tularemia. Ftt infections occur naturally in the U.S. and Ftt was developed as a bioweapon. Thus, there is a need for vaccines that protect against this deadly pathogen. Although a live vaccine strain of Francisella tularensis (LVS) exists, LVS fails to generate long-lived protective immunity against modest challenge doses of Ftt. We recently identified an important role for high avidity CD4 + T cells in short-term protection and hypothesized that expanding this pool of cells would improve overall vaccine efficacy with regard to longevity and challenge dose. In support of our hypothesis, application of a prime/boost vaccination strategy increased the pool of high avidity CD4 + T cells which correlated with improved survival following challenge with either increased doses of virulent Ftt or at late time points after vaccination. In summary, we demonstrate that both epitope selection and vaccination strategies that expand antigen-specific T cells correlate with superior immunity to Ftt as measured by survival. [ABSTRACT FROM AUTHOR]

Numerous studies have demonstrated that administration of antigen (Ag)-pulsed dendritic cells (DCs) is an effective strategy for enhancing immunity to tumors and infectious disease organisms. However, the generation and/or isolation of DCs can require substantial time and expense. Therefore, using inactivated F. tularensis (i Ft ) Ag as a model immunogen, we first sought to determine if DCs could be replaced with peripheral blood mononuclear cells (PBMCs) during the ex-vivo pulse phase and still provide protection against Ft infection. Follow up studies were then conducted using the S. pneumoniae ( Sp ) vaccine Prevnar ®13 as the Ag in the pulse phase followed by immunization and Sp challenge. In both cases, we demonstrate that PBMCs can be used in place of DCs when pulsing with i Ft and/or Prevnar ®13 ex vivo and re-administering the Ag-pulsed PBMCs as a vaccine. In addition, utilization of the i.n. route for Ag-pulsed PBMC administration is superior to use of the i.v. route in the case of Sp immunization, as well as when compared to direct injection of Prevnar ®13 vaccine i.m. or i.n. Furthermore, this PBMC-based vaccine strategy provides a more marked and enduring protective immune response and is also capable of serving as a multi-organism vaccine platform. The potential for this ex-vivo vaccine strategy to provide a simpler, less time consuming, and less expensive approach to DC-based vaccines and vaccination in general is also discussed. [ABSTRACT FROM AUTHOR]

Respiratory infection of mice with Francisella novicida has recently been used as a model for the highly virulent human pathogen Francisella tularensis . Similar to F. tularensis , even small doses of F. novicida administered by respiratory routes are lethal for inbred laboratory mice. This feature obviously limits study of infection-induced immunity. Parenteral sublethal infections of mice with F. novicida are feasible, but the resulting immune responses are incompletely characterized. Here we use parenteral intradermal (i.d.) and intraperitoneal (i.p.) F. novicida infections of C57BL/6J mice to determine the role of B cells in controlling primary and secondary F. novicida infections. Despite developing comparable levels of F. novicida -primed T cells, B cell knockout mice were much more susceptible to both primary i.d. infection and secondary i.p. challenge than wild type (normal) C57BL/6J mice. Transfer of F. novicida -immune sera to either wild type C57BL/6J mice or to B cell knockout mice did not appreciably impact survival of subsequent lethal F. novicida challenge. However, F. novicida -immune mice that were depleted of T cells after priming but just before challenge survived and cleared secondary i.p. F. novicida challenge. Collectively these results indicate that B cells, if not serum antibodies, play a major role in controlling F. novicida infections in mice. [ABSTRACT FROM AUTHOR]

This paper presents simple, fast, and sensitive detection of multiple biothreat agents by paper-based vertical flow colorimetric sandwich immunoassay for detection of Yersinia pestis (LcrV and F1) and Francisella tularensis (lipopolysaccharide; LPS) antigens using a vertical flow immunoassay (VFI) prototype with portable syringe pump and a new membrane holder. The capture antibody (cAb) printing onto nitrocellulose membrane and gold-labelled detection antibody (dAb) were optimized to enhance the assay sensitivity and specificity. Even though the paper pore size was relaxed from previous 0.1 μm to the current 0.45 μm for serum samples, detection limits as low as 0.050 ng/mL for LcrV and F1, and 0.100 ng/mL for FtLPS have been achieved in buffer and similarly in diluted serum (with LcrV and F1 LODs remained the same and LPS LOD reduced to 0.250 ng/mL). These were 40, 80, and 50X (20X for LPS in serum) better than those from lateral flow configuration. Furthermore, the comparison of multiplex format demonstrated low cross-reactivity and equal sensitivity to that of the singleplex assay. The optimized VFI platform thus provides a portable and rapid on-site monitoring system for multiplex biothreat detection with the potential for high sensitivity, specificity, reproducibility, and multiplexing capability, supporting its utility in remote and resource-limited settings. [ABSTRACT FROM AUTHOR]

