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Sample records for agent francisella tularensis

  1. Francisella tularensis as a potential agent of bioterrorism?

    PubMed

    Maurin, Max

    2015-02-01

    Francisella tularensis is a category A bioterrorism agent. It is the etiological agent of tularemia, a zoonotic disease found throughout the northern hemisphere. The intentional spread of F. tularensis aerosols would probably lead to severe and often fatal pneumonia cases, but also secondary cases from contaminated animals and environments. We are not ready to face such a situation. No vaccine is currently available. A few antibiotics are active against F. tularensis, but strains resistant to these antibiotics could be used in the context of bioterrorism. We need new therapeutic strategies to fight against category A bioterrorism agents, including development of new drugs inhibiting F. tularensis growth and/or virulence, or enhancing the host response to infection by this pathogen.

  2. Francisella tularensis Bacteremia

    PubMed Central

    Haristoy, X.; Lozniewski, A.; Tram, C.; Simeon, D.; Bevanger, L.; Lion, C.

    2003-01-01

    Bacteremia caused by Francisella tularensis is rare and has been reported mainly in the United States and infrequently in Europe. We report herein the first case of bacteremic F. tularensis pneumonia in an immunocompetent individual in southern Europe. PMID:12791928

  3. The use of resazurin as a novel antimicrobial agent against Francisella tularensis.

    PubMed

    Schmitt, Deanna M; O'Dee, Dawn M; Cowan, Brianna N; Birch, James W-M; Mazzella, Leanne K; Nau, Gerard J; Horzempa, Joseph

    2013-01-01

    The highly infectious and deadly pathogen, Francisella tularensis, is classified by the CDC as a Category A bioterrorism agent. Inhalation of a single bacterium results in an acute pneumonia with a 30-60% mortality rate without treatment. Due to the prevalence of antibiotic resistance, there is a strong need for new types of antibacterial drugs. Resazurin is commonly used to measure bacterial and eukaryotic cell viability through its reduction to the fluorescent product resorufin. When tested on various bacterial taxa at the recommended concentration of 44 μM, a potent bactericidal effect was observed against various Francisella and Neisseria species, including the human pathogens type A F. tularensis (Schu S4) and N. gonorrhoeae. As low as 4.4 μM resazurin was sufficient for a 10-fold reduction in F. tularensis growth. In broth culture, resazurin was reduced to resorufin by F. tularensis. Resorufin also suppressed the growth of F. tularensis suggesting that this compound is the biologically active form responsible for decreasing the viability of F. tularensis LVS bacteria. Replication of F. tularensis in primary human macrophages and non-phagocytic cells was abolished following treatment with 44 μM resazurin indicating this compound could be an effective therapy for tularemia in vivo. PMID:24367766

  4. ECO-EPIZOOTIOLOGIC STUDY OF FRANCISELLA TULARENSIS, THE AGENT OF TULAREMIA, IN QUÉBEC WILDLIFE.

    PubMed

    Gabriele-Rivet, Vanessa; Ogden, Nicholas; Massé, Ariane; Antonation, Kym; Corbett, Cindi; Dibernardo, Antonia; Lindsay, L Robbin; Leighton, Patrick A; Arsenault, Julie

    2016-04-28

    In Canada, Francisella tularensis , the zoonotic bacterial agent of tularemia, affects mostly snowshoe hares ( Lepus americanus ), muskrats ( Ondatra zibethicus ), and beavers ( Castor canadensis ). Despite numerous studies, the ecologic cycle and natural reservoirs of F. tularensis are not clearly defined. We conducted a cross-sectional study to estimate the prevalence of F. tularensis in snowshoe hares, muskrats, and coyotes ( Canis latrans ) in four regions of Québec, Canada, and to describe the risk of infection in relation to host and environmental characteristics at three spatial scales. Between October 2012 and April 2013, trappers captured 345 snowshoe hares, 411 muskrats, and 385 coyotes. Blood samples were tested by microagglutination tests, and DNA extracts of liver, kidney, lung, and spleen of snowshoe hares and muskrats were tested by real-time PCR to detect past and active infection to F. tularensis , respectively. Individual host characteristics, including body condition, age, and sex, were evaluated as risk factors of infection, along with ecologic characteristics of the location of capture extracted from geographic databases. Prevalences of antibody to F. tularensis and 95% confidence intervals were 2.9% (1.4-5.1%) in coyotes, 0.6% (0.1-2.1%) in hares, and 0% (0.0-0.9%) in muskrats. Francisella tularensis DNA was not detected by real-time PCR in the pools of four organs from muskrats and hares, but F. tularensis type AI was detected during testing of the individual organs of two antibody-positive hares. Exact logistic regression analyses showed that age was a significant predictor of antibody detection in coyotes, as were the proportion of forest and the proportion of area considered as suitable habitat for hares in the environment around the location of capture of the coyotes. Our results suggest a terrestrial cycle of F. tularensis in the regions studied.

  5. ECO-EPIZOOTIOLOGIC STUDY OF FRANCISELLA TULARENSIS, THE AGENT OF TULAREMIA, IN QUÉBEC WILDLIFE.

    PubMed

    Gabriele-Rivet, Vanessa; Ogden, Nicholas; Massé, Ariane; Antonation, Kym; Corbett, Cindi; Dibernardo, Antonia; Lindsay, L Robbin; Leighton, Patrick A; Arsenault, Julie

    2016-04-28

    In Canada, Francisella tularensis , the zoonotic bacterial agent of tularemia, affects mostly snowshoe hares ( Lepus americanus ), muskrats ( Ondatra zibethicus ), and beavers ( Castor canadensis ). Despite numerous studies, the ecologic cycle and natural reservoirs of F. tularensis are not clearly defined. We conducted a cross-sectional study to estimate the prevalence of F. tularensis in snowshoe hares, muskrats, and coyotes ( Canis latrans ) in four regions of Québec, Canada, and to describe the risk of infection in relation to host and environmental characteristics at three spatial scales. Between October 2012 and April 2013, trappers captured 345 snowshoe hares, 411 muskrats, and 385 coyotes. Blood samples were tested by microagglutination tests, and DNA extracts of liver, kidney, lung, and spleen of snowshoe hares and muskrats were tested by real-time PCR to detect past and active infection to F. tularensis , respectively. Individual host characteristics, including body condition, age, and sex, were evaluated as risk factors of infection, along with ecologic characteristics of the location of capture extracted from geographic databases. Prevalences of antibody to F. tularensis and 95% confidence intervals were 2.9% (1.4-5.1%) in coyotes, 0.6% (0.1-2.1%) in hares, and 0% (0.0-0.9%) in muskrats. Francisella tularensis DNA was not detected by real-time PCR in the pools of four organs from muskrats and hares, but F. tularensis type AI was detected during testing of the individual organs of two antibody-positive hares. Exact logistic regression analyses showed that age was a significant predictor of antibody detection in coyotes, as were the proportion of forest and the proportion of area considered as suitable habitat for hares in the environment around the location of capture of the coyotes. Our results suggest a terrestrial cycle of F. tularensis in the regions studied. PMID:26967133

  6. Comparative review of Francisella tularensis and Francisella novicida.

    PubMed

    Kingry, Luke C; Petersen, Jeannine M

    2014-01-01

    Francisella tularensis is the causative agent of the acute disease tularemia. Due to its extreme infectivity and ability to cause disease upon inhalation, F. tularensis has been classified as a biothreat agent. Two subspecies of F. tularensis, tularensis and holarctica, are responsible for tularemia in humans. In comparison, the closely related species F. novicida very rarely causes human illness and cases that do occur are associated with patients who are immune compromised or have other underlying health problems. Virulence between F. tularensis and F. novicida also differs in laboratory animals. Despite this varying capacity to cause disease, the two species share ~97% nucleotide identity, with F. novicida commonly used as a laboratory surrogate for F. tularensis. As the F. novicida U112 strain is exempt from U.S. select agent regulations, research studies can be carried out in non-registered laboratories lacking specialized containment facilities required for work with virulent F. tularensis strains. This review is designed to highlight phenotypic (clinical, ecological, virulence, and pathogenic) and genomic differences between F. tularensis and F. novicida that warrant maintaining F. novicida and F. tularensis as separate species. Standardized nomenclature for F. novicida is critical for accurate interpretation of experimental results, limiting clinical confusion between F. novicida and F. tularensis and ensuring treatment efficacy studies utilize virulent F. tularensis strains. PMID:24660164

  7. Bacillus anthracis, Francisella tularensis and Yersinia pestis. The most important bacterial warfare agents - review.

    PubMed

    Pohanka, M; Skládal, P

    2009-01-01

    There are three most important bacterial causative agents of serious infections that could be misused for warfare purposes: Bacillus anthracis (the causative agent of anthrax) is the most frequently mentioned one; however, Fracisella tularensis (causing tularemia) and Yersinia pestis (the causative agent of plague) are further bacterial agents enlisted by Centers for Disease Control and Prevention into the category A of potential biological weapons. This review intends to summarize basic information about these bacterial agents. Military aspects of their pathogenesis and the detection techniques suitable for field use are discussed.

  8. Francisella tularensis subsp. tularensis Group A.I, United States

    PubMed Central

    Birdsell, Dawn N.; Johansson, Anders; Öhrman, Caroline; Kaufman, Emily; Molins, Claudia; Pearson, Talima; Gyuranecz, Miklós; Naumann, Amber; Vogler, Amy J.; Myrtennäs, Kerstin; Larsson, Pär; Forsman, Mats; Sjödin, Andreas; Gillece, John D.; Schupp, James; Petersen, Jeannine M.; Keim, Paul

    2014-01-01

    We used whole-genome analysis and subsequent characterization of geographically diverse strains using new genetic signatures to identify distinct subgroups within Francisella tularensis subsp. tularensis group A.I: A.I.3, A.I.8, and A.I.12. These subgroups exhibit complex phylogeographic patterns within North America. The widest distribution was observed for A.I.12, which suggests an adaptive advantage. PMID:24755401

  9. Comparative Transcriptional Analyses of Francisella tularensis and Francisella novicida

    PubMed Central

    Waldo, Robert H.; Belland, Robert J.; Klose, Karl E.

    2016-01-01

    Francisella tularensis is composed of a number of subspecies with varied geographic distribution, host ranges, and virulence. In view of these marked differences, comparative functional genomics may elucidate some of the molecular mechanism(s) behind these differences. In this study a shared probe microarray was designed that could be used to compare the transcriptomes of Francisella tularensis subsp. tularensis Schu S4 (Ftt), Francisella tularensis subsp. holarctica OR960246 (Fth), Francisella tularensis subsp. holarctica LVS (LVS), and Francisella novicida U112 (Fn). To gain insight into expression differences that may be related to the differences in virulence of these subspecies, transcriptomes were measured from each strain grown in vitro under identical conditions, utilizing a shared probe microarray. The human avirulent Fn strain exhibited high levels of transcription of genes involved in general metabolism, which are pseudogenes in the human virulent Ftt and Fth strains, consistent with the process of genome decay in the virulent strains. Genes encoding an efflux system (emrA2 cluster of genes), siderophore (fsl operon), acid phosphatase, LPS synthesis, polyamine synthesis, and citrulline ureidase were all highly expressed in Ftt when compared to Fn, suggesting that some of these may contribute to the relative high virulence of Ftt. Genes expressed at a higher level in Ftt when compared to the relatively less virulent Fth included genes encoding isochorismatases, cholylglycine hydrolase, polyamine synthesis, citrulline ureidase, Type IV pilus subunit, and the Francisella Pathogenicity Island protein PdpD. Fth and LVS had very few expression differences, consistent with the derivation of LVS from Fth. This study demonstrated that a shared probe microarray designed to detect transcripts in multiple species/subspecies of Francisella enabled comparative transcriptional analyses that may highlight critical differences that underlie the relative pathogenesis of

  10. Comparative Transcriptional Analyses of Francisella tularensis and Francisella novicida.

    PubMed

    Sarva, Siva T; Waldo, Robert H; Belland, Robert J; Klose, Karl E

    2016-01-01

    Francisella tularensis is composed of a number of subspecies with varied geographic distribution, host ranges, and virulence. In view of these marked differences, comparative functional genomics may elucidate some of the molecular mechanism(s) behind these differences. In this study a shared probe microarray was designed that could be used to compare the transcriptomes of Francisella tularensis subsp. tularensis Schu S4 (Ftt), Francisella tularensis subsp. holarctica OR960246 (Fth), Francisella tularensis subsp. holarctica LVS (LVS), and Francisella novicida U112 (Fn). To gain insight into expression differences that may be related to the differences in virulence of these subspecies, transcriptomes were measured from each strain grown in vitro under identical conditions, utilizing a shared probe microarray. The human avirulent Fn strain exhibited high levels of transcription of genes involved in general metabolism, which are pseudogenes in the human virulent Ftt and Fth strains, consistent with the process of genome decay in the virulent strains. Genes encoding an efflux system (emrA2 cluster of genes), siderophore (fsl operon), acid phosphatase, LPS synthesis, polyamine synthesis, and citrulline ureidase were all highly expressed in Ftt when compared to Fn, suggesting that some of these may contribute to the relative high virulence of Ftt. Genes expressed at a higher level in Ftt when compared to the relatively less virulent Fth included genes encoding isochorismatases, cholylglycine hydrolase, polyamine synthesis, citrulline ureidase, Type IV pilus subunit, and the Francisella Pathogenicity Island protein PdpD. Fth and LVS had very few expression differences, consistent with the derivation of LVS from Fth. This study demonstrated that a shared probe microarray designed to detect transcripts in multiple species/subspecies of Francisella enabled comparative transcriptional analyses that may highlight critical differences that underlie the relative pathogenesis of

  11. Metapopulation structure for perpetuation of Francisella tularensis tularensis

    PubMed Central

    2009-01-01

    Background Outbreaks of Type A tularemia due to Francisella tularensis tularensis are typically sporadic and unstable, greatly hindering identification of the determinants of perpetuation and human risk. Martha's Vineyard, Massachusetts has experienced an outbreak of Type A tularemia which has persisted for 9 years. This unique situation has allowed us to conduct long-term eco-epidemiologic studies there. Our hypothesis is that the agent of Type A tularemia is perpetuated as a metapopulation, with many small isolated natural foci of transmission. During times of increased transmission, the foci would merge and a larger scale epizootic would occur, with greater likelihood that humans become exposed. Methods We sampled questing dog ticks from two natural foci on the island and tested them for tularemia DNA. We determined whether the force of transmission differed between the two foci. In addition, we examined the population structure of F. tularensis from ticks by variable number tandem repeat (VNTR) analysis, which allowed estimates of diversity, linkage disequilibrium, and eBURST analysis. Results The prevalence of tularemia DNA in ticks from our two field sites was markedly different: one site was stable over the course of the study yielding as many as 5.6% positive ticks. In contrast, infected ticks from the comparison site markedly increased in prevalence, from 0.4% in 2003 to 3.9% in 2006. Using 4 VNTR loci, we documented 75 different haplotypes (diversity = 0.91). eBURST analysis indicates that the stable site was essentially clonal, but the comparison site contained multiple unrelated lineages. The general bacterial population is evolving clonally (multilocus disequilibrium) and the bacteria in the two sites are reproductively isolated. Conclusion Even within an isolated island, tularemia natural foci that are no more than 15 km apart are uniquely segregated. One of our sites has stable transmission and the other is emergent. The population structure at the

  12. Detection of Francisella tularensis by the polymerase chain reaction.

    PubMed

    Junhui, Z; Ruifu, Y; Jianchun, L; Songle, Z; Meiling, C; Fengxiang, C; Hong, C

    1996-12-01

    Francisella tularensis is the causative agent of tularaemia. Effective antibiotic treatment of tularaemia is now available, but the early diagnosis of tularaemia remains a problem. Four primers (three pairs) were designed to detect F. tularensis by the polymerase chain reaction (PCR), based on the previously published nucleotide sequence of T-cell epitopes of a F. tularensis membrane protein. Amplification of purified F. tularensis chromosomal DNA with the three pairs of primers resulted in three different products with sizes consistent with those predicted from sequence data (211 bp, 347 bp and 568 bp). The specificity of the PCR was confirmed as no amplification was detected with a range of other bacteria. The sensitivity of the PCR was determined with limiting dilution PCR and viable counts. The preliminary application of the PCR to the detection of F. tularensis in aerosols and experimentally infected mice was investigated. Comparison of the results with those from traditional culture indicated that PCR was more sensitive. The animal challenge test showed that, 24 h after inoculation with 15 cfu of F. tularensis, 38 (82.6%) of 46 blood samples were positive by PCR, whereas only 22 (47.8%) were positive by culture. The results showed that PCR is a helpful tool for the detection of F. tularensis in blood, liver and spleen which should enable the rapid confirmation of clinical diagnoses of tularaemia. PMID:8958253

  13. Nucleotide sequences specific to Francisella tularensis and methods for the detection of Francisella tularensis

    DOEpatents

    McCready, Paula M.; Radnedge, Lyndsay; Andersen, Gary L.; Ott, Linda L.; Slezak, Thomas R.; Kuczmarski, Thomas A.; Vitalis, Elizabeth A

    2007-02-06

    Described herein is the identification of nucleotide sequences specific to Francisella tularensis that serves as a marker or signature for identification of this bacterium. In addition, forward and reverse primers and hybridization probes derived from these nucleotide sequences that are used in nucleotide detection methods to detect the presence of the bacterium are disclosed.

  14. Nucleotide sequences specific to Francisella tularensis and methods for the detection of Francisella tularensis

    DOEpatents

    McCready, Paula M.; Radnedge, Lyndsay; Andersen, Gary L.; Ott, Linda L.; Slezak, Thomas R.; Kuczmarski, Thomas A.; Vitalis, Elizabeth A

    2009-02-24

    Described herein is the identification of nucleotide sequences specific to Francisella tularensis that serves as a marker or signature for identification of this bacterium. In addition, forward and reverse primers and hybridization probes derived from these nucleotide sequences that are used in nucleotide detection methods to detect the presence of the bacterium are disclosed.

  15. Mechanisms of Heme Utilization by Francisella tularensis

    PubMed Central

    Lindgren, Helena; Lindgren, Lena; Golovliov, Igor; Sjöstedt, Anders

    2015-01-01

    Francisella tularensis is a highly virulent facultative intracellular pathogen causing the severe disease tularemia in mammals. As for other bacteria, iron is essential for its growth but very few mechanisms for iron acquisition have been identified. Here, we analyzed if and how F. tularensis can utilize heme, a major source of iron in vivo. This is by no means obvious since the bacterium lacks components of traditional heme-uptake systems. We show that SCHU S4, the prototypic strain of subspecies tularensis, grew in vitro with heme as the sole iron source. By screening a SCHU S4 transposon insertion library, 16 genes were identified as important to efficiently utilize heme, two of which were required to avoid heme toxicity. None of the identified genes appeared to encode components of a potential heme-uptake apparatus. Analysis of SCHU S4 deletion mutants revealed that each of the components FeoB, the siderophore system, and FupA, contributed to the heme-dependent growth. In the case of the former two systems, iron acquisition was impaired, whereas the absence of FupA did not affect iron uptake but led to abnormally high binding of iron to macromolecules. Overall, the present study demonstrates that heme supports growth of F. tularensis and that the requirements for the utilization are highly complex and to some extent novel. PMID:25756756

  16. Symbiosis with Francisella tularensis provides resistance to pathogens in the silkworm.

    PubMed

    Suzuki, Jin; Uda, Akihiko; Watanabe, Kenta; Shimizu, Takashi; Watarai, Masahisa

    2016-01-01

    Francisella tularensis, the causative agent of tularemia, is a highly virulent facultative intracellular pathogen found in a wide range of animals, including arthropods, and environments. This bacterium has been known for over 100 years, but the lifestyle of F. tularensis in natural reservoirs remains largely unknown. Thus, we established a novel natural host model for F. tularensis using the silkworm (Bombyx mori), which is an insect model for infection by pathogens. F. tularensis established a symbiosis with silkworms, and bacteria were observed in the hemolymph. After infection with F. tularensis, the induction of melanization and nodulation, which are immune responses to bacterial infection, were inhibited in silkworms. Pre-inoculation of silkworms with F. tularensis enhanced the expression of antimicrobial peptides and resistance to infection by pathogenic bacteria. These results suggest that silkworms acquire host resistance via their symbiosis with F. tularensis, which may have important fitness benefits in natural reservoirs. PMID:27507264

  17. Symbiosis with Francisella tularensis provides resistance to pathogens in the silkworm

    PubMed Central

    Suzuki, Jin; Uda, Akihiko; Watanabe, Kenta; Shimizu, Takashi; Watarai, Masahisa

    2016-01-01

    Francisella tularensis, the causative agent of tularemia, is a highly virulent facultative intracellular pathogen found in a wide range of animals, including arthropods, and environments. This bacterium has been known for over 100 years, but the lifestyle of F. tularensis in natural reservoirs remains largely unknown. Thus, we established a novel natural host model for F. tularensis using the silkworm (Bombyx mori), which is an insect model for infection by pathogens. F. tularensis established a symbiosis with silkworms, and bacteria were observed in the hemolymph. After infection with F. tularensis, the induction of melanization and nodulation, which are immune responses to bacterial infection, were inhibited in silkworms. Pre-inoculation of silkworms with F. tularensis enhanced the expression of antimicrobial peptides and resistance to infection by pathogenic bacteria. These results suggest that silkworms acquire host resistance via their symbiosis with F. tularensis, which may have important fitness benefits in natural reservoirs. PMID:27507264

  18. Recombinase Polymerase Amplification Assay for Rapid Detection of Francisella tularensis

    PubMed Central

    Euler, Milena; Wang, Yongjie; Otto, Peter; Tomaso, Herbert; Escudero, Raquel; Anda, Pedro; Hufert, Frank T.

    2012-01-01

    Several real-time PCR approaches to develop field detection for Francisella tularensis, the infectious agent causing tularemia, have been explored. We report the development of a novel qualitative real-time isothermal recombinase polymerase amplification (RPA) assay for use on a small ESEQuant Tube Scanner device. The analytical sensitivity and specificity were tested using a plasmid standard and DNA extracts from infected rabbit tissues. The assay showed a performance comparable to real-time PCR but reduced the assay time to 10 min. The rapid RPA method has great application potential for field use or point-of-care diagnostics. PMID:22518861

  19. Recombinase polymerase amplification assay for rapid detection of Francisella tularensis.

    PubMed

    Euler, Milena; Wang, Yongjie; Otto, Peter; Tomaso, Herbert; Escudero, Raquel; Anda, Pedro; Hufert, Frank T; Weidmann, Manfred

    2012-07-01

    Several real-time PCR approaches to develop field detection for Francisella tularensis, the infectious agent causing tularemia, have been explored. We report the development of a novel qualitative real-time isothermal recombinase polymerase amplification (RPA) assay for use on a small ESEQuant Tube Scanner device. The analytical sensitivity and specificity were tested using a plasmid standard and DNA extracts from infected rabbit tissues. The assay showed a performance comparable to real-time PCR but reduced the assay time to 10 min. The rapid RPA method has great application potential for field use or point-of-care diagnostics.

  20. Construction of a bioluminescence reporter plasmid for Francisella tularensis.

    PubMed

    Bina, Xiaowen R; Miller, Mark A; Bina, James E

    2010-11-01

    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. PMID:20620161

  1. Identifying Francisella tularensis genes required for growth in host cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Technical Abstract: Francisella tularensis is a highly virulent Gram negative intracellular pathogen capable of infecting a vast diversity of hosts, ranging from amoebae to humans. A hallmark of F. tularensis virulence is its ability to quickly grow to high densities within a diverse set of host cel...

  2. Multiple Francisella tularensis subspecies and clades, tularemia outbreak, Utah.

    PubMed

    Petersen, Jeannine M; Carlson, Jennifer K; Dietrich, Gabrielle; Eisen, Rebecca J; Coombs, Jana; Janusz, Aimee M; Summers, Jodee; Beard, C Ben; Mead, Paul S

    2008-12-01

    In July 2007, a deer fly-associated outbreak of tularemia occurred in Utah. Human infections were caused by 2 clades (A1 and A2) of Francisella tularensis subsp. tularensis. Lagomorph carcasses from the area yielded evidence of infection with A1 and A2, as well as F. tularensis subsp. holarctica. These findings indicate that multiple subspecies and clades can cause disease in a localized outbreak of tularemia. PMID:19046524

  3. The Francisella Tularensis Proteome and its Recognition by Antibodies

    PubMed Central

    Kilmury, Sara L. N.; Twine, Susan M.

    2011-01-01

    Francisella tularensis is the causative agent of a spectrum of diseases collectively known as tularemia. The extreme virulence of the pathogen in humans, combined with the low infectious dose and the ease of dissemination by aerosol have led to concerns about its abuse as a bioweapon. Until recently, nothing was known about the virulence mechanisms and even now, there is still a relatively poor understanding of pathogen virulence. Completion of increasing numbers of Francisella genome sequences, combined with comparative genomics and proteomics studies, are contributing to the knowledge in this area. Tularemia may be treated with antibiotics, but there is currently no licensed vaccine. An attenuated strain, the Live Vaccine Strain (LVS) has been used to vaccinate military and at risk laboratory personnel, but safety concerns mean that it is unlikely to be licensed by the FDA for general use. Little is known about the protective immunity induced by vaccination with LVS, in humans or animal models. Immunoproteomics studies with sera from infected humans or vaccinated mouse strains, are being used in gel-based or proteome microarray approaches to give insight into the humoral immune response. In addition, these data have the potential to be exploited in the identification of new diagnostic or protective antigens, the design of next generation live vaccine strains, and the development of subunit vaccines. Herein, we briefly review the current knowledge from Francisella comparative proteomics studies and then focus upon the findings from immunoproteomics approaches. PMID:21687770

  4. Francisella tularensis infection in a stone marten (Martes foina) without classic pathological lesions consistent with tularemia.

    PubMed

    Origgi, Francesco C; Wu, Natacha; Pilo, Paola

    2013-07-01

    The current report describes the isolation and typing of a strain of Francisella tularensis, the causative agent of tularemia, from the spleen of a stone marten (Martes foina) showing no classic lesions consistent with the disease. The identification of this bacterium, belonging to the World Health Organization risk 3 category and considered to have a low infectious dose, could be performed only because of an ongoing project screening F. tularensis in the environment sensu lato. The findings described herein should alert diagnostic laboratories of the possible presence of F. tularensis in clinical samples in countries where tularemia is endemic even in cases with no consistent anamnesis and from unsuspected animal species.

  5. Modeling early events in Francisella tularensis pathogenesis

    PubMed Central

    Gillard, Joseph J.; Laws, Thomas R.; Lythe, Grant; Molina-París, Carmen

    2014-01-01

    Computational models can provide valuable insights into the mechanisms of infection and be used as investigative tools to support development of medical treatments. We develop a stochastic, within-host, computational model of the infection process in the BALB/c mouse, following inhalational exposure to Francisella tularensis SCHU S4. The model is mechanistic and governed by a small number of experimentally verifiable parameters. Given an initial dose, the model generates bacterial load profiles corresponding to those produced experimentally, with a doubling time of approximately 5 h during the first 48 h of infection. Analytical approximations for the mean number of bacteria in phagosomes and cytosols for the first 24 h post-infection are derived and used to verify the stochastic model. In our description of the dynamics of macrophage infection, the number of bacteria released per rupturing macrophage is a geometrically-distributed random variable. When combined with doubling time, this provides a distribution for the time taken for infected macrophages to rupture and release their intracellular bacteria. The mean and variance of these distributions are determined by model parameters with a precise biological interpretation, providing new mechanistic insights into the determinants of immune and bacterial kinetics. Insights into the dynamics of macrophage suppression and activation gained by the model can be used to explore the potential benefits of interventions that stimulate macrophage activation. PMID:25566509

  6. A Combined Enrichment and Aptamer Pulldown Assay for Francisella tularensis Detection in Food and Environmental Matrices

    PubMed Central

    Enomoto, Shinichiro; Borewicz, Klaudyna; Abdallah, Ahmed; Isaacson, Richard E.; Sreevatsan, Srinand

    2014-01-01

    Francisella tularensis, a Gram-negative bacterium and causative agent of tularemia, is categorized as a Class A select agent by the Centers for Disease Control and Prevention due to its ease of dissemination and ability to cause disease. Oropharyngeal and gastrointestinal tularemia may occur due to ingestion of contaminated food and water. Despite the concern to public health, little research is focused on F. tularensis detection in food and environmental matrices. Current diagnostics rely on host responses and amplification of F. tularensis genetic elements via Polymerase Chain Reaction; however, both tools are limited by development of an antibody response and limit of detection, respectively. During our investigation to develop an improved culture medium to aid F. tularensis diagnostics, we found enhanced F. tularensis growth using the spent culture filtrate. Addition of the spent culture filtrate allowed for increased detection of F. tularensis in mixed cultures of food and environmental matrices. Ultraperformance liquid chromatography (UPLC)/MS analysis identified several unique chemicals within the spent culture supernatant of which carnosine had a matching m/z ratio. Addition of 0.625 mg/mL of carnosine to conventional F. tularensis medium increased the growth of F. tularensis at low inoculums. In order to further enrich F. tularensis cells, we developed a DNA aptamer cocktail to physically separate F. tularensis from other bacteria present in food and environmental matrices. The combined enrichment steps resulted in a detection range of 1–106 CFU/mL (starting inoculums) in both soil and lettuce backgrounds. We propose that the two-step enrichment process may be utilized for easy field diagnostics and subtyping of suspected F. tularensis contamination as well as a tool to aid in basic research of F. tularensis ecology. PMID:25536105

  7. Preservation of viable Francisella tularensis for forensic analysis

    SciTech Connect

    Valentine, Nancy B.; Wunschel, Sharon C.; Valdez, Catherine O.; Kreuzer-Martin, Helen W.; Bartholomew, Rachel A.; Straub, Tim M.; Wahl, Karen L.

    2011-01-01

    As a preservation solution, (1%) ammonium chloride may be preferred over other conventionally used storage solutions because of its compatibility with analytical techniques such as Mass Spectrometry. In this study, ammonium chloride performed as well or better than phosphate buffered saline with Tween or Butterfields/Tween for preserving Francisella tularensis novicida.

  8. 21 CFR 866.3280 - Francisella tularensis serological reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Francisella tularensis serological reagents. 866.3280 Section 866.3280 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  9. 21 CFR 866.3280 - Francisella tularensis serological reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Francisella tularensis serological reagents. 866.3280 Section 866.3280 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  10. 21 CFR 866.3280 - Francisella tularensis serological reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Francisella tularensis serological reagents. 866.3280 Section 866.3280 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  11. 21 CFR 866.3280 - Francisella tularensis serological reagents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Francisella tularensis serological reagents. 866.3280 Section 866.3280 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  12. 21 CFR 866.3280 - Francisella tularensis serological reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Francisella tularensis serological reagents. 866.3280 Section 866.3280 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  13. Water as Source of Francisella tularensis Infection in Humans, Turkey

    PubMed Central

    Kilic, Selcuk; Birdsell, Dawn N.; Karagöz, Alper; Çelebi, Bekir; Bakkaloglu, Zekiye; Arikan, Muzaffer; Sahl, Jason W.; Mitchell, Cedar; Rivera, Andrew; Maltinsky, Sara; Keim, Paul; Üstek, Duran; Durmaz, Rıza

    2015-01-01

    Francisella tularensis DNA extractions and isolates from the environment and humans were genetically characterized to elucidate environmental sources that cause human tularemia in Turkey. Extensive genetic diversity consistent with genotypes from human outbreaks was identified in environmental samples and confirmed water as a source of human tularemia in Turkey. PMID:26583383

  14. Water as Source of Francisella tularensis Infection in Humans, Turkey.

    PubMed

    Kilic, Selcuk; Birdsell, Dawn N; Karagöz, Alper; Çelebi, Bekir; Bakkaloglu, Zekiye; Arikan, Muzaffer; Sahl, Jason W; Mitchell, Cedar; Rivera, Andrew; Maltinsky, Sara; Keim, Paul; Üstek, Duran; Durmaz, Rıza; Wagner, David M

    2015-12-01

    Francisella tularensis DNA extractions and isolates from the environment and humans were genetically characterized to elucidate environmental sources that cause human tularemia in Turkey. Extensive genetic diversity consistent with genotypes from human outbreaks was identified in environmental samples and confirmed water as a source of human tularemia in Turkey.

  15. Deletion of the Bacillus anthracis capB homologue in Francisella tularensis subspecies tularensis generates an attenuated strain that protects mice against virulent tularaemia.

    PubMed

    Michell, Stephen L; Dean, Rachel E; Eyles, Jim E; Hartley, Margaret Gill; Waters, Emma; Prior, Joann L; Titball, Richard W; Oyston, Petra C F

    2010-11-01

    As there is currently no licensed vaccine against Francisella tularensis, the causative agent of tularaemia, the bacterium is an agent of concern as a potential bioweapon. Although F. tularensis has a low infectious dose and high associated mortality, it possesses few classical virulence factors. An analysis of the F. tularensis subspecies tularensis genome sequence has revealed the presence of a region containing genes with low sequence homology to part of the capBCADE operon of Bacillus anthracis. We have generated an isogenic capB mutant of F. tularensis subspecies tularensis SchuS4 and shown it to be attenuated. Furthermore, using BALB/c mice, we have demonstrated that this capB strain affords protection against significant homologous challenge with the wild-type strain. These data have important implications for the development of a defined and efficacious tularaemia vaccine.

  16. Identification of Francisella tularensis by whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry: fast, reliable, robust, and cost-effective differentiation on species and subspecies levels.

    PubMed

    Seibold, E; Maier, T; Kostrzewa, M; Zeman, E; Splettstoesser, W

    2010-04-01

    Francisella tularensis, the causative agent of tularemia, is a potential agent of bioterrorism. The phenotypic discrimination of closely related, but differently virulent, Francisella tularensis subspecies with phenotyping methods is difficult and time-consuming, often producing ambiguous results. As a fast and simple alternative, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was applied to 50 different strains of the genus Francisella to assess its ability to identify and discriminate between strains according to their designated species and subspecies. Reference spectra from five representative strains of Francisella philomiragia, Francisella tularensis subsp. tularensis, Francisella tularensis subsp. holarctica, Francisella tularensis subsp. mediasiatica, and Francisella tularensis subsp. novicida were established and evaluated for their capability to correctly identify Francisella species and subspecies by matching a collection of spectra from 45 blind-coded Francisella strains against a database containing the five reference spectra and 3,287 spectra from other microorganisms. As a reference method for identification of strains from the genus Francisella, 23S rRNA gene sequencing was used. All strains were correctly identified, with both methods showing perfect agreement at the species level as well as at the subspecies level. The identification of Francisella strains by MALDI-TOF MS and subsequent database matching was reproducible using biological replicates, different culture media, different cultivation times, different serial in vitro passages of the same strain, different preparation protocols, and different mass spectrometers.

  17. Francisella tularensis detection using magnetic labels and a magnetic biosensor based on frequency mixing

    NASA Astrophysics Data System (ADS)

    Meyer, Martin H. F.; Krause, Hans-Joachim; Hartmann, Markus; Miethe, Peter; Oster, Jürgen; Keusgen, Michael

    2007-04-01

    A biosensor that uses resonant coils with a special frequency-mixing technique and magnetic beads as detectable labels has been established for the detection of Francisella tularensis, the causative agent for tularemia. The detection principle is based on a sandwich immunoassay using an anti-Ft antibody for immunofiltration immobilized to ABICAP ® polyethylene filters, and biotinylated with streptavidin-coated magnetic beads as labels. The linear detection range of this biosensor was found to be 10 4-10 6 cfu F. tularensis lipopolysaccharide (LPS) per ml. Tested sample matrices were physiological PBS buffer and rabbit serum.

  18. Formulation and Stabilization of Francisella tularensis Live Vaccine Strain

    PubMed Central

    OHTAKE, SATOSHI; MARTIN, RUSSELL A.; SAXENA, ATUL; LECHUGA-BALLESTEROS, DAVID; SANTIAGO, ARACELI E; BARRY, EILEEN M.; TRUONG-LE, VU

    2012-01-01

    Francisella tularensis live vaccine strain (F. tularensis LVS), a promising vaccine candidate for protection against F. tularensis exposure, is a particularly thermolabile vaccine and difficult to stabilize sufficiently for storage under refrigerated conditions. Our preliminary data show that F. tularensis LVS can be stabilized in the dried state using foam drying, a modified freeze drying method, with sugar-based formulations. The process was conducted under mild drying conditions, which resulted in a good titer retention following processing. The inclusion of osmolytes in the growth media resulted in an acceleration of growth kinetics, although no change in osmotolerance was observed. The optimized F. tularensis formulation, which contained trehalose, gelatin, and Pluronic F68 demonstrated stability for approximately 1.5 weeks at 37°C (i.e., time required for the vaccine to decrease in potency by 1 log10 colony forming unit) and for 12 weeks at 25°C. At refrigerator storage condition (4°C), stabilized F. tularensis LVS vaccine exhibited no activity loss for at least 12 weeks. This stabilization method utilizes conventional freeze dryers and pharmaceutically approved stabilizers, and thus can be readily implemented at many manufacturing sites for large-scale production of stabilized vaccines. The improved heat stability of the F. tularensis LVS could mitigate risks of vaccine potency loss during long-term storage, shipping, and distribution. PMID:21491457

  19. Interactions of Francisella tularensis with Alveolar Type II Epithelial Cells and the Murine Respiratory Epithelium

    PubMed Central

    Faron, Matthew; Fletcher, Joshua R.; Rasmussen, Jed A.; Apicella, Michael A.; Jones, Bradley D.

    2015-01-01

    Francisella tularensis is classified as a Tier 1 select agent by the CDC due to its low infectious dose and the possibility that the organism can be used as a bioweapon. The low dose of infection suggests that Francisella is unusually efficient at evading host defenses. Although ~50 cfu are necessary to cause human respiratory infection, the early interactions of virulent Francisella with the lung environment are not well understood. To provide additional insights into these interactions during early Francisella infection of mice, we performed TEM analysis on mouse lungs infected with F. tularensis strains Schu S4, LVS and the O-antigen mutant Schu S4 waaY::TrgTn. For all three strains, the majority of the bacteria that we could detect were observed within alveolar type II epithelial cells at 16 hours post infection. Although there were no detectable differences in the amount of bacteria within an infected cell between the three strains, there was a significant increase in the amount of cellular debris observed in the air spaces of the lungs in the Schu S4 waaY::TrgTn mutant compared to either the Schu S4 or LVS strain. We also studied the interactions of Francisella strains with human AT-II cells in vitro by characterizing the ability of these three strains to invade and replicate within these cells. Gentamicin assay and confocal microscopy both confirmed that F. tularensis Schu S4 replicated robustly within these cells while F. tularensis LVS displayed significantly lower levels of growth over 24 hours, although the strain was able to enter these cells at about the same level as Schu S4 (1 organism per cell), as determined by confocal imaging. The Schu S4 waaY::TrgTn mutant that we have previously described as attenuated for growth in macrophages and mouse virulence displayed interesting properties as well. This mutant induced significant airway inflammation (cell debris) and had an attenuated growth phenotype in the human AT-II cells. These data extend our

  20. Francisella tularensis: No Evidence for Transovarial Transmission in the Tularemia Tick Vectors Dermacentor reticulatus and Ixodes ricinus

    PubMed Central

    Genchi, Marco; Prati, Paola; Vicari, Nadia; Manfredini, Andrea; Sacchi, Luciano; Clementi, Emanuela; Bandi, Claudio; Epis, Sara; Fabbi, Massimo

    2015-01-01

    Background Tularemia is a zoonosis caused by the Francisella tularensis, a highly infectious Gram-negative coccobacillus. Due to easy dissemination, multiple routes of infection, high environmental contamination and morbidity and mortality rates, Francisella is considered a potential bioterrorism threat and classified as a category A select agent by the CDC. Tick bites are among the most prevalent modes of transmission, and ticks have been indicated as a possible reservoir, although their reservoir competence has yet to be defined. Tick-borne transmission of F. tularensis was recognized in 1923, and transstadial transmission has been demonstrated in several tick species. Studies on transovarial transmission, however, have reported conflicting results. Objective The aim of this study was to evaluate the role of ticks as reservoirs for Francisella, assessing the transovarial transmission of F. tularensis subsp. holarctica in ticks, using experimentally-infected females of Dermacentor reticulatus and Ixodes ricinus. Results Transmission electron microscopy and fluorescence in situ hybridization showed F. tularensis within oocytes. However, cultures and bioassays of eggs and larvae were negative; in addition, microscopy techniques revealed bacterial degeneration/death in the oocytes. Conclusions These results suggest that bacterial death might occur in oocytes, preventing the transovarial transmission of Francisella. We can speculate that Francisella does not have a defined reservoir, but that rather various biological niches (e.g. ticks, rodents), that allow the bacterium to persist in the environment. Our results, suggesting that ticks are not competent for the bacterium vertical transmission, are congruent with this view. PMID:26244842

  1. Live Attenuated Francisella novicida Vaccine Protects against Francisella tularensis Pulmonary Challenge in Rats and Non-human Primates

    PubMed Central

    Chu, Ping; Cunningham, Aimee L.; Yu, Jieh-Juen; Nguyen, Jesse Q.; Barker, Jeffrey R.; Lyons, C. Rick; Wilder, Julie; Valderas, Michelle; Sherwood, Robert L.; Arulanandam, Bernard P.; Klose, Karl E.

    2014-01-01

    Francisella tularensis causes the disease tularemia. Human pulmonary exposure to the most virulent form, F. tularensis subsp. tularensis (Ftt), leads to high morbidity and mortality, resulting in this bacterium being classified as a potential biothreat agent. However, a closely-related species, F. novicida, is avirulent in healthy humans. No tularemia vaccine is currently approved for human use. We demonstrate that a single dose vaccine of a live attenuated F. novicida strain (Fn iglD) protects against subsequent pulmonary challenge with Ftt using two different animal models, Fischer 344 rats and cynomolgus macaques (NHP). The Fn iglD vaccine showed protective efficacy in rats, as did a Ftt iglD vaccine, suggesting no disadvantage to utilizing the low human virulent Francisella species to induce protective immunity. Comparison of specific antibody profiles in vaccinated rat and NHP sera by proteome array identified a core set of immunodominant antigens in vaccinated animals. This is the first report of a defined live attenuated vaccine that demonstrates efficacy against pulmonary tularemia in a NHP, and indicates that the low human virulence F. novicida functions as an effective tularemia vaccine platform. PMID:25340543

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

    PubMed

    Rabadi, Seham M; Sanchez, Belkys C; Varanat, Mrudula; Ma, Zhuo; Catlett, Sally V; Melendez, Juan Andres; Malik, Meenakshi; Bakshi, Chandra Shekhar

    2016-03-01

    Francisella tularensis, the causative agent of a fatal human disease known as tularemia, has been used in the bioweapon programs of several countries in the past, and now it is considered a potential bioterror agent. Extreme infectivity and virulence of F. tularensis is due to its ability to evade immune detection and to suppress the host's innate immune responses. However, Francisella-encoded factors and mechanisms responsible for causing immune suppression are not completely understood. Macrophages and neutrophils generate reactive oxygen species (ROS)/reactive nitrogen species as a defense mechanism for the clearance of phagocytosed microorganisms. ROS serve a dual role; at high concentrations they act as microbicidal effector molecules that destroy intracellular pathogens, and at low concentrations they serve as secondary signaling messengers that regulate the expression of various inflammatory mediators. We hypothesized that the antioxidant defenses of F. tularensis maintain redox homeostasis in infected macrophages to prevent activation of redox-sensitive signaling components that ultimately result in suppression of pro-inflammatory cytokine production and macrophage microbicidal activity. We demonstrate that antioxidant enzymes of F. tularensis prevent the activation of redox-sensitive MAPK signaling components, NF-κB signaling, and the production of pro-inflammatory cytokines by inhibiting the accumulation of ROS in infected macrophages. We also report that F. tularensis inhibits ROS-dependent autophagy to promote its intramacrophage survival. Collectively, this study reveals novel pathogenic mechanisms adopted by F. tularensis to modulate macrophage innate immune functions to create an environment permissive for its intracellular survival and growth. PMID:26644475

  3. Members of the Francisella tularensis Phagosomal Transporter Subfamily of Major Facilitator Superfamily Transporters Are Critical for Pathogenesis

    PubMed Central

    Marohn, Mark E.; Santiago, Araceli E.; Shirey, Kari Ann; Lipsky, Michael; Vogel, Stefanie N.

    2012-01-01

    Francisella tularensis is the causative agent of tularemia. Due to its aerosolizable nature and low infectious dose, F. tularensis is classified as a category A select agent and, therefore, is a priority for vaccine development. Survival and replication in macrophages and other cell types are critical to F. tularensis pathogenesis, and impaired intracellular survival has been linked to a reduction in virulence. The F. tularensis genome is predicted to encode 31 major facilitator superfamily (MFS) transporters, and the nine-member Francisella phagosomal transporter (Fpt) subfamily possesses homology with virulence factors in other intracellular pathogens. We hypothesized that these MFS transporters may play an important role in F. tularensis pathogenesis and serve as good targets for attenuation and vaccine development. Here we show altered intracellular replication kinetics and attenuation of virulence in mice infected with three of the nine Fpt mutant strains compared with wild-type (WT) F. tularensis LVS. The vaccination of mice with these mutant strains was protective against a lethal intraperitoneal challenge. Additionally, we observed pronounced differences in cytokine profiles in the livers of mutant-infected mice, suggesting that alterations in in vivo cytokine responses are a major contributor to the attenuation observed for these mutant strains. These results confirm that this subset of MFS transporters plays an important role in the pathogenesis of F. tularensis and suggest that a focus on the development of attenuated Fpt subfamily MFS transporter mutants is a viable strategy toward the development of an efficacious vaccine. PMID:22508856

  4. TaqMan Real-Time PCR Assays for Single-Nucleotide Polymorphisms Which Identify Francisella tularensis and Its Subspecies and Subpopulations

    PubMed Central

    Birdsell, Dawn N.; Vogler, Amy J.; Buchhagen, Jordan; Clare, Ashley; Kaufman, Emily; Naumann, Amber; Driebe, Elizabeth; Wagner, David M.; Keim, Paul S.

    2014-01-01

    Francisella tularensis, the etiologic agent of tularemia and a Class A Select Agent, is divided into three subspecies and multiple subpopulations that differ in virulence and geographic distribution. Given these differences, there is a need to rapidly and accurately determine if a strain is F. tularensis and, if it is, assign it to subspecies and subpopulation. We designed TaqMan real-time PCR genotyping assays using eleven single nucleotide polymorphisms (SNPs) that were potentially specific to closely related groups within the genus Francisella, including numerous subpopulations within F. tularensis species. We performed extensive validation studies to test the specificity of these SNPs to particular populations by screening the assays across a set of 565 genetically and geographically diverse F. tularensis isolates and an additional 21 genetic near-neighbor (outgroup) isolates. All eleven assays correctly determined the genetic groups of all 565 F. tularensis isolates. One assay differentiates F. tularensis, F. novicida, and F. hispaniensis from the more genetically distant F. philomiragia and Francisella-like endosymbionts. Another assay differentiates F. tularensis isolates from near neighbors. The remaining nine assays classify F. tularensis-confirmed isolates into F. tularensis subspecies and subpopulations. The genotyping accuracy of these nine assays diminished when tested on outgroup isolates (i.e. non F. tularensis), therefore a hierarchical approach of assay usage is recommended wherein the F. tularensis-specific assay is used before the nine downstream assays. Among F. tularensis isolates, all eleven assays were highly sensitive, consistently amplifying very low concentrations of DNA. Altogether, these eleven TaqMan real-time PCR assays represent a highly accurate, rapid, and sensitive means of identifying the species, subspecies, and subpopulation of any F. tularensis isolate if used in a step-wise hierarchical scheme. These assays would be very

  5. Interaction of Francisella tularensis bacterial cells with dynamic speckles

    NASA Astrophysics Data System (ADS)

    Ulianova, Onega V.; Ulyanov, Sergey S.; Sazanova, Elena V.; Zudina, Irina; Zhang, Zhihong; Sibo, Zhou; Luo, Qingming

    2006-08-01

    Influence of low-coherent speckles on the colonies grows is investigated. It has been demonstrated that effects of light on the inhibition of cells (Francisella Tularensis) are caused by speckle dynamics. The regimes of illumination of cell suspension with purpose of devitalization of hazard bacteria, caused very dangerous infections, such as tularemia, are found. Mathematical model of interaction of low-coherent laser radiation with bacteria suspension has been proposed. Computer simulations of the processes of laser-cells interaction have been carried out. Role of coherence of light in the processes of laser-cell interaction is analyzed.

  6. Survey of Francisella tularensis in Wild Animals in Japan in Areas Where Tularemia is Endemic.

    PubMed

    Hotta, Akitoyo; Tanabayashi, Kiyoshi; Fujita, Osamu; Shindo, Junji; Park, Chu-Ho; Kudo, Noboru; Hatai, Hitoshi; Oyamada, Toshifumi; Yamamoto, Yoshie; Takano, Ai; Kawabata, Hiroki; Sharma, Neekun; Uda, Akihiko; Yamada, Akio; Morikawa, Shigeru

    2016-09-21

    Samples taken from 428 wild animals and 126 ticks, collected from a tularemia-endemic area in Japan between 2005 and 2013, were analyzed for the presence of Francisella tularensis. F. tularensis was isolated from a Japanese hare carcass whereas the samples from live animals and ticks were negative for F. tularensis by real-time PCR. Our results suggest that F. tularensis is still present in Japan although its prevalence is considerably low even in areas where tularemia is endemic.

  7. Francisella tularensis Strain Typing Using Multiple-Locus, Variable-Number Tandem Repeat Analysis

    PubMed Central

    Farlow, Jason; Smith, Kimothy L.; Wong, Jane; Abrams, Michelle; Lytle, Michael; Keim, Paul

    2001-01-01

    Francisella tularensis, the etiological agent of tularemia, is found throughout the Northern hemisphere. After analyzing the F. tularensis genomic sequence for potential variable-number tandem repeats (VNTRs), we developed a multilocus VNTR analysis (MLVA) typing system for this pathogen. Variation was detected at six VNTR loci in a set of 56 isolates from California, Oklahoma, Arizona, and Oregon and the F. tularensis live vaccine strain. PCR assays revealed diversity at these loci with total allele numbers ranging from 2 to 20, and Nei's diversity index values ranging from 0.36 to 0.93. Cluster analysis identified two genetically distinct groups consistent with the current biovar classification system of F. tularensis. These findings suggest that these VNTR markers are useful for identifying F. tularensis isolates at this taxonomic level. In this study, biovar B isolates were less diverse than those in biovar A, possibly reflecting the history of tularemia in North America. Seven isolates from a recent epizootic in Maricopa County, Ariz., were identical at all VNTR marker loci. Their identity, even at a hypervariable VNTR locus, indicates a common source of infection. This demonstrates the applicability of MLVA for rapid characterization and identification of outbreak isolates. Future construction of reference databases will allow faster outbreak tracking as well as providing a foundation for deciphering global genetic relationships. PMID:11526148

  8. The Protease Locus of Francisella tularensis LVS Is Required for Stress Tolerance and Infection in the Mammalian Host

    PubMed Central

    He, Lihong; Nair, Manoj Kumar Mohan; Chen, Yuling; Liu, Xue; Zhang, Mengyun; Hazlett, Karsten R. O.

    2016-01-01

    Francisella tularensis is the causative agent of tularemia and a category A potential agent of bioterrorism, but the pathogenic mechanisms of F. tularensis are largely unknown. Our previous transposon mutagenesis screen identified 95 lung infectivity-associated F. tularensis genes, including those encoding the Lon and ClpP proteases. The present study validates the importance of Lon and ClpP in intramacrophage growth and infection of the mammalian host by using unmarked deletion mutants of the F. tularensis live vaccine strain (LVS). Further experiments revealed that lon and clpP are also required for F. tularensis tolerance to stressful conditions. A quantitative proteomic comparison between heat-stressed LVS and the isogenic Lon-deficient mutant identified 29 putative Lon substrate proteins. The follow-up protein degradation experiments identified five substrates of the F. tularensis Lon protease (FTL578, FTL663, FTL1217, FTL1228, and FTL1957). FTL578 (ornithine cyclodeaminase), FTL663 (heat shock protein), and FTL1228 (iron-sulfur activator complex subunit SufD) have been previously described as virulence-associated factors in F. tularensis. Identification of these Lon substrates has thus provided important clues for further understanding of the F. tularensis stress response and pathogenesis. The high-throughput approach developed in this study can be used for systematic identification of the Lon substrates in other prokaryotic and eukaryotic organisms. PMID:26902724

  9. Pathology of inhalational Francisella tularensis spp. tularensis SCHU S4 infection in African green monkeys (Chlorocebus aethiops).

    PubMed

    Twenhafel, N A; Alves, D A; Purcell, B K

    2009-07-01

    Tularemia, caused by Francisella tularensis, is a sporadic zoonotic disease with the potential to be an agent of biowarfare or bioterrorism. We describe here the gross, histologic, immunohistochemical, and ultrastructural findings in a group of 5 African green monkeys (AGMs) that received an average inhaled dose of 729 colony-forming units of F. tularensis and died or were euthanatized between days 7 and 11 post infection. Clinical changes were evident by 48 hours post infection, and key physiologic abnormalities included increases in body temperature, heart rate, peak cardiac pressure, and mean blood pressure. Prominent gross changes in all cases included numerous pinpoint to 1-cm, well-demarcated, necrotic foci present consistently in the lungs, mediastinal lymph nodes, and spleen but also seen in the heart, mediastinum, diaphragm, liver, urinary bladder, urethra, and mesentery. The lungs, mediastinal lymph nodes, and spleen were most severely affected, with as much as 50% of the tissue replaced by necrotic foci. Histologic changes in all tissues consisted of well-delineated foci of necrosis and neutrophilic and histiocytic inflammation, with varying amounts of hemorrhage, edema, fibrin, and vasculitis. Some lesions were immature pyogranulomas. Strong immunoreactivity was identified primarily within macrophages. Ultrastructurally, bacteria were present within cytoplasmic vacuoles of alveolar macrophages, many of which were degenerate. In summary, AGMs infected with F. tularensis by aerosol develop lethal multisystemic disease that particularly targets the lungs and lymphoid tissues. Thus, AGMs should serve as a suitable and reliable animal model for further studies of tularemia.

  10. UV-C Inactivation of Francisella tularensis Utah-112 on agar surfaces, stainless steel, and foods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Francisella tularensis has been identified as a microorganism of concern in the field of food security. There is currently very little information on the ability to inactivate F. tularensis on foods using non-thermal processing technologies. The ability of ultraviolet light (UV-C) to inactivate F....

  11. Optimal swab processing recovery method for detection of bioterrorism-related Francisella tularensis by real-time PCR.

    PubMed

    Walker, Roblena E; Petersen, Jeannine M; Stephens, Kenyatta W; Dauphin, Leslie A

    2010-10-01

    Francisella tularensis, the etiological agent of tularemia, is regarded as a potential bioterrorism agent. The advent of bioterrorism has heightened awareness of the need for validated methods for processing environmental samples. In this study we determined the optimal method for processing environmental swabs for the recovery and subsequent detection of F. tularensis by the use of real-time PCR assays. Four swab processing recovery methods were compared: heat, sonication, vortexing, and the Swab Extraction Tube System (SETS). These methods were evaluated using cotton, foam, polyester and rayon swabs spiked with six pathogenic strains of F. tularensis. Real-time PCR analysis using a multi-target 5'nuclease assay for F. tularensis showed that the use of the SETS method resulted in the best limit of detection when evaluated using multiple strains of F. tularensis. We demonstrated also that the efficiency of F. tularensis recovery from swab specimens was not equivalent for all swab processing methodologies and, thus, that this variable can affect real-time PCR assay sensitivity. The effectiveness of the SETS method was independent of the automated DNA extraction method and real-time PCR platforms used. In conclusion, diagnostic laboratories can now potentially incorporate the SETS method into specimen processing protocols for the rapid and efficient detection of F. tularensis by real-time PCR during laboratory bioterrorism-related investigations.

  12. Francisella tularensis live vaccine strain folate metabolism and pseudouridine synthase gene mutants modulate macrophage caspase-1 activation.

    PubMed

    Ulland, Tyler K; Janowski, Ann M; Buchan, Blake W; Faron, Matthew; Cassel, Suzanne L; Jones, Bradley D; Sutterwala, Fayyaz S

    2013-01-01

    Francisella tularensis is a Gram-negative bacterium and the causative agent of the disease tularemia. Escape of F. tularensis from the phagosome into the cytosol of the macrophage triggers the activation of the AIM2 inflammasome through a mechanism that is not well understood. Activation of the AIM2 inflammasome results in autocatalytic cleavage of caspase-1, resulting in the processing and secretion of interleukin-1β (IL-1β) and IL-18, which play a crucial role in innate immune responses to F. tularensis. We have identified the 5-formyltetrahydrofolate cycloligase gene (FTL_0724) as being important for F. tularensis live vaccine strain (LVS) virulence. Infection of mice in vivo with a F. tularensis LVS FTL_0724 mutant resulted in diminished mortality compared to infection of mice with wild-type LVS. The FTL_0724 mutant also induced increased inflammasome-dependent IL-1β and IL-18 secretion and cytotoxicity in macrophages in vitro. In contrast, infection of macrophages with a F. tularensis LVS rluD pseudouridine synthase (FTL_0699) mutant resulted in diminished IL-1β and IL-18 secretion from macrophages in vitro compared to infection of macrophages with wild-type LVS. In addition, the FTL_0699 mutant was not attenuated in vivo. These findings further illustrate that F. tularensis LVS possesses numerous genes that influence its ability to activate the inflammasome, which is a key host strategy to control infection with this pathogen in vivo.

  13. Francisella tularensis Live Vaccine Strain Folate Metabolism and Pseudouridine Synthase Gene Mutants Modulate Macrophage Caspase-1 Activation

    PubMed Central

    Ulland, Tyler K.; Janowski, Ann M.; Buchan, Blake W.; Faron, Matthew; Cassel, Suzanne L.; Jones, Bradley D.

    2013-01-01

    Francisella tularensis is a Gram-negative bacterium and the causative agent of the disease tularemia. Escape of F. tularensis from the phagosome into the cytosol of the macrophage triggers the activation of the AIM2 inflammasome through a mechanism that is not well understood. Activation of the AIM2 inflammasome results in autocatalytic cleavage of caspase-1, resulting in the processing and secretion of interleukin-1β (IL-1β) and IL-18, which play a crucial role in innate immune responses to F. tularensis. We have identified the 5-formyltetrahydrofolate cycloligase gene (FTL_0724) as being important for F. tularensis live vaccine strain (LVS) virulence. Infection of mice in vivo with a F. tularensis LVS FTL_0724 mutant resulted in diminished mortality compared to infection of mice with wild-type LVS. The FTL_0724 mutant also induced increased inflammasome-dependent IL-1β and IL-18 secretion and cytotoxicity in macrophages in vitro. In contrast, infection of macrophages with a F. tularensis LVS rluD pseudouridine synthase (FTL_0699) mutant resulted in diminished IL-1β and IL-18 secretion from macrophages in vitro compared to infection of macrophages with wild-type LVS. In addition, the FTL_0699 mutant was not attenuated in vivo. These findings further illustrate that F. tularensis LVS possesses numerous genes that influence its ability to activate the inflammasome, which is a key host strategy to control infection with this pathogen in vivo. PMID:23115038

  14. Deletion of ripA Alleviates Suppression of the Inflammasome and MAPK by Francisella tularensis

    PubMed Central

    Huang, Max Tze-Han; Mortensen, Brittany L.; Taxman, Debra J.; Craven, Robin R.; Taft-Benz, Sharon; Kijek, Todd M.; Fuller, James R.; Davis, Beckley K.; Allen, Irving Coy; Brickey, Willie June; Gris, Denis; Wen, Haitao; Kawula, Thomas H.; Ting, Jenny Pan-Yun

    2015-01-01

    Francisella tularensis is a facultative intracellular pathogen and potential biothreat agent. Evasion of the immune response contributes to the extraordinary virulence of this organism although the mechanism is unclear. Whereas wild-type strains induced low levels of cytokines, an F. tularensis ripA deletion mutant (LVSΔripA) provoked significant release of IL-1β, IL-18, and TNF-α by resting macrophages. IL-1β and IL-18 secretion was dependent on inflammasome components pyrin-caspase recruitment domain/apoptotic speck-containing protein with a caspase recruitment domain and caspase-1, and the TLR/IL-1R signaling molecule MyD88 was required for inflammatory cytokine synthesis. Complementation of LVSΔripA with a plasmid encoding ripA restored immune evasion. Similar findings were observed in a human monocytic line. The presence of ripA nearly eliminated activation of MAPKs including ERK1/2, JNK, and p38, and pharmacologic inhibitors of these three MAPKs reduced cytokine induction by LVSΔripA. Animals infected with LVSΔripA mounted a stronger IL-1β and TNF-α response than that of mice infected with wild-type live vaccine strain. This analysis revealed novel immune evasive mechanisms of F. tularensis. PMID:20921527

  15. The phylogeographic pattern of Francisella tularensis in Sweden indicates a Scandinavian origin of Eurosiberian tularaemia.

    PubMed

    Karlsson, Edvin; Svensson, Kerstin; Lindgren, Petter; Byström, Mona; Sjödin, Andreas; Forsman, Mats; Johansson, Anders

    2013-02-01

    Previous studies of the causative agent of tularaemia, Francisella tularensis have identified phylogeographic patterns suggestive of environmental maintenance reservoirs. To investigate the phylogeography of tularaemia in Sweden, we selected 163 clinical isolates obtained during 1995-2009 in 10 counties and sequenced one isolate's genome to identify new genetic markers. An improved typing scheme based on two indels and nine SNPs was developed using hydrolysis or TaqMan MGB probe assays. The results showed that much of the known global genetic diversity of F. tularensis subsp. holarctica is present in Sweden. Thirteen of the 163 isolates belonged to a new genetic group that is basal to all other known members of the major genetic clade B.I, which is spread across the Eurosiberian region. One hundred and twenty-five of the 163 Swedish isolates belonged to B.I, but individual clades' frequencies differed from county to county (P < 0.001). Subsequent analyses revealed a correlation between genotype variation over time and recurrent outbreaks at specific places, supporting the 'maintenance reservoir' environmental maintenance hypothesis. Most importantly, the findings reveal the presence of diverse source populations of F. tularensis subsp. holarctica in Sweden and suggest a historical spread of the disease from Scandinavia to other parts of Eurosiberia.

  16. Automated microfluidically controlled electrochemical biosensor for the rapid and highly sensitive detection of Francisella tularensis.

    PubMed

    Dulay, Samuel B; Gransee, Rainer; Julich, Sandra; Tomaso, Herbert; O'Sullivan, Ciara K

    2014-09-15

    Tularemia is a highly infectious zoonotic disease caused by a Gram-negative coccoid rod bacterium, Francisella tularensis. Tularemia is considered as a life-threatening potential biological warfare agent due to its high virulence, transmission, mortality and simplicity of cultivation. In the work reported here, different electrochemical immunosensor formats for the detection of whole F. tularensis bacteria were developed and their performance compared. An anti-Francisella antibody (FB11) was used for the detection that recognises the lipopolysaccharide found in the outer membrane of the bacteria. In the first approach, gold-supported self-assembled monolayers of a carboxyl terminated bipodal alkanethiol were used to covalently cross-link with the FB11 antibody. In an alternative second approach F(ab) fragments of the FB11 antibody were generated and directly chemisorbed onto the gold electrode surface. The second approach resulted in an increased capture efficiency and higher sensitivity. Detection limits of 4.5 ng/mL for the lipopolysaccharide antigen and 31 bacteria/mL for the F. tularensis bacteria were achieved. Having demonstrated the functionality of the immunosensor, an electrode array was functionalised with the antibody fragment and integrated with microfluidics and housed in a tester set-up that facilitated complete automation of the assay. The only end-user intervention is sample addition, requiring less than one-minute hands-on time. The use of the automated microfluidic set-up not only required much lower reagent volumes but also the required incubation time was considerably reduced and a notable increase of 3-fold in assay sensitivity was achieved with a total assay time from sample addition to read-out of less than 20 min. PMID:24747573

  17. Francisella tularensis RipA Protein Topology and Identification of Functional Domains

    PubMed Central

    Mortensen, Brittany L.; Fuller, James R.; Taft-Benz, Sharon; Collins, Edward J.

    2012-01-01

    Francisella tularensis is a Gram-negative coccobacillus and is the etiological agent of the disease tularemia. Expression of the cytoplasmic membrane protein RipA is required for Francisella replication within macrophages and other cell types; however, the function of this protein remains unknown. RipA is conserved among all sequenced Francisella species, and RipA-like proteins are present in a number of individual strains of a wide variety of species scattered throughout the prokaryotic kingdom. Cross-linking studies revealed that RipA forms homoligomers. Using a panel of RipA-green fluorescent protein and RipA-PhoA fusion constructs, we determined that RipA has a unique topology within the cytoplasmic membrane, with the N and C termini in the cytoplasm and periplasm, respectively. RipA has two significant cytoplasmic domains, one composed roughly of amino acids 1 to 50 and the second flanked by the second and third transmembrane domains and comprising amino acids 104 to 152. RipA functional domains were identified by measuring the effects of deletion mutations, amino acid substitution mutations, and spontaneously arising intragenic suppressor mutations on intracellular replication, induction of interleukin-1β (IL-1β) secretion by infected macrophages, and oligomer formation. Results from these experiments demonstrated that each of the cytoplasmic domains and specific amino acids within these domains are required for RipA function. PMID:22267515

  18. Rapid High Resolution Genotyping of Francisella tularensis by Whole Genome Sequence Comparison of Annotated Genes (“MLST+”)

    PubMed Central

    Mellmann, Alexander; Höppner, Sebastian; Splettstoesser, Wolf D.; Harmsen, Dag

    2015-01-01

    The zoonotic disease tularemia is caused by the bacterium Francisella tularensis. This pathogen is considered as a category A select agent with potential to be misused in bioterrorism. Molecular typing based on DNA-sequence like canSNP-typing or MLVA has become the accepted standard for this organism. Due to the organism’s highly clonal nature, the current typing methods have reached their limit of discrimination for classifying closely related subpopulations within the subspecies F. tularensis ssp. holarctica. We introduce a new gene-by-gene approach, MLST+, based on whole genome data of 15 sequenced F. tularensis ssp. holarctica strains and apply this approach to investigate an epidemic of lethal tularemia among non-human primates in two animal facilities in Germany. Due to the high resolution of MLST+ we are able to demonstrate that three independent clones of this highly infectious pathogen were responsible for these spatially and temporally restricted outbreaks. PMID:25856198

  19. Gallium Potentiates the Antibacterial Effect of Gentamicin against Francisella tularensis

    PubMed Central

    Lindgren, Helena

    2015-01-01

    The reasons why aminoglycosides are bactericidal have not been not fully elucidated, and evidence indicates that the cidal effects are at least partly dependent on iron. We demonstrate that availability of iron markedly affects the susceptibility of the facultative intracellular bacterium Francisella tularensis strain SCHU S4 to the aminoglycoside gentamicin. Specifically, the intracellular depots of iron were inversely correlated to gentamicin susceptibility, whereas the extracellular iron concentrations were directly correlated to the susceptibility. Further proof of the intimate link between iron availability and antibiotic susceptibility were the findings that a ΔfslA mutant, which is defective for siderophore-dependent uptake of ferric iron, showed enhanced gentamicin susceptibility and that a ΔfeoB mutant, which is defective for uptake of ferrous iron, displayed complete growth arrest in the presence of gentamicin. Based on the aforementioned findings, it was hypothesized that gallium could potentiate the effect of gentamicin, since gallium is sequestered by iron uptake systems. The ferrozine assay demonstrated that the presence of gallium inhibited >70% of the iron uptake. Addition of gentamicin and/or gallium to infected bone marrow-derived macrophages showed that both 100 μM gallium and 10 μg/ml of gentamicin inhibited intracellular growth of SCHU S4 and that the combined treatment acted synergistically. Moreover, treatment of F. tularensis-infected mice with gentamicin and gallium showed an additive effect. Collectively, the data demonstrate that SCHU S4 is dependent on iron to minimize the effects of gentamicin and that gallium, by inhibiting the iron uptake, potentiates the bactericidal effect of gentamicin in vitro and in vivo. PMID:26503658

  20. Gallium Potentiates the Antibacterial Effect of Gentamicin against Francisella tularensis.

    PubMed

    Lindgren, Helena; Sjöstedt, Anders

    2015-10-26

    The reasons why aminoglycosides are bactericidal have not been not fully elucidated, and evidence indicates that the cidal effects are at least partly dependent on iron. We demonstrate that availability of iron markedly affects the susceptibility of the facultative intracellular bacterium Francisella tularensis strain SCHU S4 to the aminoglycoside gentamicin. Specifically, the intracellular depots of iron were inversely correlated to gentamicin susceptibility, whereas the extracellular iron concentrations were directly correlated to the susceptibility. Further proof of the intimate link between iron availability and antibiotic susceptibility were the findings that a ΔfslA mutant, which is defective for siderophore-dependent uptake of ferric iron, showed enhanced gentamicin susceptibility and that a ΔfeoB mutant, which is defective for uptake of ferrous iron, displayed complete growth arrest in the presence of gentamicin. Based on the aforementioned findings, it was hypothesized that gallium could potentiate the effect of gentamicin, since gallium is sequestered by iron uptake systems. The ferrozine assay demonstrated that the presence of gallium inhibited >70% of the iron uptake. Addition of gentamicin and/or gallium to infected bone marrow-derived macrophages showed that both 100 μM gallium and 10 μg/ml of gentamicin inhibited intracellular growth of SCHU S4 and that the combined treatment acted synergistically. Moreover, treatment of F. tularensis-infected mice with gentamicin and gallium showed an additive effect. Collectively, the data demonstrate that SCHU S4 is dependent on iron to minimize the effects of gentamicin and that gallium, by inhibiting the iron uptake, potentiates the bactericidal effect of gentamicin in vitro and in vivo.

  1. Small Molecule Control of Virulence Gene Expression in Francisella tularensis

    PubMed Central

    Charity, James C.; Blalock, LeeAnn T.; Costante-Hamm, Michelle M.; Kasper, Dennis L.; Dove, Simon L.

    2009-01-01

    In Francisella tularensis, the SspA protein family members MglA and SspA form a complex that associates with RNA polymerase (RNAP) to positively control the expression of virulence genes critical for the intramacrophage growth and survival of the organism. Although the association of the MglA-SspA complex with RNAP is evidently central to its role in controlling gene expression, the molecular details of how MglA and SspA exert their effects are not known. Here we show that in the live vaccine strain of F. tularensis (LVS), the MglA-SspA complex works in concert with a putative DNA-binding protein we have called PigR, together with the alarmone guanosine tetraphosphate (ppGpp), to regulate the expression of target genes. In particular, we present evidence that MglA, SspA, PigR and ppGpp regulate expression of the same set of genes, and show that mglA, sspA, pigR and ppGpp null mutants exhibit similar intramacrophage growth defects and are strongly attenuated for virulence in mice. We show further that PigR interacts directly with the MglA-SspA complex, suggesting that the central role of the MglA and SspA proteins in the control of virulence gene expression is to serve as a target for a transcription activator. Finally, we present evidence that ppGpp exerts its effects by promoting the interaction between PigR and the RNAP-associated MglA-SspA complex. Through its responsiveness to ppGpp, the contact between PigR and the MglA-SspA complex allows the integration of nutritional cues into the regulatory network governing virulence gene expression. PMID:19876386

  2. Re-emergence of tularemia in Germany: Presence of Francisella tularensis in different rodent species in endemic areas

    PubMed Central

    Kaysser, Philipp; Seibold, Erik; Mätz-Rensing, Kerstin; Pfeffer, Martin; Essbauer, Sandra; Splettstoesser, Wolf D

    2008-01-01

    Background Tularemia re-emerged in Germany starting in 2004 (with 39 human cases from 2004 to 2007) after over 40 years of only sporadic human infections. The reasons for this rise in case numbers are unknown as is the possible reservoir of the etiologic agent Francisella (F.) tularensis. No systematic study on the reservoir situation of F. tularensis has been published for Germany so far. Methods We investigated three areas six to ten months after the initial tularemia outbreaks for the presence of F. tularensis among small mammals, ticks/fleas and water. The investigations consisted of animal live-trapping, serologic testing, screening by real-time-PCR and cultivation. Results A total of 386 small mammals were trapped. F. tularensis was detected in five different rodent species with carrier rates of 2.04, 6.94 and 10.87% per trapping area. None of the ticks or fleas (n = 432) tested positive for F. tularensis. We were able to demonstrate F. tularensis-specific DNA in one of 28 water samples taken in one of the outbreak areas. Conclusion The findings of our study stress the need for long-term surveillance of natural foci in order to get a better understanding of the reasons for the temporal and spatial patterns of tularemia in Germany. PMID:19014635

  3. Deletion of IglH in virulent Francisella tularensis subsp. holarctica FSC200 strain results in attenuation and provides protection against the challenge with the parental strain.

    PubMed

    Straskova, Adela; Cerveny, Lukas; Spidlova, Petra; Dankova, Vera; Belcic, Davor; Santic, Marina; Stulik, Jiri

    2012-02-01

    Francisella tularensis, the causative agent of tularemia, is a highly infectious intracellular pathogen with no licensed vaccine available today. The recent search for genome sequences involved in F. tularensis virulence mechanisms led to the identification of the 30-kb region defined as a Francisella pathogenicity island (FPI). In our previous iTRAQ study we described the concerted upregulation of some FPI proteins in different F. tularensis strains cultivated under stress conditions. Among them we identified the IglH protein whose role in Francisella virulence has not been characterized yet. In this work we deleted the iglH gene in a European clinical isolate of F. tularensis subsp. holarctica FSC200. We showed that the iglH gene is necessary for intracellular growth and escape of F. tularensis from phagosomes. We also showed that the iglH mutant is avirulent in a mouse model of infection and persists in the organs for about three weeks after infection. Importantly, mice vaccinated by infection with the iglH mutant were protected against subcutaneous challenge with the fully virulent parental FSC200 strain. This is the first report of a defined subsp. holarctica FPI deletion strain that provides protective immunity against subsequent subcutaneous challenge with a virulent isolate of F. tularensis subsp. holarctica.

  4. Identification of immunoreactive antigens in membrane proteins enriched fraction from Francisella tularensis LVS.

    PubMed

    Janovská, Sylva; Pávková, Ivona; Hubálek, Martin; Lenco, Juraj; Macela, Ales; Stulík, Jirí

    2007-02-15

    Francisella tularensis is a Gram-negative, facultative intracellular bacterium causing disease in many mammalian species. The low infectious dose of F. tularensis and the ease of air-borne transmission are the main features responsible for the classification of this bacterium as a potential biological weapon. The live attenuated strain of F. tularensis live vaccine strain (LVS) is currently only effective vaccine against tularemia, however, this type of vaccine has not been approved for human use. In the presented study, sub-immunoproteome analysis was performed to search for new immunogenic proteins of Francisella tularensis LVS grown under different conditions. By this approach 35 immunoreactive antigens were identified, 19 of them showed to be novel immunogens. In conclusion, sub-immunoproteome analysis resulted in successful identification of novel immunoreactive proteins. PMID:17241671

  5. Rapid dissemination of Francisella tularensis and the effect of route of infection

    PubMed Central

    Ojeda, Sandra S; Wang, Zheng J; Mares, Chris A; Chang, Tingtung A; Li, Qun; Morris, Elizabeth G; Jerabek, Paul A; Teale, Judy M

    2008-01-01

    Background Francisella tularensis subsp. tularensis is classified as a Category A bioweapon that is capable of establishing a lethal infection in humans upon inhalation of very few organisms. However, the virulence mechanisms of this organism are not well characterized. Francisella tularensis subsp. novicida, which is an equally virulent subspecies in mice, was used in concert with a microPET scanner to better understand its temporal dissemination in vivo upon intranasal infection and how such dissemination compares with other routes of infection. Adult mice were inoculated intranasally with F. tularensis subsp. novicida radiolabeled with 64Cu and imaged by microPET at 0.25, 2 and 20 hours post-infection. Results 64Cu labeled F. tularensis subsp. novicida administered intranasally or intratracheally were visualized in the respiratory tract and stomach at 0.25 hours post infection. By 20 hours, there was significant tropism to the lung compared with other tissues. In contrast, the images of radiolabeled F. tularensis subsp. novicida when administered intragastrically, intradermally, intraperitoneally and intravenouslly were more generally limited to the gastrointestinal system, site of inoculation, liver and spleen respectively. MicroPET images correlated with the biodistribution of isotope and bacterial burdens in analyzed tissues. Conclusion Our findings suggest that Francisella has a differential tissue tropism depending on the route of entry and that the virulence of Francisella by the pulmonary route is associated with a rapid bacteremia and an early preferential tropism to the lung. In addition, the use of the microPET device allowed us to identify the cecum as a novel site of colonization of Francisella tularensis subsp. novicida in mice. PMID:19068128

  6. Francisella tularensis type A Strains Cause the Rapid Encystment of Acanthamoeba castellanii and Survive in Amoebal Cysts for Three Weeks post Infection

    SciTech Connect

    El-Etr, S H; Margolis, J; Monack, D; Robison, R; Cohen, M; Moore, E; Rasley, A

    2009-07-28

    Francisella tularensis, the causative agent of the zoonotic disease tularemia, has recently gained increased attention due to the emergence of tularemia in geographical areas where the disease has been previously unknown, and the organism's potential as a bioterrorism agent. Although F. tularensis has an extremely broad host range, the bacterial reservoir in nature has not been conclusively identified. In this study, the ability of virulent F. tularensis strains to survive and replicate in the amoeba Acanthamoeba castellanii was explored. We observe that A. castellanii trophozoites rapidly encyst in response to F. tularensis infection and that this rapid encystment phenotype (REP) is caused by factor(s) secreted by amoebae and/or F. tularensis into the co-culture media. Further, our results indicate that in contrast to LVS, virulent strains of F. tularensis can survive in A. castellanii cysts for at least 3 weeks post infection and that induction of rapid amoeba encystment is essential for survival. In addition, our data indicate that pathogenic F. tularensis strains block lysosomal fusion in A. castellanii. Taken together, these data suggest that the interactions between F. tularensis strains and amoeba may play a role in the environmental persistence of F. tularensis.

  7. Molecular Investigation of Francisella-Like Endosymbiont in Ticks and Francisella tularensis in Ixodid Ticks and Mosquitoes in Turkey.

    PubMed

    Duzlu, Onder; Yildirim, Alparslan; Inci, Abdullah; Gumussoy, Kadir Semih; Ciloglu, Arif; Onder, Zuhal

    2016-01-01

    This study was carried out to investigate the molecular prevalence of Francisella-like endosymbionts (FLEs) and Francisella tularensis in ticks (Acari: Ixodidae) and mosquitoes in Turkey. Genomic DNA pools were constructed from a total of 1477 adult hard ticks of Rhipicephalus (Rh.) annulatus, Rh. turanicus, Rh. sanguineus, Rh. bursa, Haemaphysalis (Hae.) parva, Hae. sulcata, Hyalomma marginatum marginatum, H. anatolicum anatolicum, H. anatolicum excavatum, H. detritum detritum, H. dromedarii, Dermacentor marginatus, and Ixodes ricinus species, which were collected from several barns, cattle, and people. Genomic DNA was also extracted from pools consisting of 6203 adult female mosquito species belonging to Aedes vexans, Culex (Cx.) pipiens, Cx. hortensis, Cx. theileri, Culiseta annulata, and Anopheles maculipennis species. Conventional PCR and TaqMan probe-based real- time PCR targeting the 16S rRNA gene for FLEs and the lpnA gene for F. tularensis, respectively, were performed on the DNA isolates obtained. FLEs and F. tularensis were not found in any genomic DNA pools constructed from ixodid ticks and mosquitos. This study represents the first investigation of F. tularensis and FLEs in potential vector ticks and mosquitoes by molecular methods in Turkey. The present study provides useful insights into the molecular epidemiology of F. tularensis and FLEs. One of the major conclusions of the study is that tularemia outbreaks may be essentially due to direct transmission from the environment (especially from water) in Turkey and not to vector-borne transmission. PMID:26741324

  8. Molecular Investigation of Francisella-Like Endosymbiont in Ticks and Francisella tularensis in Ixodid Ticks and Mosquitoes in Turkey.

    PubMed

    Duzlu, Onder; Yildirim, Alparslan; Inci, Abdullah; Gumussoy, Kadir Semih; Ciloglu, Arif; Onder, Zuhal

    2016-01-01

    This study was carried out to investigate the molecular prevalence of Francisella-like endosymbionts (FLEs) and Francisella tularensis in ticks (Acari: Ixodidae) and mosquitoes in Turkey. Genomic DNA pools were constructed from a total of 1477 adult hard ticks of Rhipicephalus (Rh.) annulatus, Rh. turanicus, Rh. sanguineus, Rh. bursa, Haemaphysalis (Hae.) parva, Hae. sulcata, Hyalomma marginatum marginatum, H. anatolicum anatolicum, H. anatolicum excavatum, H. detritum detritum, H. dromedarii, Dermacentor marginatus, and Ixodes ricinus species, which were collected from several barns, cattle, and people. Genomic DNA was also extracted from pools consisting of 6203 adult female mosquito species belonging to Aedes vexans, Culex (Cx.) pipiens, Cx. hortensis, Cx. theileri, Culiseta annulata, and Anopheles maculipennis species. Conventional PCR and TaqMan probe-based real- time PCR targeting the 16S rRNA gene for FLEs and the lpnA gene for F. tularensis, respectively, were performed on the DNA isolates obtained. FLEs and F. tularensis were not found in any genomic DNA pools constructed from ixodid ticks and mosquitos. This study represents the first investigation of F. tularensis and FLEs in potential vector ticks and mosquitoes by molecular methods in Turkey. The present study provides useful insights into the molecular epidemiology of F. tularensis and FLEs. One of the major conclusions of the study is that tularemia outbreaks may be essentially due to direct transmission from the environment (especially from water) in Turkey and not to vector-borne transmission.

  9. Characterization of Francisella tularensis Schu S4 defined mutants as live-attenuated vaccine candidates.

    PubMed

    Santiago, Araceli E; Mann, Barbara J; Qin, Aiping; Cunningham, Aimee L; Cole, Leah E; Grassel, Christen; Vogel, Stefanie N; Levine, Myron M; Barry, Eileen M

    2015-08-01

    Francisella tularensis (Ft), the etiological agent of tularemia and a Tier 1 select agent, has been previously weaponized and remains a high priority for vaccine development. Ft tularensis (type A) and Ft holarctica (type B) cause most human disease. We selected six attenuating genes from the live vaccine strain (LVS; type B), F. novicida and other intracellular bacteria: FTT0507, FTT0584, FTT0742, FTT1019c (guaA), FTT1043 (mip) and FTT1317c (guaB) and created unmarked deletion mutants of each in the highly human virulent Ft strain Schu S4 (Type A) background. FTT0507, FTT0584, FTT0742 and FTT1043 Schu S4 mutants were not attenuated for virulence in vitro or in vivo. In contrast, Schu S4 gua mutants were unable to replicate in murine macrophages and were attenuated in vivo, with an i.n. LD50 > 10(5) CFU in C57BL/6 mice. However, the gua mutants failed to protect mice against lethal challenge with WT Schu S4, despite demonstrating partial protection in rabbits in a previous study. These results contrast with the highly protective capacity of LVS gua mutants against a lethal LVS challenge in mice, and underscore differences between these strains and the animal models in which they are evaluated, and therefore have important implications for vaccine development.

  10. EmrA1 Membrane Fusion Protein of Francisella tularensis LVS is required for Resistance to Oxidative Stress, Intramacrophage Survival and Virulence in Mice

    PubMed Central

    Ma, Zhuo; Banik, Sukalyani; Rane, Harshita; Mora, Vanessa T.; Rabadi, Seham M.; Doyle, Christopher R.; Thanassi, David G.; Bakshi, Chandra Shekhar; Malik, Meenakshi

    2014-01-01

    Francisella tularensis is a Category A Biodefense agent that causes a fatal human disease known as tularemia. The pathogenicity of F. tularensis depends on its ability to persist inside host immune cells primarily by resisting an attack from host-generated reactive oxygen and nitrogen species (ROS/RNS). Based on the ability of F. tularensis to resist high ROS/RNS levels, we have hypothesized that additional unknown factors act in conjunction with known antioxidant defenses to render ROS resistance. By screening a transposon insertion library of F. tularensis LVS in the presence of hydrogen peroxide, we have identified an oxidant sensitive mutant in putative EmrA1 (FTL_0687) secretion protein. The results demonstrate that the emrA1 mutant is highly sensitive to oxidants and several antimicrobial agents, and exhibits diminished intramacrophage growth that can be restored to wild type F. tularensis LVS levels either by transcomplementation, inhibition of ROS generation, or infection in NADPH oxidase deficient (gp91Phox−/−) macrophages. The emrA1 mutant is attenuated for virulence, which is restored by infection in gp91Phox−/− mice. Further, EmrA1 contributes to oxidative stress resistance by affecting secretion of Francisella antioxidant enzymes SodB and KatG. This study exposes unique links between transporter activity and the antioxidant defense mechanisms of F. tularensis. PMID:24397487

  11. Immunodetection of inactivated Francisella tularensis bacteria by using a quartz crystal microbalance with dissipation monitoring.

    PubMed

    Kleo, K; Schäfer, D; Klar, S; Jacob, D; Grunow, R; Lisdat, F

    2012-08-01

    Francisella tularensis are very small, gram-negative bacteria which are capable of infecting a number of mammals. As a highly pathogenic species, it is a potential bioterrorism agent. In this work we demonstrate a fast immunological detection system for whole F. tularensis bacteria. The technique is based on a quartz crystal microbalance with dissipation monitoring (QCMD), which uses sensor chips modified by a specific antibody. This antibody is useful as a capture molecule to capture the lipopolysaccharide structure on the surface of the bacterial cell wall. The QCMD technique is combined with a microfluidic system and allows the label-free online detection of the binding of whole bacteria to the sensor surface in a wide dynamic concentration range. A detection limit of about 4 × 10(3) colony-forming units per milliliter can be obtained. Furthermore, a rather short analysis time and a clear discrimination against other bacteria can be achieved. Additionally, we demonstrate two possibilities for specific and significant signal enhancement by using antibody-functionalized gold nanoparticles or an enzymatic precipitation reaction. These additional steps can be seen as further proof of the specificity and validity. PMID:22722745

  12. Rapid diagnosis and quantification of Francisella tularensis in organs of naturally infected common squirrel monkeys (Saimiri sciureus).

    PubMed

    Abril, Carlos; Nimmervoll, Helena; Pilo, Paola; Brodard, Isabelle; Korczak, Bozena; Markus, Seiler; Miserez, Raymond; Frey, Joachim

    2008-02-01

    Francisella tularensis, a small Gram-negative facultative intracellular bacterium, is the causative agent of tularaemia, a severe zoonotic disease transmitted to humans mostly by vectors such as ticks, flies and mosquitoes. The disease is endemic in many parts of the northern hemisphere. Among animals, the most affected species belong to rodents and lagomorphs, in particular hares. However, in the recent years, many cases of tularaemia among small monkeys in zoos were reported. We have developed a real-time PCR that allows to quantify F. tularensis in tissue samples. Using this method, we identified the spleen and the kidney as the most heavily infected organ containing up to 400 F. tularensis bacteria per simian host cell in two common squirrel monkeys (Saimiri sciureus) from a zoo that died of tularaemia. In other organs such as the brain, F. tularensis was detected at much lower titres. The strain that caused the infection was identified as F. tularensis subsp. holarctica biovar I, which is susceptible to erythromycin. The high number of F. tularensis present in soft organs such as spleen, liver and kidney represents a high risk for persons handling such carcasses and explains the transmission of the disease to a pathologist during post-mortem analysis. Herein, we show that real-time PCR allows a reliable and rapid diagnosis of F. tularensis directly from tissue samples of infected animals, which is crucial in order to attempt accurate prophylactic measures, especially in cases where humans or other animals have been exposed to this highly contagious pathogen. PMID:17875369

  13. Needle-Free Delivery of Acetalated Dextran-Encapsulated AR-12 Protects Mice from Francisella tularensis Lethal Challenge

    PubMed Central

    Hoang, Ky V.; Curry, Heather; Collier, Michael A.; Borteh, Hassan; Bachelder, Eric M.; Schlesinger, Larry S.; Gunn, John S.

    2016-01-01

    Francisella tularensis causes tularemia and is a potential biothreat. Given the limited antibiotics for treating tularemia and the possible use of antibiotic-resistant strains as a biowarfare agent, new antibacterial agents are needed. AR-12 is an FDA-approved investigational new drug (IND) compound that induces autophagy and has shown host-directed, broad-spectrum activity in vitro against Salmonella enterica serovar Typhimurium and F. tularensis. We have shown that AR-12 encapsulated within acetalated dextran (Ace-DEX) microparticles (AR-12/MPs) significantly reduces host cell cytotoxicity compared to that with free AR-12, while retaining the ability to control S. Typhimurium within infected human macrophages. In the present study, the toxicity and efficacy of AR-12/MPs in controlling virulent type A F. tularensis SchuS4 infection were examined in vitro and in vivo. No significant toxicity of blank MPs or AR-12/MPs was observed in lung histology sections when the formulations were given intranasally to uninfected mice. In histology sections from the lungs of intranasally infected mice treated with the formulations, increased macrophage infiltration was observed for AR-12/MPs, with or without suboptimal gentamicin treatment, but not for blank MPs, soluble AR-12, or suboptimal gentamicin alone. AR-12/MPs dramatically reduced the burden of F. tularensis in infected human macrophages, in a manner similar to that of free AR-12. However, in vivo, AR-12/MPs significantly enhanced the survival of F. tularensis SchuS4-infected mice compared to that seen with free AR-12. In combination with suboptimal gentamicin treatment, AR-12/MPs further improved the survival of F. tularensis SchuS4-infected mice. These studies provide support for Ace-DEX-encapsulated AR-12 as a promising new therapeutic agent for tularemia. PMID:26787696

  14. [An oropharyngeal tularemia case diagnosed by the isolation of Francisella tularensis on human blood agar].

    PubMed

    Ozel, Gönül; Arslan, Ilker Burak; Yeşilyurt, Murat; Celebi, Bekir; Kılıç, Selçuk

    2010-10-01

    Tularemia which is a multisystem disease of humans and some animals, is endemic in North America, some parts of Europe and Asia. The causative agent, Francisella tularensis, is a fastidious gram-negative, intracellular bacterium which requires supplementation with sulphydryl compounds (cysteine, cystine, thiosulphate, isoVitaleX) for growth on common laboratory media. In this report, a case of oropharyngeal tularemia diagnosed by the isolation of the causative agent on non-selective-common microbiological agar, has been presented. The patient was from Yozgat located in central Anatolia where tularemia has not been reported so far. Forty-two years old male was admitted to the hospital with two weeks history of sudden onset fever, headache, generalized aches, sore throat, and cervical tender lump on the left. Physical examination revealed bilateral exudative tonsillitis and tender posterior cervical lymphadenopathy. He has been empirically treated with amoxicilin-clavulanic acid for 7 days with initial diagnosis of acute tonsillopharyngitis. However, he was admitted to the hospital since the symptoms persisted and swelling increased despite antibiotic therapy. Microscopical examination of the Gram and Ehrlich-Ziehl-Neelsen stained smears prepared from the surgically drained lymph node revealed PMNL, with no evidence of bacteria. Routine cultures of the lymph node material yielded growth of gram-negative coccobacilli only on human blood agar and the cultures were negative for pyogenic bacteria, acid-fast organisms and fungi. Pathologic examination of the drainage material revealed suppurative inflammation. Lymph node aspirate and serum samples of the patient together with the isolated strain were sent to reference laboratory for further investigation in accordance to the clinical and laboratory findings compatible with tularemia. The isolate was confirmed as F.tularensis by slide agglutination and direct immunofluorescence antibody tests, and identified as F.tularensis

  15. [An oropharyngeal tularemia case diagnosed by the isolation of Francisella tularensis on human blood agar].

    PubMed

    Ozel, Gönül; Arslan, Ilker Burak; Yeşilyurt, Murat; Celebi, Bekir; Kılıç, Selçuk

    2010-10-01

    Tularemia which is a multisystem disease of humans and some animals, is endemic in North America, some parts of Europe and Asia. The causative agent, Francisella tularensis, is a fastidious gram-negative, intracellular bacterium which requires supplementation with sulphydryl compounds (cysteine, cystine, thiosulphate, isoVitaleX) for growth on common laboratory media. In this report, a case of oropharyngeal tularemia diagnosed by the isolation of the causative agent on non-selective-common microbiological agar, has been presented. The patient was from Yozgat located in central Anatolia where tularemia has not been reported so far. Forty-two years old male was admitted to the hospital with two weeks history of sudden onset fever, headache, generalized aches, sore throat, and cervical tender lump on the left. Physical examination revealed bilateral exudative tonsillitis and tender posterior cervical lymphadenopathy. He has been empirically treated with amoxicilin-clavulanic acid for 7 days with initial diagnosis of acute tonsillopharyngitis. However, he was admitted to the hospital since the symptoms persisted and swelling increased despite antibiotic therapy. Microscopical examination of the Gram and Ehrlich-Ziehl-Neelsen stained smears prepared from the surgically drained lymph node revealed PMNL, with no evidence of bacteria. Routine cultures of the lymph node material yielded growth of gram-negative coccobacilli only on human blood agar and the cultures were negative for pyogenic bacteria, acid-fast organisms and fungi. Pathologic examination of the drainage material revealed suppurative inflammation. Lymph node aspirate and serum samples of the patient together with the isolated strain were sent to reference laboratory for further investigation in accordance to the clinical and laboratory findings compatible with tularemia. The isolate was confirmed as F.tularensis by slide agglutination and direct immunofluorescence antibody tests, and identified as F.tularensis

  16. Complement sensitivity and factor H binding of European Francisella tularensis ssp. holarctica strains in selected animal species.

    PubMed

    Kreizinger, Zsuzsa; Bhide, Mangesh; Bencurova, Elena; Dolinska, Saskia; Gyuranecz, Miklós

    2015-09-01

    Francisella tularensis is a Gram-negative bacterium, the causative agent of the zoonotic disease tularaemia. The bacterium has developed several extracellular and intracellular strategies to evade the hosts' innate and adaptive immune responses. The aims of the study were to examine complement sensitivity of wild and attenuated F. tularensis ssp. holarctica strains in animal hosts of distinct sensitivity to the bacterium, to compare the complement-evading ability of wild strains of different phylogeographic background, and to examine the role of factor H in the host-pathogen interactions. Complement sensitivity assays were carried out on various F. tularensis ssp. holarctica wild strains and on the attenuated live vaccine strain (LVS) with sera of the highly sensitive house mouse (Mus musculus), the moderately sensitive European brown hare (Lepus europaeus) and the relatively resistant cattle (Bos taurus). Specific binding of complement regulator factor H to bacterial membrane proteins was examined by Western blot assays. All wild strains interacted with the hosts' complement system and showed no significant differences in their survivability. The attenuated LVS was resistant to serum killing in mouse, but was lysed in the sera of hare and cattle. Direct binding of factor H to F. tularensis membrane proteins was not detected.

  17. Francisella tularensis harvests nutrients derived via ATG5-independent autophagy to support intracellular growth.

    PubMed

    Steele, Shaun; Brunton, Jason; Ziehr, Benjamin; Taft-Benz, Sharon; Moorman, Nathaniel; Kawula, Thomas

    2013-08-01

    Francisella tularensis is a highly virulent intracellular pathogen that invades and replicates within numerous host cell types including macrophages, hepatocytes and pneumocytes. By 24 hours post invasion, F. tularensis replicates up to 1000-fold in the cytoplasm of infected cells. To achieve such rapid intracellular proliferation, F. tularensis must scavenge large quantities of essential carbon and energy sources from the host cell while evading anti-microbial immune responses. We found that macroautophagy, a eukaryotic cell process that primarily degrades host cell proteins and organelles as well as intracellular pathogens, was induced in F. tularensis infected cells. F. tularensis not only survived macroautophagy, but optimal intracellular bacterial growth was found to require macroautophagy. Intracellular growth upon macroautophagy inhibition was rescued by supplying excess nonessential amino acids or pyruvate, demonstrating that autophagy derived nutrients provide carbon and energy sources that support F. tularensis proliferation. Furthermore, F. tularensis did not require canonical, ATG5-dependent autophagy pathway induction but instead induced an ATG5-independent autophagy pathway. ATG5-independent autophagy induction caused the degradation of cellular constituents resulting in the release of nutrients that the bacteria harvested to support bacterial replication. Canonical macroautophagy limits the growth of several different bacterial species. However, our data demonstrate that ATG5-independent macroautophagy may be beneficial to some cytoplasmic bacteria by supplying nutrients to support bacterial growth.

  18. Francisella tularensis Harvests Nutrients Derived via ATG5-Independent Autophagy to Support Intracellular Growth

    PubMed Central

    Ziehr, Benjamin; Taft-Benz, Sharon; Moorman, Nathaniel; Kawula, Thomas

    2013-01-01

    Francisella tularensis is a highly virulent intracellular pathogen that invades and replicates within numerous host cell types including macrophages, hepatocytes and pneumocytes. By 24 hours post invasion, F. tularensis replicates up to 1000-fold in the cytoplasm of infected cells. To achieve such rapid intracellular proliferation, F. tularensis must scavenge large quantities of essential carbon and energy sources from the host cell while evading anti-microbial immune responses. We found that macroautophagy, a eukaryotic cell process that primarily degrades host cell proteins and organelles as well as intracellular pathogens, was induced in F. tularensis infected cells. F. tularensis not only survived macroautophagy, but optimal intracellular bacterial growth was found to require macroautophagy. Intracellular growth upon macroautophagy inhibition was rescued by supplying excess nonessential amino acids or pyruvate, demonstrating that autophagy derived nutrients provide carbon and energy sources that support F. tularensis proliferation. Furthermore, F. tularensis did not require canonical, ATG5-dependent autophagy pathway induction but instead induced an ATG5-independent autophagy pathway. ATG5-independent autophagy induction caused the degradation of cellular constituents resulting in the release of nutrients that the bacteria harvested to support bacterial replication. Canonical macroautophagy limits the growth of several different bacterial species. However, our data demonstrate that ATG5-independent macroautophagy may be beneficial to some cytoplasmic bacteria by supplying nutrients to support bacterial growth. PMID:23966861

  19. Proteomic analysis of bronchoalveolar lavage fluid proteins from mice infected with Francisella tularensis ssp novicida

    PubMed Central

    Varnum, Susan M.; Webb-Robertson, Bobbie-Jo M.; Pounds, Joel G.; Moore, Ronald J.; Smith, Richard D.; Frevert, Charles W.; Skerrett, Shawn J.; Wunschel, David

    2012-01-01

    Francisella tularensis causes the zoonosis tularemia in humans and is one of the most virulent bacterial pathogens. We utilized a global proteomic approach to characterize protein changes in bronchoalveolar lavage fluid from mice exposed to one of three organisms, F. tularensis ssp. novicida, an avirulent mutant of F. tularensis ssp. novicida (F.t. novicida-ΔmglA); and Pseudomonas aeruginosa. The composition of BALF proteins was altered following infection, including proteins involved in neutrophil activation, oxidative stress and inflammatory responses. Components of the innate immune response were induced including the acute phase response and the complement system, however the timing of their induction varied. Francisella tularensis ssp. novicida infected mice do not appear to have an effective innate immune response in the first hours of infection, however within 24 hours they show an upregulation of innate immune response proteins. This delayed response is in contrast to P. aeruginosa infected animals which show an early innate immune response. Likewise, F.t. novicida-ΔmglA infection initiates an early innate immune response, however this response is dimished by 24 hours. Finally, this study identifies several candidate biomarkers, including Chitinase 3-like-1 (CHI3L1 or YKL-40) and peroxiredoxin 1, that are associated with F. tularensis ssp. novicida but not P. aeruginosa infection. PMID:22663564

  20. Proteomic analysis of bronchoalveolar lavage fluid proteins from mice infected with Francisella tularensis ssp novicida

    SciTech Connect

    Varnum, Susan M.; Webb-Robertson, Bobbie-Jo M.; Pounds, Joel G.; Moore, Ronald J.; Smith, Richard D.; Frevert, Charles; Skerret, Shawn J.; Wunschel, David S.

    2012-07-06

    Francisella tularensis causes the zoonosis tularemia in humans and is one of the most virulent bacterial pathogens. We utilized a global proteomic approach to characterize protein changes in bronchoalveolar lavage fluid from mice exposed to one of three organisms, F. tularensis ssp. novicida, an avirulent mutant of F. tularensis ssp. novicida (F.t. novicida-ΔmglA); and Pseudomonas aeruginosa. The composition of BALF proteins was altered following infection, including proteins involved in neutrophil activation, oxidative stress and inflammatory responses. Components of the innate immune response were induced including the acute phase response and the complement system, however the timing of their induction varied. Francisella tularensis ssp. novicida infected mice do not appear to have an effective innate immune response in the first hours of infection, however within 24 hours they show an upregulation of innate immune response proteins. This delayed response is in contrast to P. aeruginosa infected animals which show an early innate immune response. Likewise, F.t. novicida-ΔmglA infection initiates an early innate immune response, however this response is dimished by 24 hours. Finally, this study identifies several candidate biomarkers, including Chitinase 3-like-1 (CHI3L1 or YKL-40) and peroxiredoxin 1, that are associated with F. tularensis ssp. novicida but not P. aeruginosa infection.