The study aimed to detect Francisella tularensis (F. tularensis) in water samples and to investigate the seroreactivity of sheep to tularemia in endemic areas where human tularemia cases have been reported in Ankara, Turkey. For the isolation of F. tularensis , 50 water samples were collected from rural areas of 5 regions of Ankara (Turkey) and selectively cultured on Francis medium supplemented with 8–9 % sheep blood and antibiotics (100 IU/ml penicillin G, 100 mg/L cycloheximide, 80,000 U/L polymixin B). No F. tularensis isolate was cultivated from the water samples. To determine the seroreactivity of sheep to tularemia , 1006 sheep blood samples were collected from the regions, where human tularemia is endemic. A microagglutination test (MAT) identified significant antibody titers, ranging from 1/20–1/640 in 181 (17.99 %) of the investigated sheep sera. Further investigation is required in order to evaluate and confirm a possible epidemiologic relationship between human outbreaks and probable role of sheep or other sources. • In this study, detection of the presence of the agent in possible reservoirs in hot spots for the disease tularemia. • Although culture positivity could not be obtained from water, seroreactivity was detected in sheep. • Significant antibody titer (17.99 %) was found by the microagglutination test. • Simultaneous sampling is needed from both end hosts and reservoirs at a time of an outbreak. [ABSTRACT FROM AUTHOR]

MALDI-TOF MS is a relatively new technology which has revolutionized the way microorganisms are identified. The technique provides specific biomarker profiles which can be employed for rapid, accurate and cost-effective microbial identification. The focus of this mini-review is on the current status and new advances in the application of MALDI-TOF MS for identification of highly pathogenic bacteria (HPB). Data from selected studies with HPB of the genera Bacillus , Burkholderia , Brucella , Francisella and Yersinia are reviewed and molecular assignments of HPB-specific high abundance biomarkers are presented. Furthermore, experiences from an European inter-laboratory ring trial on HPB are outlined. [ABSTRACT FROM AUTHOR]

Several bacterial pathogens interact with their host through protein secretion effectuated by a type VI secretion system (T6SS). Francisella tularensis is a highly pathogenic intracellular bacterium that causes the disease tularemia. Proteins encoded by the Francisella pathogenicity island (FPI), which constitute a type VI secretion system, are essential for the virulence of the bacterium and a key mechanism behind this is the escape from the phagosome followed by productive cytosolic replication. It has been shown that T6SS in Francisella is distinct since all putative substrates of F. tularensis T6SS, except for VgrG, are unique to the species. Many of the FPI proteins are secreted into the macrophage cytosol and this is dependent on the functional components of DotU, VgrG, IglC and IglG. In addition, PdpC seems to have a regulatory role for the expression of iglABCD. Since previous results showed peculiar phenotypes of the Δ pdp C and Δ igl G mutants in mouse macrophages, their unique behavior was characterized in human monocyte-derived macrophages (HMDM) in this study. Our results show that both Δ pdpC and Δ iglG mutants of the live vaccine strain (LVS) of F. tularensis did not replicate within HMDMs. The Δ pdpC mutant did not escape from the Francisella containing phagosome (FCP), neither caused cytopathogenicity in primary macrophages and was attenuated in a mouse model. Interestingly, the Δ iglG mutant escaped from the HMDMs FCP and also caused pathological changes in the spleen and liver tissues of intradermally infected C57BL/6 mice. The Δ iglG mutant, with its unique phenotype, is a potential vaccine candidate. [ABSTRACT FROM AUTHOR]

Francisella tularensis is the cause of the zoonotic disease tularemia. In Sweden and Scandinavia, epidemiological studies have implicated mosquitoes as a vector. Prior research has demonstrated the presence of Francisella DNA in infected mosquitoes but has not shown definitive transmission of tularemia from a mosquito to a mammalian host. We hypothesized that antimicrobial peptides, an important component of the innate immune system of higher organisms, may play a role in mosquito host-defense to Francisella . We established that Francisella sp. are susceptible to two cecropin antimicrobial peptides derived from the mosquito Aedes albopictus as well as Culex pipiens . We also demonstrated induced expression of Aedes albopictus antimicrobial peptide genes by Francisella infection C6/36 mosquito cell line. We demonstrate that mosquito antimicrobial peptides act against Francisella by disrupting the cellular membrane of the bacteria. Thus, it is possible that antimicrobial peptides may play a role in the inability of mosquitoes to establish an effective natural transmission of tularemia. [ABSTRACT FROM AUTHOR]