  1. Keep an Ear Out for Francisella tularensis: Otomastoiditis Cases after Canyoneering

    PubMed Central

    Guerpillon, Brice; Boibieux, Andre; Guenne, Clemence; Ploton, Christine; Ferry, Tristan; Maurin, Max; Forestier, Emmanuel; Dauwalder, Olivier; Manipoud, Patrick; Ltaïef-Boudrigua, Aicha; Gürkov, Robert; Vandenesch, Francois; Bouchiat, Coralie

    2016-01-01

    We report here three unusual cases of otomastoiditis due to Francisella tularensis, complicated by cervical abscesses and persistent hearing loss, plus facial paralysis for one patient. Intriguingly, the three patients had practiced canyoneering independently in the same French river, between 2009 and 2014, several days before clinical symptoms onset. The results point out that fresh water exposure may be a potential contamination route for tularemia. Besides, due to the frequent complications and sequelae, we believe that F. tularensis should be considered as a possible etiology in case of otitis media, failure of the conventional antibiotic treatment, and suspicious exposure of the bacteria. PMID:26973838

  2. Francisella tularensis subsp. tularensis Induces a Unique Pulmonary Inflammatory Response: Role of Bacterial Gene Expression in Temporal Regulation of Host Defense Responses

    PubMed Central

    Walters, Kathie-Anne; Olsufka, Rachael; Kuestner, Rolf E.; Cho, Ji Hoon; Li, Hong; Zornetzer, Gregory A.; Wang, Kai; Skerrett, Shawn J.; Ozinsky, Adrian

    2013-01-01

    Pulmonary exposure to Francisella tularensis is associated with severe lung pathology and a high mortality rate. The lack of induction of classical inflammatory mediators, including IL1-β and TNF-α, during early infection has led to the suggestion that F. tularensis evades detection by host innate immune surveillance and/or actively suppresses inflammation. To gain more insight into the host response to Francisella infection during the acute stage, transcriptomic analysis was performed on lung tissue from mice exposed to virulent (Francisella tularensis ssp tularensis SchuS4). Despite an extensive transcriptional response in the lungs of animals as early as 4 hrs post-exposure, Francisella tularensis was associated with an almost complete lack of induction of immune-related genes during the initial 24 hrs post-exposure. This broad subversion of innate immune responses was particularly evident when compared to the pulmonary inflammatory response induced by other lethal (Yersinia pestis) and non-lethal (Legionella pneumophila, Pseudomonas aeruginosa) pulmonary infections. However, the unique induction of a subset of inflammation-related genes suggests a role for dysregulation of lymphocyte function and anti-inflammatory pathways in the extreme virulence of Francisella. Subsequent activation of a classical inflammatory response 48 hrs post-exposure was associated with altered abundance of Francisella-specific transcripts, including those associated with bacterial surface components. In summary, virulent Francisella induces a unique pulmonary inflammatory response characterized by temporal regulation of innate immune pathways correlating with altered bacterial gene expression patterns. This study represents the first simultaneous measurement of both host and Francisella transcriptome changes that occur during in vivo infection and identifies potential bacterial virulence factors responsible for regulation of host inflammatory pathways. PMID:23690939

  3. Francisella tularensis bacteria associated with feline tularemia in the United States.

    PubMed

    Larson, Marilynn A; Fey, Paul D; Hinrichs, Steven H; Iwen, Peter C

    2014-12-01

    Tularemia in the United States was examined by reviewing 106 Francisella tularensis isolates, mostly from Nebraska, collected during 1998-2012: 48% of Nebraska cases were cat-associated; 7/8 human cases were caused by subtype A.I. A vaccine is needed to reduce feline-associated tularemia, and cat owners should protect against bites/scratches and limit their pet's outdoor access.

  4. From the Outside-In: The Francisella tularensis Envelope and Virulence

    PubMed Central

    Rowe, Hannah M.; Huntley, Jason F.

    2015-01-01

    Francisella tularensis is a highly-infectious bacterium that causes the rapid, and often lethal disease, tularemia. Many studies have been performed to identify and characterize the virulence factors that F. tularensis uses to infect a wide variety of hosts and host cell types, evade immune defenses, and induce severe disease and death. This review focuses on the virulence factors that are present in the F. tularensis envelope, including capsule, LPS, outer membrane, periplasm, inner membrane, secretion systems, and various molecules in each of aforementioned sub-compartments. Whereas, no single bacterial molecule or molecular complex single-handedly controls F. tularensis virulence, we review here how diverse bacterial systems work in conjunction to subvert the immune system, attach to and invade host cells, alter phagosome/lysosome maturation pathways, replicate in host cells without being detected, inhibit apoptosis, and induce host cell death for bacterial release and infection of adjacent cells. Given that the F. tularensis envelope is the outermost layer of the bacterium, we highlight herein how many of these molecules directly interact with the host to promote infection and disease. These and future envelope studies are important to advance our collective understanding of F. tularensis virulence mechanisms and offer targets for future vaccine development efforts. PMID:26779445

  5. Role of primary human alveolar epithelial cells in host defense against Francisella tularensis infection.

    PubMed

    Gentry, Megan; Taormina, Joanna; Pyles, Richard B; Yeager, Linsey; Kirtley, Michelle; Popov, Vsevolod L; Klimpel, Gary; Eaves-Pyles, Tonyia

    2007-08-01

    Francisella tularensis, an intracellular pathogen, is highly virulent when inhaled. Alveolar epithelial type I (ATI) and type II (ATII) cells line the majority of the alveolar surface and respond to inhaled pathogenic bacteria via cytokine secretion. We hypothesized that these cells contribute to the lung innate immune response to F. tularensis. Results demonstrated that the live vaccine strain (LVS) contacted ATI and ATII cells by 2 h following intranasal inoculation of mice. In culture, primary human ATI or ATII cells, grown on transwell filters, were stimulated on the apical (AP) surface with virulent F. tularensis Schu 4 or LVS. Basolateral (BL) conditioned medium (CM), collected 6 and 24 h later, was added to the BL surfaces of transwell cultures of primary human pulmonary microvasculature endothelial cells (HPMEC) prior to the addition of polymorphonuclear leukocytes (PMNs) or dendritic cells (DCs) to the AP surface. HPMEC responded to S4- or LVS-stimulated ATII, but not ATI, CM as evidenced by PMN and DC migration. Analysis of the AP and BL ATII CM revealed that both F. tularensis strains induced various levels of a variety of cytokines via NF-kappaB activation. ATII cells pretreated with an NF-kappaB inhibitor prior to F. tularensis stimulation substantially decreased interleukin-8 secretion, which did not occur through Toll-like receptor 2, 2/6, 4, or 5 stimulation. These data indicate a crucial role for ATII cells in the innate immune response to F. tularensis. PMID:17502386

  6. Role of Primary Human Alveolar Epithelial Cells in Host Defense against Francisella tularensis Infection▿

    PubMed Central

    Gentry, Megan; Taormina, Joanna; Pyles, Richard B.; Yeager, Linsey; Kirtley, Michelle; Popov, Vsevolod L.; Klimpel, Gary; Eaves-Pyles, Tonyia

    2007-01-01

    Francisella tularensis, an intracellular pathogen, is highly virulent when inhaled. Alveolar epithelial type I (ATI) and type II (ATII) cells line the majority of the alveolar surface and respond to inhaled pathogenic bacteria via cytokine secretion. We hypothesized that these cells contribute to the lung innate immune response to F. tularensis. Results demonstrated that the live vaccine strain (LVS) contacted ATI and ATII cells by 2 h following intranasal inoculation of mice. In culture, primary human ATI or ATII cells, grown on transwell filters, were stimulated on the apical (AP) surface with virulent F. tularensis Schu 4 or LVS. Basolateral (BL) conditioned medium (CM), collected 6 and 24 h later, was added to the BL surfaces of transwell cultures of primary human pulmonary microvasculature endothelial cells (HPMEC) prior to the addition of polymorphonuclear leukocytes (PMNs) or dendritic cells (DCs) to the AP surface. HPMEC responded to S4- or LVS-stimulated ATII, but not ATI, CM as evidenced by PMN and DC migration. Analysis of the AP and BL ATII CM revealed that both F. tularensis strains induced various levels of a variety of cytokines via NF-κB activation. ATII cells pretreated with an NF-κB inhibitor prior to F. tularensis stimulation substantially decreased interleukin-8 secretion, which did not occur through Toll-like receptor 2, 2/6, 4, or 5 stimulation. These data indicate a crucial role for ATII cells in the innate immune response to F. tularensis. PMID:17502386

  7. Temperature-Dependent Gentamicin Resistance of Francisella tularensis is Mediated by Uptake Modulation

    PubMed Central

    Loughman, Kathleen; Hall, Jesse; Knowlton, Samantha; Sindeldecker, Devin; Gilson, Tricia; Schmitt, Deanna M.; Birch, James W.-M.; Gajtka, Tara; Kobe, Brianna N.; Florjanczyk, Aleksandr; Ingram, Jenna; Bakshi, Chandra S.; Horzempa, Joseph

    2016-01-01

    Gentamicin (Gm) is an aminoglycoside commonly used to treat bacterial infections such as tularemia – the disease caused by Francisella tularensis. In addition to being pathogenic, F. tularensis is found in environmental niches such as soil where this bacterium likely encounters Gm producers (Micromonospora sp.). Here we show that F. tularensis exhibits increased resistance to Gm at ambient temperature (26°C) compared to mammalian body temperature (37°C). To evaluate whether F. tularensis was less permeable to Gm at 26°C, a fluorescent marker [Texas Red (Tr)] was conjugated with Gm, yielding Tr-Gm. Bacteria incubated at 26°C showed reduced fluorescence compared to those at 37°C when exposed to Tr-Gm suggesting that uptake of Gm was reduced at 26°C. Unconjugated Gm competitively inhibited uptake of Tr-Gm, demonstrating that this fluorescent compound was taken up similarly to unconjugated Gm. Lysates of F. tularensis bacteria incubated with Gm at 37°C inhibited the growth of Escherichia coli significantly more than lysates from bacteria incubated at 26°C, further indicating reduced uptake at this lower temperature. Other facultative pathogens (Listeria monocytogenes and Klebsiella pneumoniae) exhibited increased resistance to Gm at 26°C suggesting that the results generated using F. tularensis may be generalizable to diverse bacteria. Regulation of the uptake of antibiotics provides a mechanism by which facultative pathogens survive alongside antibiotic-producing microbes in nature. PMID:26858709

  8. Septic pneumonic tularaemia caused by Francisella tularensis subsp. holarctica biovar II.

    PubMed

    Fritzsch, Joerg; Splettstoesser, Wolf D

    2010-09-01

    This case of pneumonic tularaemia elucidates two aspects: it is believed to be the first documented case of bacteraemia caused by Francisella tularensis subsp. holarctica biovar II; furthermore, it illustrates the remission of septic pneumonic tularaemia without appropriate anti-infective therapy. A blood culture from a patient with community-acquired pneumonia was found to be positive for F. tularensis subsp. holarctica biovar II after 10 days of cultivation. Meanwhile, the patient had been treated with ceftriaxone, followed by sultamicillin and clindamycin. The patient continued suffering from fever of up to 40.7 degrees C and rising C-reactive protein (CRP) for 4 days before the fever and CRP declined. The isolated strain was later tested and found to be resistant to the antibiotics used. The present case underlines that F. tularensis subsp. holarctica infections may cause severe symptoms but mostly have a favourable outcome.

  9. A response regulator promotes Francisella tularensis intramacrophage growth by repressing an anti-virulence factor.

    PubMed

    Ramsey, Kathryn M; Dove, Simon L

    2016-08-01

    The orphan response regulator PmrA is essential for the intramacrophage growth and survival of Francisella tularensis. PmrA was thought to promote intramacrophage growth by binding directly to promoters on the Francisella Pathogenicity Island (FPI) and positively regulating the expression of FPI genes, which encode a Type VI secretion system required for intramacrophage growth. Using both ChIP-Seq and RNA-Seq we identify those regions of the F. tularensis chromosome occupied by PmrA and those genes that are regulated by PmrA. We find that PmrA associates with 252 distinct regions of the F. tularensis chromosome, but exerts regulatory effects at only a few of these locations. Rather than by functioning directly as an activator of FPI gene expression we present evidence that PmrA promotes intramacrophage growth by repressing the expression of a single target gene we refer to as priM (PmrA-repressed inhibitor of intramacrophage growth). Our findings thus indicate that the role of PmrA in facilitating intracellular growth is to repress a previously unknown anti-virulence factor. PriM is the first bacterially encoded factor to be described that can interfere with the intramacrophage growth and survival of F. tularensis. PMID:27169554

  10. Activities of Murine Peripheral Blood Lymphocytes Provide Immune Correlates That Predict Francisella tularensis Vaccine Efficacy

    PubMed Central

    Mittereder, Lara; Kennett, Nikki J.

    2016-01-01

    We previously identified potential correlates of vaccine-induced protection against Francisella tularensis using murine splenocytes and further demonstrated that the relative levels of gene expression varied significantly between tissues. In contrast to splenocytes, peripheral blood leukocytes (PBLs) represent a means to bridge vaccine efficacy in animal models to that in humans. Here we take advantage of this easily accessible source of immune cells to investigate cell-mediated immune responses against tularemia, whose sporadic incidence makes clinical trials of vaccines difficult. Using PBLs from mice vaccinated with F. tularensis Live Vaccine Strain (LVS) and related attenuated strains, we combined the control of in vitro Francisella replication within macrophages with gene expression analyses. The in vitro functions of PBLs, particularly the control of intramacrophage LVS replication, reflected the hierarchy of in vivo protection conferred by LVS-derived vaccines. Moreover, several genes previously identified by the evaluation of splenocytes were also found to be differentially expressed in immune PBLs. In addition, more extensive screening identified additional potential correlates of protection. Finally, expression of selected genes in mouse PBLs obtained shortly after vaccination, without ex vivo restimulation, was different among vaccine groups, suggesting a potential tool to monitor efficacious vaccine-induced immune responses against F. tularensis. Our studies demonstrate that murine PBLs can be used productively to identify potential correlates of protection against F. tularensis and to expand and refine a comprehensive set of protective correlates. PMID:26810039

  11. Needle-Free Delivery of Acetalated Dextran-Encapsulated AR-12 Protects Mice from Francisella tularensis Lethal Challenge.

    PubMed

    Hoang, Ky V; Curry, Heather; Collier, Michael A; Borteh, Hassan; Bachelder, Eric M; Schlesinger, Larry S; Gunn, John S; Ainslie, Kristy M

    2016-04-01

    Francisella tularensiscauses tularemia and is a potential biothreat. Given the limited antibiotics for treating tularemia and the possible use of antibiotic-resistant strains as a biowarfare agent, new antibacterial agents are needed. AR-12 is an FDA-approved investigational new drug (IND) compound that induces autophagy and has shown host-directed, broad-spectrum activityin vitroagainstSalmonella entericaserovar Typhimurium andF. tularensis We have shown that AR-12 encapsulated within acetalated dextran (Ace-DEX) microparticles (AR-12/MPs) significantly reduces host cell cytotoxicity compared to that with free AR-12, while retaining the ability to controlS.Typhimurium within infected human macrophages. In the present study, the toxicity and efficacy of AR-12/MPs in controlling virulent type AF. tularensisSchuS4 infection were examinedin vitroandin vivo No significant toxicity of blank MPs or AR-12/MPs was observed in lung histology sections when the formulations were given intranasally to uninfected mice. In histology sections from the lungs of intranasally infected mice treated with the formulations, increased macrophage infiltration was observed for AR-12/MPs, with or without suboptimal gentamicin treatment, but not for blank MPs, soluble AR-12, or suboptimal gentamicin alone. AR-12/MPs dramatically reduced the burden ofF. tularensisin infected human macrophages, in a manner similar to that of free AR-12. However,in vivo, AR-12/MPs significantly enhanced the survival ofF. tularensisSchuS4-infected mice compared to that seen with free AR-12. In combination with suboptimal gentamicin treatment, AR-12/MPs further improved the survival ofF. tularensisSchuS4-infected mice. These studies provide support for Ace-DEX-encapsulated AR-12 as a promising new therapeutic agent for tularemia.

  12. Case Report of Low Virulence Francisella tularensis Presented as Severe Bacteremic Pneumonia

    PubMed Central

    Su, Ting-Yi; Shie, Shian-Sen; Chia, Ju-Hsin; Huang, Ching-Tai

    2016-01-01

    Abstract Tularemia is a zoonotic infection seen primarily in the Northern Hemisphere. It is caused by the bacteria Francisella tularensis. Although the ulceroglandular form of the disease is the more common manifestation of infection, F tularensis is known to cause pneumonia. F tularensis has two predominant subspecies, namely subsp. tularensis (type A) and subsp. holarctica (type B). Type B tularemia is considered to be much less virulent than type A and barely caused lethal disease and pneumonia. We reported a case with a 68-year-old man immune-compromised patient diagnosed with bacteremic pneumonia engendered by type B tularemia with initial presentation of high fever, pneumonia with pleural effusion; the diagnosis was performed using 16S rRNA gene sequence analysis. The patient's fever, pneumonia, and pleural effusion were resolved with appropriate antibiotics for tularemia. This case involving severe bacteremic pneumonia in an immune-compromised patient is rare. This case suggests that low virulence F tularensis should be included in the differential diagnoses of bacteremic pneumonia for endemic tularemia. PMID:27175638

  13. FmvB: A Francisella tularensis Magnesium-Responsive Outer Membrane Protein that Plays a Role in Virulence

    PubMed Central

    Wu, Xiaojun; Ren, Guoping; Gunning, William T.; Weaver, David A.; Kalinoski, Andrea L.; Khuder, Sadik A.; Huntley, Jason F.

    2016-01-01

    Francisella tularensis is the causative agent of the lethal disease tularemia. Despite decades of research, little is understood about why F. tularensis is so virulent. Bacterial outer membrane proteins (OMPs) are involved in various virulence processes, including protein secretion, host cell attachment, and intracellular survival. Many pathogenic bacteria require metals for intracellular survival and OMPs often play important roles in metal uptake. Previous studies identified three F. tularensis OMPs that play roles in iron acquisition. In this study, we examined two previously uncharacterized proteins, FTT0267 (named fmvA, for Francisella metal and virulence) and FTT0602c (fmvB), which are homologs of the previously studied F. tularensis iron acquisition genes and are predicted OMPs. To study the potential roles of FmvA and FmvB in metal acquisition and virulence, we first examined fmvA and fmvB expression following pulmonary infection of mice, finding that fmvB was upregulated up to 5-fold during F. tularensis infection of mice. Despite sequence homology to previously-characterized iron-acquisition genes, FmvA and FmvB do not appear to be involved iron uptake, as neither fmvA nor fmvB were upregulated in iron-limiting media and neither ΔfmvA nor ΔfmvB exhibited growth defects in iron limitation. However, when other metals were examined in this study, magnesium-limitation significantly induced fmvB expression, ΔfmvB was found to express significantly higher levels of lipopolysaccharide (LPS) in magnesium-limiting medium, and increased numbers of surface protrusions were observed on ΔfmvB in magnesium-limiting medium, compared to wild-type F. tularensis grown in magnesium-limiting medium. RNA sequencing analysis of ΔfmvB revealed the potential mechanism for increased LPS expression, as LPS synthesis genes kdtA and wbtA were significantly upregulated in ΔfmvB, compared with wild-type F. tularensis. To provide further evidence for the potential role of FmvB in

  14. FmvB: A Francisella tularensis Magnesium-Responsive Outer Membrane Protein that Plays a Role in Virulence.

    PubMed

    Wu, Xiaojun; Ren, Guoping; Gunning, William T; Weaver, David A; Kalinoski, Andrea L; Khuder, Sadik A; Huntley, Jason F

    2016-01-01

    Francisella tularensis is the causative agent of the lethal disease tularemia. Despite decades of research, little is understood about why F. tularensis is so virulent. Bacterial outer membrane proteins (OMPs) are involved in various virulence processes, including protein secretion, host cell attachment, and intracellular survival. Many pathogenic bacteria require metals for intracellular survival and OMPs often play important roles in metal uptake. Previous studies identified three F. tularensis OMPs that play roles in iron acquisition. In this study, we examined two previously uncharacterized proteins, FTT0267 (named fmvA, for Francisella metal and virulence) and FTT0602c (fmvB), which are homologs of the previously studied F. tularensis iron acquisition genes and are predicted OMPs. To study the potential roles of FmvA and FmvB in metal acquisition and virulence, we first examined fmvA and fmvB expression following pulmonary infection of mice, finding that fmvB was upregulated up to 5-fold during F. tularensis infection of mice. Despite sequence homology to previously-characterized iron-acquisition genes, FmvA and FmvB do not appear to be involved iron uptake, as neither fmvA nor fmvB were upregulated in iron-limiting media and neither ΔfmvA nor ΔfmvB exhibited growth defects in iron limitation. However, when other metals were examined in this study, magnesium-limitation significantly induced fmvB expression, ΔfmvB was found to express significantly higher levels of lipopolysaccharide (LPS) in magnesium-limiting medium, and increased numbers of surface protrusions were observed on ΔfmvB in magnesium-limiting medium, compared to wild-type F. tularensis grown in magnesium-limiting medium. RNA sequencing analysis of ΔfmvB revealed the potential mechanism for increased LPS expression, as LPS synthesis genes kdtA and wbtA were significantly upregulated in ΔfmvB, compared with wild-type F. tularensis. To provide further evidence for the potential role of FmvB in

  15. Francisella tularensis Subtype A.II Genomic Plasticity in Comparison with Subtype A.I

    PubMed Central

    Larson, Marilynn A.; Nalbantoglu, Ufuk; Sayood, Khalid; Zentz, Emily B.; Bartling, Amanda M.; Francesconi, Stephen C.; Fey, Paul D.; Dempsey, Michael P.; Hinrichs, Steven H.

    2015-01-01

    Although Francisella tularensis is considered a monomorphic intracellular pathogen, molecular genotyping and virulence studies have demonstrated important differences within the tularensis subspecies (type A). To evaluate genetic variation within type A strains, sequencing and assembly of a new subtype A.II genome was achieved for comparison to other completed F. tularensis type A genomes. In contrast with the F. tularensis A.I strains (SCHU S4, FSC198, NE061598, and TI0902), substantial genomic variation was observed between the newly sequenced F. tularensis A.II strain (WY-00W4114) and the only other publically available A.II strain (WY96-3418). Genome differences between WY-00W4114 and WY96-3418 included three major chromosomal translocations, 1580 indels, and 286 nucleotide substitutions of which 159 were observed in predicted open reading frames and 127 were located in intergenic regions. The majority of WY-00W4114 nucleotide deletions occurred in intergenic regions, whereas most of the insertions and substitutions occurred in predicted genes. Of the nucleotide substitutions, 48 (30%) were synonymous and 111 (70%) were nonsynonymous. WY-00W4114 and WY96-3418 nucleotide polymorphisms were predominantly G/C to A/T allelic mutations, with WY-00W4114 having more A+T enrichment. In addition, the A.II genomes contained a considerably higher number of intact genes and longer repetitive sequences, including transposon remnants than the A.I genomes. Together these findings support the premise that F. tularensis A.II may have a fitness advantage compared to the A.I subtype due to the higher abundance of functional genes and repeated chromosomal sequences. A better understanding of the selective forces driving F. tularensis genetic diversity and plasticity is needed. PMID:25918839

  16. Biochemical studies on Francisella tularensis RelA in (p)ppGpp biosynthesis

    PubMed Central

    Wilkinson, Rachael C.; Batten, Laura E.; Wells, Neil J.; Oyston, Petra C.F.; Roach, Peter L.

    2015-01-01

    The bacterial stringent response is induced by nutrient deprivation and is mediated by enzymes of the RSH (RelA/SpoT homologue; RelA, (p)ppGpp synthetase I; SpoT, (p)ppGpp synthetase II) superfamily that control concentrations of the ‘alarmones’ (p)ppGpp (guanosine penta- or tetra-phosphate). This regulatory pathway is present in the vast majority of pathogens and has been proposed as a potential anti-bacterial target. Current understanding of RelA-mediated responses is based on biochemical studies using Escherichia coli as a model. In comparison, the Francisella tularensis RelA sequence contains a truncated regulatory C-terminal region and an unusual synthetase motif (EXSD). Biochemical analysis of F. tularensis RelA showed the similarities and differences of this enzyme compared with the model RelA from Escherichia coli. Purification of the enzyme yielded a stable dimer capable of reaching concentrations of 10 mg/ml. In contrast with other enzymes from the RelA/SpoT homologue superfamily, activity assays with F. tularensis RelA demonstrate a high degree of specificity for GTP as a pyrophosphate acceptor, with no measurable turnover for GDP. Steady state kinetic analysis of F. tularensis RelA gave saturation activity curves that best fitted a sigmoidal function. This kinetic profile can result from allosteric regulation and further measurements with potential allosteric regulators demonstrated activation by ppGpp (5′,3′-dibisphosphate guanosine) with an EC50 of 60±1.9 μM. Activation of F. tularensis RelA by stalled ribosomal complexes formed with ribosomes purified from E. coli MRE600 was observed, but interestingly, significantly weaker activation with ribosomes isolated from Francisella philomiragia. PMID:26450927

  17. Biochemical studies on Francisella tularensis RelA in (p)ppGpp biosynthesis.

    PubMed

    Wilkinson, Rachael C; Batten, Laura E; Wells, Neil J; Oyston, Petra C F; Roach, Peter L

    2015-10-08

    The bacterial stringent response is induced by nutrient deprivation and is mediated by enzymes of the RSH (RelA/SpoT homologue; RelA, (p)ppGpp synthetase I; SpoT, (p)ppGpp synthetase II) superfamily that control concentrations of the 'alarmones' (p)ppGpp (guanosine penta- or tetra-phosphate). This regulatory pathway is present in the vast majority of pathogens and has been proposed as a potential anti-bacterial target. Current understanding of RelA-mediated responses is based on biochemical studies using Escherichia coli as a model. In comparison, the Francisella tularensis RelA sequence contains a truncated regulatory C-terminal region and an unusual synthetase motif (EXSD). Biochemical analysis of F. tularensis RelA showed the similarities and differences of this enzyme compared with the model RelA from Escherichia coli. Purification of the enzyme yielded a stable dimer capable of reaching concentrations of 10 mg/ml. In contrast with other enzymes from the RelA/SpoT homologue superfamily, activity assays with F. tularensis RelA demonstrate a high degree of specificity for GTP as a pyrophosphate acceptor, with no measurable turnover for GDP. Steady state kinetic analysis of F. tularensis RelA gave saturation activity curves that best fitted a sigmoidal function. This kinetic profile can result from allosteric regulation and further measurements with potential allosteric regulators demonstrated activation by ppGpp (5',3'-dibisphosphate guanosine) with an EC50 of 60±1.9 μM. Activation of F. tularensis RelA by stalled ribosomal complexes formed with ribosomes purified from E. coli MRE600 was observed, but interestingly, significantly weaker activation with ribosomes isolated from Francisella philomiragia.

  18. [Multilocus VNTR-typing of Francisella tularensis strains].

    PubMed

    Vodop'ianov, A S; Vodop'ianov, S O; Pavlovich, N V; Mishan'kin, B N

    2004-01-01

    In the analysis of F. tularensis genome with the use of the specially developed program "DNA" a great number of loci containing tandem repeats were found. For analysis, 3 of them were selected and designated as FtA, FtB, FtC. The study of DNA of 40 F. tularensis strains in the polymerase chain reaction with specific primers to these loci a great variability in the number of repeats was established, the presence of 17 alleles being found in locus FtA, 5 alleles in locus FtB and 5 alleles in locus FtC. The strains under study formed 24 variants of genotypes, whose occurrence varied from 0.025 to 0.125. Taking into account the variability of the detected loci and a great number of potential loci VNTR in the genome, further development of this method will facilitate the creation of local and general data bases of the strains, thus ensuring more effective genetic typing of F. tularensis. PMID:15188553

  19. Characterization of Tetratricopeptide Repeat-Like Proteins in Francisella tularensis and Identification of a Novel Locus Required for Virulence

    PubMed Central

    Dankova, Vera; Balonova, Lucie; Straskova, Adela; Spidlova, Petra; Putzova, Daniela; Kijek, Todd; Bozue, Joel; Cote, Christopher; Mou, Sherry; Worsham, Patricia; Szotakova, Barbora; Stulik, Jiri

    2014-01-01

    Francisella tularensis is a highly infectious bacterium that causes the potentially lethal disease tularemia. This extremely virulent bacterium is able to replicate in the cytosolic compartments of infected macrophages. To invade macrophages and to cope with their intracellular environment, Francisella requires multiple virulence factors, which are still being identified. Proteins containing tetratricopeptide repeat (TPR)-like domains seem to be promising targets to investigate, since these proteins have been reported to be directly involved in virulence-associated functions of bacterial pathogens. Here, we studied the role of the FTS_0201, FTS_0778, and FTS_1680 genes, which encode putative TPR-like proteins in Francisella tularensis subsp. holarctica FSC200. Mutants defective in protein expression were prepared by TargeTron insertion mutagenesis. We found that the locus FTS_1680 and its ortholog FTT_0166c in the highly virulent Francisella tularensis type A strain SchuS4 are required for proper intracellular replication, full virulence in mice, and heat stress tolerance. Additionally, the FTS_1680-encoded protein was identified as a membrane-associated protein required for full cytopathogenicity in macrophages. Our study thus identifies FTS_1680/FTT_0166c as a new virulence factor in Francisella tularensis. PMID:25245806

  20. Rapid Focused Sequencing: A Multiplexed Assay for Simultaneous Detection and Strain Typing of Bacillus anthracis, Francisella tularensis, and Yersinia pestis

    PubMed Central

    Zolotova, Anna; Tan, Eugene; Selden, Richard F.

    2013-01-01

    Background The intentional release of Bacillus anthracis in the United States in 2001 has heightened concern about the use of pathogenic microorganisms in bioterrorism attacks. Many of the deadliest bacteria, including the Class A Select Agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis, are highly infectious via the pulmonary route when released in aerosolized form. Hence, rapid, sensitive, and reliable methods for detection of these biothreats and characterization of their potential impact on the exposed population are of critical importance to initiate and support rapid military, public health, and clinical responses. Methodology/Principal Findings We have developed microfluidic multiplexed PCR and sequencing assays based on the simultaneous interrogation of three pathogens per assay and ten loci per pathogen. Microfluidic separation of amplified fluorescently labeled fragments generated characteristic electrophoretic signatures for identification of each agent. The three sets of primers allowed significant strain typing and discrimination from non-pathogenic closely-related species and environmental background strains based on amplicon sizes alone. Furthermore, sequencing of the 10 amplicons per pathogen, termed “Rapid Focused Sequencing,” allowed an even greater degree of strain discrimination and, in some cases, can be used to determine virulence. Both amplification and sequencing assays were performed in microfluidic biochips developed for fast thermal cycling and requiring 7 µL per reaction. The 30-plex sequencing assay resulted in genotypic resolution of 84 representative strains belonging to each of the three biothreat species. Conclusions/Significance The microfluidic multiplexed assays allowed identification and strain differentiation of the biothreat agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis and clear discrimination from closely-related species and several environmental background strains. The

  1. Early Interactions of Murine Macrophages with Francisella tularensis Map to Mouse Chromosome 19

    PubMed Central

    Fink, Avner; Hassan, Musa A.; Okan, Nihal A.; Sheffer, Michal; Camejo, Ana; Saeij, Jeroen P. J.

    2016-01-01

    ABSTRACT Differences among individuals in susceptibility to infectious diseases can be modulated by host genetics. Much of the research in this field has aimed to identify loci within the host genome that are associated with these differences. In mice, A/J (AJ) and C57BL/6J (B6) mice show differential susceptibilities to various pathogens, including the intracellular pathogen Francisella tularensis. Because macrophages are the main initial target during F. tularensis infection, we explored early interactions of macrophages from these two mouse strains with F. tularensis as well as the genetic factors underlying these interactions. Our results indicate that bacterial interactions with bone marrow-derived macrophages (BMDMs) during early stages of infection are different in the AJ and B6 strains. During these early stages, bacteria are more numerous in B6 than in AJ macrophages and display differences in trafficking and early transcriptional response within these macrophages. To determine the genetic basis for these differences, we infected BMDMs isolated from recombinant inbred (RI) mice derived from reciprocal crosses between AJ and B6, and we followed early bacterial counts within these macrophages. Quantitative trait locus (QTL) analysis revealed a locus on chromosome 19 that is associated with early differences in bacterial counts in AJ versus B6 macrophages. QTL analysis of published data that measured the differential susceptibilities of the same RI mice to an in vivo challenge with F. tularensis confirmed the F. tularensis susceptibility QTL on chromosome 19. Overall, our results show that early interactions of macrophages with F. tularensis are dependent on the macrophage genetic background. PMID:26980837

  2. Glycosylation of DsbA in Francisella tularensis subsp. tularensis▿†

    PubMed Central

    Thomas, Rebecca M.; Twine, Susan M.; Fulton, Kelly M.; Tessier, Luc; Kilmury, Sara L. N.; Ding, Wen; Harmer, Nicholas; Michell, Stephen L.; Oyston, Petra C. F.; Titball, Richard W.; Prior, Joann L.

    2011-01-01

    In Francisella tularensis subsp. tularensis, DsbA has been shown to be an essential virulence factor and has been observed to migrate to multiple protein spots on two-dimensional electrophoresis gels. In this work, we show that the protein is modified with a 1,156-Da glycan moiety in O-linkage. The results of mass spectrometry studies suggest that the glycan is a hexasaccharide, comprised of N-acetylhexosamines, hexoses, and an unknown monosaccharide. Disruption of two genes within the FTT0789-FTT0800 putative polysaccharide locus, including a galE homologue (FTT0791) and a putative glycosyltransferase (FTT0798), resulted in loss of glycan modification of DsbA. The F. tularensis subsp. tularensis ΔFTT0798 and ΔFTT0791::Cm mutants remained virulent in the murine model of subcutaneous tularemia. This indicates that glycosylation of DsbA does not play a major role in virulence under these conditions. This is the first report of the detailed characterization of the DsbA glycan and putative role of the FTT0789-FTT0800 gene cluster in glycan biosynthesis. PMID:21803994

  3. Chemical Synthesis and Immunological Evaluation of the Inner-Core Oligosaccharide of Francisella tularensis

    PubMed Central

    Boltje, Thomas J.; Zhong, Wei; Park, Jin; Wolfert, Margreet A.; Chen, Wangxue

    2012-01-01

    Francisella tularensis, which is a gram negative bacterium that causes tularemia, has been classified by the Center for Disease Control and Prevention (CDC) as a category A bioweapon. The development of vaccines, immunotherapeutics and diagnostics for F. tularensis requires a detailed knowledge of the saccharide structures that can be recognized by protective antibodies. We have synthesized the inner core region of the lipopolysaccharide (LPS) of F. tularensis to probe antigenic responses elicited by a live and subunit vaccine. The successful preparation of the target compound relied on the use of a disaccharide which was modified by the orthogonal protecting groups diethylisopropylsilyl (DEIPS), 2-naphthylmethyl (Nap), allyl ether (All) and levulinoyl (Lev) ester. The ability to remove the protecting groups in different orders made it possible to establish the optimal glycosylations sequence to prepare a highly crowded 1,2,3-cis configured branching point. A variety of different methods were exploited to control anomeric selectivities of the glycosylations. A comparison of the 1H NMR spectra of isolated material and the synthetic derivative confirmed the reported structural assignment of the inner core oligosaccharide of F. tularensis. The observation that immunizations with LPS lead to antibody responses to the inner core saccharides provides an impetus to further explore this compound as a vaccine candidate. PMID:22867268

  4. Multifaceted effects of Francisella tularensis on human neutrophil function and lifespan.

    PubMed

    Kinkead, Lauren C; Allen, Lee-Ann H

    2016-09-01

    Francisella tularensis in an intracellular bacterial pathogen that causes a potentially lethal disease called tularemia. Studies performed nearly 100 years ago revealed that neutrophil accumulation in infected tissues correlates directly with the extent of necrotic damage during F. tularensis infection. However, the dynamics and details of bacteria-neutrophil interactions have only recently been studied in detail. Herein, we review current understanding regarding the mechanisms that recruit neutrophils to F. tularensis-infected lungs, opsonization and phagocytosis, evasion and inhibition of neutrophil defense mechanisms, as well as the ability of F. tularensis to prolong neutrophil lifespan. In addition, we discuss distinctive features of the bacterium, including its ability to act at a distance to alter overall neutrophil responsiveness to exogenous stimuli, and the evidence which suggests that macrophages and neutrophils play distinct roles in tularemia pathogenesis, such that macrophages are major vehicles for intracellular growth and dissemination, whereas neutrophils drive tissue destruction by dysregulation of the inflammatory response. PMID:27558340

  5. Phylogeographical pattern of Francisella tularensis in a nationwide outbreak of tularaemia in Norway, 2011.

    PubMed

    Afset, J E; Larssen, K W; Bergh, K; Larkeryd, A; Sjodin, A; Johansson, A; Forsman, M

    2015-05-14

    In 2011, a nationwide outbreak of tularaemia occurred in Norway with 180 recorded cases. It was associated with the largest peak in lemming density seen in 40 years. Francisella tularensis was isolated from 18 patients. To study the geographical distribution of F.tularensis genotypes in Norway and correlate genotype with epidemiology and clinical presentation,we performed whole genome sequencing of patient isolates. All 18 genomes from the outbreak carried genetic signatures of F. tularensis subsp. holarctica and were assigned to genetic clades using canonical single nucleotide polymorphisms. Ten isolates were assigned to major genetic clade B.6 (subclade B.7),seven to clade B.12, and one to clade B.4. The B.6 subclade B.7 was most common in southern and central Norway, while clade B.12 was evenly distributed between the southern, central and northern parts of the country. There was no association between genotype and clinical presentation of tularaemia, time of year or specimen type. We found extensive sequence similarity with F. tularensis subsp. holarctica genomes from high-endemic tularaemia areas in Sweden.Finding nearly identical genomes across large geographical distances in Norway and Sweden imply a life cycle of the bacterium without replication between the outbreaks and raise new questions about long-range migration mechanisms.

  6. [Investigation of the presence of Francisella tularensis by culture, serology and molecular methods in mice of Thrace Region, Turkey].

    PubMed

    Unal Yilmaz, Gülizar; Gurcan, Saban; Ozkan, Beytullah; Karadenizli, Aynur

    2014-04-01

    Tularemia is a disease that has been reported in Turkey since 1936. Although mice are considered to have a role in the transmission of Francisella tularensis to man, this has not been exactly confirmed yet. The aim of this study was to investigate the presence of F. tularensis in mice by using culture, serology and molecular methods. For this purpose, four villages (Edirne-Demirkoy, Kirklareli-Kaynarca, Tekirdag-Muzruplu, Tekirdag-Sinanli) were selected in Thrace Region of Turkey where tularemia cases had been reported previously. A total of 126 live-catch mouse traps were established in warehouses, barns, areas near wells, water tanks and creeks in the villages in December 2012. Traps were kept overnight and the next day the animals collected were identified at species-level. The live-captured mice were anesthetized and their heart blood samples were obtained. Subsequently, liver and spleen tissues were removed from every mouse under aseptic conditions in the class-2 safety cabinet. These tissues were cultivated in Francis medium containing 5% sheep blood, 0.1% cystein, 1% glucose and incubated for seven days in both normal atmosphere and 5% carbondioxide incubator at 37°C. Tularemia microagglutination test was performed by using the sera which were obtained from live-captured mice. Finally, DNAs were isolated from both liver and spleen tissues of mice, and real-time polymerase chain reaction (Tularemia RT-PCR; Public Health Agency of Turkey, Ankara) were performed. In our study, a total of 19 mice were captured and of these 11 were alive. Ten mice were identified as Apodemus flavicollis, seven were Mus macedonicus and two were Mus musculus. There were no Francisella tularensis isolation in the cultures of mice liver and spleen tissues. Serological tests yielded negative results for 10 mice whose serum samples could be obtained. In RT-PCR, positivity were detected in spleen tissues of two mice which were captured from Kaynarca where first tularemia cases in

  7. [Investigation of the presence of Francisella tularensis by culture, serology and molecular methods in mice of Thrace Region, Turkey].

    PubMed

    Unal Yilmaz, Gülizar; Gurcan, Saban; Ozkan, Beytullah; Karadenizli, Aynur

    2014-04-01

    Tularemia is a disease that has been reported in Turkey since 1936. Although mice are considered to have a role in the transmission of Francisella tularensis to man, this has not been exactly confirmed yet. The aim of this study was to investigate the presence of F. tularensis in mice by using culture, serology and molecular methods. For this purpose, four villages (Edirne-Demirkoy, Kirklareli-Kaynarca, Tekirdag-Muzruplu, Tekirdag-Sinanli) were selected in Thrace Region of Turkey where tularemia cases had been reported previously. A total of 126 live-catch mouse traps were established in warehouses, barns, areas near wells, water tanks and creeks in the villages in December 2012. Traps were kept overnight and the next day the animals collected were identified at species-level. The live-captured mice were anesthetized and their heart blood samples were obtained. Subsequently, liver and spleen tissues were removed from every mouse under aseptic conditions in the class-2 safety cabinet. These tissues were cultivated in Francis medium containing 5% sheep blood, 0.1% cystein, 1% glucose and incubated for seven days in both normal atmosphere and 5% carbondioxide incubator at 37°C. Tularemia microagglutination test was performed by using the sera which were obtained from live-captured mice. Finally, DNAs were isolated from both liver and spleen tissues of mice, and real-time polymerase chain reaction (Tularemia RT-PCR; Public Health Agency of Turkey, Ankara) were performed. In our study, a total of 19 mice were captured and of these 11 were alive. Ten mice were identified as Apodemus flavicollis, seven were Mus macedonicus and two were Mus musculus. There were no Francisella tularensis isolation in the cultures of mice liver and spleen tissues. Serological tests yielded negative results for 10 mice whose serum samples could be obtained. In RT-PCR, positivity were detected in spleen tissues of two mice which were captured from Kaynarca where first tularemia cases in

  8. B-Cell Epitopes in GroEL of Francisella tularensis

    PubMed Central

    Lu, Zhaohua; Rynkiewicz, Michael J.; Madico, Guillermo; Li, Sheng; Yang, Chiou-Ying; Perkins, Hillary M.; Sompuram, Seshi R.; Kodela, Vani; Liu, Tong; Morris, Timothy; Wang, Daphne; Roche, Marly I.; Seaton, Barbara A.; Sharon, Jacqueline

    2014-01-01

    The chaperonin protein GroEL, also known as heat shock protein 60 (Hsp60), is a prominent antigen in the human and mouse antibody response to the facultative intracellular bacterium Francisella tularensis (Ft), the causative agent of tularemia. In addition to its presumed cytoplasmic location, FtGroEL has been reported to be a potential component of the bacterial surface and to be released from the bacteria. In the current study, 13 IgG2a and one IgG3 mouse monoclonal antibodies (mAbs) specific for FtGroEL were classified into eleven unique groups based on shared VH-VL germline genes, and seven crossblocking profiles revealing at least three non-overlapping epitope areas in competition ELISA. In a mouse model of respiratory tularemia with the highly pathogenic Ft type A strain SchuS4, the Ab64 and N200 IgG2a mAbs, which block each other’s binding to and are sensitive to the same two point mutations in FtGroEL, reduced bacterial burden indicating that they target protective GroEL B-cell epitopes. The Ab64 and N200 epitopes, as well as those of three other mAbs with different crossblocking profiles, Ab53, N3, and N30, were mapped by hydrogen/deuterium exchange–mass spectrometry (DXMS) and visualized on a homology model of FtGroEL. This model was further supported by its experimentally-validated computational docking to the X-ray crystal structures of Ab64 and Ab53 Fabs. The structural analysis and DXMS profiles of the Ab64 and N200 mAbs suggest that their protective effects may be due to induction or stabilization of a conformational change in FtGroEL. PMID:24968190

  9. Pullulanase Is Necessary for the Efficient Intracellular Growth of Francisella tularensis

    PubMed Central

    Takimoto, Kazuhiro; Deyu, Tian; Koyama, Yuuki; Park, Eun-sil; Fujita, Osamu; Hotta, Akitoyo; Morikawa, Shigeru

    2016-01-01

    Pullulanase, an enzyme that catalyzes the hydrolysis of polysaccharides, has been identified in a broad range of organisms, including bacteria, yeasts, fungi, and animals. The pullulanase (pulB; FTT_0412c) of F. tularensis subspecies tularensis Schu S4 is considered to be a homologue of the type I pullulanase (pulA) of the other Francisella subspecies. The significance of Francisella pullulanase has been obscure until now. In the present study, we characterized a recombinant PulB of F. tularensis SCHU P9, which was expressed as a his-tagged protein in Escherichia coli. The recombinant PulB was confirmed to be a type I pullulanase by its enzymatic activity in vitro. A pulB gene knockout mutant of F. tularensis SCHU P9 (ΔpulB) was constructed using the TargeTron Knockout system and plasmid pKEK1140 to clarify the function of PulB during the growth of F. tularensis in macrophages. The intracellular growth of the ΔpulB mutant in murine macrophage J774.1 cells was significantly reduced compared with that of the parental strain SCHU P9. Expression of PulB in ΔpulB, using an expression plasmid, resulted in the complementation of the reduced growth in macrophages, suggesting that PulB is necessary for the efficient growth of F. tularensis in macrophages. To assess the role of PulB in virulence, the knockout and parent bacterial strains were used to infect C57BL/6J mice. Histopathological analyses showed that tissues from ΔpulB-infected mice showed milder lesions compared to those from SCHU P9-infected mice. However, all mice infected with SCHU P9 and ΔpulB showed the similar levels of bacterial loads in their tissues. The results suggest that PulB plays a significant role in bacterial growth within murine macrophage but does not contribute to bacterial virulence in vivo. PMID:27448164

  10. Structural and Biological Evaluation of a Novel Series of Benzimidazole Inhibitors of Francisella tularensis Enoyl-ACP Reductase (FabI)

    PubMed Central

    Mehboob, Shahila; Song, Jinhua; Hevener, Kirk E; Su, Pin-Chih; Boci, Teuta; Brubaker, Libby; Truong, Lena; Mistry, Tina; Deng, Jiangping; Cook, James L; Santarsiero, Bernard D; Ghosh, Arun K; Johnson, Michael E

    2015-01-01

    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 S. 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. PMID:25677657

  11. Thermal resistance of Francisella tularensis in infant formula and fruit juices.

    PubMed

    Day, J B; Trujillo, S; Hao, Y Y D; Whiting, R C

    2008-11-01

    Francisella tularensis is a gram-negative bacterium that can cause gastrointestinal or oropharyngeal tularemia from ingestion of contaminated food or water. Despite the potential for accidental or intentional contamination of foods with F. tularensis, little information exists on the thermal stability of this organism in food matrices. In the present study, the thermal resistance of the live vaccine strain of F. tularensis in four food products (liquid infant formula, apple juice, mango juice, and orange juice) was investigated. D-values ranged from 12 s (57.5 degrees C) to 580 s (50 degrees C) in infant formula with a z-value of 4.37 degrees C. D-values in apple juice ranged from 8 s (57.5 degrees C) to 59 s (50 degrees C) with a z-value of 9.17 degrees C. The live vaccine strain did not survive at temperatures above 55 degrees C in mango juice and orange juice (>6-log inactivation). D-values at 55 to 47.5 degrees C were 15 to 59 s in mango juice and 16 to 105 s in orange juice with z-values of 9.28 and 12.30 degrees C, respectively. These results indicate that current pasteurization parameters used for destroying common foodborne bacterial pathogens are adequate for eliminating F. tularensis in the four foods tested. This study is the first to determine thermal inactivation of F. tularensis in specific foods and will permit comparisons with the thermal inactivation data of other more traditional foodborne pathogens.

  12. PATHOGENESIS AND IMMUNE RESPONSES OF FRANCISELLA TULARENSIS STRAINS IN WILD-CAUGHT COTTONTAIL RABBITS (SYLVILAGUS SPP.).

    PubMed

    Brown, Vienna R; Adney, Danielle R; Bielefeldt-Ohmann, Helle; Gordy, Paul W; Felix, Todd A; Olea-Popelka, Francisco J; Bowen, Richard A

    2015-07-01

    Francisella tularensis is a highly virulent, zoonotic bacterium that causes significant natural disease and is of concern as an organism for bioterrorism. Serologic testing of wildlife is frequently used to monitor spatial patterns of infection and to quantify exposure. Cottontail rabbits (Sylvilagus spp.) are a natural reservoir for F. tularensis in the US, although very little work has been done experimentally to determine how these animals respond to infection; thus, information gathered from field samples can be difficult to interpret. We characterized clinical disease, bacteremia, pathology, and antibody kinetics of North American cottontail rabbits experimentally infected with five strains of F. tularensis. Rabbits were infected with four field strains, including MA00-2987 (type A1b), WY96-3418 (type A2), KY99-3387, and OR96-0246 (type B), and with SchuS4 (type A1a), a widely used, virulent laboratory strain. Infection with the different strains of the bacterium resulted in varied patterns of clinical disease, gross pathology, and histopathology. Each of the type A strains were highly virulent, with rabbits succumbing to infection 3-13 d after infection. At necropsy, numerous microabscesses were observed in the livers and spleens of most rabbits, associated with high bacterial organ burdens. In contrast, most rabbits infected with type B strains developed mild fever and became lethargic, but the disease was infrequently lethal. Those rabbits infected with type B strains that survived past 14 d developed a robust humoral immune response, and F. tularensis was not isolated from liver, spleen, or lung of those animals. Understanding F. tularensis infection in a natural reservoir species can guide serosurveillance and generate new insights into environmental maintenance of this pathogen.

  13. Towards Development of Improved Serodiagnostics for Tularemia by Use of Francisella tularensis Proteome Microarrays.

    PubMed

    Nakajima, Rie; Escudero, Raquel; Molina, Douglas M; Rodríguez-Vargas, Manuela; Randall, Arlo; Jasinskas, Algis; Pablo, Jozelyn; Felgner, Philip L; AuCoin, David P; Anda, Pedro; Davies, D Huw

    2016-07-01

    Tularemia in humans is caused mainly by two subspecies of the Gram-negative facultative anaerobe Francisella tularensis: F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B). The current serological test for tularemia is based on agglutination of whole organisms, and the reactive antigens are not well understood. Previously, we profiled the antibody responses in type A and B tularemia cases in the United States using a proteome microarray of 1,741 different proteins derived from the type A strain Schu S4. Fifteen dominant antigens able to detect antibodies to both types of infection were identified, although these were not validated in a different immunoassay format. Since type A and B subspecies are closely related, we hypothesized that Schu S4 antigens would also have utility for diagnosing type B tularemia caused by strains from other geographic locations. To test this, we probed the Schu S4 array with sera from 241 type B tularemia cases in Spain. Despite there being no type A strains in Spain, we confirmed the responses against some of the same potential serodiagnostic antigens reported previously, as well as determined the responses against additional potential serodiagnostic antigens. Five potential serodiagnostic antigens were evaluated on immunostrips, and two of these (FTT1696/GroEL and FTT0975/conserved hypothetical protein) discriminated between the Spanish tularemia cases and healthy controls. We conclude that antigens from the type A strain Schu S4 are suitable for detection of antibodies from patients with type B F. tularensis infections and that these can be used for the diagnosis of tularemia in a deployable format, such as the immunostrip.

  14. Simultaneous real-time PCR detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis.

    PubMed

    Skottman, T; Piiparinen, H; Hyytiäinen, H; Myllys, V; Skurnik, M; Nikkari, S

    2007-03-01

    This report describes the development of in-house real-time PCR assays using minor groove binding probes for simultaneous detection of the Bacillus anthracis pag and cap genes, the Francisella tularensis 23 KDa gene, as well as the Yersinia pestis pla gene. The sensitivities of these assays were at least 1 fg, except for the assay targeting the Bacillus anthracis cap gene, which showed a sensitivity of 10 fg when total DNA was used as a template in a serial dilution. The clinical value of the Bacillus anthracis- and Francisella tularensis-specific assays was demonstrated by successful amplification of DNA from cases of cow anthrax and hare tularemia, respectively. No cross-reactivity between these species-specific assays or with 39 other bacterial species was noted. These assays may provide a rapid tool for the simultaneous detection and identification of the three category A bacterial species listed as biological threats by the Centers for Disease Control and Prevention. PMID:17294160

  15. Comparison of bone marrow-derived and mucosal mast cells in controlling intramacrophage Francisella tularensis replication

    PubMed Central

    Hunter, Colleen; Rodriguez, Annette; Yu, Jieh-Juen; Chambers, James; Guentzel, M Neal; Arulanandam, Bernard

    2014-01-01

    Although the importance of mast cells (MCs) in response to allergens has been characterized extensively, the contribution of these cells in host defense against bacterial pathogens is not well understood. Previously, we have demonstrated that the release of interleukin-4 by bone marrow-derived MCs inhibits intramacrophage replication of Francisella tularensis live vaccine strain (LVS). Because pneumonic tularemia is one of the several manifestations of infection by Francisella, it is important to determine whether MCs present in mucosal tissues, i.e. the lung, exhibit similar effects on LVS replication. On the basis of this rationale, we phenotypically compared mucosal mast cells (MMCs) to traditional bone marrow-derived MCs. Both cell types exhibited similar levels of cell surface expression of fragment crystal epsilon receptor I (FcεRI), mast/ stem cell growth factor receptor (c-Kit) and major histocompatibility complex I (MHCI), as well as patterns of granulation. MMCs exhibited a comparable, but somewhat greater uptake of fluorescent-labeled beads compared with MCs, suggesting an increased phagocytic ability. MCs and MMCs co-cultured with primary macrophages exhibited comparable significant decreases in LVS replication compared with macrophages cultured alone. Collectively, these results suggest that MMCs are phenotypically similar to MCs and appear equally effective in the control of intramacrophage F. tularensis LVS replication. PMID:22688822

  16. NMR Structure of Francisella tularensis Virulence Determinant Reveals Structural Homology to Bet v1 Allergen Proteins.

    PubMed

    Zook, James; Mo, Gina; Sisco, Nicholas J; Craciunescu, Felicia M; Hansen, Debra T; Baravati, Bobby; Cherry, Brian R; Sykes, Kathryn; Wachter, Rebekka; Van Horn, Wade D; Fromme, Petra

    2015-06-01

    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.

  17. Protection Afforded by Fluoroquinolones in Animal Models of Respiratory Infections with Bacillus anthracis, Yersinia pestis, and Francisella tularensis.

    PubMed

    Peterson, Johnny W; Moen, Scott T; Healy, Daniel; Pawlik, Jennifer E; Taormina, Joanna; Hardcastle, Jason; Thomas, John M; Lawrence, William S; Ponce, Cindy; Chatuev, Bagram M; Gnade, Bryan T; Foltz, Sheri M; Agar, Stacy L; Sha, Jian; Klimpel, Gary R; Kirtley, Michelle L; Eaves-Pyles, Tonyia; Chopra, Ashok K

    2010-01-01

    Successful treatment of inhalation anthrax, pneumonic plague and tularemia can be achieved with fluoroquinolone antibiotics, such as ciprofloxacin and levofloxacin, and initiation of treatment is most effective when administered as soon as possible following exposure. Bacillus anthracis Ames, Yersinia pestis CO92, and Francisella tularensis SCHU S4 have equivalent susceptibility in vitro to ciprofloxacin and levofloxacin (minimal inhibitory concentration is 0.03 μg/ml); however, limited information is available regarding in vivo susceptibility of these infectious agents to the fluoroquinolone antibiotics in small animal models. Mice, guinea pig, and rabbit models have been developed to evaluate the protective efficacy of antibiotic therapy against these life-threatening infections. Our results indicated that doses of ciprofloxacin and levofloxacin required to protect mice against inhalation anthrax were approximately 18-fold higher than the doses of levofloxacin required to protect against pneumonic plague and tularemia. Further, the critical period following aerosol exposure of mice to either B. anthracis spores or Y. pestis was 24 h, while mice challenged with F. tularensis could be effectively protected when treatment was delayed for as long as 72 h postchallenge. In addition, it was apparent that prolonged antibiotic treatment was important in the effective treatment of inhalation anthrax in mice, but short-term treatment of mice with pneumonic plague or tularemia infections were usually successful. These results provide effective antibiotic dosages in mice, guinea pigs, and rabbits and lay the foundation for the development and evaluation of combinational treatment modalities. PMID:21127743

  18. Protection Afforded by Fluoroquinolones in Animal Models of Respiratory Infections with Bacillus anthracis, Yersinia pestis, and Francisella tularensis

    PubMed Central

    Peterson, Johnny W; Moen, Scott T; Healy, Daniel; Pawlik, Jennifer E; Taormina, Joanna; Hardcastle, Jason; Thomas, John M; Lawrence, William S; Ponce, Cindy; Chatuev, Bagram M; Gnade, Bryan T; Foltz, Sheri M; Agar, Stacy L; Sha, Jian; Klimpel, Gary R; Kirtley, Michelle L; Eaves-Pyles, Tonyia; Chopra, Ashok K

    2010-01-01

    Successful treatment of inhalation anthrax, pneumonic plague and tularemia can be achieved with fluoroquinolone antibiotics, such as ciprofloxacin and levofloxacin, and initiation of treatment is most effective when administered as soon as possible following exposure. Bacillus anthracis Ames, Yersinia pestis CO92, and Francisella tularensis SCHU S4 have equivalent susceptibility in vitro to ciprofloxacin and levofloxacin (minimal inhibitory concentration is 0.03 μg/ml); however, limited information is available regarding in vivo susceptibility of these infectious agents to the fluoroquinolone antibiotics in small animal models. Mice, guinea pig, and rabbit models have been developed to evaluate the protective efficacy of antibiotic therapy against these life-threatening infections. Our results indicated that doses of ciprofloxacin and levofloxacin required to protect mice against inhalation anthrax were approximately 18-fold higher than the doses of levofloxacin required to protect against pneumonic plague and tularemia. Further, the critical period following aerosol exposure of mice to either B. anthracis spores or Y. pestis was 24 h, while mice challenged with F. tularensis could be effectively protected when treatment was delayed for as long as 72 h postchallenge. In addition, it was apparent that prolonged antibiotic treatment was important in the effective treatment of inhalation anthrax in mice, but short-term treatment of mice with pneumonic plague or tularemia infections were usually successful. These results provide effective antibiotic dosages in mice, guinea pigs, and rabbits and lay the foundation for the development and evaluation of combinational treatment modalities. PMID:21127743

  19. Visualization of murine intranasal dosing efficiency using luminescent Francisella tularensis: effect of instillation volume and form of anesthesia.

    PubMed

    Miller, Mark A; Stabenow, Jennifer M; Parvathareddy, Jyothi; Wodowski, Andrew J; Fabrizio, Thomas P; Bina, Xiaowen R; Zalduondo, Lillian; Bina, James E

    2012-01-01

    Intranasal instillation is a widely used procedure for pneumonic delivery of drugs, vaccine candidates, or infectious agents into the respiratory tract of research mice. However, there is a paucity of published literature describing the efficiency of this delivery technique. In this report we have used the murine model of tularemia, with Francisella tularensis live vaccine strain (FTLVS) infection, to evaluate the efficiency of pneumonic delivery via intranasal dosing performed either with differing instillation volumes or different types of anesthesia. FTLVS was rendered luminescent via transformation with a reporter plasmid that constitutively expressed the Photorhabdus luminescens lux operon from a Francisella promoter. We then used an IVIS Spectrum whole animal imaging system to visualize FT dissemination at various time points following intranasal instillation. We found that instillation of FT in a dose volume of 10 µl routinely resulted in infection of the upper airways but failed to initiate infection of the pulmonary compartment. Efficient delivery of FT into the lungs via intranasal instillation required a dose volume of 50 µl or more. These studies also demonstrated that intranasal instillation was significantly more efficient for pneumonic delivery of FTLVS in mice that had been anesthetized with inhaled (isoflurane) vs. parenteral (ketamine/xylazine) anesthesia. The collective results underscore the need for researchers to consider both the dose volume and the anesthesia type when either performing pneumonic delivery via intranasal instillation, or when comparing studies that employed this technique. PMID:22384012

  20. Francisella tularensis Live Vaccine Strain deficient in capB and overexpressing the fusion protein of IglA, IglB, and IglC from the bfr promoter induces improved protection against F. tularensis respiratory challenge.

    PubMed

    Jia, Qingmei; Bowen, Richard; Lee, Bai-Yu; Dillon, Barbara Jane; Masleša-Galić, Saša; Horwitz, Marcus A

    2016-09-22

    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 ΔcapB 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 ΔcapB, we developed recombinant LVS ΔcapB (rLVS ΔcapB) 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 ΔcapB/bfr-iglA, iglB, iglC, and iglABC express more IglA, IglB, IglC or IglABC than parental LVS ΔcapB 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 ΔcapB/bfr-iglC or bfr-iglABC secrete greater amounts of interferon-gamma and/or interleukin-17 than those from mice immunized with LVS ΔcapB, comparable to those from LVS-immunized mice. Mice immunized with rLVS ΔcapB/bfr-iglA, iglB, iglC or iglABC produce serum antibodies at levels similar to LVS-immunized mice. Mice immunized intradermally with rLVS ΔcapB/bfr-iglABC and challenged intranasally with virulent F. tularensis Schu S4 survive longer than sham- and LVS ΔcapB-immunized mice. Mice immunized intranasally with rLVS ΔcapB/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

  1. Live attenuated mutants of Francisella tularensis protect rabbits against aerosol challenge with a virulent type A strain.

    PubMed

    Reed, Douglas S; Smith, Le'kneitah P; Cole, Kelly Stefano; Santiago, Araceli E; Mann, Barbara J; Barry, Eileen M

    2014-05-01

    Francisella tularensis, a Gram-negative bacterium, is the causative agent of tularemia. No licensed vaccine is currently available for protection against tularemia, although an attenuated strain, dubbed the live vaccine strain (LVS), is given to at-risk laboratory personnel as an investigational new drug (IND). In an effort to develop a vaccine that offers better protection, recombinant attenuated derivatives of a virulent type A strain, SCHU S4, were evaluated in New Zealand White (NZW) rabbits. Rabbits vaccinated via scarification with the three attenuated derivatives (SCHU S4 ΔguaBA, ΔaroD, and ΔfipB strains) or with LVS developed a mild fever, but no weight loss was detected. Twenty-one days after vaccination, all vaccinated rabbits were seropositive for IgG to F. tularensis lipopolysaccharide (LPS). Thirty days after vaccination, all rabbits were challenged with aerosolized SCHU S4 at doses ranging from 50 to 500 50% lethal doses (LD50). All rabbits developed fevers and weight loss after challenge, but the severity was greater for mock-vaccinated rabbits. The ΔguaBA and ΔaroD SCHU S4 derivatives provided partial protection against death (27 to 36%) and a prolonged time to death compared to results for the mock-vaccinated group. In contrast, LVS and the ΔfipB strain both prolonged the time to death, but there were no survivors from the challenge. This is the first demonstration of vaccine efficacy against aerosol challenge with virulent type A F. tularensis in a species other than a rodent since the original work with LVS in the 1960s. The ΔguaBA and ΔaroD SCHU S4 derivatives warrant further evaluation and consideration as potential vaccines for tularemia and for identification of immunological correlates of protection.

  2. Biochemical responses and oxidative stress in Francisella tularensis infection: a European brown hare model

    PubMed Central

    2011-01-01

    Background The aim of the present study was to investigate biochemical and oxidative stress responses to experimental F. tularensis infection in European brown hares, an important source of human tularemia infections. Methods For these purposes we compared the development of an array of biochemical parameters measured in blood plasma using standard procedures of dry chemistry as well as electrochemical devices following a subcutaneous infection with a wild Francisella tularensis subsp. holarctica strain (a single dose of 2.6 × 109 CFU pro toto). Results Subcutaneous inoculation of a single dose with 2.6 × 109 colony forming units of a wild F. tularensis strain pro toto resulted in the death of two out of five hares. Plasma chemistry profiles were examined on days 2 to 35 post-infection. When compared to controls, the total protein, urea, lactate dehydrogenase, aspartate aminotransferase and alanine aminotransferase were increased, while albumin, glucose and amylase were decreased. Both uric and ascorbic acids and glutathione dropped on day 2 and then increased significantly on days 6 to 12 and 6 to 14 post-inoculation, respectively. There was a two-fold increase in lipid peroxidation on days 4 to 8 post-inoculation. Conclusions Contrary to all expectations, the present study demonstrates that the European brown hare shows relatively low susceptibility to tularemia. Therefore, the circumstances of tularemia in hares under natural conditions should be further studied. PMID:21232117

  3. Expression, purification and crystallization of class C acid phosphatases from Francisella tularensis and Pasteurella multocida

    PubMed Central

    Singh, Harkewal; Felts, Richard L.; Ma, Li; Malinski, Thomas J.; Calcutt, Michael J.; Reilly, Thomas J.; Tanner, John J.

    2009-01-01

    Class C nonspecific acid phosphatases are bacterial enzymes that are secreted across the cytoplasmic membrane and hydrolyze a variety of phosphomono­esters at acidic pH. These enzymes are of interest for the development of improved vaccines and clinical diagnostic methods. In one case, the category A pathogen Francisella tularensis, the class C phosphatase plays a role in bacterial fitness. Here, the cloning, expression, purification and crystallization methods for the class C acid phosphatases from F. tularensis and Pasteurella multocida are reported. Crystals of the F. tularensis enzyme diffracted to 2.0 Å resolution and belonged to space group C2221, with one enzyme molecule in the asymmetric unit. Crystals of the P. multocida enzyme diffracted to 1.85 Å resolution and belonged to space group C2, with three molecules in the asymmetric unit. Diffraction patterns from crystals of the P. multocida enzyme exhibited multiple interpenetrating reciprocal-space lattices, indicating epitaxial twinning. Despite this aberrance, autoindexing was robust and the data could be satisfactorily processed to 1.85 Å resolution using MOSFLM and SCALA. PMID:19255471

  4. Francisella tularensis - Immune Cell Activator, Suppressor, or Stealthy Evader: The Evolving View from the Petri Dish

    PubMed Central

    Holland, Kristen M.; Rosa, Sarah J.; Hazlett, Karsten R.O.

    2016-01-01

    One of the hallmarks of pulmonary tularemia, which results from inhalation of Francisella tularensis - a significant bioterrorism concern, is the lack of an acute TH1-biased inflammatory response in the early phase of disease (days 1–3) despite significant bacterial loads. In an effort to understand this apparent hypo-responsiveness, many laboratories have utilized in vitro cell-based models as tools to probe the nature and consequences of host cell interactions with F. tularensis. The first uses of this model suggested that mammalian host cells recognize this bacterium principally through TLR2 to evoke a robust, classical TH1-biased cytokine response including TNF, IL-6, IL-1β, and IFN-γ. Others used this model in concert with a variety of non-genetic perturbations of the bacterial-host cell interaction and suggested that F. tularensis actively-suppressed the cellular response. Consistent with this notion, others engaged this model to assess isogenic mutants and, in many cases, found the mutant bacteria to be more pro-inflammatory than their WT counter-parts. Frequently, these observations were interpreted as evidence for the immunosuppressive function of the gene of interest. However, recently appreciated roles of the health of the bacterium and the impact of host factors have refined this model to suggest a “stealthy” mode of bacterial-host cell interaction (rather than one involving active suppression) consistent with the observations during early phase disease.

  5. Francisella tularensis 2-C-Methyl-D-Erythritol 4-Phosphate Cytidylyltransferase: Kinetic Characterization and Phosphoregulation

    PubMed Central

    Tsang, Arthur; Seidle, Heather; Jawaid, Safdar; Zhou, Weidong; Smith, Clint; Couch, Robin D.

    2011-01-01

    Deliberate and natural outbreaks of infectious disease, the prevalence of antibiotic resistant strains, and the ease by which antibiotic resistant bacteria can be intentionally engineered all underscore the necessity of effective vaccines and continued development of novel antimicrobial/antiviral therapeutics. Isoprenes, a group of molecules fundamentally involved in a variety of crucial biological functions, are derived from either the mevalonic acid (MVA) or methylerythritol phosphate (MEP) pathway. While mammals utilize the MVA pathway, many bacteria utilize the MEP pathway, highlighting the latter as an attractive target for antibiotic development. In this report we describe the cloning and characterization of Francisella tularensis MEP cytidylyltransferase, a MEP pathway enzyme and potential target for antibiotic development. Size exclusion chromatography indicates the protein exists as a dimer in solution. Enzyme assays produced an apparent , , , , and a . The enzyme exhibits a strict preference for Mg+2 as a divalent cation and CTP as the nucleotide. Titanium dioxide chromatography-tandem mass spectrometry identified Thr141 as a site of phosphorylation. T141D and T141E site-directed mutants are catalytically inactive, suggesting a mechanism for post-translational control of metabolic flux through the F. tularensis MEP pathway. Overall, our study suggests that MEP cytidylyltransferase is an excellent target for the development of novel antibiotics against F. tularensis. PMID:21694781

  6. Francisella tularensis alters human neutrophil gene expression: insights into the molecular basis of delayed neutrophil apoptosis.

    PubMed

    Schwartz, Justin T; Bandyopadhyay, Sarmistha; Kobayashi, Scott D; McCracken, Jenna; Whitney, Adeline R; Deleo, Frank R; Allen, Lee-Ann H

    2013-01-01

    We demonstrated recently that Francisella tularensis profoundly impairs human neutrophil apoptosis, but how this is achieved is largely unknown. Herein we used human oligonucleotide microarrays to test the hypothesis that changes in neutrophil gene expression contribute to this phenotype, and now demonstrate that F. tularensis live vaccine strain (LVS) caused significant changes in neutrophil gene expression over a 24-hour time period relative to the uninfected controls. Of approximately 47,000 genes analyzed, 3,435 were significantly up- or downregulated by LVS, including 365 unique genes associated with apoptosis and cell survival. Specific targets in this category included genes asso-ciated with the intrinsic and extrinsic apoptotic pathways (CFLAR, TNFAIP3, TNFRSF10D, SOD2, BCL2A1, BIRC4, PIM2, TNFSF10, TNFRSF10C, CASP2 and CASP8) and genes that act via the NFĸB pathway and other mechanisms to prolong cell viability (NFKB1, NFKB2 and RELA, IL1B, CAST, CDK2,GADD45B, BCL3, BIRC3, CDK2, IL1A, PBEF1, IL6, CXCL1, CCL4 and VEGF). The microarray data were confirmed by qPCR and pathway analysis. Moreover, we demonstrate that the X-linked inhibitor of apoptosis protein remained abundant in polymorphonuclear leukocytes over 48 h of LVS infection, whereas BAX mRNA and protein were progressively downregulated. These data strongly suggest that antiapoptotic and prosurvival mechanisms collaborate to sustain the viability of F. tularensis--infected neutrophils. PMID:22986450

  7. Francisella tularensis - Immune Cell Activator, Suppressor, or Stealthy Evader: The Evolving View from the Petri Dish

    PubMed Central

    Holland, Kristen M.; Rosa, Sarah J.; Hazlett, Karsten R.O.

    2016-01-01

    One of the hallmarks of pulmonary tularemia, which results from inhalation of Francisella tularensis - a significant bioterrorism concern, is the lack of an acute TH1-biased inflammatory response in the early phase of disease (days 1–3) despite significant bacterial loads. In an effort to understand this apparent hypo-responsiveness, many laboratories have utilized in vitro cell-based models as tools to probe the nature and consequences of host cell interactions with F. tularensis. The first uses of this model suggested that mammalian host cells recognize this bacterium principally through TLR2 to evoke a robust, classical TH1-biased cytokine response including TNF, IL-6, IL-1β, and IFN-γ. Others used this model in concert with a variety of non-genetic perturbations of the bacterial-host cell interaction and suggested that F. tularensis actively-suppressed the cellular response. Consistent with this notion, others engaged this model to assess isogenic mutants and, in many cases, found the mutant bacteria to be more pro-inflammatory than their WT counter-parts. Frequently, these observations were interpreted as evidence for the immunosuppressive function of the gene of interest. However, recently appreciated roles of the health of the bacterium and the impact of host factors have refined this model to suggest a “stealthy” mode of bacterial-host cell interaction (rather than one involving active suppression) consistent with the observations during early phase disease. PMID:27695643

  8. Natural History of Francisella tularensis in Aerosol-Challenged BALB/c Mice

    PubMed Central

    Chuvala, Lara; Riggins, Renaldo; Cirz, Ryan; Cass, Robert; Louie, Arnold; Drusano, G. L.

    2016-01-01

    The objective of this study was to evaluate the natural history and pathogenesis of Francisella tularensis in a murine model of inhalational tularemia with the SchuS4 strain. Before the efficacy of antimicrobials could be assessed in this model, further model development was required to determine the optimal time to start therapy. This study helped define the time course of infection after aerosol challenge by quantifying the presence of bacteria in lung, blood, and spleen at multiple harvest points. In this study, mice were infected via a targeted inhaled dose of 100 50% lethal doses (LD50s) (LD50 = 300 CFU) of F. tularensis by whole-body aerosol. At 1, 24, 36, 48, 60, 72, 75, 78, 81, 84, 87, and 90 h postchallenge, groups of 15 animals were sacrificed and blood, lung, and splenic tissue samples were harvested, homogenized, plated, and incubated to evaluate the bacterial load in those tissues. It was determined that of the 3 sample types harvested, splenic tissue provided the most consistent bacterial counts, which steadily increased with the progressing infection. Further, it was determined that lung samples from all (15/15) animals were positive for infection at 75 h postaerosolization and that 14/15 animals had positive splenic tissue counts. Bacterial levels in blood were not predictive of treatment initiation. For future therapeutic evaluation studies in this model using F. tularensis (SchuS4), it was determined that therapy should be initiated at 75 h postchallenge and validated by spleen involvement. PMID:26824958

  9. Structural and Enzymatic Analyses Reveal the Binding Mode of a Novel Series of Francisella tularensis Enoyl Reductase (FabI) Inhibitors

    SciTech Connect

    Mehboob, Shahila; Hevener, Kirk E.; Truong, Kent; Boci, Teuta; Santarsiero, Bernard D.; Johnson, Michael E.

    2012-10-10

    Because of structural and mechanistic differences between eukaryotic and prokaryotic fatty acid synthesis enzymes, the bacterial pathway, FAS-II, is an attractive target for the design of antimicrobial agents. We have previously reported the identification of a novel series of benzimidazole compounds with particularly good antibacterial effect against Francisella tularensis, a Category A biowarfare pathogen. Herein we report the crystal structure of the F. tularensis FabI enzyme in complex with our most active benzimidazole compound bound with NADH. The structure reveals that the benzimidazole compounds bind to the substrate site in a unique conformation that is distinct from the binding motif of other known FabI inhibitors. Detailed inhibition kinetics have confirmed that the compounds possess a novel inhibitory mechanism that is unique among known FabI inhibitors. These studies could have a strong impact on future antimicrobial design efforts and may reveal new avenues for the design of FAS-II active antibacterial compounds.

  10. Structural and enzymatic analyses reveal the binding mode of a novel series of Francisella tularensis enoyl reductase (FabI) inhibitors

    PubMed Central

    Mehboob, Shahila; Hevener, Kirk E; Truong, Kent; Boci, Teuta; Santarsiero, Bernard D; Johnson, Michael E

    2012-01-01

    Due to structural and mechanistic differences between eukaryotic and prokaryotic fatty acid synthesis enzymes, the bacterial pathway, FAS-II, is an attractive target for the design of antimicrobial agents. We have previously reported the identification of a novel series of benzimidazole compounds with particularly good antibacterial effect against Francisella tularensis, a Category A biowarfare pathogen. Herein we report the crystal structure of the F. tularensis FabI enzyme in complex with our most active benzimidazole compound bound with NADH. The structure reveals that the benzimidazole compounds bind to the substrate site in a unique conformation that is distinct from the binding motif of other known FabI inhibitors. Detailed inhibition kinetics have confirmed that the compounds possess a novel inhibitory mechanism that is unique among known FabI inhibitors. These studies could have a strong impact on future antimicrobial design efforts and may reveal new avenues for the design of FAS-II active antibacterial compounds. PMID:22642319

  11. Francisella tularensis Catalase Restricts Immune Function by Impairing TRPM2 Channel Activity.

    PubMed

    Shakerley, Nicole L; Chandrasekaran, Akshaya; Trebak, Mohamed; Miller, Barbara A; Melendez, J Andrés

    2016-02-19

    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 Ca(2+) 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 Ca(2+) concentration that did not increase further in response to H2O2. Additionally, ΔkatG-infected macrophages displayed limited Ca(2+) 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 Ca(2+). Basal increases in cytosolic Ca(2+) and insensitivity to H2O2-mediated Ca(2+) entry in ΔkatG-infected cells are reversed by the Ca(2+) channel inhibitors 2-aminoethyl diphenylborinate and SKF-96365. 2-Aminoethyl diphenylborinate but not SKF-96365 abrogated ΔkatG-dependent increases in macrophage actin remodeling and IL-6 secretion, suggesting a role for H2O2-mediated Ca(2+) entry through the transient receptor potential melastatin 2 (TRPM2) channel in macrophages. Indeed, increases in basal Ca(2+), 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 Ca(2+) signaling and limit host immune function. PMID:26679996

  12. Francisella tularensis Catalase Restricts Immune Function by Impairing TRPM2 Channel Activity.

    PubMed

    Shakerley, Nicole L; Chandrasekaran, Akshaya; Trebak, Mohamed; Miller, Barbara A; Melendez, J Andrés

    2016-02-19

    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 Ca(2+) 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 Ca(2+) concentration that did not increase further in response to H2O2. Additionally, ΔkatG-infected macrophages displayed limited Ca(2+) 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 Ca(2+). Basal increases in cytosolic Ca(2+) and insensitivity to H2O2-mediated Ca(2+) entry in ΔkatG-infected cells are reversed by the Ca(2+) channel inhibitors 2-aminoethyl diphenylborinate and SKF-96365. 2-Aminoethyl diphenylborinate but not SKF-96365 abrogated ΔkatG-dependent increases in macrophage actin remodeling and IL-6 secretion, suggesting a role for H2O2-mediated Ca(2+) entry through the transient receptor potential melastatin 2 (TRPM2) channel in macrophages. Indeed, increases in basal Ca(2+), 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 Ca(2+) signaling and limit host immune function.

  13. Crystallization of a newly discovered histidine acid phosphatase from Francisella tularensis

    SciTech Connect

    Felts, Richard L.; Reilly, Thomas J.; Calcutt, Michael J.; Tanner, John J.

    2006-01-01

    A histidine acid phosphatase from the CDC Category A pathogen F. tularensis has been crystallized in space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = 61.96, c = 210.78 Å. A 1.75 Å resolution data set was collected at Advanced Light Source beamline 4.2.2. Francisella tularensis is a highly infectious bacterial pathogen that is considered by the Centers for Disease Control and Prevention to be a potential bioterrorism weapon. Here, the crystallization of a 37.2 kDa phosphatase encoded by the genome of F. tularensis subsp. holarctica live vaccine strain is reported. This enzyme shares 41% amino-acid sequence identity with Legionella pneumophila major acid phosphatase and contains the RHGXRXP motif that is characteristic of the histidine acid phosphatase family. Large diffraction-quality crystals were grown in the presence of Tacsimate, HEPES and PEG 3350. The crystals belong to space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = 61.96, c = 210.78 Å. The asymmetric unit is predicted to contain one protein molecule, with a solvent content of 53%. A 1.75 Å resolution native data set was recorded at beamline 4.2.2 of the Lawrence Berkeley National Laboratory Advanced Light Source. Molecular-replacement trials using the human prostatic acid phosphatase structure as the search model (28% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of F. tularensis histidine acid phosphatase will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative.

  14. Lymphotoxin-α Plays Only a Minor Role in Host Resistance to Respiratory Infection with Virulent Type A Francisella tularensis in Mice

    PubMed Central

    Zhang, Deng; KuoLee, Rhonda; Harris, Greg; Zhang, Qinxian; Conlan, J. Wayne; Chen, Wangxue

    2008-01-01

    This study examined the role of lymphotoxin (LT)-α in host defense against airborne infection with Francisella tularensis, a gram-negative facultative intracellular bacterium and the causative agent of tularemia. Following a low-dose aerosol infection with the highly virulent type A strain of F. tularensis, mice deficient in LTα (LTα−/−) consistently harbored approximately 10-fold fewer bacteria in their spleens at day 2 and 10-fold more bacteria in their lungs at day 4 than LTα+/+ mice. However, the mortality and median time to death 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 generally similar between LTα−/− and LTα+/+ mice. These data suggest that although LTα does not contribute significantly to the resistance and host responses of mice to airborne type A F. tularensis infection, it does play a subtle role in the multiplication/dissemination of F. tularensis. PMID:18769490

  15. Adaptation of Francisella tularensis to the Mammalian Environment Is Governed by Cues Which Can Be Mimicked In Vitro▿ †

    PubMed Central

    Hazlett, Karsten R. O.; Caldon, Seth D.; McArthur, Debbie G.; Cirillo, Kerry A.; Kirimanjeswara, Girish S.; Magguilli, Micheal L.; Malik, Meenakshi; Shah, Aaloki; Broderick, Scott; Golovliov, Igor; Metzger, Dennis W.; Rajan, Krishna; Sellati, Timothy J.; Loegering, Daniel J.

    2008-01-01

    The intracellular bacterium Francisella tularensis survives in mammals, arthropods, and freshwater amoeba. It was previously established that the conventional media used for in vitro propagation of this microbe do not yield bacteria that mimic those harvested from infected mammals; whether these in vitro-cultivated bacteria resemble arthropod- or amoeba-adapted Francisella is unknown. As a foundation for our goal of identifying F. tularensis outer membrane proteins which are expressed during mammalian infection, we first sought to identify in vitro cultivation conditions that induce the bacterium's infection-derived phenotype. We compared Francisella LVS grown in brain heart infusion broth (BHI; a standard microbiological medium rarely used in Francisella research) to that grown in Mueller-Hinton broth (MHB; the most widely used F. tularensis medium, used here as a negative control) and macrophages (a natural host cell, used here as a positive control). BHI- and macrophage-grown F. tularensis cells showed similar expression of MglA-dependent and MglA-independent proteins; expression of the MglA-dependent proteins was repressed by the supraphysiological levels of free amino acids present in MHB. We observed that during macrophage infection, protein expression by intracellular bacteria differed from that by extracellular bacteria; BHI-grown bacteria mirrored the latter, while MHB-grown bacteria resembled neither. Naïve macrophages responding to BHI- and macrophage-grown bacteria produced markedly lower levels of proinflammatory mediators than those in cells exposed to MHB-grown bacteria. In contrast to MHB-grown bacteria, BHI-grown bacteria showed minimal delay during intracellular replication. Cumulatively, our findings provide compelling evidence that growth in BHI yields bacteria which recapitulate the phenotype of Francisella organisms that have emerged from macrophages. PMID:18644878

  16. Mast cell toll-like receptor 2 signaling is crucial for effective killing of Francisella tularensis1

    PubMed Central

    Rodriguez, Annette R.; Yu, Jieh-Juen; Guentzel, M. N.; Navara, Christopher S.; Klose, Karl E.; Forsthuber, Thomas G.; Chambers, James P.; Berton, Michael T.; Arulanandam, Bernard P

    2012-01-01

    Toll-like receptor (TLR) signaling is critical for early host defense against pathogens, but the contribution of mast cell TLR-mediated mechanisms and subsequent effector functions during pulmonary infection is largely unknown. We have previously demonstrated that mast cells, through the production of IL-4, effectively control Francisella tularensis replication. In this study, the highly human virulent strain of F. tularensis SCHU S4 and the Live Vaccine Strain (LVS) were utilized to investigate the contribution of mast cell-TLR regulation of Francisella. Mast cells required TLR2 for effective bacterial killing, regulation of the hydrolytic enzyme cathepsin L, and for coordination and trafficking of MHCII and lysosomal associated membrane protein 2 (LAMP2). Infected TLR2−/− mast cells, in contrast to WT and TLR4−/−, lacked detectable IL-4 and displayed increased cell death with a 2–3 log increase of F. tularensis replication, but could be rescued with recombinant IL-4 treatment. Importantly, MHCII and LAMP2 localization with labeled F. tularensis in the lungs was greater in WT than in TLR2−/− mice. These results provide evidence for the important effector contribution of mast cells and TLR2-mediated signaling on early innate processes in the lung following pulmonary F. tularensis infection and provide additional insight into possible mechanisms by which intracellular pathogens modulate respiratory immune defenses. PMID:22529298

  17. Electrochemiluminescence (ECL) immunosensor for detection of Francisella tularensis on screen-printed gold electrode array.

    PubMed

    Spehar-Délèze, Anna-Maria; Julich, Sandra; Gransee, Rainer; Tomaso, Herbert; Dulay, Samuel B; O'Sullivan, Ciara K

    2016-10-01

    An electrochemiluminescence (ECL) immunosensor for the rapid detection of the Francisella tularensis pathogen using whole antibodies or antibody fragments as capture biomolecule is described. A sandwich immunoassay was used with either lipopolysaccharide (LPS) or the whole inactivated bacterial cell (LVS) as a target, while Ru(bpy)3 (2+)-encapsulated silicate nanoparticles were linked to the secondary antibody and used as ECL labels. The assay was performed in a fluidic chip housed in a custom-built black box incorporating electronics, optics and fluidics. The obtained limit of detection for LPS was 0.4 ng/mL, while for the LVS it was 70 and 45 bacteria/mL when the capturing molecule was the whole antibody and the antibody F(ab) fragment, respectively. PMID:27255102

  18. Target discrimination by surface-immobilized molecular beacons designed to detect Francisella tularensis.

    PubMed

    Ramachandran, Akhilesh; Flinchbaugh, James; Ayoubi, Patricia; Olah, Glenn A; Malayer, Jerry R

    2004-02-15

    A molecular beacon (MB) array was designed based on unique regions of the 16S rRNA of the bacterium Francisella tularensis. Nucleic acid molecular beacons undergo a spontaneous fluorogenic conformational change when they hybridize to specific complementary targets. The array was printed on aldehyde glass or hydrogel slides and evaluated for functioning in presence of complementary oligonucleotide sequences, single-nucleotide mismatch sequences and multiple nucleotide mismatch sequences. Discriminating true target from mismatched targets was found to be dependent on type, number, and location of mismatches within the beacon (i.e. located in the stem or loop regions). Optimal conditions for molecular beacon deposition, and target hybridization were determined for oligonucleotide target mismatch discrimination. The beacon array was stable upon recharging by exposure to an alkaline solution, and repeatedly used. In addition, performance of the beacon array biosensor was compared with molecular beacons in homogeneous solution.

  19. Biochemical and structural characterization of polyphosphate kinase 2 from the intracellular pathogen Francisella tularensis

    PubMed Central

    Batten, Laura E.; Parnell, Alice E.; Wells, Neil J.; Murch, Amber L.; Oyston, Petra C. F.; Roach, Peter L.

    2015-01-01

    The metabolism of polyphosphate is important for the virulence of a wide range of pathogenic bacteria and the enzymes of polyphosphate metabolism have been proposed as an anti-bacterial target. In the intracellular pathogen Francisella tularensis, the product of the gene FTT1564 has been identified as a polyphosphate kinase from the polyphosphate kinase 2 (PPK2) family. The isogenic deletion mutant was defective for intracellular growth in macrophages and was attenuated in mice, indicating an important role for polyphosphate in the virulence of Francisella. Herein, we report the biochemical and structural characterization of F. tularensis polyphosphate kinase (FtPPK2) with a view to characterizing the enzyme as a novel target for inhibitors. Using an HPLC-based activity assay, the substrate specificity of FtPPK2 was found to include purine but not pyrimidine nts. The activity was also measured using 31P-NMR. FtPPK2 has been crystallized and the structure determined to 2.23 Å (1 Å=0.1 nm) resolution. The structure consists of a six-stranded parallel β-sheet surrounded by 12 α-helices, with a high degree of similarity to other members of the PPK2 family and the thymidylate kinase superfamily. Residues proposed to be important for substrate binding and catalysis have been identified in the structure, including a lid-loop and the conserved Walker A and B motifs. The ΔFTT1564 strain showed significantly increased sensitivity to a range of antibiotics in a manner independent of the mode of action of the antibiotic. This combination of biochemical, structural and microbiological data provide a sound foundation for future studies targeting the development of PPK2 small molecule inhibitors. PMID:26582818

  20. [Development of a novel Francisella tularensis antigen stained with tetrazolium-blue for tularemia microagglutination test].

    PubMed

    Celebi, Bekir; Kılıç, Selçuk

    2013-07-01

    The isolation of Francisella tularensis in cultures is the reference method for the laboratory diagnosis of tularemia. However, due to the limitations such as the low sensitivity and need for high safety level and equipped laboratories, serologic methods are frequently used as diagnostic tools. F.tularensis-specific antibodies may be demonstrated by several methods, however microagglutination test (MA) remains the most common method with its high sensitivity and specificity. The aim of this study was to develop a novel MA test antigen prepared from whole F.tularensis cells and stained with tetrazolium-blue for more clear and easier evaluation. F.tularensis NCTC 10857 strain was cultured on the cysteine heart agar supplemented with 9% sheep blood and bacterial cells were harvested by scraping, collected in physiological saline (PS) and centrifuged at 1500 rpm for 10 minutes. For preparing stock antigen suspension cell concentration was adjusted to OD600=1.5, spectrophotometrically. Tetrazolium-blue solution (BTC [3,3'-(3,3'-Dimethoxy[1,1'-biphenyl]-4,4'-diyl) bis [2,5-diphenyl-2H-tetrazolium dichloride], T4375 Sigma-Aldrich) at the final concentration of 1% was added to cell suspension and incubated at 37°C for 5 hours for absorption. Then, the living cells were chemically inactivated by formaldehyde. Repeating centrifugations were performed to discard excess dye and formaline, then 0.4% formaline saline was added on the sediment. Optimum concentration of this novel antigen (BTC-Ag) for MA test was determined by plate titration method by using standard serum sample with a known MA titer (1/128). The performance of BTC-Ag in MA test was evaluated by using 100 patient sera positive for F.tularensis antibodies, and 100 tularemia negative patient sera (of them 50 were seropositive for brucellosis). All of the results were compared with standard MA test in which safranin-O stained antigen (SO-Ag) was used. There was 100% agreement between the two tests performed with

  1. Border Patrol Gone Awry: Lung NKT Cell Activation by Francisella tularensis Exacerbates Tularemia-Like Disease.

    PubMed

    Hill, Timothy M; Gilchuk, Pavlo; Cicek, Basak B; Osina, Maria A; Boyd, Kelli L; Durrant, Douglas M; Metzger, Dennis W; Khanna, Kamal M; Joyce, Sebastian

    2015-06-01

    The respiratory mucosa is a major site for pathogen invasion and, hence, a site requiring constant immune surveillance. The type I, semi-invariant natural killer T (NKT) cells are enriched within the lung vasculature. Despite optimal positioning, the role of NKT cells in respiratory infectious diseases remains poorly understood. Hence, we assessed their function in a murine model of pulmonary tularemia--because tularemia is a sepsis-like proinflammatory disease and NKT cells are known to control the cellular and humoral responses underlying sepsis. Here we show for the first time that respiratory infection with Francisella tularensis live vaccine strain resulted in rapid accumulation of NKT cells within the lung interstitium. Activated NKT cells produced interferon-γ and promoted both local and systemic proinflammatory responses. Consistent with these results, NKT cell-deficient mice showed reduced inflammatory cytokine and chemokine response yet they survived the infection better than their wild type counterparts. Strikingly, NKT cell-deficient mice had increased lymphocytic infiltration in the lungs that organized into tertiary lymphoid structures resembling induced bronchus-associated lymphoid tissue (iBALT) at the peak of infection. Thus, NKT cell activation by F. tularensis infection hampers iBALT formation and promotes a systemic proinflammatory response, which exacerbates severe pulmonary tularemia-like disease in mice.

  2. Complete genome sequence of Francisella tularensis subspecies holarctica FTNF002-00.

    PubMed

    Barabote, Ravi D; Xie, Gary; Brettin, Thomas S; Hinrichs, Steven H; Fey, Paul D; Jay, Justin J; Engle, Jennifer L; Godbole, Shubhada D; Noronha, Jyothi M; Scheuermann, Richard H; Zhou, Liwei W; Lion, Christine; Dempsey, Michael P

    2009-09-16

    Francisella tularensis subspecies holarctica FTNF002-00 strain was originally obtained from the first known clinical case of bacteremic F. tularensis pneumonia in Southern Europe isolated from an immunocompetent individual. The FTNF002-00 complete genome contains the RD(23) deletion and represents a type strain for a clonal population from the first epidemic tularemia outbreak in Spain between 1997-1998. Here, we present the complete sequence analysis of the FTNF002-00 genome. The complete genome sequence of FTNF002-00 revealed several large as well as small genomic differences with respect to two other published complete genome sequences of F. tularensis subsp. holarctica strains, LVS and OSU18. The FTNF002-00 genome shares >99.9% sequence similarity with LVS and OSU18, and is also approximately 5 MB smaller by comparison. The overall organization of the FTNF002-00 genome is remarkably identical to those of LVS and OSU18, except for a single 3.9 kb inversion in FTNF002-00. Twelve regions of difference ranging from 0.1-1.5 kb and forty-two small insertions and deletions were identified in a comparative analysis of FTNF002-00, LVS, and OSU18 genomes. Two small deletions appear to inactivate two genes in FTNF002-00 causing them to become pseudogenes; the intact genes encode a protein of unknown function and a drug:H(+) antiporter. In addition, we identified ninety-nine proteins in FTNF002-00 containing amino acid mutations compared to LVS and OSU18. Several non-conserved amino acid replacements were identified, one of which occurs in the virulence-associated intracellular growth locus subunit D protein. Many of these changes in FTNF002-00 are likely the consequence of direct selection that increases the fitness of this subsp. holarctica clone within its endemic population. Our complete genome sequence analyses lay the foundation for experimental testing of these possibilities.

  3. Mouse Models of Aerosol-Acquired Tularemia Caused by Francisella tularensis Types A and B

    PubMed Central

    Fritz, David L; England, Marilyn J; Miller, Lynda; Waag, David M

    2014-01-01

    After preliminary assessment of virulence in AKR/J, DBA/1, BALB/c, and C57BL/6 mice, we investigated histopathologic changes in BALB/c and C57BL/6 mice infected with type A (strain SCHU S4) or type B (strain 425) Francisella tularensis by aerosol exposure. In mice exposed to type A infection, changes in histologic presentation were not apparent until day 3 after infection, when pyogranulomatous inflammation was detected in spleens and livers of BALB/c mice, and in lungs and spleens of C57BL/6 mice. Histopathologic changes were most severe and widespread in both mouse strains on day 5 after infection and seemed to completely resolve within 22 d of challenge. BALB/c mice were more resistant than C57BL/6 mice in lethal-dose calculations, but C57BL/6 mice cleared the infection more rapidly. Mice similarly challenged with type B F. tularensis also developed histopathologic signs of infection beginning on day 3. The most severe changes were noted on day 8 and were characterized by granulomatous or pyogranulomatous infiltrations of the lungs. Unlike type A infection, lesions due to type B did not resolve over time and remained 3 wk after infection. In type B, but not type A, infection we noted extensive inflammation of the heart muscle. Although no microorganisms were found in tissues of type A survivors beyond 9 d after infection, mice surviving strain 425 infection had a low level of residual infection at 3 wk after challenge. The histopathologic presentation of tularemia caused by F. tularensis types A and B in BALB/c and C57BL/6 mice bears distinct similarities to tularemia in humans. PMID:25402174

  4. Mouse models of aerosol-acquired tularemia caused by Francisella tularensis types A and B.

    PubMed

    Fritz, David L; England, Marilyn J; Miller, Lynda; Waag, David M

    2014-10-01

    After preliminary assessment of virulence in AKR/J, DBA/1, BALB/c, and C57BL/6 mice, we investigated histopathologic changes in BALB/c and C57BL/6 mice infected with type A (strain SCHU S4) or type B (strain 425) Francisella tularensis by aerosol exposure. In mice exposed to type A infection, changes in histologic presentation were not apparent until day 3 after infection, when pyogranulomatous inflammation was detected in spleens and livers of BALB/c mice, and in lungs and spleens of C57BL/6 mice. Histopathologic changes were most severe and widespread in both mouse strains on day 5 after infection and seemed to completely resolve within 22 d of challenge. BALB/c mice were more resistant than C57BL/6 mice in lethal-dose calculations, but C57BL/6 mice cleared the infection more rapidly. Mice similarly challenged with type B F. tularensis also developed histopathologic signs of infection beginning on day 3. The most severe changes were noted on day 8 and were characterized by granulomatous or pyogranulomatous infiltrations of the lungs. Unlike type A infection, lesions due to type B did not resolve over time and remained 3 wk after infection. In type B, but not type A, infection we noted extensive inflammation of the heart muscle. Although no microorganisms were found in tissues of type A survivors beyond 9 d after infection, mice surviving strain 425 infection had a low level of residual infection at 3 wk after challenge. The histopathologic presentation of tularemia caused by F. tularensis types A and B in BALB/c and C57BL/6 mice bears distinct similarities to tularemia in humans.

  5. [Evaluation of a newly-developed ready-to-use commercial PCR kit for the molecular diagnosis of Francisella tularensis].

    PubMed

    Celebi, Bekir; Kılıç, Selçuk; Yeşilyurt, Murat; Acar, Bülent

    2014-01-01

    Tularemia is a rare zoonotic infection, however, considerations of tularemia as a biological weapon and several recent major epidemics have caused renewed interest in this disease. Laboratory diagnosis of tularemia is done in the presence of appropriate epidemiological data, by the demonstration of specific antibodies in the serum samples obtained with 1-2 week intervals following the development of symptoms. It is an a posteriori analysis with limited use for prompt diagnosis of the patient during the early symptomatic phase and deliberate release of biological agents. Limitations in both culture and serology have led to substantial research in the development of new diagnostic techniques. Several PCR methods for tularemia have been developed, both for conventional and real-time polymerase chain reaction (rtPCR). However, PCR methods are hard to be deployed in remote endemic areas that lack sufficient infrastructure. Recently a "Toolbox" which includes all instruments, equipments and solutions [DNA4U® Bacteria Genomic DNA Isolation Kit, CubeCycler® (Personal Thermal Cycler), PCR4U® Bioterrorism Agents Detection Kit, electrophoresis tank, power supply, ready-agarose gel and electrophoresis buffer] necessary for conventional PCR, was developed for the identification of bioterrorism agents in the field. In this study we aimed to evaluate the efficacy of a ready-to-use commercial PCR kit (Nanobiz, Ankara, Turkey) targeting the tul4 gene, for the diagnosis of tularemia and to compare the results with an in-house conventional PCR and a rtPCR test. We applied the assay to a collection of four F.tularensis standard strains, 15 field isolates (from humans, animals, water), 13 non-Francisella strains which are phylogenetically related to F.tularensis and a total of 60 lymph node aspirates obtained from suspected tularemia cases. Compared to the in-house PCR method used in our laboratory, the sensitivity, specificity, positive and negative predictive values of Nanobiz PCR

  6. Nicotinamide mononucleotide synthetase is the key enzyme for an alternative route of NAD biosynthesis in Francisella tularensis

    PubMed Central

    Sorci, Leonardo; Martynowski, Dariusz; Rodionov, Dmitry A.; Eyobo, Yvonne; Zogaj, Xhavit; Klose, Karl E.; Nikolaev, Evgeni V.; Magni, Giulio; Zhang, Hong; Osterman, Andrei L.

    2009-01-01

    Enzymes involved in the last 2 steps of nicotinamide adenine dinucleotide (NAD) cofactor biosynthesis, which catalyze the adenylylation of the nicotinic acid mononucleotide (NaMN) precursor to nicotinic acid dinucleotide (NaAD) followed by its amidation to NAD, constitute promising drug targets for the development of new antibiotics. These enzymes, NaMN adenylyltransferase (gene nadD) and NAD synthetase (gene nadE), respectively, are indispensable and conserved in nearly all bacterial pathogens. However, a comparative genome analysis of Francisella tularensis allowed us to predict the existence of an alternative route of NAD synthesis in this category A priority pathogen, the causative agent of tularaemia. In this route, the amidation of NaMN to nicotinamide mononucleotide (NMN) occurs before the adenylylation reaction, which converts this alternative intermediate to the NAD cofactor. The first step is catalyzed by NMN synthetase, which was identified and characterized in this study. A crystal structure of this enzyme, a divergent member of the NadE family, was solved at 1.9-Å resolution in complex with reaction products, providing a rationale for its unusual substrate preference for NaMN over NaAD. The second step is performed by NMN adenylyltransferase of the NadM family. Here, we report validation of the predicted route (NaMN → NMN → NAD) in F. tularensis including mathematical modeling, in vitro reconstitution, and in vivo metabolite analysis in comparison with a canonical route (NaMN → NaAD → NAD) of NAD biosynthesis as represented by another deadly bacterial pathogen, Bacillus anthracis. PMID:19204287

  7. Genomic analyses of Francisella tularensis strains confirm disease transmission from drinking water sources, Turkey, 2008, 2009 and 2012.

    PubMed

    Karadenizli, A; Forsman, M; Şimşek, H; Taner, M; Öhrman, C; Myrtennäs, K; Lärkeryd, A; Johansson, A; Özdemir, L; Sjödin, A

    2015-05-28

    Waterborne epidemics of tularaemia caused by Francisella tularensis are increasingly reported in Turkey. We have used whole genome sequencing to investigate if F. tularensis isolated from patients could be traced back to drinking water sources. Tonsil swabs from 33 patients diagnosed with oropharyngeal tularaemia in three outbreaks and 140 water specimens were analysed. F. tularensis subsp. holarctica was confirmed by microagglutination and PCR in 12 patients and five water specimens. Genomic analysis of three pairs of patient and water isolates from outbreaks in Sivas, Çorum, and Kocaeli showed the isolates to belong to two new clusters of the F. tularensis B.12 genetic clade. The clusters were defined by 19 and 15 single nucleotide polymorphisms (SNPs) in a multiple alignment based on 507 F. tularensis genomes. One synonymous SNP was chosen as a new canonical SNP (canSNP) for each cluster for future use in diagnostic assays. No SNP was identified between the genomes from the patient–water pair of isolates from Kocaeli, one SNP between the pair of isolates from Sivas, whereas the pair from Çorum differed at seven SNPs. These results illustrate the power of whole genome sequencing for tracing F. tularensis patient isolates back to their environmental source.