A safer and more effective vaccine than the unlicensed Francisella tularensis Live Vaccine Strain (LVS) is needed to protect against the biowarfare agent F. tularensis . Previously, we developed an LVS Δ capB mutant that is significantly safer than LVS and provides potent protective immunity against F. tularensis respiratory challenge when administered intranasally but limited protection when administered intradermally unless as part of a prime-boost vaccination strategy. To improve the immunogenicity and efficacy of LVS Δ capB , we developed recombinant LVS Δ capB (rLVS Δ capB ) strains overexpressing various F. tularensis Francisella Pathogenicity Island (FPI) proteins - IglA, IglB and IglC, and a fusion protein (IglABC) comprising immunodominant epitopes of IglA, IglB, and IglC downstream of different Francisella promoters, including the bacterioferritin ( bfr ) promoter. We show that rLVS Δ capB / bfr-iglA, iglB, iglC, and iglABC express more IglA, IglB, IglC or IglABC than parental LVS Δ capB in broth and in human macrophages, and stably express FPI proteins in macrophages and mice absent antibiotic selection. In response to IglC and heat-inactivated LVS, spleen cells from mice immunized intradermally with rLVS Δ capB / bfr-iglC or bfr-iglABC secrete greater amounts of interferon-gamma and/or interleukin-17 than those from mice immunized with LVS Δ capB , comparable to those from LVS-immunized mice. Mice immunized with rLVS Δ capB / bfr-iglA , iglB , iglC or iglABC produce serum antibodies at levels similar to LVS-immunized mice. Mice immunized intradermally with rLVS Δ capB / bfr-iglABC and challenged intranasally with virulent F. tularensis Schu S4 survive longer than sham- and LVS Δ capB -immunized mice. Mice immunized intranasally with rLVS Δ capB / bfr-iglABC - but not with LVS - just before or after respiratory challenge with F. tularensis Schu S4 are partially protected; protection is correlated with induction of a strong innate immune response. Thus, rLVS Δ capB / bfr-iglABC shows improved immunogenicity and protective efficacy compared with parental LVS Δ capB and, in contrast to LVS, has partial efficacy as immediate pre- and post-exposure prophylaxis. [ABSTRACT FROM AUTHOR]

Tularemia is a zoonotic disease that has been reported in many countries of the Northern Hemisphere. However, in some countries, such as Iran, this disease has been neglected by the health care system, and it is under-reported. Here we report an unusual case of ulceroglandular tularemia occurring in a 35-year-old woman who presented with a skin lesion of the left flank, inguinal lymphadenopathy, and an abdominal abscess. The serological and real-time PCR tests for tularemia were positive for this patient, and infection by Francisella tularensis subsp. holarctica was confirmed. It is necessary to implement various educational programs to increase the awareness of physicians with tularemia. [ABSTRACT FROM AUTHOR]

• Roe deer and other wild animals contribute to the diffusion of ticks. • Roe deer may be source of zoonotic VBPs. • New VBPs may occur as consequence of high presence of ticks. Environmental changes, due to climatic emergency and to anthropogenic activities severely impact on the epidemiology of vector borne diseases, mostly when transmitted by ticks. The data about the distribution of microorganisms responsible for them in roe deer (Capreolus capreolus) population living in Italy are scanty and completely lacking in Tuscany, so a molecular survey was carried out to estimate the prevalence of some zoonotic tick-borne pathogens in roe deer, and ticks removed from them, living in areas of Central Italy with high risk of arthropod exposure. Spleen samples from 72 roe deer were tested by PCR for Anaplasma phagocytophilum, Borrelia burgdorferi s.l., Francisella tularensis and piroplasms. Moreover, 345 ticks were removed from 65 roe deer, morphologically or molecularly identified and grouped into 162 pools that were submitted to PCR for detecting the same pathogens. Forty-six (63.88%) roe deer were positive for at least one investigated pathogen: 43 (59.72%) for A. phagocytophilum , 2 (2.78%) for Babesia capreoli , 1 (1.39%) for B. burgdorferi , and 1 (1.39%) for Babesia sp. No animals were PCR positive for F. tularensis. All ticks were identified as Ixodes ricinus. Seventy-six (46.91%) tick pools showed DNA of one or more pathogens: 66 (40.74%) were positive for A. phagocytophilum , 22 (13.58%) for B. burgodorferi s.l., 6 (3.70%) for B. venatorum and 3 (1.85%) for B. capreoli. No pools were positive for F. tularensis. Two or three pathogens were detected in 23 (14.19%) pools. Environmental changes, due to climatic emergency and to anthropogenic activities, severely impact on the epidemiology of vector borne diseases, mostly when transmitted by ticks. [Display omitted] [ABSTRACT FROM AUTHOR]