  8. Francisella tularensis LVS surface and membrane proteins as targets of effective post-exposure immunization for tularemia.

    PubMed

    Chandler, Jeffrey C; Sutherland, Marjorie D; Harton, Marisa R; Molins, Claudia R; Anderson, Rebecca V; Heaslip, Darragh G; Bosio, Catharine M; Belisle, John T

    2015-02-01

    Francisella tularensis causes disease (tularemia) in a large number of mammals, including man. We previously demonstrated enhanced efficacy of conventional antibiotic therapy for tularemia by postexposure passive transfer of immune sera developed against a F. tularensis LVS membrane protein fraction (MPF). However, the protein composition of this immunogenic fraction was not defined. Proteomic approaches were applied to define the protein composition and identify the immunogens of MPF. MPF consisted of at least 299 proteins and 2-D Western blot analyses using sera from MPF-immunized and F. tularensis LVS-vaccinated mice coupled to liquid chromatography-tandem mass spectrometry identified 24 immunoreactive protein spots containing 45 proteins. A reverse vaccinology approach that applied labeling of F. tularensis LVS surface proteins and bioinformatics was used to reduce the complexity of potential target immunogens. Bioinformatics analyses of the immunoreactive proteins reduced the number of immunogen targets to 32. Direct surface labeling of F. tularensis LVS resulted in the identification of 31 surface proteins. However, only 13 of these were reactive with MPF and/or F. tularensis LVS immune sera. Collectively, this use of orthogonal proteomic approaches reduced the complexity of potential immunogens in MPF by 96% and allowed for prioritization of target immunogens for antibody-based immunotherapies against tularemia.

  9. Protective B-cell epitopes of Francisella tularensis O-polysaccharide in a mouse model of respiratory tularaemia.

    PubMed

    Lu, Zhaohua; Madico, Guillermo; Roche, Marly I; Wang, Qi; Hui, Julia H; Perkins, Hillary M; Zaia, Joseph; Costello, Catherine E; Sharon, Jacqueline

    2012-07-01

    Antibodies to the lipopolysaccharide (LPS) of Francisella tularensis have been shown to be protective against respiratory tularaemia in mouse models, and we have previously described mouse monoclonal antibodies (mAbs) to non-overlapping terminal and internal epitopes of the F. tularensis LPS O-polysaccharide (OAg). In the current study, we used F. tularensis LPS oligosaccharides of defined OAg repeat length as molecular rulers in competition ELISA to demonstrate that the epitope targeted by the terminal OAg-binding mAb FB11 is contained within one tetrasaccharide repeat whereas the epitope targeted by the internal OAg-binding mAb Ab52 spans two tetrasaccharide repeats. Both mAbs conferred survival to BALB/c mice infected intranasally with the F. tularensis type B live vaccine strain and prolonged survival of BALB/c mice infected intranasally with the highly virulent F. tularensis type A strain SchuS4. The protective effects correlated with reduced bacterial burden in mAb-treated infected mice. These results indicate that an oligosaccharide with two OAg tetrasaccharide repeats covers both terminal and internal protective OAg epitopes, which may inform the design of vaccines for tularaemia. Furthermore, the FB11 and Ab52 mAbs could serve as reporters to monitor the response of vaccine recipients to protective B-cell epitopes of F. tularensis OAg.

  10. Development and evaluation of an up-converting phosphor technology-based lateral flow assay for rapid detection of Francisella tularensis.

    PubMed

    Hua, Fei; Zhang, Pingping; Zhang, Fuli; Zhao, Yong; Li, Chunfeng; Sun, Chongyun; Wang, Xiaochen; Yang, Ruifu; Wang, Chengbin; Yu, Ailian; Zhou, Lei

    2015-11-26

    Francisella tularensis is a potential biowarfare/bioterrorism agent and zoonotic pathogen that causes tularemia; thus, surveillance of F. tularensis and first-level emergency response using point-of-care testing (POCT) are essential. The UPT-LF POCT assay was established to quantitatively detect F. tularensis within 15 min, and the sensitivity of the assay was 10(4) CFU · mL(-1) (100 CFU/test). The linear quantitative range covered five orders of magnitude, and the coefficients of variation were less than 10%. Except Shigella dysenteriae, UPT-LF showed excellent specificity to four strains that are also potential biowarfare/bioterrorism agents and 13 food-borne pathogenic strains. Samples with pH 2-13, high ion strengths (≥ 2 mol · L(-1) solution of KCl and NaCl), high viscosities (≤ 50 mg · mL(-1) PEG20000 or ≥ 20% glycerol), and high concentrations of biomacromolecules (≥ 400 mg · mL(-1) bovine serum albumin or ≥ 80 mg · mL(-1) casein) showed little influence on the assay. For practical utilization, the tolerance limits for seven powders and eight viscera were determined, and operation errors of liquid measurement demonstrated a minor influence on the strip. Ftu-UPT-LF is a candidate POCT method because of its excellent sensitivity, specificity, and stability in complex samples, as well as low operation error.

  11. Development and evaluation of an up-converting phosphor technology-based lateral flow assay for rapid detection of Francisella tularensis

    PubMed Central

    Hua, Fei; Zhang, Pingping; Zhang, Fuli; Zhao, Yong; Li, Chunfeng; Sun, Chongyun; Wang, Xiaochen; Yang, Ruifu; Wang, Chengbin; Yu, Ailian; Zhou, Lei

    2015-01-01

    Francisella tularensis is a potential biowarfare/bioterrorism agent and zoonotic pathogen that causes tularemia; thus, surveillance of F. tularensis and first-level emergency response using point-of-care testing (POCT) are essential. The UPT-LF POCT assay was established to quantitatively detect F. tularensis within 15 min, and the sensitivity of the assay was 104 CFU · mL−1 (100 CFU/test). The linear quantitative range covered five orders of magnitude, and the coefficients of variation were less than 10%. Except Shigella dysenteriae, UPT-LF showed excellent specificity to four strains that are also potential biowarfare/bioterrorism agents and 13 food-borne pathogenic strains. Samples with pH 2–13, high ion strengths (≥2 mol · L−1 solution of KCl and NaCl), high viscosities (≤50 mg · mL−1 PEG20000 or ≥20% glycerol), and high concentrations of biomacromolecules (≥400 mg · mL−1 bovine serum albumin or ≥80 mg · mL−1 casein) showed little influence on the assay. For practical utilization, the tolerance limits for seven powders and eight viscera were determined, and operation errors of liquid measurement demonstrated a minor influence on the strip. Ftu-UPT-LF is a candidate POCT method because of its excellent sensitivity, specificity, and stability in complex samples, as well as low operation error. PMID:26608358

  12. FipB, an Essential Virulence Factor of Francisella tularensis subsp. tularensis, Has Dual Roles in Disulfide Bond Formation

    PubMed Central

    Qin, Aiping; Zhang, Yan; Clark, Melinda E.; Rabideau, Meaghan M.; Millan Barea, Luis R.

    2014-01-01

    FipB, an essential virulence factor of Francisella tularensis, is a lipoprotein with two conserved domains that have similarity to disulfide bond formation A (DsbA) proteins and the amino-terminal dimerization domain of macrophage infectivity potentiator (Mip) proteins, which are proteins with peptidyl-prolyl cis/trans isomerase activity. This combination of conserved domains is unusual, so we further characterized the enzymatic activity and the importance of the Mip domain and lipid modification in virulence. Unlike typical DsbA proteins, which are oxidases, FipB exhibited both oxidase and isomerase activities. FipA, which also shares similarity with Mip proteins, potentiated the isomerase activity of FipB in an in vitro assay and within the bacteria, as measured by increased copper sensitivity. To determine the importance of the Mip domain and lipid modification of FipB, mutants producing FipB proteins that lacked either the Mip domain or the critical cysteine necessary for lipid modification were constructed. Both strains replicated within host cells and retained virulence in mice, though there was some attenuation. FipB formed surface-exposed dimers that were sensitive to dithiothreitol (DTT), dependent on the Mip domain and on at least one cysteine in the active site of the DsbA-like domain. However, these dimers were not essential for virulence, because the Mip deletion mutant, which failed to form dimers, was still able to replicate intracellularly and retained virulence in mice. Thus, the Mip domains of FipB and FipA impart additional isomerase functionality to FipB, but only the DsbA-like domain and oxidase activity are essential for its critical virulence functions. PMID:25092026

  13. Use of a zwitterionic detergent for the restoration of the antibody binding capacity of immunoblotted Francisella tularensis lipopolysaccharide.

    PubMed

    Fulop, M J; Webber, T; Manchee, R J

    1992-05-15

    A method for the partial restoration of the antibody binding capacity of Francisella tularensis lipopolysaccharide (LPS) following denaturation (dissociation) in boiling sodium dodecyl sulfate (SDS) is described. The method relies on the presence of a zwitterionic detergent in the matrix of an SDS-polyacrylamide gel and in the transfer buffer during an immunoblot. F. tularensis LPS, which had lost its earlier capacity to bind to a particular monoclonal antibody in the normal blot procedure, did bind following the addition of the zwitterionic detergent to the polyacrylamide gel and transfer buffer. A number of detergents were tested but most success in restoring antibody binding was achieved with Zwittergent 3-08. This simple modification to the immunoblot procedure proved helpful in identifying a monoclonal antibody specific to hot phenol-extracted F. tularensis LPS.

  14. Ubiquitous Promoter-Localization of Essential Virulence Regulators in Francisella tularensis

    PubMed Central

    Ramsey, Kathryn M.; Osborne, Melisa L.; Vvedenskaya, Irina O.; Su, Cathy; Nickels, Bryce E.; Dove, Simon L.

    2015-01-01

    Francisella tularensis is a Gram-negative bacterium whose ability to replicate within macrophages and cause disease is strictly dependent upon the coordinate activities of three transcription regulators called MglA, SspA, and PigR. MglA and SspA form a complex that associates with RNA polymerase (RNAP), whereas PigR is a putative DNA-binding protein that functions by contacting the MglA-SspA complex. Most transcription activators that bind the DNA are thought to occupy only those promoters whose activities they regulate. Here we show using chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-Seq) that PigR, MglA, and SspA are found at virtually all promoters in F. tularensis and not just those of regulated genes. Furthermore, we find that the ability of PigR to associate with promoters is dependent upon the presence of MglA, suggesting that interaction with the RNAP-associated MglA-SspA complex is what directs PigR to promoters in F. tularensis. Finally, we present evidence that the ability of PigR (and thus MglA and SspA) to positively control the expression of genes is dictated by a specific 7 base pair sequence element that is present in the promoters of regulated genes. The three principal regulators of virulence gene expression in F. tularensis therefore function in a non-classical manner with PigR interacting with the RNAP-associated MglA-SspA complex at the majority of promoters but only activating transcription from those that contain a specific sequence element. Our findings reveal how transcription factors can exert regulatory effects at a restricted set of promoters despite being associated with most or all. This distinction between occupancy and regulatory effect uncovered by our data may be relevant to the study of RNAP-associated transcription regulators in other pathogenic bacteria. PMID:25830507

  15. Metabolism-Directed Structure Optimization of Benzimidazole-Based Francisella tularensis Enoyl-Reductase (FabI) Inhibitors

    PubMed Central

    Zhang, Yan-Yan; Liu, Yong; Mehboob, Shahila; Song, Jin-Hua; Boci, Teuta; Johnson, Michael E.; Ghosh, Arun K.; Jeong, Hyunyoung

    2015-01-01

    FabI is a potential antibiotic target against Francisella tularensis, which has been classified as a Category A biowarfare agent of high risk to public health. Our previous work demonstrated that N-benzyl benzimidazole compounds possess promising FabI inhibitory activity, but their druggability properties including metabolic stability are unknown. The objective of this study was to characterize structure-metabolism relationships of a series of N-benzyl benzimidazole compounds to guide chemical optimization for better metabolic stability. To this end, metabolic stability data were obtained for 22 initial lead compounds using mouse hepatic microsomes. Metabolic hotspots on the benzimidazole core structure as well as the benzyl ring were identified and verified by metabolite identification studies of 4 model compounds. Interestingly, the proposed structure-metabolism relationships did not apply to 9 newly synthesized cyclopentane or oxacyclopentane derivatives of N-benzyl benzimidazole. Subsequently, in silico quantitative structure-property relationship models were developed. Four molecular descriptors representing molecular polarity/polarisability, symmetry and size were identified to best explain variability in metabolic stability of different compounds. Multi-linear and nonlinear regression models based on the selected molecular descriptors were developed and validated. The structure-metabolism relationships for N-benzyl benzimidazole compounds should help optimization of N-benzyl benzimidazole compounds for better pharmacokinetic behavior. PMID:24171690

  16. Activation of macrophages for destruction of Francisella tularensis: identification of cytokines, effector cells, and effector molecules.

    PubMed Central

    Fortier, A H; Polsinelli, T; Green, S J; Nacy, C A

    1992-01-01

    Francisella tularensis live vaccine strain (LVS) was grown in culture with nonadherent resident, starch-elicited, or Proteose Peptone-elicited peritoneal cells. Numbers of bacteria increased 4 logs over the input inoculum in 48 to 72 h. Growth rates were faster in inflammatory cells than in resident cells: generation times for the bacterium were 3 h in inflammatory cells and 6 h in resident macrophages. LVS-infected macrophage cultures treated with lymphokines did not support growth of the bacterium, although lymphokines alone had no inhibitory effects on replication of LVS in culture medium devoid of cells. Removal of gamma interferon (IFN-gamma) by immunoaffinity precipitation rendered lymphokines ineffective for induction of macrophage anti-LVS activity, and recombinant IFN-gamma stimulated both resident and inflammatory macrophage populations to inhibit LVS growth in vitro. Inflammatory macrophages were more sensitive to effects of IFN-gamma: half-maximal activity was achieved at 5 U/ml for inflammatory macrophages and 20 U/ml for resident macrophages. IFN-gamma-induced anti-LVS activity correlated with the production of nitrite (NO2-), an oxidative end product of L-arginine-derived nitric oxide (NO). Anti-LVS activity and nitrite production were both completely inhibited by the addition of either the L-arginine analog NG-monomethyl-L-arginine or anti-tumor necrosis factor antibodies to activated macrophage cultures. Thus, macrophages can be activated by IFN-gamma to suppress the growth of F. tularensis by generation of toxic levels of NO, and inflammatory macrophages are substantially more sensitive to activation activities of IFN-gamma for this effector reaction than are more differentiated resident cells. PMID:1541555

  17. Identification of Mechanisms for Attenuation of the FSC043 Mutant of Francisella tularensis SCHU S4

    PubMed Central

    Lindgren, Marie; Tancred, Linda; Golovliov, Igor; Conlan, Wayne; Twine, Susan M.

    2014-01-01

    Previously, we identified a spontaneous, essentially avirulent mutant, FSC043, of the highly virulent strain SCHU S4 of Francisella tularensis subsp. tularensis. We have now characterized the phenotype of the mutant and the mechanisms of its attenuation in more detail. Genetic and proteomic analyses revealed that the pdpE gene and most of the pdpC gene were very markedly downregulated and, as previously demonstrated, that the strain expressed partially deleted and fused fupA and fupB genes. FSC043 showed minimal intracellular replication and induced no cell cytotoxicity. The mutant showed delayed phagosomal escape; at 18 h, colocalization with LAMP-1 was 80%, indicating phagosomal localization, whereas the corresponding percentages for SCHU S4 and the ΔfupA mutant were <10%. However, a small subset of the FSC043-infected cells contained up to 100 bacteria with LAMP-1 colocalization of around 30%. The unusual intracellular phenotype was similar to that of the ΔpdpC and ΔpdpC ΔpdpE mutants. Complementation of FSC043 with the intact fupA and fupB genes did not affect the phenotype, whereas complementation with the pdpC and pdpE genes restored intracellular replication and led to marked virulence. Even higher virulence was observed after complementation with both double-gene constructs. After immunization with the FSC043 strain, moderate protection against respiratory challenge with the SCHU S4 strain was observed. In summary, FSC043 showed a highly unusual intracellular phenotype, and based on our findings, we hypothesize that the mutation in the pdpC gene makes an essential contribution to the phenotype. PMID:24935978

  18. Purification and Biophysical Characterization of the CapA Membrane Protein FTT0807 from Francisella tularensis

    PubMed Central

    2015-01-01

    The capA gene (FTT0807) from Francisella tularensis subsp. tularensis SCHU S4 encodes a 44.4 kDa integral membrane protein composed of 403 amino acid residues that is part of an apparent operon that encodes at least two other membrane proteins, CapB, and CapC, which together play a critical role in the virulence and pathogenesis of this bacterium. The capA gene was overexpressed in Escherichia coli as a C-terminal His6-tagged fusion with a folding reporter green fluorescent protein (frGFP). Purification procedures using several detergents were developed for the fluorescing and membrane-bound product, yielding approximately 30 mg of pure protein per liter of bacterial culture. Dynamic light scattering indicated that CapA-frGFP was highly monodisperse, with a size that was dependent upon both the concentration and choice of detergent. Circular dichroism showed that CapA-frGFP was stable over the range of 3–9 for the pH, with approximately half of the protein having well-defined α-helical and β-sheet secondary structure. The addition of either sodium chloride or calcium chloride at concentrations producing ionic strengths above 0.1 M resulted in a small increase of the α-helical content and a corresponding decrease in the random-coil content. Secondary-structure predictions on the basis of the analysis of the sequence indicate that the CapA membrane protein has two transmembrane helices with a substantial hydrophilic domain. The hydrophilic domain is predicted to contain a long disordered region of 50–60 residues, suggesting that the increase of α-helical content at high ionic strength could arise because of electrostatic interactions involving the disordered region. CapA is shown to be an inner-membrane protein and is predicted to play a key cellular role in the assembly of polysaccharides. PMID:24593131

  19. Detection of Francisella tularensis-specific antibodies in patients with tularemia by a novel competitive enzyme-linked immunosorbent assay.

    PubMed

    Sharma, Neekun; Hotta, Akitoyo; Yamamoto, Yoshie; Fujita, Osamu; Uda, Akihiko; Morikawa, Shigeru; Yamada, Akio; Tanabayashi, Kiyoshi

    2013-01-01

    A novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies against Francisella tularensis in humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed against F. tularensis lipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivated F. tularensis-immunized rabbits, and F. tularensis-infected mice. Antibodies against F. tularensis were successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R(2) = 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species. PMID:23114700

  20. Detection of Francisella tularensis-Specific Antibodies in Patients with Tularemia by a Novel Competitive Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Sharma, Neekun; Hotta, Akitoyo; Yamamoto, Yoshie; Fujita, Osamu; Uda, Akihiko; Morikawa, Shigeru; Yamada, Akio

    2013-01-01

    A novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies against Francisella tularensis in humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed against F. tularensis lipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivated F. tularensis-immunized rabbits, and F. tularensis-infected mice. Antibodies against F. tularensis were successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R2 = 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species. PMID:23114700

  1. Detection of Francisella tularensis-specific antibodies in patients with tularemia by a novel competitive enzyme-linked immunosorbent assay.

    PubMed

    Sharma, Neekun; Hotta, Akitoyo; Yamamoto, Yoshie; Fujita, Osamu; Uda, Akihiko; Morikawa, Shigeru; Yamada, Akio; Tanabayashi, Kiyoshi

    2013-01-01

    A novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies against Francisella tularensis in humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed against F. tularensis lipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivated F. tularensis-immunized rabbits, and F. tularensis-infected mice. Antibodies against F. tularensis were successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R(2) = 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species.

  2. Electrochemical detection of Francisella tularensis genomic DNA using solid-phase recombinase polymerase amplification.

    PubMed

    del Río, Jonathan Sabaté; Yehia Adly, Nouran; Acero-Sánchez, Josep Lluis; Henry, Olivier Y F; O'Sullivan, Ciara K

    2014-04-15

    Solid-phase isothermal DNA amplification was performed exploiting the homology protein recombinase A (recA). The system was primarily tested on maleimide activated microtitre plates as a proof-of-concept and later translated to an electrochemical platform. In both cases, forward primer for Francisella tularensis holarctica genomic DNA was surface immobilised via a thiol or an amino moiety and then elongated during the recA mediated amplification, carried out in the presence of specific target sequence and reverse primers. The formation of the subsequent surface tethered amplicons was either colorimetrically or electrochemically monitored using a horseradish peroxidase (HRP)-labelled DNA secondary probe complementary to the elongated strand. The amplification time was optimised to amplify even low amounts of DNA copies in less than an hour at a constant temperature of 37°C, achieving a limit of detection of 1.3×10(-13) M (4×10(6) copies in 50 μL) for the colorimetric assay and 3.3×10(-14) M (2×10(5) copies in 10 μL) for the chronoamperometric assay. The system was demonstrated to be highly specific with negligible cross-reactivity with non-complementary targets or primers.

  3. Establishment of lethal inhalational infection with Francisella tularensis (tularaemia) in the common marmoset (Callithrix jacchus).

    PubMed

    Nelson, Michelle; Lever, Mark S; Savage, Victoria L; Salguero, Francisco Javier; Pearce, Peter C; Stevens, Daniel J; Simpson, Andrew J H

    2009-04-01

    Susceptibility and lethality studies of inhalational tularaemia were undertaken using the common marmoset (Callithrix jacchus) to determine its suitability as a non-human primate model. Pairs of marmosets were exposed to varying challenge doses of Francisella tularensis by the airborne route and monitored for up to 14 days postchallenge (p.c.). Lethal infection was achieved following a retained dose of less than 10 bacterial colony-forming units (CFU). However, precise LD(50) determination was not possible. The model was characterized using a target challenge dose of approximately 100 CFU. Increased core body temperature was the first indicator of disease, at approximately 2.5 days p.c. Overt clinical signs were first observed 12-18 h after the temperature increase. Significantly decreased activity was observed after approximately 3 days. All animals succumbed to infection between 4.5 and 7 days p.c. At postmortem examination, gross pathology was evident in the liver, spleen and lungs of all animals and high bacterial numbers were detected in all the organs assessed. Bacteraemia was demonstrated in all animals postmortem. Histopathological observations included severe suppurative bronchopneumonia, severe multifocal pyogranulomatous hepatitis, splenitis and lymphadenitis. Tularaemia disease progression in the common marmoset therefore appears to be consistent with the disease seen in humans and other animal models. The common marmoset may therefore be considered a suitable model for further studies of inhalational tularaemia.

  4. B1a cells enhance susceptibility to infection with virulent Francisella tularensis via modulation of NK/NKT cell responses*

    PubMed Central

    Crane, Deborah D.; Griffin, Amanda J.; Wehrly, Tara D.; Bosio, Catharine M.

    2013-01-01

    B1a cells are an important source of natural antibodies, antibodies directed against T-independent antigens, and are a primary source of IL-10. Bruton's tyrosine kinase (btk) is a cytoplasmic kinase that is essential for mediating signals from the B cell receptor and is critical for development of B1a cells. Consequentially, animals lacking btk have few B1a cells, minimal antibody responses, and can preferentially generate Th1 type immune responses following infection. B1a cells have been shown to aid in protection against infection with attenuated Francisella tularensis but their role in infection mediated by fully virulent F. tularensis is not known. Therefore, we utilized mice with defective btk (XID mice) to determine the contribution of B1a cells in defense against the virulent, F. tularensis ssp. tularensis strain SchuS4. Surprisingly, XID mice displayed increased resistance to pulmonary infection with F. tularensis. Specifically, XID mice had enhanced clearance of bacteria from the lung and spleen and significantly greater survival of infection compared to wild type controls. We revealed that resistance to infection in XID mice was associated with decreased numbers of IL-10 producing B1a cells and concomitant increased numbers of IL-12 producing macrophages and IFN-γ producing NK/NKT cells. Adoptive transfer of wild type B1a cells into XID mice reversed the control of bacterial replication. Similarly, depletion of NK/NKT cells also increased bacterial burdens in XID mice. Together, our data suggest B cell-NK/NKT cell crosstalk is a critical pivot controlling survival of infection with virulent F. tularensis. PMID:23378429

  5. About three cases of ulceroglandular tularemia, is this the re-emergence of Francisella tularensis in Belgium?

    PubMed

    Dupont, E; Van Eeckhoudt, S; Thissen, X; Ausselet, N; Fretin, D; Stefanescu, I; Glupczynski, Y; Delaere, B

    2015-10-01

    Tularemia is a zoonosis caused by Francisella tularensis that can be transmitted by several ways to human being and cause different clinical manifestations. We report three clinical cases of tularemia with ulceroglandular presentation in young males acquired during outdoor activities in Southern Belgium. Confirmation of the diagnosis was established by serology. Only three cases of tularemia have been reported in Belgium between 1950 and 2012 by the National Reference Laboratory CODA-CERVA (Ref Lab CODA-CERVA) but re-emergence of tularemia is established in several European countries and F. tularensis is also well known to be present in animal reservoirs and vectors in Belgium. The diagnosis of tularemia has to be considered in case of suggestive clinical presentation associated with epidemiological risk factors.

  6. Generating Isogenic Deletions (Knockouts) in Francisella tularensis, a Highly-infectious and Fastidious Gram-negative Bacterium

    PubMed Central

    Huntley, Jason F.

    2015-01-01

    Generating bacterial gene deletion mutants, also known as knockouts (KOs), is a powerful tool to investigate individual gene functions. However, fastidious bacteria such as Francisella tularensis (F. tularensis) often are difficult to genetically manipulate. Indeed, many different approaches have been tested to generate F. tularensis mutants. First, Tn5-based EZ::TN transposons have been successfully used to generate transposon libraries in F. tularensis (Qin and Mann, 2006; Weiss et al., 2007). However, creating a comprehensive transposon library with saturating mutations can be laborious, screening for gene disruption requires high-throughput assays where known phenotypes can be measured, and transposons may not completely inactivate the gene of interest or may alter downstream gene expression. Second, group II introns (also referred to as Targetron) have been used to inactivate F. tularensis genes of interest (Rodriguez et al., 2008; Rodriguez et al., 2009). Targetron functions by forming a complex between plasmid-encoded RNA and chromosomal DNA, followed by group II intron insertion into the gene of interest. The main advantage of Targetron is that it does not require an antibiotic resistance marker. However, as noted for transposons, targetron gene insertions may not eliminate all gene functions or may affect downstream gene expression. Third, homologous recombination can be used to completely replace the chromosomal target gene with a selectable marker, such as an antibiotic resistance marker. This classical genetic technique has been used in many F. tularensis studies (Ramakrishnan et al., 2008; Ren et al., 2014; Mohapatra et al., 2008; Robertson et al., 2013). To accomplish this, a suicide plasmid is engineered to include a selectable marker flanked by regions upstream and downstream of the gene of interest. This KO plasmid can be delivered into host bacteria by many methods, including electroporation, chemical transformation, or conjugation. Here, we

  7. The Role of the Francisella Tularensis Pathogenicity Island in Type VI Secretion, Intracellular Survival, and Modulation of Host Cell Signaling

    PubMed Central

    Bröms, Jeanette E.; Sjöstedt, Anders; Lavander, Moa

    2010-01-01

    Francisella tularensis is a highly virulent gram-negative intracellular bacterium that causes the zoonotic disease tularemia. Essential for its virulence is the ability to multiply within host cells, in particular monocytic cells. The bacterium has developed intricate means to subvert host immune mechanisms and thereby facilitate its intracellular survival by preventing phagolysosomal fusion followed by escape into the cytosol, where it multiplies. Moreover, it targets and manipulates numerous host cell signaling pathways, thereby ameliorating the otherwise bactericidal capacity. Many of the underlying molecular mechanisms still remain unknown but key elements, directly or indirectly responsible for many of the aforementioned mechanisms, rely on the expression of proteins encoded by the Francisella pathogenicity island (FPI), suggested to constitute a type VI secretion system. We here describe the current knowledge regarding the components of the FPI and the roles that have been ascribed to them. PMID:21687753

  8. Novel engineered cationic antimicrobial peptides display broad-spectrum activity against Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei.

    PubMed

    Abdelbaqi, Suha; Deslouches, Berthony; Steckbeck, Jonathan; Montelaro, Ronald; Reed, Douglas S

    2016-02-01

    Broad-spectrum antimicrobials are needed to effectively treat patients infected in the event of a pandemic or intentional release of a pathogen prior to confirmation of the pathogen's identity. Engineered cationic antimicrobial peptides (eCAPs) display activity against a number of bacterial pathogens including multi-drug-resistant strains. Two lead eCAPs, WLBU2 and WR12, were compared with human cathelicidin (LL-37) against three highly pathogenic bacteria: Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei. Both WLBU2 and WR12 demonstrated bactericidal activity greater than that of LL-37, particularly against F. tularensis and Y. pestis. Only WLBU2 had bactericidal activity against B. pseudomallei. WLBU2, WR12 and LL-37 were all able to inhibit the growth of the three bacteria in vitro. Because these bacteria can be facultative intracellular pathogens, preferentially infecting macrophages and dendritic cells, we evaluated the activity of WLBU2 against F. tularensis in an ex vivo infection model with J774 cells, a mouse macrophage cell line. In that model WLBU2 was able to achieve greater than 50% killing of F. tularensis at a concentration of 12.5 μM. These data show the therapeutic potential of eCAPs, particularly WLBU2, as a broad-spectrum antimicrobial for treating highly pathogenic bacterial infections. PMID:26673248

  9. Recovery of Francisella tularensis from soil samples by filtration and detection by real-time PCR and cELISA.

    PubMed

    Sellek, Ricela; Jimenez, Oscar; Aizpurua, Carmen; Fernandez-Frutos, Begoña; De Leon, Patricia; Camacho, Maite; Fernandez-Moreira, Daniel; Ybarra, Carmen; Carlos Cabria, Juan

    2008-03-01

    The aim of this study was to develop a specific and highly sensitive method able to detect very low concentrations of Francisella tularensis in soil samples by real-time PCR (qPCR) with SYBR Green I. tul4 gene, which encodes the 17-kDa protein (TUL4) in F. tularensis strains, was amplified using a LightCycler (LC) device. We achieved a detection limit of 0.69 fg of genomic DNA from F. tularensis subp. holarctica live vaccine strain (LVS), corresponding to a value less than 3.4 genome equivalents per reaction. The qPCR was shown to be specific, highly sensitive and reproducible. In addition, we evaluated 2 new methods for recovering bacteria from soil based on 1-step filtration using glass fiber filters and PVDF filters. These filtration methods enabled us to recover F. tularensis efficiently from soil samples. As few as 50 CFU per 0.5 g of soil were detected by qPCR. Capture enzyme-linked immunosorbent assay (cELISA) allowed us to detect and quantify the amount of bacteria recovered from soil by an immunological method. Although qPCR was more sensitive than cELISA, we did not observe substantial differences in the amount of bacteria quantified by both methods.

  10. Francisella tularensis Schu S4 O-antigen and capsule biosynthesis gene mutants induce early cell death in human macrophages.

    PubMed

    Lindemann, Stephen R; Peng, Kaitian; Long, Matthew E; Hunt, Jason R; Apicella, Michael A; Monack, Denise M; Allen, Lee-Ann H; Jones, Bradley D

    2011-02-01

    Francisella tularensis is capable of rampant intracellular growth and causes a potentially fatal disease in humans. Whereas many mutational studies have been performed with avirulent strains of Francisella, relatively little has been done with strains that cause human disease. We generated a near-saturating transposon library in the virulent strain Schu S4, which was subjected to high-throughput screening by transposon site hybridization through primary human macrophages, negatively selecting 202 genes. Of special note were genes in a locus of the Francisella chromosome, FTT1236, FTT1237, and FTT1238. Mutants with mutations in these genes demonstrated significant sensitivity to complement-mediated lysis compared with wild-type Schu S4 and exhibited marked defects in O-antigen and capsular polysaccharide biosynthesis. In the absence of complement, these mutants were phagocytosed more efficiently by macrophages than wild-type Schu S4 and were capable of phagosomal escape but exhibited reduced intracellular growth. Microscopic and quantitative analyses of macrophages infected with mutant bacteria revealed that these macrophages exhibited signs of cell death much earlier than those infected with Schu S4. These data suggest that FTT1236, FTT1237, and FTT1238 are important for polysaccharide biosynthesis and that the Francisella O antigen, capsule, or both are important for avoiding the early induction of macrophage death and the destruction of the replicative niche.

  11. New protein fold revealed by a 1.65 Å resolution crystal structure of Francisella tularensis pathogenicity island protein IglC

    PubMed Central

    Sun, Ping; Austin, Brian P.; Schubot, Florian D.; Waugh, David S.

    2007-01-01

    Francisella tularensis is a highly infectious Gram-negative intracellular pathogen that causes the fulminating disease tularemia and is considered to be a potential bioweapon. F. tularensis pathogenicity island proteins play a key role in modulating phagosome biogenesis and subsequent bacterial escape into the cytoplasm of macrophages. The 23 kDa pathogenicity island protein IglC is essential for the survival and proliferation of F. tularensis in macrophages. Seeking to gain some insight into its function, we determined the crystal structure of IglC at 1.65 Å resolution. IglC adopts a β-sandwich conformation that exhibits no similarity with any known protein structure. PMID:17905833

  12. Francisella tularensis IglG Belongs to a Novel Family of PAAR-Like T6SS Proteins and Harbors a Unique N-terminal Extension Required for Virulence

    PubMed Central

    Mosnier, Amandine; Hologne, Maggy; Martin, Amandine; Lindgren, Lena; Punginelli, Claire; Lays, Claire; Walker, Olivier; Charbit, Alain; Telouk, Philippe; Conlan, Wayne; Terradot, Laurent; Sjöstedt, Anders; Henry, Thomas

    2016-01-01

    The virulence of Francisella tularensis, the etiological agent of tularemia, relies on an atypical type VI secretion system (T6SS) encoded by a genomic island termed the Francisella Pathogenicity Island (FPI). While the importance of the FPI in F. tularensis virulence is clearly established, the precise role of most of the FPI-encoded proteins remains to be deciphered. In this study, using highly virulent F. tularensis strains and the closely related species F. novicida, IglG was characterized as a protein featuring a unique α-helical N-terminal extension and a domain of unknown function (DUF4280), present in more than 250 bacterial species. Three dimensional modeling of IglG and of the DUF4280 consensus protein sequence indicates that these proteins adopt a PAAR-like fold, suggesting they could cap the T6SS in a similar way as the recently described PAAR proteins. The newly identified PAAR-like motif is characterized by four conserved cysteine residues, also present in IglG, which may bind a metal atom. We demonstrate that IglG binds metal ions and that each individual cysteine is required for T6SS-dependent secretion of IglG and of the Hcp homologue, IglC and for the F. novicida intracellular life cycle. In contrast, the Francisella-specific N-terminal α-helical extension is not required for IglG secretion, but is critical for F. novicida virulence and for the interaction of IglG with another FPI-encoded protein, IglF. Altogether, our data suggest that IglG is a PAAR-like protein acting as a bi-modal protein that may connect the tip of the Francisella T6SS with a putative T6SS effector, IglF. PMID:27602570

  13. Francisella tularensis IglG Belongs to a Novel Family of PAAR-Like T6SS Proteins and Harbors a Unique N-terminal Extension Required for Virulence.

    PubMed

    Rigard, Mélanie; Bröms, Jeanette E; Mosnier, Amandine; Hologne, Maggy; Martin, Amandine; Lindgren, Lena; Punginelli, Claire; Lays, Claire; Walker, Olivier; Charbit, Alain; Telouk, Philippe; Conlan, Wayne; Terradot, Laurent; Sjöstedt, Anders; Henry, Thomas

    2016-09-01

    The virulence of Francisella tularensis, the etiological agent of tularemia, relies on an atypical type VI secretion system (T6SS) encoded by a genomic island termed the Francisella Pathogenicity Island (FPI). While the importance of the FPI in F. tularensis virulence is clearly established, the precise role of most of the FPI-encoded proteins remains to be deciphered. In this study, using highly virulent F. tularensis strains and the closely related species F. novicida, IglG was characterized as a protein featuring a unique α-helical N-terminal extension and a domain of unknown function (DUF4280), present in more than 250 bacterial species. Three dimensional modeling of IglG and of the DUF4280 consensus protein sequence indicates that these proteins adopt a PAAR-like fold, suggesting they could cap the T6SS in a similar way as the recently described PAAR proteins. The newly identified PAAR-like motif is characterized by four conserved cysteine residues, also present in IglG, which may bind a metal atom. We demonstrate that IglG binds metal ions and that each individual cysteine is required for T6SS-dependent secretion of IglG and of the Hcp homologue, IglC and for the F. novicida intracellular life cycle. In contrast, the Francisella-specific N-terminal α-helical extension is not required for IglG secretion, but is critical for F. novicida virulence and for the interaction of IglG with another FPI-encoded protein, IglF. Altogether, our data suggest that IglG is a PAAR-like protein acting as a bi-modal protein that may connect the tip of the Francisella T6SS with a putative T6SS effector, IglF. PMID:27602570

  14. [A water-borne tularemia outbreak caused by Francisella tularensis subspecies holarctica in Central Anatolia region].

    PubMed

    Ulu Kılıç, Ayşegül; Kılıç, Selçuk; Sencan, Irfan; Ciçek Şentürk, Gönül; Gürbüz, Yunus; Tütüncü, Emin Ediz; Celebi, Bekir; Kıcıman, Özlem; Ergönül, Önder

    2011-04-01

    In this study, we investigated a waterborne tularemia outbreak occured in Kadiozu, a village of Cerkes county of Cankiri province (located in North-west part of central Anatolia, Turkey) between 18 November 2009-24 December 2009. Active surveillance was conducted to determine clinical characteristics and risk factors of cases after two patients from the same village had been diagnosed as oropharyngeal tularemia. All villagers were examined, and clinical specimens from cases and water samples which may be the source of outbreak in the field investigations were taken. Cases were in the form of oropharyngeal, glandular and pneumonic. Polymerase chain reaction (PCR) and cultures were conducted from lymph node aspirates, throat swabs taken from cases and samples from water sources of epidemic zone. All serum samples taken from the villagers were screened for F.tularensis antibodies with microagglutination test (MAT). Oropharyngeal tularemia was diagnosed in 11 patients, glandular form in 3 patients and pneumonic form in one patient according to clinical and laboratory results. Age of the patients ranged between 6-75 years old (mean age: 52.5 years) and thirty one of them (54.7%) were female. MAT titers ranged between 1/160 and 1/5120 in cases of tularemia. Causative agent was grown in the cultures of two patients (including a throat swab and a lymph node aspirate). F.tularensis DNA was shown by PCR in a throat swab and four lymph node aspirates. F.tularensis was also detected by PCR in the water sample obtained from one of the spring water commonly used by villagers. Only one of the lymph node samples obtained from two different patients, was positive by direct fluorescent antibody method. Causative agent was defined as F.tularensis subsp. holarctica by conventional and also molecular methods. Patients were treated with aminoglycoside (streptomycin, gentamicin, amikacin) or quinolone (ciprofloxacin, levofloxacin) antibiotics. Treatment failure was observed in five

  15. Francisella tularensis novicida proteomic and transcriptomic data integration and annotation based on semantic web technologies

    PubMed Central

    Anwar, Nadia; Hunt, Ela

    2009-01-01

    Background This paper summarises the lessons and experiences gained from a case study of the application of semantic web technologies to the integration of data from the bacterial species Francisella tularensis novicida (Fn). Fn data sources are disparate and heterogeneous, as multiple laboratories across the world, using multiple technologies, perform experiments to understand the mechanism of virulence. It is hard to integrate these data sources in a flexible manner that allows new experimental data to be added and compared when required. Results Public domain data sources were combined in RDF. Using this connected graph of database cross references, we extended the annotations of an experimental data set by superimposing onto it the annotation graph. Identifiers used in the experimental data automatically resolved and the data acquired annotations in the rest of the RDF graph. This happened without the expensive manual annotation that would normally be required to produce these links. This graph of resolved identifiers was then used to combine two experimental data sets, a proteomics experiment and a transcriptomic experiment studying the mechanism of virulence through the comparison of wildtype Fn with an avirulent mutant strain. Conclusion We produced a graph of Fn cross references which enabled the combination of two experimental datasets. Through combination of these data we are able to perform queries that compare the results of the two experiments. We found that data are easily combined in RDF and that experimental results are easily compared when the data are integrated. We conclude that semantic data integration offers a convenient, simple and flexible solution to the integration of published and unpublished experimental data. PMID:19796400

  16. Characterization of the O-antigen Polymerase (Wzy) of Francisella tularensis*

    PubMed Central

    Kim, Tae-Hyun; Sebastian, Shite; Pinkham, Jessica T.; Ross, Robin A.; Blalock, LeeAnn T.; Kasper, Dennis L.

    2010-01-01

    The O-antigen polymerase of Gram-negative bacteria has been difficult to characterize. Herein we report the biochemical and functional characterization of the protein product (Wzy) of the gene annotated as the putative O-antigen polymerase, which is located in the O-antigen biosynthetic locus of Francisella tularensis. In silico analysis (homology searching, hydropathy plotting, and codon usage assessment) strongly suggested that Wzy is an O-antigen polymerase whose function is to catalyze the addition of newly synthesized O-antigen repeating units to a glycolipid consisting of lipid A, inner core polysaccharide, and one repeating unit of the O-polysaccharide (O-PS). To characterize the function of the Wzy protein, a non-polar deletion mutant of wzy was generated by allelic replacement, and the banding pattern of O-PS was observed by immunoblot analysis of whole-cell lysates obtained by SDS-PAGE and stained with an O-PS-specific monoclonal antibody. These immunoblot analyses showed that O-PS of the wzy mutant expresses only one repeating unit of O-antigen. Further biochemical characterization of the subcellular fractions of the wzy mutant demonstrated that (as is characteristic of O-antigen polymerase mutants) the low molecular weight O-antigen accumulates in the periplasm of the mutant. Site-directed mutagenesis based on protein homology and topology, which was carried out to locate a catalytic residue of the protein, showed that modification of specific residues (Gly176, Asp177, Gly323, and Tyr324) leads to a loss of O-PS polymerization. Topology models indicate that these amino acids most likely lie in close proximity on the bacterial surface. PMID:20605777

  17. Serosurveillance for Francisella tularensis among wild animals in Japan using a newly developed competitive enzyme-linked immunosorbent assay.

    PubMed

    Sharma, Neekun; Hotta, Akitoyo; Yamamoto, Yoshie; Uda, Akihiko; Fujita, Osamu; Mizoguchi, Toshio; Shindo, Junji; Park, Chun-Ho; Kudo, Noboru; Hatai, Hitoshi; Oyamada, Toshifumi; Yamada, Akio; Morikawa, Shigeru; Tanabayashi, Kiyoshi

    2014-04-01

    Tularemia, a highly infectious zoonotic disease caused by Francisella tularensis, occurs sporadically in Japan. However, little is known about the prevalence of the disease in wild animals. A total of 632 samples obtained from 150 Japanese black bears, 142 Japanese hares, 120 small rodents, 97 rats, 53 raptors, 26 Japanese monkeys, 21 Japanese raccoon dogs, 20 masked palm civets, and three Japanese red foxes between 2002 and 2010 were investigated for the presence of antibodies to F. tularensis by competitive enzyme-linked immunosorbent assay (cELISA) and the commonly used microagglutination (MA) test. Seropositive cELISA and MA results were obtained in 23 and 18 Japanese black bears, three and two Japanese raccoon dogs, and two and one small rodents, respectively. All MA-positive samples (n=21) were also positive by cELISA. Six of seven samples that were only positive by cELISA were confirmed to be antibody-positive by western blot analysis. These findings suggest that cELISA is a highly sensitive and useful test for serosurveillance of tularemia among various species of wild animals. Because this is the first study to detect F. tularensis-seropositive Japanese raccoon dogs, these could join Japanese black bears as sentinel animals for tularemia in the wild in Japan. Further continuous serosurveillance for F. tularensis in various species of wild animals using appropriate methods such as cELISA is important to assess the risks of human exposure and to improve our understanding of the ecology of F. tularensis in the wild. PMID:24689989

  18. Kinetic Characterization and Phosphoregulation of the Francisella tularensis 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase)

    PubMed Central

    Jawaid, Safdar; Seidle, Heather; Zhou, Weidong; Abdirahman, Hafsa; Abadeer, Maher; Hix, Joseph H.; van Hoek, Monique L.; Couch, Robin D.

    2009-01-01

    Deliberate and natural outbreaks of infectious disease underscore the necessity of effective vaccines and antimicrobial/antiviral therapeutics. The prevalence of antibiotic resistant strains and the ease by which antibiotic resistant bacteria can be intentionally engineered further highlights the need for continued development of novel antibiotics against new bacterial targets. Isoprenes are a class of molecules fundamentally involved in a variety of crucial biological functions. Mammalian cells utilize the mevalonic acid pathway for isoprene biosynthesis, whereas many bacteria utilize the methylerythritol phosphate (MEP) pathway, making the latter an attractive target for antibiotic development. In this report we describe the cloning and characterization of Francisella tularensis MEP synthase, a MEP pathway enzyme and potential target for antibiotic development. In vitro growth-inhibition assays using fosmidomycin, an inhibitor of MEP synthase, illustrates the effectiveness of MEP pathway inhibition with F. tularensis. To facilitate drug development, F. tularensis MEP synthase was cloned, expressed, purified, and characterized. Enzyme assays produced apparent kinetic constants (KMDXP = 104 µM, KMNADPH = 13 µM, kcatDXP = 2 s−1, kcatNADPH = 1.3 s−1), an IC50 for fosmidomycin of 247 nM, and a Ki for fosmidomycin of 99 nM. The enzyme exhibits a preference for Mg+2 as a divalent cation. Titanium dioxide chromatography-tandem mass spectrometry identified Ser177 as a site of phosphorylation. S177D and S177E site-directed mutants are inactive, suggesting a mechanism for post-translational control of metabolic flux through the F. tularensis MEP pathway. Overall, our study suggests that MEP synthase is an excellent target for the development of novel antibiotics against F. tularensis. PMID:20011597

  19. Development of a multitarget real-time TaqMan PCR assay for enhanced detection of Francisella tularensis in complex specimens.

    PubMed

    Versage, Jessica L; Severin, Darlena D M; Chu, May C; Petersen, Jeannine M

    2003-12-01

    Tularemia is the zoonotic disease caused by the gram-negative coccobacillus Francisella tularensis. Its wide distribution in the environment poses a challenge for understanding the transmission, ecology, and epidemiology of the disease. F. tularensis is also considered a potential biological weapon due to its extreme infectivity. We have developed a multitarget real-time TaqMan PCR assay capable of rapidly and accurately detecting F. tularensis in complex specimens. Targeted regions included the ISFtu2 element and the 23kDa, fopA, and tul4 genes. Analysis of the four TaqMan assays demonstrated that three (ISFtu2, 23kDa, and tul4) performed within our established criterion of a detection limit of one organism. The combined use of the three assays was highly specific, displaying no cross-reactivity with the non-Francisella bacteria tested and capable of differentially diagnosing both F. tularensis and Francisella philomiragia. When the multitarget TaqMan assay (ISFtu2, 23kDa, and tul4) was compared to culturing, using environmentally contaminated specimens, the TaqMan PCR assay was significantly more sensitive than culturing (P tularensis. PMID:14662930

  20. Identification of a Small Molecule That Modifies MglA/SspA Interaction and Impairs Intramacrophage Survival of Francisella tularensis

    PubMed Central

    Wrench, Algevis P.; Gardner, Christopher L.; Gonzalez, Claudio F.; Lorca, Graciela L.

    2013-01-01

    The transcription factors MglA and SspA of Francisella tularensis form a heterodimer complex and interact with the RNA polymerase to regulate the expression of the Francisella pathogenicity island (FPI) genes. These genes are essential for this pathogen’s virulence and survival within host cells. In this study, we used a small molecule screening to identify quinacrine as a thermal stabilizing compound for F. tularensis SCHU S4 MglA and SspA. A bacterial two-hybrid system was used to analyze the in vivo effect of quinacrine on the heterodimer complex. The results show that quinacrine affects the interaction between MglA and SspA, indicated by decreased β-galactosidase activity. Further in vitro analyses, using size exclusion chromatography, indicated that quinacrine does not disrupt the heterodimer formation, however, changes in the alpha helix content were confirmed by circular dichroism. Structure-guided site-directed mutagenesis experiments indicated that quinacrine makes contact with amino acid residues Y63 in MglA, and K97 in SspA, both located in the “cleft” of the interacting surfaces. In F. tularensis subsp. novicida, quinacrine decreased the transcription of the FPI genes, iglA, iglD, pdpD and pdpA. As a consequence, the intramacrophage survival capabilities of the bacteria were affected. These results support use of the MglA/SspA interacting surface, and quinacrine’s chemical scaffold, for the design of high affinity molecules that will function as therapeutics for the treatment of Tularemia. PMID:23372736

  1. 3-substituted indole inhibitors against Francisella tularensis FabI identified by structure-based virtual screening.

    PubMed

    Hu, Xin; Compton, Jaimee R; Abdulhameed, Mohamed Diwan M; Marchand, Charles L; Robertson, Kelly L; Leary, Dagmar H; Jadhav, Ajit; Hershfield, Jeremy R; Wallqvist, Anders; Friedlander, Arthur M; Legler, Patricia M

    2013-07-11

    In this study, we describe novel inhibitors against Francisella tularensis SchuS4 FabI identified from structure-based in silico screening with integrated molecular dynamics simulations to account for induced fit of a flexible loop crucial for inhibitor binding. Two 3-substituted indoles, 54 and 57, preferentially bound the NAD(+) form of the enzyme and inhibited growth of F. tularensis SchuS4 at concentrations near that of their measured Ki. While 57 was species-specific, 54 showed a broader spectrum of growth inhibition against F. tularensis , Bacillus anthracis , and Staphylococcus aureus . Binding interaction analysis in conjunction with site-directed mutagenesis revealed key residues and elements that contribute to inhibitor binding and species specificity. Mutation of Arg-96, a poorly conserved residue opposite the loop, was unexpectedly found to enhance inhibitor binding in the R96G and R96M variants. This residue may affect the stability and closure of the flexible loop to enhance inhibitor (or substrate) binding. PMID:23815100

  2. Prior Inoculation with Type B Strains of Francisella tularensis Provides Partial Protection against Virulent Type A Strains in Cottontail Rabbits.

    PubMed

    Brown, Vienna R; Adney, Danielle R; Olea-Popelka, Francisco; Bowen, Richard A

    2015-01-01

    Francisella tularensis is a highly virulent bacterium that is capable of causing severe disease (tularemia) in a wide range of species. This organism is characterized into two distinct subspecies: tularensis (type A) and holarctica (type B) which vary in several crucial ways, with some type A strains having been found to be considerably more virulent in humans and laboratory animals. Cottontail rabbits have been widely implicated as a reservoir species for this subspecies; however, experimental inoculation in our laboratory revealed type A organisms to be highly virulent, resulting in 100% mortality following challenge with 50-100 organisms. Inoculation of cottontail rabbits with the same number of organisms from type B strains of bacteria was found to be rarely lethal and to result in a robust humoral immune response. The objective of this study was to characterize the protection afforded by a prior challenge with type B strains against a later inoculation with a type A strain in North American cottontail rabbits (Sylvilagus spp). Previous infection with a type B strain of organism was found to lengthen survival time and in some cases prevent death following inoculation with a type A2 strain of F. tularensis. In contrast, inoculation of a type A1b strain was uniformly lethal in cottontail rabbits irrespective of a prior type B inoculation. These findings provide important insight about the role cottontail rabbits may play in environmental maintenance and transmission of this organism.

  3. Francisella tularensis Modulates a Distinct Subset of Regulatory Factors and Sustains Mitochondrial Integrity to Impair Human Neutrophil Apoptosis.

    PubMed

    McCracken, Jenna M; Kinkead, Lauren C; McCaffrey, Ramona L; Allen, Lee-Ann H

    2016-01-01

    Tularemia is a disease characterized by profound neutrophil accumulation and tissue destruction. The causative organism, Francisella tularensis, is a facultative intracellular bacterium that replicates in neutrophil cytosol, inhibits caspase activation and profoundly prolongs cell lifespan. Here, we identify unique features of this infection and provide fundamental insight into the mechanisms of apoptosis inhibition. Mitochondria are critical regulators of neutrophil apoptosis. We demonstrate that F. tularensis significantly inhibits Bax translocation and Bid processing during 24-48 h of infection, and in this manner sustains mitochondrial integrity. Downstream of mitochondria, X-linked inhibitor of apoptosis protein (XIAP) and proliferating cell nuclear antigen (PCNA) inhibit caspase-9 and caspase-3 by direct binding. Notably, we find that PCNA disappeared rapidly and selectively from infected cells, thereby demonstrating that it is not essential for neutrophil survival, whereas upregulation of calpastatin correlated with diminished calpain activity and reduced XIAP degradation. In addition, R-roscovitine is a cyclin-dependent kinase inhibitor developed for the treatment of cancer; it also induces neutrophil apoptosis and can promote the resolution of several infectious and inflammatory disorders. We confirm the ability of R-roscovitine to induce neutrophil apoptosis, but also demonstrate that its efficacy is significantly impaired by F. tularensis. Collectively, our findings advance the understanding of neutrophil apoptosis and its capacity to be manipulated by pathogenic bacteria.

  4. Crystal Structures of the Histidine Acid Phosphatase from Francisella tularensis Provide Insight into Substrate Recognition

    SciTech Connect

    Singh, Harkewal; Felts, Richard L.; Schuermann, Jonathan P.; Reilly, Thomas J.; Tanner, John J.

    2009-12-01

    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-{angstrom}-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 {angstrom}, respectively. The structure of the Asp261Ala mutant enzyme complexed with the substrate 3'-AMP was determined at 1.50 {angstrom} resolution to gain insight into substrate recognition. FtHAP exhibits a two-domain fold similar to that of human prostatic acid phosphatase, consisting of an {alpha}/{beta} 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-{angstrom}-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{sub m} by factors of 7 and 10, respectively. The structural data are consistent with a role for FtHAP in scavenging phosphate from small

  5. Serosurveillance for Francisella tularensis Among Wild Animals in Japan Using a Newly Developed Competitive Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Sharma, Neekun; Hotta, Akitoyo; Yamamoto, Yoshie; Uda, Akihiko; Fujita, Osamu; Mizoguchi, Toshio; Shindo, Junji; Park, Chun-Ho; Kudo, Noboru; Hatai, Hitoshi; Oyamada, Toshifumi; Yamada, Akio; Morikawa, Shigeru

    2014-01-01

    Abstract Tularemia, a highly infectious zoonotic disease caused by Francisella tularensis, occurs sporadically in Japan. However, little is known about the prevalence of the disease in wild animals. A total of 632 samples obtained from 150 Japanese black bears, 142 Japanese hares, 120 small rodents, 97 rats, 53 raptors, 26 Japanese monkeys, 21 Japanese raccoon dogs, 20 masked palm civets, and three Japanese red foxes between 2002 and 2010 were investigated for the presence of antibodies to F. tularensis by competitive enzyme-linked immunosorbent assay (cELISA) and the commonly used microagglutination (MA) test. Seropositive cELISA and MA results were obtained in 23 and 18 Japanese black bears, three and two Japanese raccoon dogs, and two and one small rodents, respectively. All MA-positive samples (n=21) were also positive by cELISA. Six of seven samples that were only positive by cELISA were confirmed to be antibody-positive by western blot analysis. These findings suggest that cELISA is a highly sensitive and useful test for serosurveillance of tularemia among various species of wild animals. Because this is the first study to detect F. tularensis–seropositive Japanese raccoon dogs, these could join Japanese black bears as sentinel animals for tularemia in the wild in Japan. Further continuous serosurveillance for F. tularensis in various species of wild animals using appropriate methods such as cELISA is important to assess the risks of human exposure and to improve our understanding of the ecology of F. tularensis in the wild. PMID:24689989

  6. The use of matrix-assisted laser desorption ionization-time of flight mass spectrometry in the identification of Francisella tularensis

    PubMed Central

    Karatuna, Onur; Çelebi, Bekir; Can, Simge; Akyar, Işın; Kiliç, Selçuk

    2016-01-01

    Francisella tularensis is the cause of the zoonotic disease tularemia and is classified among highly pathogenic bacteria (HPB) due to its low infection dose and potential for airborne transmission. In the case of HBP, there is a pressing need for rapid, accurate and reliable identification. Phenotypic identification of Francisella species is inappropriate for clinical microbiology laboratories because it is time-consuming, hazardous and subject to variable interpretation. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was recently evaluated as a useful tool for the rapid identification of a variety of microorganisms. In this study, we evaluated the use of MALDI-TOF MS for the rapid identification of Francisella tularensis and differentiation of its subspecies. Using national collection of Francisella isolates from the National Tularemia Reference Laboratory (Public Health Institution of Turkey, Ankara), a total of 75 clinical isolates were investigated by species and subspecies-specific polymerase chain reaction (PCR) test and MALDI-TOF MS. All isolates were originally identified as F. tularensis subsp. holarctica according to region of difference 1 (RD1) subspecies-specific PCR results. For all isolates MALDI-TOF MS provided results in concordance with subspecies-specific PCR analysis. Although PCR-based methods are effective in identifying Francisella species, they are labor-intensive and take longer periods of time to obtain the results when compared with MALDI-TOF MS. MALDI-TOF MS appeared to be a rapid, reliable and cost-effective identification technique for Francisella spp. Shorter analysis time and low cost make this an appealing new option in microbiology laboratories. PMID:26773181

  7. Evaluation of the FilmArray® system for detection of Bacillus anthracis, Francisella tularensis, and Yersinia pestis

    SciTech Connect

    Seiner, Derrick R.; Colburn, Heather A.; Baird, Cheryl L.; Bartholomew, Rachel A.; Straub, Tim M.; Victry, Kristin D.; Hutchison, Janine R.; Valentine, Nancy B.; Bruckner-Lea, Cindy J.

    2013-04-29

    To evaluate the sensitivity and specificity of the Idaho Technologies FilmArray® Biothreat Panel for the detection of Bacillus anthracis (Ba), Francisella tularensis (Ft), and Yersinia pestis (Yp) DNA, and demonstrate the detection of Ba spores. Methods and Results: DNA samples from Ba, Ft and Yp strains and near-neighbors, and live Ba spores were analyzed using the Biothreat Panel, a multiplexed PCR-based assay for 17 pathogens and toxins. Sensitivity studies with DNA suggest a limit of detection of 250 genome equivalents (GEs) per sample. Furthermore, the correct call of Ft, Yp or Bacillus species was made in 63 of 72 samples tested at 25 GE or less. With samples containing 25 Ba Sterne spores, at least one of the two possible Ba markers were identified in all samples tested. We observed no cross-reactivity with near-neighbor DNAs.

  8. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of macrophage growth locus A (MglA) protein from Francisella tularensis

    SciTech Connect

    Subburaman, P.; Austin, B.P.; Shaw, G.X.; Waugh, D.S.; Ji, X.

    2010-11-03

    Francisella tularensis, a potential bioweapon, causes a rare infectious disease called tularemia in humans and animals. The macrophage growth locus A (MglA) protein from F. tularensis associates with RNA polymerase to positively regulate the expression of multiple virulence factors that are required for its survival and replication within macrophages. The MglA protein was overproduced in Escherichia coli, purified and crystallized. The crystals diffracted to 7.5 {angstrom} resolution at the Advanced Photon Source, Argonne National Laboratory and belonged to the hexagonal space group P6{sub 1} or P6{sub 5}, with unit-cell parameters a = b = 125, c = 54 {angstrom}.

  9. Structure of the Francisella tularensis enoyl-acyl carrier protein reductase (FabI) in complex with NAD+ and triclosan

    PubMed Central

    Mehboob, Shahila; Truong, Kent; Santarsiero, Bernard D.; Johnson, Michael E.

    2010-01-01

    Enoyl-acyl carrier protein reductase (FabI) catalyzes the last rate-limiting step in the elongation cycle of the fatty-acid biosynthesis pathway and has been validated as a potential antimicrobial drug target in Francisella tularensis. The development of new antibiotic therapies is important both to combat potential drug-resistant bioweapons and to address the broader societal problem of increasing antibiotic resistance among many pathogenic bacteria. The crystal structure of FabI from F. tularensis (FtuFabI) in complex with the inhibitor triclosan and the cofactor NAD+ has been solved to a resolution of 2.1 Å. Triclosan is known to effectively inhibit FabI from different organisms. Precise characterization of the mode of triclosan binding is required to develop highly specific inhibitors. Comparison of our structure with the previously determined FtuFabI structure (PDB code 2jjy) which is bound to only NAD+ reveals the conformation of the substrate-binding loop, electron density for which was missing in the earlier structure, and demonstrates a shift in the conformation of the NAD+ cofactor. This shift in the position of the phosphate groups allows more room in the active site for substrate or inhibitor to bind and be better accommodated. This information will be crucial for virtual screening studies to identify novel scaffolds for development into new active inhibitors. PMID:21045289

  10. Non FcεR-bearing Mast Cells Secrete Sufficient Interleukin-4 to Control Francisella tularensis Replication within Macrophages

    PubMed Central

    Thathiah, Prea; Sanapala, Shilpa; Rodriguez, Annette R.; Yu, Jieh-Juen; Murthy, Ashlesh K.; Guentzel, M. Neal; Forsthuber, Thomas G.; Chambers, James P.; Arulanandam, Bernard P.

    2011-01-01

    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 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. PMID:21565523

  11. Inactivation of Francisella tularensis Gene Encoding Putative ABC Transporter Has a Pleiotropic Effect upon Production of Various Glycoconjugates.

    PubMed

    Dankova, Vera; Balonova, Lucie; Link, Marek; Straskova, Adela; Sheshko, Valeria; Stulik, Jiri

    2016-02-01

    Francisella tularensis, an intracellular pathogen causing the disease tularemia, utilizes surface glycoconjugates such as lipopolysaccharide, capsule, and capsule-like complex for its protection against inhospitable conditions of the environment. Francisella species also possess a functional glycosylation apparatus by which specific proteins are O-glycosidically modified. We here created a mutant with a nonfunctional FTS_1402 gene encoding for a putative glycan flippase and studied the consequences of its disruption. The mutant strain expressed diminished glycosylation similarly to, but to a lesser extent than, that of the oligosaccharyltransferase-deficient ΔpglA mutant. In contrast to ΔpglA, inactivation of FTS_1402 had a pleiotropic effect, leading to alteration in glycosylation and, importantly, to decrease in lipopolysaccharide, capsule, and/or capsule-like complex production, which were reflected by distinct phenotypes in host-pathogen associated properties and virulence potential of the two mutant strains. Disruption of FTS_1402 resulted in enhanced sensitivity to complement-mediated lysis and reduced virulence in mice that was independent of diminished glycosylation. Importantly, the mutant strain induced a protective immune response against systemic challenge with homologous wild-type FSC200 strain. Targeted disruption of genes shared by multiple metabolic pathways may be considered a novel strategy for constructing effective live, attenuated vaccines. PMID:26815358

  12. Understanding Virulence in the Brucellae and Francisellae: Towards Efficacious Treatments for Two Potential Biothreat Agents

    SciTech Connect

    Rasley, A; Parsons, D A; El-Etr, S; Roux, C; Tsolis, R

    2009-12-30

    Francisella tularensis, Yersinia pestis and Brucellae species are highly infectious pathogens classified as select agents by the Centers for Disease Control and Prevention (CDC) with the potential for use in bioterrorism attacks. These organisms are known to be facultative intracellular pathogens that preferentially infect human monocytes. As such, understanding how the host responds to infection with these organisms is paramount in detecting and combating human disease. We have compared the ability of fully virulent strains of each pathogen and their non-pathogenic near neighbors to enter and survive inside the human monocytic cell line THP-1 and have quantified the cellular response to infection with the goal of identifying both unique and common host response patterns. We expanded the scope of these studies to include experiments with pathogenic and non-pathogenic strains of Y. pestis, the causative agent of plague. Nonpathogenic strains of each organism were impaired in their ability to survive intracellularly compared with their pathogenic counterparts. Furthermore, infection of THP-1 cells with pathogenic strains of Y. pestis and F. tularensis resulted in marked increases in the secretion of the inflammatory chemokines IL-8, RANTES, and MIP-1{beta}. In contrast, B. melitensis infection failed to elicit any significant increases in a panel of cytokines tested. These differences may underscore distinct strategies in pathogenic mechanisms employed by these pathogens.

  13. Evidence Suggesting That Francisella tularensis O-Antigen Capsule Contains a Lipid A-Like Molecule That Is Structurally Distinct from the More Abundant Free Lipid A

    PubMed Central

    Barker, Jason H.; Kaufman, Justin W.; Apicella, Michael A.; Weiss, Jerrold P.

    2016-01-01

    Francisella tularensis, the Gram-negative bacterium that causes tularemia, produces a high molecular weight capsule that is immunologically distinct from Francisella lipopolysaccharide but contains the same O-antigen tetrasaccharide. To pursue the possibility that the capsule of Francisella live vaccine strain (LVS) has a structurally unique lipid anchor, we have metabolically labeled Francisella with [14C]acetate to facilitate highly sensitive compositional analysis of capsule-associated lipids. Capsule was purified by two independent methods and yielded similar results. Autoradiographic and immunologic analysis confirmed that this purified material was largely devoid of low molecular weight LPS and of the copious amounts of free lipid A that the Francisellae accumulate. Chemical hydrolysis yielded [14C]-labeled free fatty acids characteristic of Francisella lipid A but with a different molar ratio of 3-OH C18:0 to 3-OH C16:0 and different composition of non-hydroxylated fatty acids (mainly C14:0 rather than C16:0) than that of free Francisella lipid A. Mild acid hydrolysis to induce selective cleavage of KDO-lipid A linkage yielded a [14C]-labeled product that partitioned during Bligh/Dyer extraction and migrated during thin-layer chromatography like lipid A. These findings suggest that the O-antigen capsule of Francisella contains a covalently linked and structurally distinct lipid A species. The presence of a discrete lipid A-like molecule associated with capsule raises the possibility that Francisella selectively exploits lipid A structural heterogeneity to regulate synthesis, transport, and stable bacterial surface association of the O-antigen capsular layer. PMID:27326857

  14. Evaluation of an immunochromatographic test for rapid and reliable serodiagnosis of human tularemia and detection of Francisella tularensis-specific antibodies in sera from different mammalian species.

    PubMed

    Splettstoesser, W; Guglielmo-Viret, V; Seibold, E; Thullier, P

    2010-05-01

    Tularemia is a highly contagious infectious zoonosis caused by the bacterial agent Francisella tularensis. Serology is still considered to be a cornerstone in tularemia diagnosis due to the low sensitivity of bacterial culture and the lack of standardization in PCR methodology for the direct identification of the pathogen. We developed a novel immunochromatographic test (ICT) to efficiently detect F. tularensis-specific antibodies in sera from humans and other mammalian species (nonhuman primate, pig, and rabbit). This new tool requires none or minimal laboratory equipment, and the results are obtained within 15 min. When compared to the method of microagglutination, which was shown to be more specific than the enzyme-linked immunosorbent assay, the ICT had a sensitivity of 98.3% (58 positive sera were tested) and a specificity of 96.5% (58 negative sera were tested) on human sera. On animal sera, the overall sensitivity was 100% (22 positive sera were tested) and specificity was also 100% (70 negative sera were tested). This rapid test preferentially detects IgG antibodies that may occur early in the course of human tularemia, but further evaluation with human sera is important to prove that the ICT can be a valuable field test to support a presumptive diagnosis of tularemia. The ICT can also be a useful tool to monitor successful vaccination with subunit vaccines or live vaccine strains containing lipopolysaccharide (e.g., LVS) and to detect seropositive individuals or animals in outbreak situations or in the context of epidemiologic surveillance programs in areas of endemicity as recently recommended by the World Health Organization. PMID:20220165

  15. Seroprevalence for Coxiella burnetii, Francisella tularensis, Brucella abortus and Brucella melitensis in Austrian adults: a cross-sectional survey among military personnel and civilians.

    PubMed

    Tobudic, Selma; Nedomansky, Klara; Poeppl, Wolfgang; Müller, Maria; Faas, Angelus; Mooseder, Gerhard; Allerberger, Franz; Stanek, Gerold; Burgmann, Heinz

    2014-04-01

    The prevalence of Coxiella burnetii, Francisella tularensis, Brucella abortus, and Brucella melitensis infections in Austria and the exposure risk of military personnel were assessed in an exploratory nationwide cross-sectional seroprevalence survey in 526 healthy adult individuals, 222 of which were soldiers and 304 were civilians. Screening for IgA/IgG antibodies to C. burnetii (Phase I) and IgG/IgM antibodies to C. burnetii (Phase II), and to F. tularensis was done with commercial enzyme-linked immunosorbent assays. To detect antibodies against B. abortus and B. melitensis, an in-house complement fixation test was used. Overall, 11 individuals (2.0%) showed antibodies to C. burnetii, 3 individuals (0.5%) were seropositive for F. tularensis, and one (0.3%) individual was borderline positive. All individuals positive or borderline for F. tularensis tested negative for antibodies against C. burnetii. All individuals tested negative for antibodies against B. melitensis/B. abortus. There were no significant differences between the seroprevalence of C. burnetii and F. tularensis among military personnel and civilians. Our data demonstrate serological evidence of a low rate of exposure to C. burnetii and F. tularensis among the Austrian adult population and military personnel.

  16. Targeting of a Fixed Bacterial Immunogen to Fc Receptors Reverses the Anti-Inflammatory Properties of the Gram-Negative Bacterium, Francisella tularensis, during the Early Stages of Infection

    PubMed Central

    Babadjanova, Zulfia; Wiedinger, Kari; Gosselin, Edmund J.; Bitsaktsis, Constantine

    2015-01-01

    Production of pro-inflammatory cytokines by innate immune cells at the early stages of bacterial infection is important for host protection against the pathogen. Many intracellular bacteria, including Francisella tularensis, the agent of tularemia, utilize the anti-inflammatory cytokine IL-10, to evade the host immune response. It is well established that IL-10 has the ability to inhibit robust antigen presentation by dendritic cells and macrophages, thus suppressing the generation of protective immunity. The pathogenesis of F. tularensis is not fully understood, and research has failed to develop an effective vaccine to this date. In the current study, we hypothesized that F. tularensis polarizes antigen presenting cells during the early stages of infection towards an anti-inflammatory status characterized by increased synthesis of IL-10 and decreased production of IL-12p70 and TNF-α in an IFN-ɣ-dependent fashion. In addition, F. tularensis drives an alternative activation of alveolar macrophages within the first 48 hours post-infection, thus allowing the bacterium to avoid protective immunity. Furthermore, we demonstrate that targeting inactivated F. tularensis (iFt) to Fcγ receptors (FcɣRs) via intranasal immunization with mAb-iFt complexes, a proven vaccine strategy in our laboratories, reverses the anti-inflammatory effects of the bacterium on macrophages by down-regulating production of IL-10. More specifically, we observed that targeting of iFt to FcγRs enhances the classical activation of macrophages not only within the respiratory mucosa, but also systemically, at the early stages of infection. These results provide important insight for further understanding the protective immune mechanisms generated when targeting immunogens to Fc receptors. PMID:26114641

  17. Use of a Capture-Based Pathogen Transcript Enrichment Strategy for RNA-Seq Analysis of the Francisella Tularensis LVS Transcriptome during Infection of Murine Macrophages

    PubMed Central

    Bent, Zachary W.; Brazel, David M.; Tran-Gyamfi, Mary B.; Hamblin, Rachelle Y.; VanderNoot, Victoria A.; Branda, Steven S.

    2013-01-01

    Francisella tularensis is a zoonotic intracellular pathogen that is capable of causing potentially fatal human infections. Like all successful bacterial pathogens, F. tularensis rapidly responds to changes in its environment during infection of host cells, and upon encountering different microenvironments within those cells. This ability to appropriately respond to the challenges of infection requires rapid and global shifts in gene expression patterns. In this study, we use a novel pathogen transcript enrichment strategy and whole transcriptome sequencing (RNA-Seq) to perform a detailed characterization of the rapid and global shifts in F. tularensis LVS gene expression during infection of murine macrophages. We performed differential gene expression analysis on all bacterial genes at two key stages of infection: phagosomal escape, and cytosolic replication. By comparing the F. tularensis transcriptome at these two stages of infection to that of the bacteria grown in culture, we were able to identify sets of genes that are differentially expressed over the course of infection. This analysis revealed the temporally dynamic expression of a number of known and putative transcriptional regulators and virulence factors, providing insight into their role during infection. In addition, we identified several F. tularensis genes that are significantly up-regulated during infection but had not been previously identified as virulence factors. These unknown genes may make attractive therapeutic or vaccine targets. PMID:24155975

  18. Hare-to-Human Transmission of Francisella tularensis subsp. holarctica, Germany

    PubMed Central

    Otto, Peter; Kohlmann, Rebekka; Müller, Wolfgang; Julich, Sandra; Geis, Gabriele; Gatermann, Sören G.; Peters, Martin; Wolf, Peter Johannes; Karlsson, Edvin; Forsman, Mats; Myrtennäs, Kerstin

    2015-01-01

    In November 2012, a group of 7 persons who participated in a hare hunt in North Rhine-Westphalia, Germany, acquired tularemia. Two F. tularensis subsp. holarctica isolates were cultivated from human and hare biopsy material. Both isolates belonged to the FTN002–00 genetic subclade (derived for single nucleotide polymorphisms B.10 and B.18), thus indicating likely hare-to-human transmission. PMID:25531286

  19. Levofloxacin Rescues Mice from Lethal Intra-nasal Infections with Virulent Francisella tularensis and Induces Immunity and Production of Protective Antibody

    PubMed Central

    Klimpel, Gary R.; Eaves-Pyles, Tonyia; Moen, Scott T.; Taormina, Joanna; Peterson, Johnny W.; Chopra, Ashok K.; Niesel, David W.; Carness, Paige; Haithcoat, Judith L.; Kirtley, Michelle; Ben Nasr, Abdelhakim

    2009-01-01

    The ability to protect mice against respiratory infections with virulent Francisella tularensis has been problematic and the role of antibody-versus-cell-mediated immunity controversial. In this study, we tested the hypothesis that protective immunity can develop in mice that were given antibiotic therapy following infection via the respiratory tract with Francisella tularensis SCHU S4. We show that mice infected with a lethal dose of SCHU S4, via an intra-nasal challenge, could be protected with levofloxacin treatment. This protection was evident even when levofloxacin treatment was delayed 72 hours post-infection. At early time points after levofloxacin treatment, significant numbers of bacteria could be recovered from the lungs and spleens of mice, which was followed by a dramatic disappearance of bacteria from these tissues. Mice successfully treated with levofloxacin were later shown to be almost completely resistant to rechallenge with SCHU S4 by the intra-nasal route. Serum antibody appeared to play an important role in this immunity. Normal mice, when given sera from animals protected by levofloxacin treatment, were solidly protected from a lethal intra-nasal challenge with SCHU S4. The protective antiserum contained high titers of SCHU S4 specific IgG2a, indicating that a strong Th1 response was induced following levofloxacin treatment. Thus, this study describes a potentially valuable animal model for furthering our understanding of respiratory tularemia and provides suggestive evidence that antibody can protect against respiratory infections with virulent F. tularensis. PMID:18930100

  20. Temporal Transcriptional Response during Infection of Type II Alveolar Epithelial Cells with Francisella tularensis Live Vaccine Strain (LVS) Supports a General Host Suppression and Bacterial Uptake by Macropinocytosis*

    PubMed Central

    Bradburne, Christopher E.; Verhoeven, Anne B.; Manyam, Ganiraju C.; Chaudhry, Saira A.; Chang, Eddie L.; Thach, Dzung C.; Bailey, Charles L.; van Hoek, Monique L.

    2013-01-01

    Pneumonic tularemia is caused by inhalation of Francisella tularensis, one of the most infectious microbes known. We wanted to study the kinetics of the initial and early interactions between bacterium and host cells in the lung. To do this, we examined the infection of A549 airway epithelial cells with the live vaccine strain (LVS) of F. tularensis. A549 cells were infected and analyzed for global transcriptional response at multiple time points up to 16 h following infection. At 15 min and 2 h, a strong transcriptional response was observed including cytoskeletal rearrangement, intracellular transport, and interferon signaling. However, at later time points (6 and 16 h), very little differential gene expression was observed, indicating a general suppression of the host response consistent with other reported cell lines and murine tissues. Genes for macropinocytosis and actin/cytoskeleton rearrangement were highly up-regulated and common to the 15 min and 2 h time points, suggesting the use of this method for bacterial entry into cells. We demonstrate macropinocytosis through the uptake of FITC-dextran and amiloride inhibition of Francisella LVS uptake. Our results suggest that macropinocytosis is a potential mechanism of intracellular entry by LVS and that the host cell response is suppressed during the first 2–6 h of infection. These results suggest that the attenuated Francisella LVS induces significant host cell signaling at very early time points after the bacteria's interaction with the cell. PMID:23322778

  1. Inclusion of Epitopes That Expand High-Avidity CD4+ T Cells Transforms Subprotective Vaccines to Efficacious Immunogens against Virulent Francisella tularensis.

    PubMed

    Roberts, Lydia M; Crane, Deborah D; Wehrly, Tara D; Fletcher, Joshua R; Jones, Bradley D; Bosio, Catharine M

    2016-10-01

    T cells are the immunological cornerstone in host defense against infections by intracellular bacterial pathogens, such as virulent Francisella tularensis spp. tularensis (Ftt). The general paucity of novel vaccines for Ftt during the past 60 y can, in part, be attributed to the poor understanding of immune parameters required to survive infection. Thus, we developed a strategy utilizing classical immunological tools to elucidate requirements for effective adaptive immune responses directed against Ftt. Following generation of various Francisella strains expressing well-characterized lymphocytic choriomeningitis virus epitopes, we found that survival correlated with persistence of Ag-specific CD4(+) T cells. Function of these cells was confirmed in their ability to more effectively control Ftt replication in vitro. The importance of understanding the Ag-specific response was underscored by our observation that inclusion of an epitope that elicits high-avidity CD4(+) T cells converted a poorly protective vaccine to one that engenders 100% protection. Taken together, these data suggest that improved efficacy of current tularemia vaccine platforms will require targeting appropriate Ag-specific CD4(+) T cell responses and that elucidation of Francisella epitopes that elicit high-avidity CD4(+) T cell responses, specifically in humans, will be required for successful vaccine development. PMID:27543611

  2. Slow-Onset Inhibition of the FabI Enoyl Reductase from Francisella tularensis: Residence Time and in Vivo Activity

    SciTech Connect

    Lu, H.; England, K; Ende, C; Truglio, J; Luckner, S; Reddy, B; Marlenee, N; Knudson, S; Knudson, D; et. al.

    2009-01-01

    Francisella tularensis is a highly virulent and contagious Gram-negative intracellular bacterium that causes the disease tularemia in mammals. The high infectivity and the ability of the bacterium to survive for weeks in a cool, moist environment have raised the possibility that this organism could be exploited deliberately as a potential biological weapon. Fatty acid biosynthesis (FAS-II) is essential for bacterial viability and has been validated as a target for the discovery of novel antibacterials. The FAS-II enoyl reductase ftuFabI has been cloned and expressed, and a series of diphenyl ethers have been identified that are subnanomolar inhibitors of the enzyme with MIC90 values as low as 0.00018 ?g mL-1. The existence of a linear correlation between the Ki and MIC values strongly suggests that the antibacterial activity of the diphenyl ethers results from direct inhibition of ftuFabI within the cell. The compounds are slow-onset inhibitors of ftuFabI, and the residence time of the inhibitors on the enzyme correlates with their in vivo activity in a mouse model of tularemia infection. Significantly, the rate of breakdown of the enzyme-inhibitor complex is a better predictor of in vivo activity than the overall thermodynamic stability of the complex, a concept that has important implications for the discovery of novel chemotherapeutics that normally rely on equilibrium measurements of potency.

  3. From microfluidic modules to an integrated Lab-on-a-chip system for the detection of Francisella tularensis

    NASA Astrophysics Data System (ADS)

    Hlawatsch, Nadine; Krumbholz, Marco; Prüfer, Anna; Moche, Christian; Becker, Holger; Gärtner, Claudia

    2013-05-01

    Lab-on-a-chip (LoC) systems translating the whole process of pathogen analysis to an integrated, miniaturized, and automatically functioning microfluidic platform are generally expected to be very promising future diagnostic approaches. The development of such a LoC system for the detection of bacterial pathogens applied to the example pathogen Francisella tularensis is described in this report. To allow functional testing of the whole process cascade before final device integration, various bio-analytical steps such as cell lysis, DNA extraction and purification, continuous-flow PCR and analyte detection have been adapted to unique functional microfluidic modules. As a successive step, positively tested modules for pathogen detection have been successfully assembled to an integrated chip. Moreover, technical solutions for a smooth interaction between sample input from the outer world as well as microfluidic chip and chip driving instrument have been developed. In conclusion, a full repertoire of analytical tools have been developed and successfully tested in the concerted manner of a functionally integrated microfluidic device representing a tool for future diagnostic approaches.

  4. Evaluation of the FilmArray® system for detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis

    PubMed Central

    Seiner, DR; Colburn, HA; Baird, C; Bartholomew, RA; Straub, T; Victry, K; Hutchison, JR; Valentine, N; Bruckner-Lea, CJ

    2013-01-01

    Aims To evaluate the sensitivity and specificity of the BioFire Diagnostics FilmArray® system in combination with their Biothreat Panel for the detection of Bacillus anthracis (Ba), Francisella tularensis (Ft) and Yersinia pestis (Yp) DNA, and demonstrate the detection of Ba spores. Methods and Results DNA samples from Ba, Ft and Yp strains and near-neighbours, and live Ba spores were analysed using the FilmArray® Biothreat Panel, a multiplexed PCR-based assay for 17 pathogens and toxins. Sensitivity studies with DNA indicate that the limit of detection is 250 genome equivalents (GEs) per sample or lower. Furthermore, the identification of Ft, Yp or Bacillus species was made in 63 of 72 samples tested at 25 GE or less. With samples containing 25 CFU of Ba Sterne spores, at least one of the two possible Ba markers was identified in all samples tested. We observed no cross-reactivity with near-neighbour DNAs. Conclusions Our results indicate that the FilmArray® Biothreat Panel is a sensitive and selective assay for detecting the genetic signatures of Ba, Ft and Yp. Significance and Impact of the Study The FilmArray® platform is a complete sample-to-answer system, combining sample preparation, PCR and data analysis. This system is particularly suited for biothreat testing where samples need to be analysed for multiple biothreats by operators with limited training. PMID:23279070

  5. Diversity of Francisella species in environmental samples from Martha's Vineyard, Massachusetts.

    PubMed

    Berrada, Zenda L; Telford, Sam R

    2010-02-01

    We determined whether Francisella spp. are present in water, sediment, and soil from an active tularemia natural focus on Martha's Vineyard, Massachusetts, during a multiyear outbreak of pneumonic tularemia. Environmental samples were tested by polymerase chain reaction (PCR) targeting Francisella species 16S rRNA gene and succinate dehydrogenase A (sdhA) sequences; evidence of the agent of tularemia was sought by amplification of Francisella tularensis-specific sequences for the insertion element ISFTu2, 17-kDa protein gene tul4, and the 43-kDa outer membrane protein gene fopA. Evidence of F. tularensis subsp. tularensis, the causative agent of the human infections in this outbreak, was not detected from environmental samples despite its active transmission among ticks and animals in the sampling site. Francisella philomiragia was frequently detected from a brackish-water pond using Francisella species PCR targets, and subsequently F. philomiragia was isolated from an individual brackish-water sample. Distinct Francisella sp. sequences that are closely related to F. tularensis and Francisella novicida were detected from samples collected from the brackish-water pond. We conclude that diverse Francisella spp. are present in the environment where human cases of pneumonic tularemia occur.

  6. Perforin- and Granzyme-Mediated Cytotoxic Effector Functions Are Essential for Protection against Francisella tularensis following Vaccination by the Defined F. tularensis subsp. novicida ΔfopC Vaccine Strain

    PubMed Central

    Sanapala, Shilpa; Yu, Jieh-Juen; Murthy, Ashlesh K.; Li, Weidang; Guentzel, M. Neal; Chambers, James P.; Klose, Karl E.

    2012-01-01

    A licensed vaccine against Francisella tularensis is currently not available. Two Francisella tularensis subsp. novicida (herein referred to by its earlier name, Francisella novicida) attenuated strains, the ΔiglB and ΔfopC strains, have previously been evaluated as potential vaccine candidates against pneumonic tularemia in experimental animals. F. novicida ΔiglB, a Francisella pathogenicity island (FPI) mutant, is deficient in phagosomal escape and intracellular growth, whereas F. novicida ΔfopC, lacking the outer membrane lipoprotein FopC, which is required for evasion of gamma interferon (IFN-γ)-mediated signaling, is able to escape and replicate in the cytosol. To dissect the difference in protective immune mechanisms conferred by these two vaccine strains, we examined the efficacy of the F. novicida ΔiglB and ΔfopC mutants against pulmonary live-vaccine-strain (LVS) challenge and found that both strains provided comparable protection in wild-type, major histocompatibility complex class I (MHC I) knockout, and MHC II knockout mice. However, F. novicida ΔfopC-vaccinated but not F. novicida ΔiglB-vaccinated perforin-deficient mice were more susceptible and exhibited greater bacterial burdens than similarly vaccinated wild-type mice. Moreover, perforin produced by natural killer (NK) cells and release of granzyme contributed to inhibition of LVS replication within macrophages. This NK cell-mediated LVS inhibition was enhanced with anti-F. novicida ΔfopC immune serum, suggesting antibody-dependent cell-mediated cytotoxicity (ADCC) in F. novicida ΔfopC-mediated protection. Overall, this study provides additional immunological insight into the basis for protection conferred by live attenuated F. novicida strains with different phenotypes and supports further investigation of this organism as a vaccine platform for tularemia. PMID:22493083

  7. Models Derived from In Vitro Analyses of Spleen, Liver, and Lung Leukocyte Functions Predict Vaccine Efficacy against the Francisella tularensis Live Vaccine Strain (LVS)

    PubMed Central

    De Pascalis, Roberto; Chou, Alicia Y.; Ryden, Patrik; Kennett, Nikki J.; Sjöstedt, Anders; Elkins, Karen L.

    2014-01-01

    ABSTRACT Currently, there are no licensed vaccines and no correlates of protection against Francisella tularensis, which causes tularemia. We recently demonstrated that measuring in vitro control of intramacrophage bacterial growth by murine F. tularensis-immune splenocytes, as well as transcriptional analyses, discriminated Francisella vaccines of different efficacies. Further, we identified potential correlates of protection against systemic challenge. Here, we extended this approach by studying leukocytes derived from lungs and livers of mice immunized by parenteral and respiratory routes with F. tularensis vaccines. Liver and lung leukocytes derived from intradermally and intranasally vaccinated mice controlled in vitro Francisella Live Vaccine Strain (LVS) intramacrophage replication in patterns similar to those of splenocytes. Gene expression analyses of potential correlates also revealed similar patterns in liver cells and splenocytes. In some cases (e.g., tumor necrosis factor alpha [TNF-α], interleukin 22 [IL-22], and granulocyte-macrophage colony-stimulating factor [GM-CSF]), liver cells exhibited even higher relative gene expression, whereas fewer genes exhibited differential expression in lung cells. In contrast with their strong ability to control LVS replication, splenocytes from intranasally vaccinated mice expressed few genes with a hierarchy of expression similar to that of splenocytes from intradermally vaccinated mice. Thus, the relative levels of gene expression vary between cell types from different organs and by vaccination route. Most importantly, because studies comparing cell sources and routes of vaccination supported the predictive validity of this coculture and gene quantification approach, we combined in vitro LVS replication with gene expression data to develop analytical models that discriminated between vaccine groups and successfully predicted the degree of vaccine efficacy. Thus, this strategy remains a promising means of

  8. A rapid real-time quantitative PCR assay to determine the minimal inhibitory extracellular concentration of antibiotics against an intracellular Francisella tularensis Live Vaccine Strain

    PubMed Central

    Aloni-Grinstein, Ronit; Shifman, Ohad; Lazar, Shlomi; Steinberger-Levy, Ida; Maoz, Sharon; Ber, Raphael

    2015-01-01

    Francisella tularensis is a highly virulent facultative intracellular bacterium. The lack of a safe and efficient vaccine makes antibiotics the preferred treatment. F. tularensis antibiotic susceptibility tests are based on the in vitro standard CLSI-approved microdilution method for determining the MIC. However, limited data are available regarding the minimal inhibitory extracellular concentration (MIEC) needed to eradicate intracellular bacteria. Here, we evaluated the MIEC values of various WHO-recommended antibiotics and compared the MIEC values to the established MICs. We describe a rapid 3-h quantitative PCR (qPCR) intracellular antibiogram assay, which yields comparable MIEC values to those obtained by the classical 72-h cfu assay. This rapid qPCR assay is highly advantageous in light of the slow growth rates of F. tularensis. Our results showed that the MIECs obtained for doxycycline, chloramphenicol and ciprofloxacin were indicative of intracellular activity. Gentamicin was not effective against intracellular bacteria for at least 32 h post treatment, raising the question of whether slow-penetrating gentamicin should be used for certain stages of the disease. We suggest that the qPCR intracellular antibiogram assay may be used to screen for potentially active antibiotics against intracellular F. tularensis as well as to detect strains with acquired resistance to recommended antibiotics. PMID:26579112

  9. Francisella tularensis Schu S4 Lipopolysaccharide Core Sugar and O-Antigen Mutants Are Attenuated in a Mouse Model of Tularemia

    PubMed Central

    Rasmussen, Jed A.; Post, Deborah M. B.; Gibson, Bradford W.; Lindemann, Stephen R.; Apicella, Michael A.; Meyerholz, David K.

    2014-01-01

    The virulence factors mediating Francisella pathogenesis are being investigated, with an emphasis on understanding how the organism evades innate immunity mechanisms. Francisella tularensis produces a lipopolysaccharide (LPS) that is essentially inert and a polysaccharide capsule that helps the organism to evade detection by components of innate immunity. Using an F. tularensis Schu S4 mutant library, we identified strains that are disrupted for capsule and O-antigen production. These serum-sensitive strains lack both capsule production and O-antigen laddering. Analysis of the predicted protein sequences for the disrupted genes (FTT1236 and FTT1238c) revealed similarity to those for waa (rfa) biosynthetic genes in other bacteria. Mass spectrometry further revealed that these proteins are involved in LPS core sugar biosynthesis and the ligation of O antigen to the LPS core sugars. The 50% lethal dose (LD50) values of these strains are increased 100- to 1,000-fold for mice. Histopathology revealed that the immune response to the F. tularensis mutant strains was significantly different from that observed with wild-type-infected mice. The lung tissue from mutant-infected mice had widespread necrotic debris, but the spleens lacked necrosis and displayed neutrophilia. In contrast, the lungs of wild-type-infected mice had nominal necrosis, but the spleens had widespread necrosis. These data indicate that murine death caused by wild-type strains occurs by a mechanism different from that by which the mutant strains kill mice. Mice immunized with these mutant strains displayed >10-fold protective effects against virulent type A F. tularensis challenge. PMID:24452684

  10. Downmodulation of Vaccine-Induced Immunity and Protection against the Intracellular Bacterium Francisella tularensis by the Inhibitory Receptor FcγRIIB

    PubMed Central

    Franz, Brian J.; Li, Ying; Bitsaktsis, Constantine; Iglesias, Bibiana V.; Pham, Giang; Sunagar, Raju; Kumar, Sudeep; Gosselin, Edmund J.

    2015-01-01

    Fc gamma receptor IIB (FcγRIIB) is the only Fc gamma receptor (FcγR) which negatively regulates the immune response, when engaged by antigen- (Ag-) antibody (Ab) complexes. Thus, the generation of Ag-specific IgG in response to infection or immunization has the potential to downmodulate immune protection against infection. Therefore, we sought to determine the impact of FcγRIIB on immune protection against Francisella tularensis (Ft), a Category A biothreat agent. We utilized inactivated Ft (iFt) as an immunogen. Naïve and iFt-immunized FcγRIIB knockout (KO) or wildtype (WT) mice were challenged with Ft-live vaccine strain (LVS). While no significant difference in survival between naïve FcγRIIB KO versus WT mice was observed, iFt-immunized FcγRIIB KO mice were significantly better protected than iFt-immunized WT mice. Ft-specific IgA in serum and bronchial alveolar lavage, as well as IFN-γ, IL-10, and TNF-α production by splenocytes harvested from iFt-immunized FcγRIIB KO, were also significantly elevated. In addition, iFt-immunized FcγRIIB KO mice exhibited a reduction in proinflammatory cytokine levels in vivo at 5 days after challenge, which correlates with increased survival following Ft-LVS challenge in published studies. Thus, these studies demonstrate for the first time the ability of FcγRIIB to regulate vaccine-induced IgA production and downmodulate immunity and protection. The immune mechanisms behind the above observations and their potential impact on vaccine development are discussed. PMID:25961064

  11. Functional and Structural Characterization of Francisella tularensis O-Antigen Antibodies at the Low End of Antigen Reactivity

    PubMed Central

    Lu, Zhaohua; Rynkiewicz, Michael J.; Yang, Chiou-Ying; Madico, Guillermo; Perkins, Hillary M.; Roche, Marly I.; Seaton, Barbara A.

    2014-01-01

    The O-antigen (OAg) of the Gram-negative bacterium Francisella tularensis (Ft), which is both a capsular polysaccharide and a component of lipopolysaccharide, is comprised of tetrasaccharide repeats and induces antibodies mainly against repeating internal epitopes. We previously reported on several BALB/c mouse monoclonal antibodies (MAbs) that bind to internal Ft OAg epitopes and are protective in mouse models of respiratory tularemia. We now characterize three new internal Ft OAg IgG2a MAbs, N203, N77, and N24, with 10- to 100-fold lower binding potency than previously characterized internal-OAg IgG2a MAbs, despite sharing one or more variable region germline genes with some of them. In a mouse model of respiratory tularemia with the highly virulent Ft type A strain SchuS4, the three new MAbs reduced blood bacterial burden with potencies that mirror their antigen-binding strength; the best binder of the new MAbs, N203, prolonged survival in a dose-dependent manner, but was at least 10-fold less potent than the best previously characterized IgG2a MAb, Ab52. X-ray crystallographic studies of N203 Fab showed a flexible binding site in the form of a partitioned groove, which cannot provide as many contacts to OAg as does the Ab52 binding site. These results reveal structural features of antibodies at the low end of reactivity with multi-repeat microbial carbohydrates and demonstrate that such antibodies still have substantial protective effects against infection. PMID:25171003

  12. Cooperation of both, the FKBP_N-like and the DSBA-like, domains is necessary for the correct function of FTS_1067 protein involved in Francisella tularensis virulence and pathogenesis.

    PubMed

    Senitkova, Iva; Spidlova, Petra; Stulik, Jiri

    2015-08-01

    Francisella tularensis the etiological agent of tularaemia is one of the most infectious human pathogen known. Our knowledge about its key virulence factors has increased recently but it still remains a lot to explore. One of the described essential virulence factors is membrane lipoprotein FTS_1067 (nomenclature of F. tularensis subsp. holarctica strain FSC200) with homology to the protein family of disulphide oxidoreductases DsbA. Lipoprotein consists of two different domains: the C-terminal DsbA_Com1-like domain (DSBA-like) and the N-terminal FKBP-type peptidyl-prolyl cis/trans isomerases (FKBP_N-like). To uncover the biological role of these domains, we created bacterial strain with deletion of the DSBA-like domain. This defect in gene coding for lipoprotein FTS_1067 led to high in vivo attenuation associated with the ability to induce host protective immunity. Analyses performed with the truncated recombinant protein showed that the absence of DSBA-like domain revealed the loss of thiol/disulphide oxidoreductase activity and, additionally, confirmed the role of the FKBP_N-like domain in the FTS_1067 oligomerization and chaperone-like function. Finally, we verified that only full-length form of FTS_1067 recombinant protein possesses the isomerase activity. Based on our results, we proposed that for the correct FTS_1067 protein function both domains are needed.

  13. Characterization of Francisella tularensis Schu S4 mutants identified from a transposon library screened for O-antigen and capsule deficiencies

    PubMed Central

    Rasmussen, Jed A.; Fletcher, Joshua R.; Long, Matthew E.; Allen, Lee-Ann H.; Jones, Bradley D.

    2015-01-01

    The lipopolysaccharide (LPS) and O-antigen polysaccharide capsule structures of Francisella tularensis play significant roles in helping these highly virulent bacteria avoid detection within a host. We previously created pools of F. tularensis mutants that we screened to identify strains that were not reactive to a monoclonal antibody to the O-antigen capsule. To follow up previously published work, we characterize further seven of the F. tularensis Schu S4 mutant strains identified by our screen. These F. tularensis strains carry the following transposon mutations: FTT0846::Tn5, hemH::Tn5, wbtA::Tn5, wzy::Tn5, FTT0673p/prsA::Tn5, manB::Tn5, or dnaJ::Tn5. Each of these strains displayed sensitivity to human serum, to varying degrees, when compared to wild-type F. tularensis Schu S4. By Western blot, only FTT0846::Tn5, wbtA::Tn5, wzy::Tn5, and manB::Tn5 strains did not react to the capsule and LPS O-antigen antibody 11B7, although the wzy::Tn5 strain did have a single O-antigen reactive band that was detected by the FB11 monoclonal antibody. Of these strains, manB::Tn5 and FTT0846 appear to have LPS core truncations, whereas wbtA::Tn5 and wzy::Tn5 had LPS core structures that are similar to the parent F. tularensis Schu S4. These strains were also shown to have poor growth within human monocyte derived macrophages (MDMs) and bone marrow derived macrophages (BMDMs). We examined the virulence of these strains in mice, following intranasal challenge, and found that each was attenuated compared to wild type Schu S4. Our results provide additional strong evidence that LPS and/or capsule are F. tularensis virulence factors that most likely function by providing a stealth shield that prevents the host immune system from detecting this potent pathogen. PMID:25999917

  14. Lipids Derived from Virulent Francisella tularensis Broadly Inhibit Pulmonary Inflammation via Toll-Like Receptor 2 and Peroxisome Proliferator-Activated Receptor α

    PubMed Central

    Crane, Deborah D.; Ireland, Robin; Alinger, Joshua B.; Small, Pamela

    2013-01-01

    Francisella tularensis is a Gram-negative facultative intracellular pathogen that causes an acute lethal respiratory disease in humans. The heightened virulence of the pathogen is linked to its unique ability to inhibit Toll-like receptor (TLR)-mediated inflammatory responses. The bacterial component and mechanism of this inhibition are unknown. Here we show that lipids isolated from virulent but not attenuated strains of F. tularensis are not detected by host cells, inhibit production of proinflammatory cytokines by primary macrophages in response to known TLR ligands, and suppress neutrophil recruitment in vivo. We further show that lipid-mediated inhibition of inflammation is dependent on TLR2, MyD88, and the nuclear hormone and fatty acid receptor peroxisome proliferator-activated receptor α (PPARα). Pathogen lipid-mediated interference with inflammatory responses through the engagement of TLR2 and PPARα represents a novel manipulation of host signaling pathways consistent with the ability of highly virulent F. tularensis to efficiently evade host immune responses. PMID:23925884

  15. Levofloxacin rescues mice from lethal intra-nasal infections with virulent Francisella tularensis and induces immunity and production of protective antibody.

    PubMed

    Klimpel, Gary R; Eaves-Pyles, Tonyia; Moen, Scott T; Taormina, Joanna; Peterson, Johnny W; Chopra, Ashok K; Niesel, David W; Carness, Paige; Haithcoat, Judith L; Kirtley, Michelle; Nasr, Abdelhakim Ben

    2008-12-01

    The ability to protect mice against respiratory infections with virulent Francisella tularensis has been problematic and the role of antibody-versus-cell-mediated immunity controversial. In this study, we tested the hypothesis that protective immunity can develop in mice that were given antibiotic therapy following infection via the respiratory tract with F. tularensis SCHU S4. We show that mice infected with a lethal dose of SCHU S4, via an intra-nasal challenge, could be protected with levofloxacin treatment. This protection was evident even when levofloxacin treatment was delayed 72h post-infection. At early time points after levofloxacin treatment, significant numbers of bacteria could be recovered from the lungs and spleens of mice, which was followed by a dramatic disappearance of bacteria from these tissues. Mice successfully treated with levofloxacin were later shown to be almost completely resistant to re-challenge with SCHU S4 by the intra-nasal route. Serum antibody appeared to play an important role in this immunity. Normal mice, when given sera from animals protected by levofloxacin treatment, were solidly protected from a lethal intra-nasal challenge with SCHU S4. The protective antiserum contained high titers of SCHU S4-specific IgG2a, indicating that a strong Th1 response was induced following levofloxacin treatment. Thus, this study describes a potentially valuable animal model for furthering our understanding of respiratory tularemia and provides suggestive evidence that antibody can protect against respiratory infections with virulent F. tularensis. PMID:18930100

  16. The Francisella tularensis migR, trmE, and cphA Genes Contribute to F. tularensis Pathogenicity Island Gene Regulation and Intracellular Growth by Modulation of the Stress Alarmone ppGpp

    PubMed Central

    Faron, Matthew; Fletcher, Joshua R.; Rasmussen, Jed A.; Long, Matthew E.; Allen, Lee-Ann H.