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

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

As an innate defense mechanism, macrophages produce reactive oxygen species that weaken pathogens and serve as secondary messengers involved in immune function. The Gram-negative bacterium Francisella tularensis utilizes its antioxidant armature to limit the host immune response, but the mechanism behind this suppression is not defined. Here we establish that F. tularensis limits Ca2+ entry in macrophages, thereby limiting actin reorganization and IL-6 production in a redox-dependent fashion. Wild type (live vaccine strain) or catalase-deficient F. tularensis (ΔkatG) show distinct profiles in their H2O2 scavenging rates, 1 and 0.015 pM/s, respectively. Murine alveolar macrophages infected with ΔkatG display abnormally high basal intracellular Ca2+ concentration that did not increase further in response to H2O2. Additionally, ΔkatG-infected macrophages displayed limited Ca2+ influx in response to ionomycin, as a result of ionophore H2O2 sensitivity. Exogenously added H2O2 or H2O2 generated by ΔkatG likely oxidizes ionomycin and alters its ability to transport Ca2+. Basal increases in cytosolic Ca2+ and insensitivity to H2O2-mediated Ca2+ entry in ΔkatG-infected cells are reversed by the Ca2+ channel inhibitors 2-aminoethyl diphenylborinate and SKF-96365. 2-Aminoethyl diphenylborinate but not SKF-96365 abrogatedΔkatG-dependent increases in macrophage actin remodeling and IL-6 secretion, suggesting a role for H2O2 -mediated Ca2+ entry through the transient receptor potential melastatin 2 (TRPM2) channel in macrophages. Indeed, increases in basal Ca2+, actin polymerization, and IL-6 production are reversed in TRPM2- null macrophages infected with ΔkatG. Together, our findings provide compelling evidence that F. tularensis catalase restricts reactive oxygen species to temper macrophage TRPM2-mediated Ca2+signaling and limit host immune function. [ABSTRACT FROM AUTHOR]

Tularaemia is a zoonotic disease caused by Francisella tularensis, a Gram-negative, facultative intracellular bacterium. Typically, human and animal infections are caused by F tularensis subspecies tularensis (type A) strains mainly in Canada and USA, and F tularensis subspecies holarctica (type B) strains throughout the northern hemisphere, including Europe. In the past, the epidemiological, clinical, therapeutic, and prognostic aspects of tularaemia reported in the English medical literature were mainly those that had been reported in the USA, where the disease was first described. Tularaemia has markedly changed in the past decade, and a large number of studies have provided novel data for the disease characteristics in Europe. In this Review we aim to emphasise the specific and variable aspects of tularaemia in different European countries. In particular, two natural lifecycles of F tularensis have been described in this continent, although not fully characterised, which are associated with different modes of transmission, clinical features, and public health burdens of tularaemia. [ABSTRACT FROM AUTHOR]

Francisella tularensis is a human zoonotic pathogen and the causative agent of tularemia, a severe infectious disease. Given the extreme infectivity of F. tularensis and its potential to be used as a biological warfare agent, a fast and sensitive detection method is highly desirable. Herein, we construct a novel detection platform composed of two units: (1) Magnetic beads conjugated with multiple capturing antibodies against F. tularensis for its simple and rapid separation and (2) Genetically-engineered apoferritin protein constructs conjugated with multiple quantum dots and a detection antibody against F. tularensis for the amplification of signal. We demonstrate a 10-fold increase in the sensitivity relative to traditional lateral flow devices that utilize enzyme-based detection methods. We ultimately envision the use of our novel nanoprobe detection platform in future applications that require the highly-sensitive on-site detection of high-risk pathogens. [ABSTRACT FROM AUTHOR]