    2013-01-01

    The Francisella tularensis pathogenicity island (FPI) encodes many proteins that are required for virulence. Expression of these genes depends upon the FevR (PigR) regulator and its interactions with the MglA/SspA and RNA polymerase transcriptional complex. Experiments to identify how transcription of the FPI genes is activated have led to identification of mutations within the migR, trmE, and cphA genes that decrease FPI expression. Recent data demonstrated that the small alarmone ppGpp, produced by RelA and SpoT, is important for stabilizing MglA/SspA and FevR (PigR) interactions in Francisella. Production of ppGpp is commonly known to be activated by cellular and nutritional stress in bacteria, which indicates that cellular and nutritional stresses act as important signals for FPI activation. In this work, we demonstrate that mutations in migR, trmE, or cphA significantly reduce ppGpp accumulation. The reduction in ppGpp levels was similar for each of the mutants and correlated with a corresponding reduction in iglA reporter expression. In addition, we observed that there were differences in the ability of each of these mutants to replicate within various mammalian cells, indicating that the migR, trmE, and cphA genes are likely parts of different cellular stress response pathways in Francisella. These results also indicate that different nutritional and cellular stresses exist in different mammalian cells. This work provides new information to help understand how Francisella regulates its virulence genes in response to host cell environments, and it contributes to our growing knowledge of this highly successful bacterial pathogen. PMID:23716606

  17. The Francisella tularensis migR, trmE, and cphA genes contribute to F. tularensis pathogenicity island gene regulation and intracellular growth by modulation of the stress alarmone ppGpp.

    PubMed

    Faron, Matthew; Fletcher, Joshua R; Rasmussen, Jed A; Long, Matthew E; Allen, Lee-Ann H; Jones, Bradley D

    2013-08-01

    The Francisella tularensis pathogenicity island (FPI) encodes many proteins that are required for virulence. Expression of these genes depends upon the FevR (PigR) regulator and its interactions with the MglA/SspA and RNA polymerase transcriptional complex. Experiments to identify how transcription of the FPI genes is activated have led to identification of mutations within the migR, trmE, and cphA genes that decrease FPI expression. Recent data demonstrated that the small alarmone ppGpp, produced by RelA and SpoT, is important for stabilizing MglA/SspA and FevR (PigR) interactions in Francisella. Production of ppGpp is commonly known to be activated by cellular and nutritional stress in bacteria, which indicates that cellular and nutritional stresses act as important signals for FPI activation. In this work, we demonstrate that mutations in migR, trmE, or cphA significantly reduce ppGpp accumulation. The reduction in ppGpp levels was similar for each of the mutants and correlated with a corresponding reduction in iglA reporter expression. In addition, we observed that there were differences in the ability of each of these mutants to replicate within various mammalian cells, indicating that the migR, trmE, and cphA genes are likely parts of different cellular stress response pathways in Francisella. These results also indicate that different nutritional and cellular stresses exist in different mammalian cells. This work provides new information to help understand how Francisella regulates its virulence genes in response to host cell environments, and it contributes to our growing knowledge of this highly successful bacterial pathogen.

  18. Genome characterisation of the genus Francisella reveals insight into similar evolutionary paths in pathogens of mammals and fish

    PubMed Central

    2012-01-01

    Background Prior to this study, relatively few strains of Francisella had been genome-sequenced. Previously published Francisella genome sequences were largely restricted to the zoonotic agent F. tularensis. Only limited data were available for other members of the Francisella genus, including F. philomiragia, an opportunistic pathogen of humans, F. noatunensis, a serious pathogen of farmed fish, and other less well described endosymbiotic species. Results We determined the phylogenetic relationships of all known Francisella species, including some for which the phylogenetic positions were previously uncertain. The genus Francisella could be divided into two main genetic clades: one included F. tularensis, F. novicida, F. hispaniensis and Wolbachia persica, and another included F. philomiragia and F. noatunensis. Some Francisella species were found to have significant recombination frequencies. However, the fish pathogen F. noatunensis subsp. noatunensis was an exception due to it exhibiting a highly clonal population structure similar to the human pathogen F. tularensis. Conclusions The genus Francisella can be divided into two main genetic clades occupying both terrestrial and marine habitats. However, our analyses suggest that the ancestral Francisella species originated in a marine habitat. The observed genome to genome variation in gene content and IS elements of different species supports the view that similar evolutionary paths of host adaptation developed independently in F. tularensis (infecting mammals) and F. noatunensis subsp. noatunensis (infecting fish). PMID:22727144

  19. CHLORINE INACTIVATION OF CATEGORY "A" BIO-TERRORISM AGENTS

    EPA Science Inventory

    This poster presents information on the inactivation of select bioterrorist agents. Information will be presented on chlorine disinfection of vegetative cells of Brucella suis, Brucella melitensis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis and endos...

  20. Detection of Diverse New Francisella-Like Bacteria in Environmental Samples†

    PubMed Central

    Barns, Susan M.; Grow, Christy C.; Okinaka, Richard T.; Keim, Paul; Kuske, Cheryl R.

    2005-01-01

    Following detection of putative Francisella species in aerosol samples from Houston, Texas, we surveyed soil and water samples from the area for the agent of tularemia, Francisella tularensis, and related species. The initial survey used 16S rRNA gene primers to detect Francisella species and related organisms by PCR amplification of DNA extracts from environmental samples. This analysis indicated that sequences related to Francisella were present in one water and seven soil samples. This is the first report of the detection of Francisella-related species in soil samples by DNA-based methods. Cloning and sequencing of PCR products indicated the presence of a wide variety of Francisella-related species. Sequences from two soil samples were 99.9% similar to previously reported sequences from F. tularensis isolates and may represent new subspecies. Additional analyses with primer sets developed for detection and differentiation of F. tularensis subspecies support the finding of very close relatives to known F. tularensis strains in some samples. While the pathogenicity of these organisms is unknown, they have the potential to be detected in F. tularensis-specific assays. Similarly, a potential new subspecies of Francisella philomiragia was identified. The majority of sequences obtained, while more similar to those of Francisella than to any other genus, were phylogenetically distinct from known species and formed several new clades potentially representing new species or genera. The results of this study revise our understanding of the diversity and distribution of Francisella and have implications for tularemia epidemiology and our ability to detect bioterrorist activities. PMID:16151142

  1. Detection of diverse new Francisella-like bacteria in environmental samples.

    PubMed

    Barns, Susan M; Grow, Christy C; Okinaka, Richard T; Keim, Paul; Kuske, Cheryl R

    2005-09-01

    Following detection of putative Francisella species in aerosol samples from Houston, Texas, we surveyed soil and water samples from the area for the agent of tularemia, Francisella tularensis, and related species. The initial survey used 16S rRNA gene primers to detect Francisella species and related organisms by PCR amplification of DNA extracts from environmental samples. This analysis indicated that sequences related to Francisella were present in one water and seven soil samples. This is the first report of the detection of Francisella-related species in soil samples by DNA-based methods. Cloning and sequencing of PCR products indicated the presence of a wide variety of Francisella-related species. Sequences from two soil samples were 99.9% similar to previously reported sequences from F. tularensis isolates and may represent new subspecies. Additional analyses with primer sets developed for detection and differentiation of F. tularensis subspecies support the finding of very close relatives to known F. tularensis strains in some samples. While the pathogenicity of these organisms is unknown, they have the potential to be detected in F. tularensis-specific assays. Similarly, a potential new subspecies of Francisella philomiragia was identified. The majority of sequences obtained, while more similar to those of Francisella than to any other genus, were phylogenetically distinct from known species and formed several new clades potentially representing new species or genera. The results of this study revise our understanding of the diversity and distribution of Francisella and have implications for tularemia epidemiology and our ability to detect bioterrorist activities. PMID:16151142

  2. Inactivation of F.tularensis Utah-112 on food and food contact surfaces by ultraviolet light

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Francisella tularensis is the causative agent of tularemia, a plague-like illness that affects animals and humans, and has caused large illness pandemics in the last century. It has also been used as a biological warfare agent, and tularemia can be contracted through consumption of contaminated food...

  3. Structure of the Francisella tularensis enoyl-acyl carrier protein reductase (FabI) in complex with NAD[superscript +] and triclosan

    SciTech Connect

    Mehboob, Shahila; Truong, Kent; Santarsiero, Bernard D.; Johnson, Michael E.

    2010-11-19

    Enoyl-acyl carrier protein reductase (FabI) catalyzes the last rate-limiting step in the elongation cycle of the fatty-acid biosynthesis pathway and has been validated as a potential antimicrobial drug target in Francisella tularensis. The development of new antibiotic therapies is important both to combat potential drug-resistant bioweapons and to address the broader societal problem of increasing antibiotic resistance among many pathogenic bacteria. The crystal structure of FabI from F. tularensis (FtuFabI) in complex with the inhibitor triclosan and the cofactor NAD{sup +} has been solved to a resolution of 2.1 {angstrom}. Triclosan is known to effectively inhibit FabI from different organisms. Precise characterization of the mode of triclosan binding is required to develop highly specific inhibitors. Comparison of our structure with the previously determined FtuFabI structure (PDB code 2jjy) which is bound to only NAD{sup +} reveals the conformation of the substrate-binding loop, electron density for which was missing in the earlier structure, and demonstrates a shift in the conformation of the NAD{sup +} cofactor. This shift in the position of the phosphate groups allows more room in the active site for substrate or inhibitor to bind and be better accommodated. This information will be crucial for virtual screening studies to identify novel scaffolds for development into new active inhibitors.

  4. Successful protection against tularemia in C57BL/6 mice is correlated with expansion of Francisella tularensis-specific effector T cells.

    PubMed

    Griffin, Amanda J; Crane, Deborah D; Wehrly, Tara D; Bosio, Catharine M

    2015-01-01

    Francisella tularensis is an intracellular, Gram-negative bacterium that causes the fatal disease tularemia. Currently, there are no licensed vaccines for tularemia and the requirements for protection against infection are poorly defined. To identify correlates of vaccine-induced immunity against tularemia, we compared different strains of the live vaccine strain (LVS) for their relative levels of virulence and ability to protect C57BL/6 mice against challenge with virulent F. tularensis strain SchuS4. Successful vaccination, as defined by survival of C57BL/6 mice, was correlated with significantly greater numbers of effector T cells in the spleen and lung. Further, lung cells and splenocytes from fully protected animals were more effective than lung cells and splenocytes from vaccinated but nonimmune animals in limiting intracellular replication of SchuS4 in vitro. Together, our data provide a unique model to compare efficacious vaccines to nonefficacious vaccines, which will enable comprehensive identification of host and bacterial components required for immunization against tularemia.

  5. The binding sites of monoclonal antibodies to the non-reducing end of Francisella tularensis O-antigen accommodate mainly the terminal saccharide

    PubMed Central

    Lu, Zhaohua; Rynkiewicz, Michael J; Yang, Chiou-Ying; Madico, Guillermo; Perkins, Hillary M; Wang, Qi; Costello, Catherine E; Zaia, Joseph; Seaton, Barbara A; Sharon, Jacqueline

    2013-01-01

    We have previously described two types of protective B-cell epitopes in the O-antigen (OAg) of the Gram-negative bacterium Francisella tularensis: repeating internal epitopes targeted by the vast majority of anti-OAg monoclonal antibodies (mAbs), and a non-overlapping epitope at the non-reducing end targeted by the previously unique IgG2a mAb FB11. We have now generated and characterized three mAbs specific for the non-reducing end of F. tularensis OAg, partially encoded by the same variable region germline genes, indicating that they target the same epitope. Like FB11, the new mAbs, Ab63 (IgG3), N213 (IgG3) and N62 (IgG2b), had higher antigen-binding bivalent avidity than internally binding anti-OAg mAbs, and an oligosaccharide containing a single OAg repeat was sufficient for optimal inhibition of their antigen-binding. The X-ray crystal structure of N62 Fab showed that the antigen-binding site is lined mainly by aromatic amino acids that form a small cavity, which can accommodate no more than one and a third sugar residues, indicating that N62 binds mainly to the terminal Qui4NFm residue at the nonreducing end of OAg. In efficacy studies with mice infected intranasally with the highly virulent F. tularensis strain SchuS4, N62, N213 and Ab63 prolonged survival and reduced blood bacterial burden. These results yield insights into how antibodies to non-reducing ends of microbial polysaccharides can contribute to immune protection despite the smaller size of their target epitopes compared with antibodies to internal polysaccharide regions. PMID:23844703

  6. The binding sites of monoclonal antibodies to the non-reducing end of Francisella tularensis O-antigen accommodate mainly the terminal saccharide.

    PubMed

    Lu, Zhaohua; Rynkiewicz, Michael J; Yang, Chiou-Ying; Madico, Guillermo; Perkins, Hillary M; Wang, Qi; Costello, Catherine E; Zaia, Joseph; Seaton, Barbara A; Sharon, Jacqueline

    2013-11-01

    We have previously described two types of protective B-cell epitopes in the O-antigen (OAg) of the Gram-negative bacterium Francisella tularensis: repeating internal epitopes targeted by the vast majority of anti-OAg monoclonal antibodies (mAbs), and a non-overlapping epitope at the non-reducing end targeted by the previously unique IgG2a mAb FB11. We have now generated and characterized three mAbs specific for the non-reducing end of F. tularensis OAg, partially encoded by the same variable region germline genes, indicating that they target the same epitope. Like FB11, the new mAbs, Ab63 (IgG3), N213 (IgG3) and N62 (IgG2b), had higher antigen-binding bivalent avidity than internally binding anti-OAg mAbs, and an oligosaccharide containing a single OAg repeat was sufficient for optimal inhibition of their antigen-binding. The X-ray crystal structure of N62 Fab showed that the antigen-binding site is lined mainly by aromatic amino acids that form a small cavity, which can accommodate no more than one and a third sugar residues, indicating that N62 binds mainly to the terminal Qui4NFm residue at the nonreducing end of OAg. In efficacy studies with mice infected intranasally with the highly virulent F. tularensis strain SchuS4, N62, N213 and Ab63 prolonged survival and reduced blood bacterial burden. These results yield insights into how antibodies to non-reducing ends of microbial polysaccharides can contribute to immune protection despite the smaller size of their target epitopes compared with antibodies to internal polysaccharide regions. PMID:23844703

  7. Antibodies to both terminal and internal B-cell epitopes of Francisella tularensis O-polysaccharide produced by patients with tularemia.

    PubMed

    Lu, Zhaohua; Perkins, Hillary M; Sharon, Jacqueline

    2014-02-01

    Francisella tularensis, the Gram-negative bacterium that causes tularemia, is considered a potential bioterrorism threat due to its low infectivity dose and the high morbidity and mortality from respiratory disease. We previously characterized two mouse monoclonal antibodies (MAbs) specific for the O-polysaccharide (O antigen [OAg]) of F. tularensis lipopolysaccharide (LPS): Ab63, which targets a terminal epitope at the nonreducing end of OAg, and Ab52, which targets a repeating internal OAg epitope. These two MAbs were protective in a mouse model of respiratory tularemia. To determine whether these epitope types are also targeted by humans, we tested the ability of each of 18 blood serum samples from 11 tularemia patients to inhibit the binding of Ab63 or Ab52 to F. tularensis LPS in a competition enzyme-linked immunosorbent assay (ELISA). Although all serum samples had Ab63- and Ab52-inhibitory activities, the ratios of Ab63 to Ab52 inhibitory potencies varied 75-fold. However, the variation was only 2.3-fold for sequential serum samples from the same patient, indicating different distributions of terminal- versus internal-binding antibodies in different individuals. Western blot analysis using class-specific anti-human Ig secondary antibodies showed that both terminal- and internal-binding OAg antibodies were of the IgG, IgM, and IgA isotypes. These results support the use of a mouse model to discover protective B-cell epitopes for tularemia vaccines or prophylactic/therapeutic antibodies, and they present a general strategy for interrogating the antibody responses of patients and vaccinees to microbial carbohydrate epitopes that have been characterized in experimental animals.

  8. Low dose vaccination with attenuated Francisella tularensis strain SchuS4 mutants protects against tularemia independent of the route of vaccination.

    PubMed

    Rockx-Brouwer, Dedeke; Chong, Audrey; Wehrly, Tara D; Child, Robert; Crane, Deborah D; Celli, Jean; Bosio, Catharine M

    2012-01-01

    Tularemia, caused by the gram-negative bacterium Francisella tularensis, is a severe, sometimes fatal disease. Interest in tularemia has increased over the last decade due to its history as a biological weapon. In particular, development of novel vaccines directed at protecting against pneumonic tularemia has been an important goal. Previous work has demonstrated that, when delivered at very high inoculums, administration of live, highly attenuated strains of virulent F. tularensis can protect against tularemia. However, lower vaccinating inoculums did not offer similar immunity. One concern of using live vaccines is that the host may develop mild tularemia in response to infection and use of high inoculums may contribute to this issue. Thus, generation of a live vaccine that can efficiently protect against tularemia when delivered in low numbers, e.g. <100 organisms, may address this concern. Herein we describe the ability of three defined, attenuated mutants of F. tularensis SchuS4, deleted for FTT0369c, FTT1676, or FTT0369c and FTT1676, respectively, to engender protective immunity against tularemia when delivered at concentrations of approximately 50 or fewer bacteria. Attenuated strains for use as vaccines were selected by their inability to efficiently replicate in macrophages in vitro and impaired replication and dissemination in vivo. Although all strains were defective for replication in vitro within macrophages, protective efficacy of each attenuated mutant was correlated with their ability to modestly replicate and disseminate in the host. Finally, we demonstrate the parenteral vaccination with these strains offered superior protection against pneumonic tularemia than intranasal vaccination. Together our data provides proof of principle that low dose attenuated vaccines may be a viable goal in development of novel vaccines directed against tularemia.

  9. The FupA/B protein uniquely facilitates transport of ferrous iron and siderophore-associated ferric iron across the outer membrane of Francisella tularensis live vaccine strain.

    PubMed

    Ramakrishnan, Girija; Sen, Bhaswati

    2014-02-01

    Francisella tularensis is a highly infectious Gram-negative pathogen that replicates intracellularly within the mammalian host. One of the factors associated with virulence of F. tularensis is the protein FupA that mediates high-affinity transport of ferrous iron across the outer membrane. Together with its paralogue FslE, a siderophore-ferric iron transporter, FupA supports survival of the pathogen in the host by providing access to the essential nutrient iron. The FupA orthologue in the attenuated live vaccine strain (LVS) is encoded by the hybrid gene fupA/B, the product of an intergenic recombination event that significantly contributes to attenuation of the strain. We used (55)Fe transport assays with mutant strains complemented with the different paralogues to show that the FupA/B protein of LVS retains the capacity for high-affinity transport of ferrous iron, albeit less efficiently than FupA of virulent strain Schu S4. (55)Fe transport assays using purified siderophore and siderophore-dependent growth assays on iron-limiting agar confirmed previous findings that FupA/B also contributes to siderophore-mediated ferric iron uptake. These assays further demonstrated that the LVS FslE protein is a weaker siderophore-ferric iron transporter than the orthologue from Schu S4, and may be a result of the sequence variation between the two proteins. Our results indicate that iron-uptake mechanisms in LVS differ from those in Schu S4 and that functional differences in the outer membrane iron transporters have distinct effects on growth under iron limitation.

  10. Rapid Identification and Characterization of Francisella by Molecular Biology and Other Techniques

    PubMed Central

    Lai, Xin-He; Zhao, Long-Fei; Chen, Xiao-Ming; Ren, Yi

    2016-01-01

    Francisella tularensis is the causative pathogen of tularemia and a Tier 1 bioterror agent on the CDC list. Considering the fact that some subpopulation of the F. tularensis strains is more virulent, more significantly associated with mortality, and therefore poses more threat to humans, rapid identification and characterization of this subpopulation strains is of invaluable importance. This review summarizes the up-to-date developments of assays for mainly detecting and characterizing F. tularensis and a touch of caveats of some of the assays. PMID:27335619

  11. Early pathogenesis of infection in the liver with the facultative intracellular bacteria Listeria monocytogenes, Francisella tularensis, and Salmonella typhimurium involves lysis of infected hepatocytes by leukocytes.

    PubMed

    Conlan, J W; North, R J

    1992-12-01

    The results show that Listeria monocytogenes, Francisella tularensis, and Salmonella typhimurium are facultative intracellular bacteria with a capacity to invade and grow in nonphagocytic cells in vivo. In the liver, all of these pathogens were seen to invade and to multiply extensively in hepatocytes. In all three cases, inflammatory phagocytes were rapidly marshalled to foci of infection where they appeared to cause the destruction of infected hepatocytes, thereby releasing bacteria into the extracellular space, in which presumably they could be ingested and destroyed by the phagocytes. If phagocytic cells were prevented from accumulating at foci of liver infection by treatment of the mice with a monoclonal antibody (NIMP-R10) directed against the type 3 complement receptor of myelomonocytic cells, then lysis of hepatocytes failed to occur and bacteria proliferated unrestrictedly within them. Under these circumstances, otherwise sublethal infections became rapidly lethal. These findings strongly suggest that lysis of infected hepatocytes by phagocytic cells is an important general early-defense strategy against liver infection with at least three different intracellular bacteria.

  12. β-Hydroxyacyl-acyl Carrier Protein Dehydratase (FabZ) from Francisella tularensis and Yersinia pestis: Structure Determination, Enzymatic Characterization, and Cross-Inhibition Studies.

    PubMed

    McGillick, Brian E; Kumaran, Desigan; Vieni, Casey; Swaminathan, Subramanyam

    2016-02-23

    The bacterial system for fatty acid biosynthesis (FAS) contains several enzymes whose sequence and structure are highly conserved across a vast array of pathogens. This, coupled with their low homology and difference in organization compared to the equivalent system in humans, makes the FAS pathway an excellent target for antimicrobial drug development. To this end, we have cloned, expressed, and purified the β-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from both Francisella tularensis (FtFabZ) and Yersinia pestis (YpFabZ). We also solved the crystal structures and performed an enzymatic characterization of both enzymes and several mutant forms of YpFabZ. Additionally, we have discovered two novel inhibitors of FabZ, mangostin and stictic acid, which show similar potencies against both YpFabZ and FtFabZ. Lastly, we selected several compounds from the literature that have been shown to be active against single homologues of FabZ and tested them against both YpFabZ and FtFabZ. These results have revealed clues as to which scaffolds are likely to lead to broad-spectrum antimicrobials targeted against FabZ as well as modifications to existing FabZ inhibitors that may improve potency. PMID:26818694

  13. M-Cells Contribute to the Entry of an Oral Vaccine but Are Not Essential for the Subsequent Induction of Protective Immunity against Francisella tularensis

    PubMed Central

    Cunningham, Aimee L.; Guentzel, M. Neal; Yu, Jieh-Juen; Hung, Chiung-Yu; Forsthuber, Thomas G.; Navara, Christopher S.; Yagita, Hideo; Williams, Ifor R.; Klose, Karl E.; Eaves-Pyles, Tonyia D.; Arulanandam, Bernard P.

    2016-01-01

    M-cells (microfold cells) are thought to be a primary conduit of intestinal antigen trafficking. Using an established neutralizing anti-RANKL (Receptor Activator of NF-κB Ligand) antibody treatment to transiently deplete M-cells in vivo, we sought to determine whether intestinal M-cells were required for the effective induction of protective immunity following oral vaccination with ΔiglB (a defined live attenuated Francisella novicida mutant). M-cell depleted, ΔiglB-vaccinated mice exhibited increased (but not significant) morbidity and mortality following a subsequent homotypic or heterotypic pulmonary F. tularensis challenge. No significant differences in splenic IFN-γ, IL-2, or IL-17 or serum antibody (IgG1, IgG2a, IgA) production were observed compared to non-depleted, ΔiglB-vaccinated animals suggesting complementary mechanisms for ΔiglB entry. Thus, we examined other possible routes of gastrointestinal antigen sampling following oral vaccination and found that ΔiglB co-localized to villus goblet cells and enterocytes. These results provide insight into the role of M-cells and complementary pathways in intestinal antigen trafficking that may be involved in the generation of optimal immunity following oral vaccination. PMID:27100824

  14. Neutralization of gamma interferon and tumor necrosis factor alpha blocks in vivo synthesis of nitrogen oxides from L-arginine and protection against Francisella tularensis infection in Mycobacterium bovis BCG-treated mice.

    PubMed Central

    Green, S J; Nacy, C A; Schreiber, R D; Granger, D L; Crawford, R M; Meltzer, M S; Fortier, A H

    1993-01-01

    Peritoneal cells from Mycobacterium bovis BCG-infected C3H/HeN mice produced nitrite (NO2-, an oxidative end product of nitric oxide [NO] synthesis) and inhibited the growth of Francisella tularensis, a facultative intracellular bacterium. Both NO2- production and inhibition of bacterial growth were suppressed by NG-monomethyl-L-arginine, a substrate inhibitor of nitrogen oxidation of L-arginine, and monoclonal antibodies (MAbs) to gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). Intraperitoneal injection of mice with BCG increased urinary nitrate (NO3-) excretion coincident with development of activated macrophages capable of secreting nitrogen oxides and inhibiting F. tularensis growth in vitro. Eight days after BCG inoculation, mice survived a normally lethal intraperitoneal challenge with F. tularensis. Treatment of these BCG-infected mice with MAbs to IFN-gamma or TNF-alpha at the time of BCG inoculation reduced urinary NO3- levels to those found in normal uninfected mice for up to 14 days. The same anticytokine antibody treatment abolished BCG-mediated protection against F. tularensis: mice died within 4 to 6 days. Intraperitoneal administration of anti-IFN-gamma or anti-TNF-alpha antibody 8 days after BCG infection also reduced urinary NO3- and abolished protection against F. tularensis. Isotype control (immunoglobulin G) or anti-interleukin 4 MAbs had little effect on these parameters at any time of treatment. IFN-gamma and TNF-alpha were clearly involved in the regulation of macrophage activation by BCG in vivo. Protection against F. tularensis challenge by BCG depended upon the physiological generation of reactive nitrogen oxides induced by these cytokines. PMID:8423095

  15. Mucosal Immunization with Live Attenuated Francisella novicida U112ΔiglB Protects against Pulmonary F. tularensis SCHU S4 in the Fischer 344 Rat Model

    PubMed Central

    Signarovitz, Aimee L.; Ray, Heather J.; Yu, Jieh-Juen; Guentzel, M. N.; Chambers, James P.; Klose, Karl E.; Arulanandam, Bernard P.

    2012-01-01

    The need for an efficacious vaccine against Francisella tularensis is a consequence of its low infectious dose and high mortality rate if left untreated. This study sought to characterize a live attenuated subspecies novicida-based vaccine strain (U112ΔiglB) in an established second rodent model of pulmonary tularemia, namely the Fischer 344 rat using two distinct routes of vaccination (intratracheal [i.t.] and oral). Attenuation was verified by comparing replication of U112ΔiglB with wild type parental strain U112 in F344 primary alveolar macrophages. U112ΔiglB exhibited an LD50>107 CFU compared to the wild type (LD50 = 5×106 CFU i.t.). Immunization with 107 CFU U112ΔiglB by i.t. and oral routes induced antigen-specific IFN-γ and potent humoral responses both systemically (IgG2a>IgG1 in serum) and at the site of mucosal vaccination (respiratory/intestinal compartment). Importantly, vaccination with U112ΔiglB by either i.t. or oral routes provided equivalent levels of protection (50% survival) in F344 rats against a subsequent pulmonary challenge with ∼25 LD50 (1.25×104 CFU) of the highly human virulent strain SCHU S4. Collectively, these results provide further evidence on the utility of a mucosal vaccination platform with a defined subsp. novicida U112ΔiglB vaccine strain in conferring protective immunity against pulmonary tularemia. PMID:23118885

  16. Enhancement of Vaccine Efficacy by Expression of a TLR5 Ligand in the Defined Live Attenuated Francisella tularensis subsp. novicida Strain U112▲iglB::fljB

    PubMed Central

    Cunningham, Aimee L.; Dang, Kim Minh; Yu, Jieh-Juen; Guentzel, M. Neal; Heidner, Hans; Klose, Karl E.; Arulanandam, Bernard P.

    2014-01-01

    Oral vaccination with the defined live attenuated Francisella novicida vaccine strain U112▲iglB has been demonstrated to induce protective immunity against pulmonary challenge with the highly human virulent F. tularensis strain SCHU S4. However, this vaccination regimen requires a booster dose in mice and exhibits 50% protective efficacy in the Fischer 344 rat model. To enhance the efficacy of this vaccine strain, we engineered U112▲iglB to express the Salmonella typhimurium FljB flagellin D1 domain, a TLR5 agonist. The U112▲iglB::fljB strain was highly attenuated for intracellular macrophage replication, and although the FljB protein was expressed within the cytosol, it exhibited TLR5 activation in a TLR5-expressing HEK cell line. Additionally, infection of splenocytes and lymphocytes with U112▲iglB::fljB induced significantly greater TNF-α production than infection with U112▲iglB. Oral vaccination with U112▲iglB::fljB also induced significantly greater protection than U112ΔiglB against pulmonary SCHU S4 challenge in rats. The enhanced protection was accompanied by higher IgG2a production and serum-mediated reduction of Francisella infectivity. Thus, the U112▲iglB::fljB strain may serve as a potential vaccine candidate against pneumonic tularemia. PMID:25050972

  17. Detection of a novel subspecies of Francisella noatunensis as endosymbiont of the ciliate Euplotes raikovi.

    PubMed

    Schrallhammer, Martina; Schweikert, Michael; Vallesi, Adriana; Verni, Franco; Petroni, Giulio

    2011-02-01

    Francisella are facultative intracellular bacteria causing severe disease in a broad range of animals. Two species are notable: Francisella tularensis, the causative organism of tularemia and a putative warfare agent, and Francisella noatunensis, an emerging fish pathogen causing significant losses in wild and farmed fish. Although various aspects of Francisella biology have been intensively studied, their natural reservoir in periods between massive outbreaks remains mysterious. Protists have been suspected to serve as a disguised vector of Francisella and co-culturing attempts demonstrate that some species are able to survive and multiply within protozoan cells. Here, we report the first finding of a natural occurrence of Francisella sp. as a protist endosymbiont. By molecular and morphological approaches, we identified intracellular bacteria localized in a strain of the marine ciliate Euplotes raikovi, isolated from the coast of Adriatic Sea. Phylogenetic analysis placed these endosymbionts within the genus Francisella, in close but distinct association with F. noatunensis. We suggest the establishment of a novel subspecies within F. noatunensis and propose the cytoplasmatic endosymbiont of E. raikovi as "Candidatus F. noatunensis subsp. endociliophora" subsp. nov.

  18. Francisella infections in fish and shellfish.

    PubMed

    Birkbeck, T H; Feist, S W; Verner-Jeffreys, D W

    2011-03-01

    A series of recent reports have implicated bacteria from the family Francisellaceae as the cause of disease in farmed and wild fish and shellfish species such as Atlantic cod, Gadus morhua L., tilapia, Oreochromis spp., Atlantic salmon, Salmo salar L., three-line grunt, Parapristipoma trilineatum (Thunberg), ornamental cichlid species, hybrid striped bass Morone chrysops x M. saxatilis and, recently, a shellfish species, the giant abalone, Haliotisgigantea Gmelin. The range of taxa affected will very probably rise as it is likely that there has been considerable under-reporting to date of these disease agents. In common with other Francisella species, their isolation and culture require specialized solid and liquid media containing cysteine and a source of iron. This likely restricted earlier efforts to identify them correctly as the cause of disease in aquatic animals. The most information to date relates to disease in cod, caused by F. noatunensis and tilapia, caused by F. noatunensis subsp. orientalis (also termed F. asiatica), both causing granulomatous inflammatory reactions. Mortalities in both species can be high and, as the disease can likely be transferred via live fish movements, they pose a significant threat to tilapia and cod aquaculture operations. Although the fish-pathogenic Francisella species are classified in the same genus as the human pathogens F. tularensis, causative agent of tularemia, and F. philomiragia, the risk to humans from the fish and shellfish pathogenic Francisella species is considered very low. PMID:21306585

  19. Genome Sequencing of 18 Francisella Strains To Aid in Assay Development and Testing

    PubMed Central

    Daligault, Hajnalka E.; Davenport, Karen W.; Coyne, Susan R.; Frey, Kenneth G.; Koroleva, Galina I.; Broomall, Stacey M.; Bishop-Lilly, Kimberly A.; Bruce, David C.; Chertkov, Olga; Freitas, Tracey; Jaissle, James; Ladner, Jason T.; Rosenzweig, C. Nicole; Gibbons, Henry S.; Palacios, Gustavo F.; Redden, Cassie L.; Xu, Yan; Minogue, Timothy D.; Chain, Patrick S.

    2015-01-01

    Francisella tularensis is a highly infectious bacterium with the potential to cause high fatality rates if infections are untreated. To aid in the development of rapid and accurate detection assays, we have sequenced and annotated the genomes of 18 F. tularensis and Francisella philomiragia strains. PMID:25931589

  20. Genome Sequencing of 18 Francisella Strains To Aid in Assay Development and Testing

    DOE PAGES

    Johnson, Shannon L.; Daligault, Hajnalka E.; Davenport, Karen W.; Coyne, Susan R.; Frey, Kenneth G.; Koroleva, Galina I.; Broomall, Stacey M.; Bishop-Lilly, Kimberly A.; Bruce, David C.; Chertkov, Olga; et al

    2015-04-30

    Francisella tularensis is a highly infectious bacterium that has the potential of causing high fatality rates if infections are untreated. To aid in the development of rapid and accurate detection assays, we have sequenced and annotated the genomes of 18 F. tularensis and Francisella philomiragia strains.

  1. Serological Investigation of Wild Boars (Sus scrofa) and Red Foxes (Vulpes vulpes) As Indicator Animals for Circulation of Francisella tularensis in Germany

    PubMed Central

    Chaignat, Valerie; Klimpel, Diana; Diller, Roland; Melzer, Falk; Müller, Wolfgang; Tomaso, Herbert

    2014-01-01

    Abstract Tularemia outbreaks in humans have recently been reported in many European countries, but data on the occurrence in the animal population are scarce. In North America, seroconversion of omnivores and carnivores was used as indicator for the presence of tularemia, for the European fauna, however, data are barely available. Therefore, the suitability of wild boars (Sus scrofa) and red foxes (Vulpes vulpes) as indicators for the circulation of F. tularensis in Germany was evaluated. Serum samples from 566 wild boars and 457 red foxes were collected between 1995 and 2012 in three federal states in Central Germany (Hesse, Saxony-Anhalt, and Thuringia). The overall rate of seropositive animals was 1.1% in wild boars and 7.4% in red foxes. In conclusion, serological examination of red foxes is recommended, because they can be reliably used as indicator animals for the presence of F. tularensis in the environment. PMID:24359418

  2. Serological investigation of wild boars (Sus scrofa) and red foxes (Vulpes vulpes) as indicator animals for circulation of Francisella tularensis in Germany.

    PubMed

    Otto, Peter; Chaignat, Valerie; Klimpel, Diana; Diller, Roland; Melzer, Falk; Müller, Wolfgang; Tomaso, Herbert

    2014-01-01

    Tularemia outbreaks in humans have recently been reported in many European countries, but data on the occurrence in the animal population are scarce. In North America, seroconversion of omnivores and carnivores was used as indicator for the presence of tularemia, for the European fauna, however, data are barely available. Therefore, the suitability of wild boars (Sus scrofa) and red foxes (Vulpes vulpes) as indicators for the circulation of F. tularensis in Germany was evaluated. Serum samples from 566 wild boars and 457 red foxes were collected between 1995 and 2012 in three federal states in Central Germany (Hesse, Saxony-Anhalt, and Thuringia). The overall rate of seropositive animals was 1.1% in wild boars and 7.4% in red foxes. In conclusion, serological examination of red foxes is recommended, because they can be reliably used as indicator animals for the presence of F. tularensis in the environment.

  3. Modulation of Human Airway Barrier Functions during Burkholderia thailandensis and Francisella tularensis Infection Running Title: Airway Barrier Functions during Bacterial Infections.

    PubMed

    Blume, Cornelia; David, Jonathan; Bell, Rachel E; Laver, Jay R; Read, Robert C; Clark, Graeme C; Davies, Donna E; Swindle, Emily J

    2016-01-01

    The bronchial epithelium provides protection against pathogens from the inhaled environment through the formation of a highly-regulated barrier. In order to understand the pulmonary diseases melioidosis and tularemia caused by Burkholderia thailandensis and Fransicella tularensis, respectively, the barrier function of the human bronchial epithelium were analysed. Polarised 16HBE14o- or differentiated primary human bronchial epithelial cells (BECs) were exposed to increasing multiplicities of infection (MOI) of B. thailandensis or F. tularensis Live Vaccine Strain and barrier responses monitored over 24-72 h. Challenge of polarized BECs with either bacterial species caused an MOI- and time-dependent increase in ionic permeability, disruption of tight junctions, and bacterial passage from the apical to the basolateral compartment. B. thailandensis was found to be more invasive than F. tularensis. Both bacterial species induced an MOI-dependent increase in TNF-α release. An increase in ionic permeability and TNF-α release was induced by B. thailandensis in differentiated BECs. Pretreatment of polarised BECs with the corticosteroid fluticasone propionate reduced bacterial-dependent increases in ionic permeability, bacterial passage, and TNF-α release. TNF blocking antibody Enbrel(®) reduced bacterial passage only. BEC barrier properties are disrupted during respiratory bacterial infections and targeting with corticosteroids or anti-TNF compounds may represent a therapeutic option. PMID:27527221

  4. Modulation of Human Airway Barrier Functions during Burkholderia thailandensis and Francisella tularensis Infection Running Title: Airway Barrier Functions during Bacterial Infections

    PubMed Central

    Blume, Cornelia; David, Jonathan; Bell, Rachel E.; Laver, Jay R.; Read, Robert C.; Clark, Graeme C.; Davies, Donna E.; Swindle, Emily J.

    2016-01-01

    The bronchial epithelium provides protection against pathogens from the inhaled environment through the formation of a highly-regulated barrier. In order to understand the pulmonary diseases melioidosis and tularemia caused by Burkholderia thailandensis and Fransicella tularensis, respectively, the barrier function of the human bronchial epithelium were analysed. Polarised 16HBE14o- or differentiated primary human bronchial epithelial cells (BECs) were exposed to increasing multiplicities of infection (MOI) of B. thailandensis or F. tularensis Live Vaccine Strain and barrier responses monitored over 24–72 h. Challenge of polarized BECs with either bacterial species caused an MOI- and time-dependent increase in ionic permeability, disruption of tight junctions, and bacterial passage from the apical to the basolateral compartment. B. thailandensis was found to be more invasive than F. tularensis. Both bacterial species induced an MOI-dependent increase in TNF-α release. An increase in ionic permeability and TNF-α release was induced by B. thailandensis in differentiated BECs. Pretreatment of polarised BECs with the corticosteroid fluticasone propionate reduced bacterial-dependent increases in ionic permeability, bacterial passage, and TNF-α release. TNF blocking antibody Enbrel® reduced bacterial passage only. BEC barrier properties are disrupted during respiratory bacterial infections and targeting with corticosteroids or anti-TNF compounds may represent a therapeutic option. PMID:27527221

  5. Rapid differentiation of Francisella species and subspecies by fluorescent in situ hybridization targeting the 23S rRNA

    PubMed Central

    2010-01-01

    Background Francisella (F.) tularensis is the causative agent of tularemia. Due to its low infectious dose, ease of dissemination and high case fatality rate, F. tularensis was the subject in diverse biological weapons programs and is among the top six agents with high potential if misused in bioterrorism. Microbiological diagnosis is cumbersome and time-consuming. Methods for the direct detection of the pathogen (immunofluorescence, PCR) have been developed but are restricted to reference laboratories. Results The complete 23S rRNA genes of representative strains of F. philomiragia and all subspecies of F. tularensis were sequenced. Single nucleotide polymorphisms on species and subspecies level were confirmed by partial amplification and sequencing of 24 additional strains. Fluorescent In Situ Hybridization (FISH) assays were established using species- and subspecies-specific probes. Different FISH protocols allowed the positive identification of all 4 F. philomiragia strains, and more than 40 F. tularensis strains tested. By combination of different probes, it was possible to differentiate the F. tularensis subspecies holarctica, tularensis, mediasiatica and novicida. No cross reactivity with strains of 71 clinically relevant bacterial species was observed. FISH was also successfully applied to detect different F. tularensis strains in infected cells or tissue samples. In blood culture systems spiked with F. tularensis, bacterial cells of different subspecies could be separated within single samples. Conclusion We could show that FISH targeting the 23S rRNA gene is a rapid and versatile method for the identification and differentiation of F. tularensis isolates from both laboratory cultures and clinical samples. PMID:20205957

  6. Serological survey of Bartonella spp., Borrelia burgdorferi, Brucella spp., Coxiella burnetii, Francisella tularensis, Leptospira spp., Echinococcus, Hanta-, TBE- and XMR-virus infection in employees of two forestry enterprises in North Rhine-Westphalia, Germany, 2011-2013.

    PubMed

    Jurke, Annette; Bannert, N; Brehm, K; Fingerle, V; Kempf, V A J; Kömpf, D; Lunemann, M; Mayer-Scholl, A; Niedrig, M; Nöckler, K; Scholz, H; Splettstoesser, W; Tappe, D; Fischer, Silke F

    2015-10-01

    We initiated a survey to collect basic data on the frequency and regional distribution of various zoonoses in 722 employees of forestry enterprises in the German state of North Rhine-Westphalia (NRW) from 2011 to 2013. Exposures associated with seropositivity were identified to give insight into the possible risk factors for infection with each pathogen. 41.2% of participants were found to be seropositive for anti-Bartonella IgG, 30.6% for anti-Borrelia burgdorferi IgG, 14.2% for anti-Leptospira IgG, 6.5% for anti-Coxiella burnetii IgG, 6.0% for anti-Hantavirus IgG, 4.0% for anti-Francisella tularensis IgG, 3.4% for anti-TBE-virus IgG, 1.7% for anti-Echinococcus IgG, 0.0% for anti-Brucella IgG and anti-XMRV IgG. Participants seropositive for B. burgdorferi were 3.96 times more likely to be professional forestry workers (univariable analysis: OR 3.96; 95% CI 2.60-6.04; p<0.001); and participants seropositive for Hantavirus 3.72 times more likely (univariable analysis: OR 3.72; 95% CI 1.44-9.57; p=0.007). This study found a surprisingly high percentage of participants seropositive for anti-B. henselae IgG and for anti-F. tularensis IgG. The relatively high seroprevalence for anti-Leptospira IgG seen in this study could be related to living conditions rather than to exposure at work. No specific risk for exposure to C. burnetii and Echinococcus was identified, indicating that neither forestry workers nor office workers represent a risk population and that NRW is not a typical endemic area. Forestry workers appear to have higher risk for contact with B. burgdorferi-infected ticks and a regionally diverse risk for acquiring Hantavirus-infection. The regional epidemiology of zoonoses is without question of great importance for public health. Knowledge of the regional risk factors facilitates the development of efficient prevention strategies and the implementation of such prevention measures in a sustainable manner.

  7. Identification of Francisella novicida mutants that fail to induce prostaglandin E2 synthesis by infected macrophages

    PubMed Central

    Woolard, Matthew D.; Barrigan, Lydia M.; Fuller, James R.; Buntzman, Adam S.; Bryan, Joshua; Manoil, Colin; Kawula, Thomas H.; Frelinger, Jeffrey A.

    2013-01-01

    Francisella tularensis is the causative agent of tularemia. We have previously shown that infection with F. tularensis Live Vaccine Strain (LVS) induces macrophages to synthesize prostaglandin E2 (PGE2). Synthesis of PGE2 by F. tularensis infected macrophages results in decreased T cell proliferation in vitro and increased bacterial survival in vivo. Although we understand some of the biological consequences of F. tularensis induced PGE2 synthesis by macrophages, we do not understand the cellular pathways (neither host nor bacterial) that result in up-regulation of the PGE2 biosynthetic pathway in F. tularensis infected macrophages. We took a genetic approach to begin to understand the molecular mechanisms of bacterial induction of PGE2 synthesis from infected macrophages. To identify F. tularensis genes necessary for the induction of PGE2 in primary macrophages, we infected cells with individual mutants from the closely related strain F. tularensis subspecies novicida U112 (U112) two allele mutant library. Twenty genes were identified that when disrupted resulted in U112 mutant strains unable to induce the synthesis of PGE2 by infected macrophages. Fourteen of the genes identified are located within the Francisella pathogenicity island (FPI). Genes in the FPI are required for F. tularensis to escape from the phagosome and replicate in the cytosol, which might account for the failure of U112 with transposon insertions within the FPI to induce PGE2. This implies that U112 mutant strains that do not grow intracellularly would also not induce PGE2. We found that U112 clpB::Tn grows within macrophages yet fails to induce PGE2, while U112 pdpA::Tn does not grow yet does induce PGE2. We also found that U112 iglC::Tn neither grows nor induces PGE2. These findings indicate that there is dissociation between intracellular growth and the ability of F. tularensis to induce PGE2 synthesis. These mutants provide a critical entrée into the pathways used in the host for PGE2

  8. Mouse Model of Oral Infection with Virulent Type A Francisella tularensis▿

    PubMed Central

    KuoLee, R.; Zhao, X.; Austin, J.; Harris, G.; Conlan, J. W.; Chen, W.

    2007-01-01

    Francisella tularensis is a gram-negative facultative intracellular pathogen and the causative agent of tularemia. Little is known about the immunopathogenesis of oral infection with this pathogen. Here, for the first time, we examined the susceptibility of mice to intragastric inoculation with virulent type A F. tularensis and characterized the course of infection and the associated host responses. Both immunocompetent and immunodeficient mice were relatively susceptible to intragastric inoculation of type A F. tularensis with a 50% lethal dose (LD50) of 106 organisms, which was 100,000-fold higher than the LD100 for intradermal or respiratory routes of infection. Mice deficient in gamma interferon or tumor necrosis factor receptors 1 and 2 were more susceptible than wild-type controls to oral infection with a high dose of the pathogen. After oral inoculation, F. tularensis appeared first in the mesenteric lymph nodes (MLN) and then rapidly spread to the livers and spleens, where the organism multiplied to high numbers and induced marked neutrophilic infiltration and severe tissue necrosis. Infected mice showed rapid increases in tissue cytokine mRNA expression, which peaked in the MLN at 2 days postinfection (dpi) and in the liver and spleen at 3 dpi. The levels of gamma interferon, interleukin-1β (IL-1β), IL-6, tumor necrosis factor alpha, macrophage inflammatory protein 1α, KC, interferon-inducible protein 10, and monocyte chemotactic protein 1 were elevated from day 2 postinoculation onward. Moreover, mice intradermally immunized with the live vaccine strain of F. tularensis showed little survival advantage over naive mice after oral challenge with type A F. tularensis. These results suggest that type A F. tularensis is an effective oral pathogen that can cause fatal systemic infection and could pose a public health concern, particularly to immunocompromised individuals, if ingested in contaminated water and food. PMID:17242058

  9. Alpha-1 antitrypsin is markedly decreased following pulmonary F. tularensis challenge.

    PubMed

    Chambers, James P; Yu, Jieh-Juen; Jupelli, Madhulika; Weintraub, Susan T; Lopez-Ribot, Jose L; Valdes, James J; Arulanandam, Bernard P

    2011-01-01

    Neutrophils form the first line of defense during infection and are indispensable in this function. The neutrophil elastase is a key effector molecule of the innate immune system with potent antimicrobial activity against Gram-negative bacteria, spirochaetes, and fungi. However, the release of neutrophil elastase during bacterial infection must be checked otherwise its release in the extracellular milieu will result in damage to surrounding tissues. Alpha-1 antitrypsin is a small glycoprotein clade A serpine serine protease inhibitor and has been shown to increase in humans following bacterial and viral infection. Francisella tularensis is a Gram-negative facultative intracellular bacterium and the causative agent of tularemia. Type A strains are the most virulent with an infectious dose as low as 10 colony forming units and a mortality rate of 30-60% among untreated cases of pneumonic tularemia. We report here significant reduction of this major inhibitor of the neutrophil elastase in plasma of F. tularensis LVS and F. tularensis (type A) SCHU S4 infected animals following pulmonary challenge. Associated with an imbalance of protease-antiprotease function at the alveolar level in lungs of infected animals, increased elastase activity was observed in lung lavage fluids accompanied by decrease lung function, i.e., loss of lung elastance with concomitant increase of pulmonary hysteresivity. Consistent with a competent acute phase response following F. tularensis LVS and F. tularensis (type A) SCHU S4 pulmonary challenge and proposed up-regulation of plasma haptoglobin during the course of the acute phase reaction, haptoglobin was observed significantly increased. These data suggest that unchecked neutrophil serine protease activity may arise from F. tularensis targeted reduction of plasma α(1)-antitrysin promoting lung tissue damage facilitating increased dissemination of this bacterium in infected animals. PMID:22919586

  10. Alpha-1 Antitrypsin is Markedly Decreased Following Pulmonary F. tularensis Challenge

    PubMed Central

    Chambers, James P.; Yu, Jieh-Juen; Jupelli, Madhulika; Weintraub, Susan T.; Lopez-Ribot, Jose L.; Valdes, James J.; Arulanandam, Bernard P.

    2011-01-01

    Neutrophils form the first line of defense during infection and are indispensable in this function. The neutrophil elastase is a key effector molecule of the innate immune system with potent antimicrobial activity against Gram-negative bacteria, spirochaetes, and fungi. However, the release of neutrophil elastase during bacterial infection must be checked otherwise its release in the extracellular milieu will result in damage to surrounding tissues. Alpha-1 antitrypsin is a small glycoprotein clade A serpine serine protease inhibitor and has been shown to increase in humans following bacterial and viral infection. Francisella tularensis is a Gram-negative facultative intracellular bacterium and the causative agent of tularemia. Type A strains are the most virulent with an infectious dose as low as 10 colony forming units and a mortality rate of 30–60% among untreated cases of pneumonic tularemia. We report here significant reduction of this major inhibitor of the neutrophil elastase in plasma of F. tularensis LVS and F. tularensis (type A) SCHU S4 infected animals following pulmonary challenge. Associated with an imbalance of protease–antiprotease function at the alveolar level in lungs of infected animals, increased elastase activity was observed in lung lavage fluids accompanied by decrease lung function, i.e., loss of lung elastance with concomitant increase of pulmonary hysteresivity. Consistent with a competent acute phase response following F. tularensis LVS and F. tularensis (type A) SCHU S4 pulmonary challenge and proposed up-regulation of plasma haptoglobin during the course of the acute phase reaction, haptoglobin was observed significantly increased. These data suggest that unchecked neutrophil serine protease activity may arise from F. tularensis targeted reduction of plasma α1-antitrysin promoting lung tissue damage facilitating increased dissemination of this bacterium in infected animals. PMID:22919586

  11. Long-Lasting Fever and Lymphadenitis: Think about F. tularensis

    PubMed Central

    Longo, Maria Vittoria; Jaton, Katia; Pilo, Paola; Chabanel, David; Erard, Véronique

    2015-01-01

    We report the case of glandular tularemia that developed in a man supposedly infected by a tick bite in Western Switzerland. Francisella tularensis (F. tularensis) was identified. In Europe tularemia most commonly manifests itself as ulcero-glandular or glandular disease; the diagnosis of tularemia may be delayed in glandular form where skin or mucous lesion is absent, particularly in areas which are assumed to have a low incidence of the disease. PMID:26612988

  12. Antimicrobial activity of mosquito cecropin peptides against Francisella.

    PubMed

    Kaushal, Akanksha; Gupta, Kajal; Shah, Ruhee; van Hoek, Monique L

    2016-10-01

    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. PMID:27235883

  13. [THE DEVELOPMENT OF IMMUNE ENZYME AND IMMUNE CHROMATOGRAPHIC MONOCLONAL TEST-SYSTEM FOR DETECTING TULAREMIA AGENT].