In the event of suspected releases or natural outbreaks of contagious pathogens, rapid identification of the infectious agent is essential for appropriate medical intervention and disease containment. The purpose of this study was to compare the performance of a novel portable real-time PCR thermocycler, PikoReal™, to the standard real-time PCR thermocycler, Applied Biosystems® 7300 (ABI 7300), for the detection of three high-risk biothreat bacterial pathogens: Francisella tularensis , Bacillus anthracis and Yersinia pestis . In addition, a novel confirmatory real-time PCR assay for the detection of F. tularensis is presented and validated. The results show that sensitivity of the assays, based on a dilution series, for the three infectious agents ranged from 1 to 100 fg of target DNA with both instruments. No cross-reactivity was revealed in specificity testing. Duration of the assays with the PikoReal and ABI 7300 systems were 50 and 100 min, respectively. In field testing for F. tularensis , results were obtained with the PikoReal system in 95 min, as the pre-PCR preparation, including DNA extraction, required an additional 45 min. We conclude that the PikoReal system enables highly sensitive and rapid on-site detection of biothreat agents under field conditions, and may be a more efficient alternative to conventional diagnostic methods. [ABSTRACT FROM AUTHOR]

Summary Tularemia is a potentially fatal bacterial infection caused by Francisella tularensis , and is endemic to North America and many parts of northern Europe and Asia. The outer membrane lipoprotein, Flpp3, has been identified as a virulence determinant as well as a potential subunit template for vaccine development. Here we present the first structure for the soluble domain of Flpp3 from the highly infectious Type A SCHU S4 strain, derived through high-resolution solution nuclear magnetic resonance (NMR) spectroscopy; the first structure of a lipoprotein from the genus Francisella . The Flpp3 structure demonstrates a globular protein with an electrostatically polarized surface containing an internal cavity—a putative binding site based on the structurally homologous Bet v1 protein family of allergens. NMR-based relaxation studies suggest loop regions that potentially modulate access to the internal cavity. The Flpp3 structure may add to the understanding of F. tularensis virulence and contribute to the development of effective vaccines. [ABSTRACT FROM AUTHOR]

Tularemia is a zoonotic disease caused by Francisella tularensis , which is transmitted to humans most commonly by contact with infected animals, tick bites, or inhalation of aerosolized bacteria. F. tularensis is highly infectious via the aerosol route; inhalation of as few as 10–50 organisms can cause pneumonic tularemia. Left untreated, the pneumonic form has more than >30% case-fatality rate but with early antibiotic intervention can be reduced to 3%. This study compared tularemia disease progression across three species of nonhuman primates [African green monkey (AGM), cynomolgus macaque (CM), and rhesus macaque (RM)] following aerosolized F. tularensis Schu S4 exposure. Groups of the animals exposed to various challenge doses were observed for clinical signs of infection and blood samples were analyzed to characterize the disease pathogenesis. Whereas the AGMs and CMs succumbed to disease following challenge doses of 40 and 32 colony forming units (CFU), respectively, the RM lethal dose was 276,667 CFU. Following all challenge doses that caused disease, the NHPs experienced weight loss, bacteremia, fever as early as 4 days post exposure, and tissue burden. Necrotizing-to-pyogranulomatous lesions were observed most commonly in the lung, lymph nodes, spleen, and bone marrow. Overall, the CM model consistently manifested pathological responses similar to those resulting from inhalation of F. tularensis in humans and thereby most closely emulates human tularemia disease. The RM model displayed a higher tolerance to infection and survived exposures of up to 15,593 CFU of aerosolized F. tularensis . [ABSTRACT FROM AUTHOR]

A simple, rapid and potentiostat-free method for grafting of close to monolayers of diazonium salts to anchor DNA probes for application in highly sensitive electrochemical genosensing is presented. Zn-mediated grafting is demonstrated to produce a highly organised surface facilitating enhanced sensitivity and a notably reduced detection limit as compared to electrochemical grafting. [ABSTRACT FROM AUTHOR]

Francisella tularensis , the causative agent of tularemia, presents a significant biological threat and is a Category A priority pathogen due to its potential for weaponization. The bacterial FASII pathway is a viable target for the development of novel antibacterial agents treating Gram-negative infections. Here we report the advancement of a promising series of benzimidazole FabI (enoyl-ACP reductase) inhibitors to a second-generation using a systematic, structure-guided lead optimization strategy, and the determination of several co-crystal structures that confirm the binding mode of designed inhibitors. These compounds display an improved low nanomolar enzymatic activity as well as promising low microgram/mL antibacterial activity against both F. tularensis and Staphylococcus aureus and its methicillin-resistant strain (MRSA). The improvements in activity accompanying structural modifications lead to a better understanding of the relationship between the chemical structure and biological activity that encompasses both enzymatic and whole-cell activity. [ABSTRACT FROM AUTHOR]