    PubMed

    Eremkin, A V; Elagin, G D; Petchenkin, D V; Fomenkov, O O; Bogatcheva, N V; Kitmanov, A A; Kuklina, G V; Tikhvinskaya, O V

    2016-03-01

    The immune enzyme and immunochromatographic test-systems for detecting tularemia agent were developed on the basis of selected set of monoclonal antibodies having immunochemical activity to antigens Francisella tularensis. The evaluation of sensitivity and specificity of developed test-systems demonstrated that samples provided detection of strains of F. tularensis in concentration from 5.0 x 105 mkxcm-3 to 1.0 x 106 mkxcm-3 and gave no false positive results in analysis of heterologous microorganisms in concentration of 1.0 x 108 mkxcm-3. PMID:27506111

  14. [THE DEVELOPMENT OF IMMUNE ENZYME AND IMMUNE CHROMATOGRAPHIC MONOCLONAL TEST-SYSTEM FOR DETECTING TULAREMIA AGENT].

    PubMed

    Eremkin, A V; Elagin, G D; Petchenkin, D V; Fomenkov, O O; Bogatcheva, N V; Kitmanov, A A; Kuklina, G V; Tikhvinskaya, O V

    2016-03-01

    The immune enzyme and immunochromatographic test-systems for detecting tularemia agent were developed on the basis of selected set of monoclonal antibodies having immunochemical activity to antigens Francisella tularensis. The evaluation of sensitivity and specificity of developed test-systems demonstrated that samples provided detection of strains of F. tularensis in concentration from 5.0 x 105 mkxcm-3 to 1.0 x 106 mkxcm-3 and gave no false positive results in analysis of heterologous microorganisms in concentration of 1.0 x 108 mkxcm-3.

  15. Francisella Inflammasomes: Integrated Responses to a Cytosolic Stealth Bacterium.

    PubMed

    Wallet, Pierre; Lagrange, Brice; Henry, Thomas

    2016-01-01

    Francisella tularensis is a facultative intracellular bacterium causing tularemia, a zoonotic disease. Francisella replicates in the macrophage cytosol and eventually triggers cytosolic immune responses. In murine macrophages, Francisella novicida and Francisella tularensis live vaccine strain lyse in the host cytosol and activate the cytosolic DNA receptor Aim2. Here, we review the mechanisms leading or contributing to Aim2 inflammasome activation, including the role of TLRs and of IFN signaling and the implication of the guanylate-binding proteins 2 and 5 in triggering cytosolic bacteriolysis. Furthermore, we present how this cytosolic Gram-negative bacterium escapes recognition by caspase-11 but can trigger a non-canonical caspase-8 inflammasome. In addition, we highlight the differences in inflammasome activation in murine and human cells with pyrin, NLRP3, and AIM2 involved in sensing Francisella in human phagocytes. From a bacterial prospective, we describe the hiding strategy of Francisella to escape recognition by innate sensors and to resist to bacteriolysis in the host cytosol. Finally, we discuss the inability of the inflammasome sensors to detect F. tularensis subspecies tularensis strains, making them highly pathogenic stealth microbes.

  16. FEASIBILITY OF THE AEROSOL-TO-LIQUID PARTICLE EXTRACTION SYSTEM (ALPES) FOR COLLECTION OF VIABLE FRANCISELLA SP.

    SciTech Connect

    Heitkamp, M

    2006-08-07

    Several Biowatch monitoring sites in the Houston area have tested positive for Francisella tularensis and there is a need to determine whether natural occurring Francisella-related microorganism(s) may be responsible for these observed positive reactions. The collection, culturing and characterization of Francisella-related natural microorganisms will provide the knowledge base to improve the future selectivity of Biowatch monitoring for Francisella. The aerosol-to-liquid particle extraction system (ALPES) is a high-efficiency, dual mechanism collection system that utilizes a liquid collection medium for capture of airborne microorganisms. Since the viability of microorganisms is preserved better in liquid medium than on air filters, this project was undertaken to determine whether Francisella philomiragia and Francisella tularensis LVS maintain acceptable viability in the continuous liquid recirculation, high direct current voltage and residual ozone concentrations which occur during ALPES operation. Throughout a series of preliminary trial runs with representative gram-negative and gram-positive microorganisms, several design modifications and improvements to the ALPES optimized liquid handling, electrical stability, sampling and overall performance for biological sampling. Initial testing with Francisella philomiragia showed viability was preserved better in PBS buffer than HBSS buffer. Trial runs at starting cell concentrations of 1.8 x 10{sup 6} and 2.5 x 10{sup 4} CFU/L showed less than a 1-log decrease in viability for F. philomiragia after 24 h in the ALPES. Francisella tularensis LVS (live vaccine strain) was used as a surrogate for virulent F. tularensis in ALPES trial runs conducted at starting cell concentrations of 10{sup 4}, 10{sup 5} and 10{sup 6} CFU/L. F. tularensis LVS was slow-growing and required highly selective growth media to prevent overgrowth by collected airborne microorganisms. In addition, one ALPES unit intake was HEPA filtered during

  17. Evasion of IFN-γ Signaling by Francisella novicida Is Dependent upon Francisella Outer Membrane Protein C

    PubMed Central

    Nallaparaju, Kalyan C.; Yu, Jieh-Juen; Rodriguez, Stephen A.; Zogaj, Xhavit; Manam, Srikanth; Guentzel, M. Neal; Seshu, Janakiram; Murthy, Ashlesh K.; Chambers, James P.; Klose, Karl E.; Arulanandam, Bernard P.

    2011-01-01

    Background Francisella tularensis is a Gram-negative facultative intracellular bacterium and the causative agent of the lethal disease tularemia. An outer membrane protein (FTT0918) of F. tularensis subsp. tularensis has been identified as a virulence factor. We generated a F. novicida (F. tularensis subsp. novicida) FTN_0444 (homolog of FTT0918) fopC mutant to study the virulence-associated mechanism(s) of FTT0918. Methods and Findings The ΔfopC strain phenotype was characterized using immunological and biochemical assays. Attenuated virulence via the pulmonary route in wildtype C57BL/6 and BALB/c mice, as well as in knockout (KO) mice, including MHC I, MHC II, and µmT (B cell deficient), but not in IFN-γ or IFN-γR KO mice was observed. Primary bone marrow derived macrophages (BMDM) prepared from C57BL/6 mice treated with rIFN-γ exhibited greater inhibition of intracellular ΔfopC than wildtype U112 strain replication; whereas, IFN-γR KO macrophages showed no IFN-γ-dependent inhibition of ΔfopC replication. Moreover, phosphorylation of STAT1 was downregulated by the wildtype strain, but not the fopC mutant, in rIFN-γ treated macrophages. Addition of NG-monomethyl-L-arginine, an NOS inhibitor, led to an increase of ΔfopC replication to that seen in the BMDM unstimulated with rIFN-γ. Enzymatic screening of ΔfopC revealed aberrant acid phosphatase activity and localization. Furthermore, a greater abundance of different proteins in the culture supernatants of ΔfopC than that in the wildtype U112 strain was observed. Conclusions F. novicida FopC protein facilitates evasion of IFN-γ-mediated immune defense(s) by down-regulation of STAT1 phosphorylation and nitric oxide production, thereby promoting virulence. Additionally, the FopC protein also may play a role in maintaining outer membrane stability (integrity) facilitating the activity and localization of acid phosphatases and other F. novicida cell components. PMID:21483828

  18. Identification of Genome-Wide Mutations in Ciprofloxacin-Resistant F. tularensis LVS Using Whole Genome Tiling Arrays and Next Generation Sequencing

    PubMed Central

    Jaing, Crystal J.; McLoughlin, Kevin S.; Thissen, James B.; Zemla, Adam; Vergez, Lisa M.; Bourguet, Feliza; Mabery, Shalini; Fofanov, Viacheslav Y.; Koshinsky, Heather; Jackson, Paul J.

    2016-01-01

    Francisella tularensis is classified as a Class A bioterrorism agent by the U.S. government due to its high virulence and the ease with which it can be spread as an aerosol. It is a facultative intracellular pathogen and the causative agent of tularemia. Ciprofloxacin (Cipro) is a broad spectrum antibiotic effective against Gram-positive and Gram-negative bacteria. Increased Cipro resistance in pathogenic microbes is of serious concern when considering options for medical treatment of bacterial infections. Identification of genes and loci that are associated with Ciprofloxacin resistance will help advance the understanding of resistance mechanisms and may, in the future, provide better treatment options for patients. It may also provide information for development of assays that can rapidly identify Cipro-resistant isolates of this pathogen. In this study, we selected a large number of F. tularensis live vaccine strain (LVS) isolates that survived in progressively higher Ciprofloxacin concentrations, screened the isolates using a whole genome F. tularensis LVS tiling microarray and Illumina sequencing, and identified both known and novel mutations associated with resistance. Genes containing mutations encode DNA gyrase subunit A, a hypothetical protein, an asparagine synthase, a sugar transamine/perosamine synthetase and others. Structural modeling performed on these proteins provides insights into the potential function of these proteins and how they might contribute to Cipro resistance mechanisms. PMID:27668749

  19. Francisella DnaK Inhibits Tissue-nonspecific Alkaline Phosphatase*

    PubMed Central

    Arulanandam, Bernard P.; Chetty, Senthilnath Lakshmana; Yu, Jieh-Juen; Leonard, Sean; Klose, Karl; Seshu, Janakiram; Cap, Andrew; Valdes, James J.; Chambers, James P.

    2012-01-01

    Following pulmonary infection with Francisella tularensis, we observed an unexpected but significant reduction of alkaline phosphatase, an enzyme normally up-regulated following inflammation. However, no reduction was observed in mice infected with a closely related Gram-negative pneumonic organism (Klebsiella pneumoniae) suggesting the inhibition may be Francisella-specific. In similar fashion to in vivo observations, addition of Francisella lysate to exogenous alkaline phosphatase (tissue-nonspecific isozyme) was inhibitory. Partial purification and subsequent proteomic analysis indicated the inhibitory factor to be the heat shock protein DnaK. Incubation with increasing amounts of anti-DnaK antibody reduced the inhibitory effect in a dose-dependent manner. Furthermore, DnaK contains an adenosine triphosphate binding domain at its N terminus, and addition of adenosine triphosphate enhances dissociation of DnaK with its target protein, e.g. alkaline phosphatase. Addition of adenosine triphosphate resulted in decreased DnaK co-immunoprecipitated with alkaline phosphatase as well as reduction of Francisella-mediated alkaline phosphatase inhibition further supporting the binding of Francisella DnaK to alkaline phosphatase. Release of DnaK via secretion and/or bacterial cell lysis into the extracellular milieu and inhibition of plasma alkaline phosphatase could promote an orchestrated, inflammatory response advantageous to Francisella. PMID:22923614

  20. Nanolipoprotein Particles (NLPs) as Versatile Vaccine Platforms for Co-delivery of Multiple Adjuvants with Subunit Antigens from Burkholderia spp. and F. tularensis - Annual Technical Report

    SciTech Connect

    Fischer, N. O.

    2015-04-16

    The goal of this proposal is to demonstrate that co-localization of protein subunit antigens and adjuvants on nanolipoprotein particles (NLPs) can increase the protective efficacy of recombinant subunit antigens from Burkholderia spp. and Francisella tularensis against an aerosol challenge. NLPs are are biocompatible, high-density lipoprotein mimetics that are amenable to the incorporation of multiple, chemically-disparate adjuvant and antigen molecules. We hypothesize that the ability to co-localize optimized adjuvant formulations with subunit antigens within a single particle will enhance the stimulation and activation of key immune effector cells, increasing the protective efficacy of subunit antigen-based vaccines. While Burkholderia spp. and F. tularensis subunit antigens are the focus of this proposal, we anticipate that this approach is applicable to a wide range of DOD-relevant biothreat agents. The F344 rat aerosol challenge model for F. tularensis has been successfully established at Battelle under this contract, and Year 3 efficacy studies performed at Battelle demonstrated that an NLP vaccine formulation was able to enhance survival of female F344 rats relative to naïve animals. In addition, Year 3 focused on the incorporation of multiple Burkholderia antigens (both polysaccharides and proteins) onto adjuvanted NLPs, with immunological analysis poised to begin in the next quarter.

  1. Importance of Branched-Chain Amino Acid Utilization in Francisella Intracellular Adaptation

    PubMed Central

    Gesbert, Gael; Ramond, Elodie; Tros, Fabiola; Dairou, Julien; Frapy, Eric; Barel, Monique

    2014-01-01

    Intracellular bacterial pathogens have adapted their metabolism to optimally utilize the nutrients available in infected host cells. We recently reported the identification of an asparagine transporter required specifically for cytosolic multiplication of Francisella. In the present work, we characterized a new member of the major super family (MSF) of transporters, involved in isoleucine uptake. We show that this transporter (here designated IleP) plays a critical role in intracellular metabolic adaptation of Francisella. Inactivation of IleP severely impaired intracellular F. tularensis subsp. novicida multiplication in all cell types tested and reduced bacterial virulence in the mouse model. To further establish the importance of the ileP gene in F. tularensis pathogenesis, we constructed a chromosomal deletion mutant of ileP (ΔFTL_1803) in the F. tularensis subsp. holarctica live vaccine strain (LVS). Inactivation of IleP in the F. tularensis LVS provoked comparable intracellular growth defects, confirming the critical role of this transporter in isoleucine uptake. The data presented establish, for the first time, the importance of isoleucine utilization for efficient phagosomal escape and cytosolic multiplication of Francisella and suggest that virulent F. tularensis subspecies have lost their branched-chain amino acid biosynthetic pathways and rely exclusively on dedicated uptake systems. This loss of function is likely to reflect an evolution toward a predominantly intracellular life style of the pathogen. Amino acid transporters should be thus considered major players in the adaptation of intracellular pathogens. PMID:25332124

  2. Francisella philomiragia adenitis and pulmonary nodules in a child with chronic granulomatous disease.

    PubMed

    Mailman, Timothy L; Schmidt, Matthias H

    2005-07-01

    Francisella philomiragia is a rare and opportunistic pathogen capable of producing invasive infection in patients with compromised neutrophil function and in patients that have survived a near-drowning. A case of F philomiragia adenitis and lung nodules, refractory to cephalosporin therapy, is reported in a 10-year-old boy with chronic granulomatous disease following a facial abrasion from a saltwater crab. To the authors' knowledge, this is the first Canadian clinical isolate to be reported. Genus and species identification was confirmed via 16S ribosomal RNA sequence analysis. A literature review revealed three groups at risk of F philomiragia infection: young patients with chronic granulomatous disease; adults with hematogenous malignancy; and near-drowning patients. Pneumonia, fever without an apparent source and sepsis are the main clinical presentations. Invasive procedures may be required to isolate this organism and ensure appropriate antimicrobial therapy. Limited awareness of F philomiragia has led to delayed identification, patient death and misidentification as Francisella tularensis - a biosafety level three pathogen and potential bioterrorism agent.

  3. DNA capture elements for rapid detection and identification of biological agents

    NASA Astrophysics Data System (ADS)

    Kiel, Johnathan L.; Parker, Jill E.; Holwitt, Eric A.; Vivekananda, Jeeva

    2004-08-01

    DNA capture elements (DCEs; aptamers) are artificial DNA sequences, from a random pool of sequences, selected for their specific binding to potential biological warfare agents. These sequences were selected by an affinity method using filters to which the target agent was attached and the DNA isolated and amplified by polymerase chain reaction (PCR) in an iterative, increasingly stringent, process. Reporter molecules were attached to the finished sequences. To date, we have made DCEs to Bacillus anthracis spores, Shiga toxin, Venezuelan Equine Encephalitis (VEE) virus, and Francisella tularensis. These DCEs have demonstrated specificity and sensitivity equal to or better than antibody.

  4. Detection of biological warfare agents using the polymerase chain reaction. Final report, June-August 1991

    SciTech Connect

    Mann, B.J.

    1992-09-01

    The detection of biological warfare agents is an important mission for the U.S. Army. This report explores the feasibility of using the polymerase chain reaction as a means of rapid detection of biological warfare agents. Two levels of detection are proposed. The first level is group specific detection, using primers derived from 16S rDNA sequences, to detect various groups of pathogenic bacteria. The second level is species-specific detection using primers derived from DNA sequences, unique to each pathogenic organism targeted for detection. Specific examples of Vibrio cholerae, Francisella tularensis, Yersinia pestis, Staphylococcus aureus, and Bacillus anthracis are described.

  5. Common ancestry and novel genetic traits of Francisella novicida-like isolates from North America and Australia as revealed by comparative genomic analyses.

    PubMed

    Siddaramappa, Shivakumara; Challacombe, Jean F; Petersen, Jeannine M; Pillai, Segaran; Hogg, Geoff; Kuske, Cheryl R

    2011-08-01

    Francisella novicida is a close relative of Francisella tularensis, the causative agent of tularemia. The genomes of F. novicida-like clinical isolates 3523 (Australian strain) and Fx1 (Texas strain) were sequenced and compared to F. novicida strain U112 and F. tularensis strain Schu S4. The strain 3523 chromosome is 1,945,310 bp and contains 1,854 protein-coding genes. The strain Fx1 chromosome is 1,913,619 bp and contains 1,819 protein-coding genes. NUCmer analyses revealed that the genomes of strains Fx1 and U112 are mostly colinear, whereas the genome of strain 3523 has gaps, translocations, and/or inversions compared to genomes of strains Fx1 and U112. Using the genome sequence data and comparative analyses with other members of the genus Francisella, several strain-specific genes that encode putative proteins involved in RTX toxin production, polysaccharide biosynthesis/modification, thiamine biosynthesis, glucuronate utilization, and polyamine biosynthesis were identified. The RTX toxin synthesis and secretion operon of strain 3523 contains four open reading frames (ORFs) and was named rtxCABD. Based on the alignment of conserved sequences upstream of operons involved in thiamine biosynthesis from various bacteria, a putative THI box was identified in strain 3523. The glucuronate catabolism loci of strains 3523 and Fx1 contain a cluster of nine ORFs oriented in the same direction that appear to constitute an operon. Strains U112 and Schu S4 appeared to have lost the loci for RTX toxin production, thiamine biosynthesis, and glucuronate utilization as a consequence of host adaptation and reductive evolution. In conclusion, comparative analyses provided insights into the common ancestry and novel genetic traits of these strains. PMID:21666011

  6. Common Ancestry and Novel Genetic Traits of Francisella novicida-Like Isolates from North America and Australia as Revealed by Comparative Genomic Analyses▿†

    PubMed Central

    Siddaramappa, Shivakumara; Challacombe, Jean F; Petersen, Jeannine M; Pillai, Segaran; Hogg, Geoff; Kuske, Cheryl R

    2011-01-01

    Francisella novicida is a close relative of Francisella tularensis, the causative agent of tularemia. The genomes of F. novicida-like clinical isolates 3523 (Australian strain) and Fx1 (Texas strain) were sequenced and compared to F. novicida strain U112 and F. tularensis strain Schu S4. The strain 3523 chromosome is 1,945,310 bp and contains 1,854 protein-coding genes. The strain Fx1 chromosome is 1,913,619 bp and contains 1,819 protein-coding genes. NUCmer analyses revealed that the genomes of strains Fx1 and U112 are mostly colinear, whereas the genome of strain 3523 has gaps, translocations, and/or inversions compared to genomes of strains Fx1 and U112. Using the genome sequence data and comparative analyses with other members of the genus Francisella, several strain-specific genes that encode putative proteins involved in RTX toxin production, polysaccharide biosynthesis/modification, thiamine biosynthesis, glucuronate utilization, and polyamine biosynthesis were identified. The RTX toxin synthesis and secretion operon of strain 3523 contains four open reading frames (ORFs) and was named rtxCABD. Based on the alignment of conserved sequences upstream of operons involved in thiamine biosynthesis from various bacteria, a putative THI box was identified in strain 3523. The glucuronate catabolism loci of strains 3523 and Fx1 contain a cluster of nine ORFs oriented in the same direction that appear to constitute an operon. Strains U112 and Schu S4 appeared to have lost the loci for RTX toxin production, thiamine biosynthesis, and glucuronate utilization as a consequence of host adaptation and reductive evolution. In conclusion, comparative analyses provided insights into the common ancestry and novel genetic traits of these strains. PMID:21666011

  7. Increased knowledge of Francisella genus diversity highlights the benefits of optimised DNA-based assays

    PubMed Central

    2012-01-01

    Background Recent advances in sequencing technologies offer promising tools for generating large numbers of genomes, larger typing databases and improved mapping of environmental bacterial diversity. However, DNA-based methods for the detection of Francisella were developed with limited knowledge about genetic diversity. This, together with the high sequence identity between several Francisella species, means there is a high risk of false identification and detection of the highly virulent pathogen Francisella tularensis. Moreover, phylogenetic reconstructions using single or limited numbers of marker sequences often result in incorrect tree topologies and inferred evolutionary distances. The recent growth in publicly accessible whole-genome sequences now allows evaluation of published genetic markers to determine optimal combinations of markers that minimise both time and laboratory costs. Results In the present study, we evaluated 38 previously published DNA markers and the corresponding PCR primers against 42 genomes representing the currently known diversity of the genus Francisella. The results highlight that PCR assays for Francisella tularensis are often complicated by low specificity, resulting in a high probability of false positives. A method to select a set of one to seven markers for obtaining optimal phylogenetic resolution or diagnostic accuracy is presented. Conclusions Current multiple-locus sequence-typing systems and detection assays of Francisella, could be improved by redesigning some of the primers and reselecting typing markers. The use of only a few optimally selected sequence-typing markers allows construction of phylogenetic topologies with almost the same accuracy as topologies based on whole-genome sequences. PMID:23009728

  8. Gluconeogenesis, an essential metabolic pathway for pathogenic Francisella.

    PubMed

    Brissac, Terry; Ziveri, Jason; Ramond, Elodie; Tros, Fabiola; Kock, Stephanie; Dupuis, Marion; Brillet, Magali; Barel, Monique; Peyriga, Lindsay; Cahoreau, Edern; Charbit, Alain

    2015-10-01

    Intracellular multiplication and dissemination of the infectious bacterial pathogen Francisella tularensis implies the utilization of multiple host-derived nutrients. Here, we demonstrate that gluconeogenesis constitutes an essential metabolic pathway in Francisella pathogenesis. Indeed, inactivation of gene glpX, encoding the unique fructose 1,6-bisphosphatase of Francisella, severely impaired bacterial intracellular multiplication when cells were supplemented by gluconeogenic substrates such as glycerol or pyruvate. The ΔglpX mutant also showed a severe virulence defect in the mouse model, confirming the importance of this pathway during the in vivo life cycle of the pathogen. Isotopic profiling revealed the major role of the Embden-Meyerhof (glycolysis) pathway in glucose catabolism in Francisella and confirmed the importance of glpX in gluconeogenesis. Altogether, the data presented suggest that gluconeogenesis allows Francisella to cope with the limiting glucose availability it encounters during its infectious cycle by relying on host amino acids. Hence, targeting the gluconeogenic pathway might constitute an interesting therapeutic approach against this pathogen.

  9. Francisella infections in farmed and wild aquatic organisms.

    PubMed

    Colquhoun, Duncan J; Duodu, Samuel

    2011-01-01

    Over the last 10 years or so, infections caused by bacteria belonging to a particular branch of the genus Francisella have become increasingly recognised in farmed fish and molluscs worldwide. While the increasing incidence of diagnoses may in part be due to the development and widespread availability of molecular detection techniques, the domestication of new organisms has undoubtedly instigated emergence of clinical disease in some species. Francisellosis in fish develops in a similar fashion independent of host species and is commonly characterised by the presence of multi-organ granuloma and high morbidity, with varying associated mortality levels. A number of fish species are affected including Atlantic cod, Gadus morhua; tilapia, Oreochromis sp.; Atlantic salmon, Salmo salar; hybrid striped bass, Morone chrysops × M. saxatilis and three-lined grunt, Parapristipoma trilinineatum. The disease is highly infectious and often prevalent in affected stocks. Most, if not all strains isolated from teleost fish belong to either F. noatunensis subsp. orientalis in warm water fish species or Francisella noatunensis subsp. noatunensis in coldwater fish species. The disease is quite readily diagnosed following histological examination and identification of the aetiological bacterium by culture on cysteine rich media or PCR. The available evidence may indicate a degree of host specificity for the various Francisella strains, although this area requires further study. No effective vaccine is currently available. Investigation of the virulence mechanisms and host response shows similarity to those known from Francisella tularensis infection in mammals. However, no evidence exists for zoonotic potential amongst the fish pathogenic Francisella. PMID:21385413

  10. Francisella infections in farmed and wild aquatic organisms

    PubMed Central

    2011-01-01

    Over the last 10 years or so, infections caused by bacteria belonging to a particular branch of the genus Francisella have become increasingly recognised in farmed fish and molluscs worldwide. While the increasing incidence of diagnoses may in part be due to the development and widespread availability of molecular detection techniques, the domestication of new organisms has undoubtedly instigated emergence of clinical disease in some species. Francisellosis in fish develops in a similar fashion independent of host species and is commonly characterised by the presence of multi-organ granuloma and high morbidity, with varying associated mortality levels. A number of fish species are affected including Atlantic cod, Gadus morhua; tilapia, Oreochromis sp.; Atlantic salmon, Salmo salar; hybrid striped bass, Morone chrysops × M. saxatilis and three-lined grunt, Parapristipoma trilinineatum. The disease is highly infectious and often prevalent in affected stocks. Most, if not all strains isolated from teleost fish belong to either F. noatunensis subsp. orientalis in warm water fish species or Francisella noatunensis subsp. noatunensis in coldwater fish species. The disease is quite readily diagnosed following histological examination and identification of the aetiological bacterium by culture on cysteine rich media or PCR. The available evidence may indicate a degree of host specificity for the various Francisella strains, although this area requires further study. No effective vaccine is currently available. Investigation of the virulence mechanisms and host response shows similarity to those known from Francisella tularensis infection in mammals. However, no evidence exists for zoonotic potential amongst the fish pathogenic Francisella. PMID:21385413

  11. Attenuated virulence of a Francisella mutant lacking the lipid A 4′-phosphatase

    PubMed Central

    Wang, Xiaoyuan; Ribeiro, Anthony A.; Guan, Ziqiang; Abraham, Soman N.; Raetz, Christian R. H.

    2007-01-01

    Francisella tularensis causes tularemia, a highly contagious disease of animals and humans, but the virulence features of F. tularensis are poorly defined. F. tularensis and the related mouse pathogen Francisella novicida synthesize unusual lipid A molecules lacking the 4′-monophosphate group typically found in the lipid A of Gram-negative bacteria. LpxF, a selective phosphatase located on the periplasmic surface of the inner membrane, removes the 4′-phosphate moiety in the late stages of F. novicida lipid A assembly. To evaluate the relevance of the 4′-phosphatase to pathogenesis, we constructed a deletion mutant of lpxF and compared its virulence with wild-type F. novicida. Intradermal injection of 106 wild-type but not 108 mutant F. novicida cells is lethal to mice. The rapid clearance of the lpxF mutant is associated with a stronger local cytokine response and a greater influx of neutrophils compared with wild-type. The F. novicida mutant was highly susceptible to the cationic antimicrobial peptide polymyxin. LpxF therefore represents a kind of virulence factor that confers a distinct lipid A phenotype, preventing Francisella from activating the host innate immune response and preventing the bactericidal actions of cationic peptides. Francisella lpxF mutants may be useful for immunization against tularemia. PMID:17360489

  12. Francisella asiatica as the causative agent of piscine francisellosis in cultured tilapia (Oreochromis sp.) in the United States.

    PubMed

    Soto, Esteban; Baumgartner, Wes; Wiles, Judy; Hawke, John P

    2011-07-01

    Francisella asiatica is a Gram-negative, pleomorphic, facultative intracellular, bacterial pathogen that causes acute to chronic disease in a wide variety of warm-water cultured and wild fish species. Outbreaks of francisellosis in warm water fish have been documented in Taiwan, Japan, United Kingdom, Hawaii, and Latin America (including Costa Rica) but the organism has only been reported from the United States on one occasion from hybrid striped bass in California. In 2010, the bacterium was detected from diseased tilapia by culture on cystine heart agar supplemented with hemoglobin and by utilizing an F. asiatica-specific real-time polymerase chain reaction assay. The tilapia (Oreochromis niloticus) were cultured in an indoor, closed, recirculating aquaculture facility in the Midwest of the United States. The identity of isolates recovered from diseased fish was confirmed as F. asiatica by amplification and sequence comparison of the 16S ribosomal RNA and intracellular growth locus C (iglC) gene. Gross and microscopic examination of affected tissues revealed the presence of marked anterior renomegaly and splenomegaly with severe granulomatous disease. PMID:21908332

  13. A piscirickettsiosis-like syndrome in cultured Nile tilapia in Latin America with Francisella spp. as the pathogenic agent.

    PubMed

    Mauel, M J; Soto, E; Moralis, J A; Hawke, J

    2007-03-01

    In 2004, cultured Nile tilapia Oreochromis niloticus in several Latin America farms began to succumb to a disease similar to the piscirickettsiosis-like syndrome previously reported in tilapia in Taiwan and the United States. Mortality increased during 2005; reductions in tilapia biomass ranged from 5% to 80% in individual ponds and averaged 50% overall. All ages of fish have been involved. Clinical signs include lethargy, loss of appetite, petechia, exophthalmia, and abnormal swimming behavior. Gross lesions have included splenomegaly, renomegaly, and numerous white nodules observed in the spleen, kidney, testes, heart, ovaries, and occasionally the liver. A previously unreported black granulomatous lesion was reported in up to 30% of the fillets. Histologically, granulomatous infiltrates were observed in the kidney, spleen, liver, testes, ovary, and choroid gland, and rarely in the brain and heart. A small pleomorphic bacterium was observed in Giemsa-stained blood smears and spleen imprints. The bacterium did not grow on standard microbiological media and has not been isolated in cell culture. We obtained a near-complete 16S ribosomal DNA sequence with high similarity to Francisella spp. sequences previously identified in tilapias Oreochromis spp. (Taiwan), Atlantic cod Gadus morhua (Norway), and three-line grunts Parapristipoma trilineatum (Japan). PMID:18236629

  14. Fast, sensitive point of care electrochemical molecular system for point mutation and select agent detection.

    PubMed

    MacLeod, J A; Nemeth, A C; Dicke, W C; Wang, D; Manalili Wheeler, S; Hannis, J C; Collier, G B; Drader, J J

    2016-07-01

    Point of care molecular diagnostics benefits from a portable battery-operated device capable of performing a fast turnaround using reliable inexpensive cartridges. We describe a prototype device for performing a molecular diagnostics test for clinical and biodefense samples in 16 minutes using a prototype capable of an 8 minute PCR reaction, followed by hybridization and detection on an electrochemical microarray based on the i-STAT® system. We used human buccal swabs for hemochromatosis testing including in-device DNA extraction. Additional clinical and biodefense samples included influenza A and bacterial select agents Bacillus anthracis, Yersinia pestis and Francisella tularensis. PMID:27280174

  15. Tick-related facial cellulitis caused by Francisella tularensis.

    PubMed

    Arslan, Ferhat; Karagöz, Ergenekon; Zemheri, Ebru; Vahaboglu, Haluk; Mert, Ali

    2016-06-01

    Tick-borne illnesses have diverse biological and clinical features that make recognition and appropriate treatment challenging. Arthropod-transmitted (ticks, fleas and deer flies) tularaemia remains a concern worldwide. Generally, two kinds of tularaemia manifestations, namely ulceroglandular and glandular infections, can arise from the bite of an infected arthropod vector. If the ulceroglandular or glandular form is not treated, suppuration can arise from the gland. In addition, cellulitis is rarely observed around the ulcers. In our case, with the knowledge of tick exposure to the scalp, tularaemia was not initially considered for facial cellulitis without regional lymphadenopathy and also due to apparent failure to respond to doxycycline and gentamicin therapy. Serological confirmation in the late stages of the disease suggests the importance of clinical suspicion in such rare conditions. PMID:27367325

  16. Comparison of traditional and molecular analytical methods for detecting biological agents in raw and drinking water following ultrafiltration

    USGS Publications Warehouse

    Francy, D.S.; Bushon, R.N.; Brady, A.M.G.; Bertke, E.E.; Kephart, C.M.; Likirdopulos, C.A.; Mailot, B.E.; Schaefer, F. W.; Lindquist, H.D. Alan

    2009-01-01

    Aims: To compare the performance of traditional methods to quantitative polymerase chain reaction (qPCR) for detecting five biological agents in large-volume drinking-water samples concentrated by ultrafiltration (UF). Methods and Results: Drinking-water samples (100 l) were seeded with Bacillus anthracis, Cryptospordium parvum, Francisella tularensis, Salmonella Typhi, and Vibrio cholerae and concentrated by UF. Recoveries by traditional methods were variable between samples and between some replicates; recoveries were not determined by qPCR. Francisella tularensis and V. cholerae were detected in all 14 samples after UF, B. anthracis was detected in 13, and C. parvum was detected in 9 out of 14 samples. Numbers found by qPCR after UF were significantly or nearly related to those found by traditional methods for all organisms except for C. parvum. A qPCR assay for S. Typhi was not available. Conclusions: qPCR can be used to rapidly detect biological agents after UF as well as traditional methods, but additional work is needed to improve qPCR assays for several biological agents, determine recoveries by qPCR, and expand the study to other areas. Significance and Impact of the Study: To our knowledge, this is the first study to compare the use of traditional and qPCR methods to detect biological agents in large-volume drinking-water samples. ?? 2009 The Society for Applied Microbiology.

  17. A Real-Time PCR Array for Hierarchical Identification of Francisella Isolates

    PubMed Central

    Svensson, Kerstin; Granberg, Malin; Karlsson, Linda; Neubauerova, Vera; Forsman, Mats; Johansson, Anders

    2009-01-01

    A robust, rapid and flexible real-time PCR assay for hierarchical genetic typing of clinical and environmental isolates of Francisella is presented. Typing markers were found by multiple genome and gene comparisons, from which 23 canonical single nucleotide polymorphisms (canSNPs) and 11 canonical insertion-deletion mutations (canINDELs) were selected to provide phylogenetic guidelines for classification from genus to isolate level. The specificity of the developed assay, which uses 68 wells of a 96-well real-time PCR format with a detection limit of 100 pg DNA, was assessed using 62 Francisella isolates of diverse genetic and geographical origins. It was then successfully used for typing 14 F. tularensis subsp. holarctica isolates obtained from tularemia patients in Sweden in 2008 and five more genetically diverse Francisella isolates of global origins. When applied to human ulcer specimens for direct pathogen detection the results were incomplete due to scarcity of DNA, but sufficient markers were identified to detect fine-resolution differences among F. tularensis subsp. holarctica isolates causing infection in the patients. In contrast to other real-time PCR assays for Francisella, which are typically designed for specific detection of a species, subspecies, or strain, this type of assay can be easily tailored to provide appropriate phylogenetic and/or geographical resolution to meet the objectives of the analysis. PMID:20027310

  18. Francisella novicida forms in vitro biofilms mediated by an orphan response regulator.

    PubMed

    Durham-Colleran, Meghan W; Verhoeven, Anne Brooks; van Hoek, Monique L

    2010-04-01

    Francisella tularensis is associated with water and waterways and infects many species of animals, insects, and protists. The mechanism Francisella utilizes to persist in the environment and in tick vectors is currently unknown. We have demonstrated for the first time that Francisella novicida, a model organism of F. tularensis, forms a biofilm in vitro. Selected F. novicida transposon mutants were tested for their ability to form biofilm compared to the wildtype F. novicida strain. Mutation of the putative qseB gene led to an impairment in the ability to form biofilm with no impairment in bacterial growth. A qseC mutant had impaired growth but demonstrated a marked impairment in biofilm production. Mutation in capC affected both bacterial growth and biofilm formation, but no biofilm production impairment was seen with capB or pilE mutants. A deletion mutant in the orphan response regulator FTN_1465, which we propose is the putative QseB, formed significantly less biofilm than the wildtype. When FTN_1465 was complemented back into the deletion mutant, biofilm formation was restored. Thus, the orphan response regulator FTN_1465 is an important factor in biofilm production in vitro in F. novicida. These results demonstrate that Francisella species are able to form biofilms in vitro, suggesting that biofilm formation may be important for the lifecycle of this organism.

  19. Susceptibility of Select Agents to Predation by Predatory Bacteria

    PubMed Central

    Russo, Riccardo; Chae, Richard; Mukherjee, Somdatta; Singleton, Eric J.; Occi, James L.; Kadouri, Daniel E.; Connell, Nancy D.

    2015-01-01

    Select Agents are microorganisms and toxins considered to be exploitable as biological weapons. Although infections by many Select Agents can be treated by conventional antibiotics, the risk of an emerging or engineered drug resistant strain is of great concern. One group of microorganisms that is showing potential to control drug resistant Gram-negative bacteria are the predatory bacteria from the genera Bdellovibrio spp. and Micavibrio spp. In this study, we have examined the ability of Bdellovibrio bacteriovorus (B. bacteriovorus) strain 109J, HD100 and Micavibrio aeruginosavorus (M. aeruginosavorus) ARL-13 to prey on a variety of Select Agents. Our findings demonstrate that B. bacteriovorus and M. aeruginosavorus are able to prey efficiently on Yersinia pestis and Burkholderia mallei. Modest predation was also measured in co-cultures of B. bacteriovorus and Francisella tularensis. However, neither of the predators showed predation when Burkholderia pseudomallei and Brucella melitensis were used as prey.

  20. Susceptibility of Select Agents to Predation by Predatory Bacteria

    PubMed Central

    Russo, Riccardo; Chae, Richard; Mukherjee, Somdatta; Singleton, Eric J.; Occi, James L.; Kadouri, Daniel E.; Connell, Nancy D.

    2015-01-01

    Select Agents are microorganisms and toxins considered to be exploitable as biological weapons. Although infections by many Select Agents can be treated by conventional antibiotics, the risk of an emerging or engineered drug resistant strain is of great concern. One group of microorganisms that is showing potential to control drug resistant Gram-negative bacteria are the predatory bacteria from the genera Bdellovibrio spp. and Micavibrio spp. In this study, we have examined the ability of Bdellovibrio bacteriovorus (B. bacteriovorus) strain 109J, HD100 and Micavibrio aeruginosavorus (M. aeruginosavorus) ARL-13 to prey on a variety of Select Agents. Our findings demonstrate that B. bacteriovorus and M. aeruginosavorus are able to prey efficiently on Yersinia pestis and Burkholderia mallei. Modest predation was also measured in co-cultures of B. bacteriovorus and Francisella tularensis. However, neither of the predators showed predation when Burkholderia pseudomallei and Brucella melitensis were used as prey. PMID:27682124

  1. Burkholderia Diffusible Signal Factor Signals to Francisella novicida To Disperse Biofilm and Increase Siderophore Production

    PubMed Central

    Dean, Scott N.; Chung, Myung-Chul

    2015-01-01

    In many bacteria, the ability to modulate biofilm production relies on specific signaling molecules that are either self-produced or made by neighboring microbes within the ecological niche. We analyzed the potential interspecies signaling effect of the Burkholderia diffusible signal factor (BDSF) on Francisella novicida, a model organism for Francisella tularensis, and demonstrated that BDSF both inhibits the formation and causes the dispersion of Francisella biofilm. Specificity was demonstrated for the cis versus the trans form of BDSF. Using transcriptome sequencing, quantitative reverse transcription-PCR, and activity assays, we found that BDSF altered the expression of many F. novicida genes, including genes involved in biofilm formation, such as chitinases. Using a chitinase inhibitor, the antibiofilm activity of BDSF was also shown to be chitinase dependent. In addition, BDSF caused an increase in RelA expression and increased levels of (p)ppGpp, leading to decreased biofilm production. These results support our observation that exposure of F. novicida to BDSF causes biofilm dispersal. Furthermore, BDSF upregulated the genes involved in iron acquisition (figABCD), increasing siderophore production. Thus, this study provides evidence for a potential role and mechanism of diffusible signal factor (DSF) signaling in the genus Francisella and suggests the possibility of interspecies signaling between Francisella and other bacteria. Overall, this study suggests that in response to the interspecies DSF signal, F. novicida can alter its gene expression and regulate its biofilm formation. PMID:26231649

  2. Monocyte/macrophage inflammatory response pathways to combat Francisella infection: possible therapeutic targets?

    PubMed Central

    Gillette, Devyn D.; Tridandapani, Susheela; Butchar, Jonathan P.

    2014-01-01

    Francisella tularensis can bypass and suppress host immune responses, even to the point of manipulating immune cell phenotypes and intercellular inflammatory networks. Strengthening these responses such that immune cells more readily identify and destroy the bacteria is likely to become a viable (and perhaps necessary) strategy for combating infections with Francisella, especially given the likelihood of antibiotic resistance in the foreseeable future. Monocytes and macrophages offer a niche wherein Francisella can invade and replicate, resulting in substantially higher bacterial load that can overcome the host. As such, understanding their responses to Francisella may uncover potential avenues of therapy that could promote a lowering of bacterial burden and clearance of infection. These response pathways include Toll-like Receptor 2 (TLR2), the caspase-1 inflammasome, Interferons, NADPH oxidase, Phosphatidylinositide 3-kinase (PI3K), and the Ras pathway. In this review we summarize the literature pertaining to the roles of these pathways during Francisella infection, with an emphasis on monocyte/macrophage responses. The therapeutic targeting of one or more such pathways may ultimately become a valuable tool for the treatment of tularemia, and several possibilities are discussed. PMID:24600590

  3. Burkholderia Diffusible Signal Factor Signals to Francisella novicida To Disperse Biofilm and Increase Siderophore Production.

    PubMed

    Dean, Scott N; Chung, Myung-Chul; van Hoek, Monique L

    2015-10-01

    In many bacteria, the ability to modulate biofilm production relies on specific signaling molecules that are either self-produced or made by neighboring microbes within the ecological niche. We analyzed the potential interspecies signaling effect of the Burkholderia diffusible signal factor (BDSF) on Francisella novicida, a model organism for Francisella tularensis, and demonstrated that BDSF both inhibits the formation and causes the dispersion of Francisella biofilm. Specificity was demonstrated for the cis versus the trans form of BDSF. Using transcriptome sequencing, quantitative reverse transcription-PCR, and activity assays, we found that BDSF altered the expression of many F. novicida genes, including genes involved in biofilm formation, such as chitinases. Using a chitinase inhibitor, the antibiofilm activity of BDSF was also shown to be chitinase dependent. In addition, BDSF caused an increase in RelA expression and increased levels of (p)ppGpp, leading to decreased biofilm production. These results support our observation that exposure of F. novicida to BDSF causes biofilm dispersal. Furthermore, BDSF upregulated the genes involved in iron acquisition (figABCD), increasing siderophore production. Thus, this study provides evidence for a potential role and mechanism of diffusible signal factor (DSF) signaling in the genus Francisella and suggests the possibility of interspecies signaling between Francisella and other bacteria. Overall, this study suggests that in response to the interspecies DSF signal, F. novicida can alter its gene expression and regulate its biofilm formation. PMID:26231649

  4. Mutations of Francisella novicida that Alter the Mechanism of Its Phagocytosis by Murine Macrophages

    PubMed Central

    Lai, Xin-He; Shirley, Renee L.; Crosa, Lidia; Kanistanon, Duangjit; Tempel, Rebecca; Ernst, Robert K.; Gallagher, Larry A.; Manoil, Colin; Heffron, Fred

    2010-01-01

    Infection with the bacterial pathogen Francisella tularensis tularensis (F. tularensis) causes tularemia, a serious and debilitating disease. Francisella tularensis novicida strain U112 (abbreviated F. novicida), which is closely related to F. tularensis, is pathogenic for mice but not for man, making it an ideal model system for tularemia. Intracellular pathogens like Francisella inhibit the innate immune response, thereby avoiding immune recognition and death of the infected cell. Because activation of inflammatory pathways may lead to cell death, we reasoned that we could identify bacterial genes involved in inhibiting inflammation by isolating mutants that killed infected cells faster than the wild-type parent. We screened a comprehensive transposon library of F. novicida for mutant strains that increased the rate of cell death following infection in J774 macrophage-like cells, as compared to wild-type F. novicida. Mutations in 28 genes were identified as being hypercytotoxic to both J774 and primary macrophages of which 12 were less virulent in a mouse infection model. Surprisingly, we found that F. novicida with mutations in four genes (lpcC, manB, manC and kdtA) were taken up by and killed macrophages at a much higher rate than the parent strain, even upon treatment with cytochalasin D (cytD), a classic inhibitor of macrophage phagocytosis. At least 10-fold more mutant bacteria were internalized by macrophages as compared to the parent strain if the bacteria were first fixed with formaldehyde, suggesting a surface structure is required for the high phagocytosis rate. However, bacteria were required to be viable for macrophage toxicity. The four mutant strains do not make a complete LPS but instead have an exposed lipid A. Interestingly, other mutations that result in an exposed LPS core were not taken up at increased frequency nor did they kill host cells more than the parent. These results suggest an alternative, more efficient macrophage uptake mechanism

  5. Metabolic Network Analysis-Based Identification of Antimicrobial Drug Targets in Category A Bioterrorism Agents

    PubMed Central

    Ahn, Yong-Yeol; Lee, Deok-Sun; Burd, Henry; Blank, William; Kapatral, Vinayak

    2014-01-01

    The 2001 anthrax mail attacks in the United States demonstrated the potential threat of bioterrorism, hence driving the need to develop sophisticated treatment and diagnostic protocols to counter biological warfare. Here, by performing flux balance analyses on the fully-annotated metabolic networks of multiple, whole genome-sequenced bacterial strains, we have identified a large number of metabolic enzymes as potential drug targets for each of the three Category A-designated bioterrorism agents including Bacillus anthracis, Francisella tularensis and Yersinia pestis. Nine metabolic enzymes- belonging to the coenzyme A, folate, phosphatidyl-ethanolamine and nucleic acid pathways common to all strains across the three distinct genera were identified as targets. Antimicrobial agents against some of these enzymes are available. Thus, a combination of cross species-specific antibiotics and common antimicrobials against shared targets may represent a useful combinatorial therapeutic approach against all Category A bioterrorism agents. PMID:24454817

  6. Metabolic network analysis-based identification of antimicrobial drug targets in category A bioterrorism agents.

    PubMed

    Ahn, Yong-Yeol; Lee, Deok-Sun; Burd, Henry; Blank, William; Kapatral, Vinayak

    2014-01-01

    The 2001 anthrax mail attacks in the United States demonstrated the potential threat of bioterrorism, hence driving the need to develop sophisticated treatment and diagnostic protocols to counter biological warfare. Here, by performing flux balance analyses on the fully-annotated metabolic networks of multiple, whole genome-sequenced bacterial strains, we have identified a large number of metabolic enzymes as potential drug targets for each of the three Category A-designated bioterrorism agents including Bacillus anthracis, Francisella tularensis and Yersinia pestis. Nine metabolic enzymes- belonging to the coenzyme A, folate, phosphatidyl-ethanolamine and nucleic acid pathways common to all strains across the three distinct genera were identified as targets. Antimicrobial agents against some of these enzymes are available. Thus, a combination of cross species-specific antibiotics and common antimicrobials against shared targets may represent a useful combinatorial therapeutic approach against all Category A bioterrorism agents.

  7. Genetic diversity within the genus Francisella as revealed by comparative analyses of the genomes of two North American isolates from environmental sources

    PubMed Central

    2012-01-01

    Background Francisella tularensis is an intracellular pathogen that causes tularemia in humans and the public health importance of this bacterium has been well documented in recent history. Francisella philomiragia, a distant relative of F. tularensis, is thought to constitute an environmental lineage along with Francisella novicida. Nevertheless, both F. philomiragia and F. novicida have been associated with human disease, primarily in immune-compromised individuals. To understand the genetic relationships and evolutionary contexts among different lineages within the genus Francisella, the genome of Francisella spp. strain TX07-7308 was sequenced and compared to the genomes of F. philomiragia strains ATCC 25017 and 25015, F. novicida strain U112, and F. tularensis strain Schu S4. Results The size of strain ATCC 25017 chromosome was 2,045,775 bp and contained 1,983 protein-coding genes. The size of strain TX07-7308 chromosome was 2,035,931 bp and contained 1,980 protein-coding genes. Pairwise BLAST comparisons indicated that strains TX07-7308 and ATCC 25017 contained 1,700 protein coding genes in common. NUCmer analyses revealed that the chromosomes of strains TX07-7308 and ATCC 25017 were mostly collinear except for a few gaps, translocations, and/or inversions. Using the genome sequence data and comparative analyses with other members of the genus Francisella (e.g., F. novicida strain U112 and F. tularensis strain Schu S4), several strain-specific genes were identified. Strains TX07-7308 and ATCC 25017 contained an operon with six open reading frames encoding proteins related to enzymes involved in thiamine biosynthesis that was absent in F. novicida strain U112 and F. tularensis strain Schu S4. Strain ATCC 25017 contained an operon putatively involved in lactose metabolism that was absent in strain TX07-7308, F. novicida strain U112, and F. tularensis strain Schu S4. In contrast, strain TX07-7308 contained an operon putatively involved in glucuronate metabolism

  8. Comparison of bacterial culture and polymerase chain reaction (PCR) for the detection of F. tularensis subsp. holarctica in wild animals.

    PubMed

    Sting, Reinhard; Runge, Martin; Eisenberg, Tobias; Braune, Silke; Müller, Wolfgang; Otto, Peter

    2013-01-01

    Detection of the zoonotic pathogen Francisella tularensis subsp. holarctica (EF tularensis) in wild animals with culture techniques as well as polymerase chain reaction were compared and discussed on the basis of the investigation of 60 animals. The samples originated from 55 European brown hares (Lepus europaeus), two red foxes (Vulpes vulpes) and one each from a wild rabbit (Oryctolagus cuniculus), a European beaver (Castor fiber), and a lemur (Lemur catta). When comparing the growth of 28 F. tularensis isolates on the cysteine blood agar and the modified Martin-Lewis-agar used in this study, cultivation was successful for 26 isolates on both media, but for two isolates only on the cysteine blood agar. Out of 43 carcasses 19 tested positive in bacteriological culture and PCR. Two culture positive samples of tonsils originating from foxes could not be confirmed by PCR, although PCR was positive in 22 samples that missed growth of F. tularensis. Comparative studies on cultural detection of E. tularensis were performed on samples of 16 hares from lung, spleen, liver and gut and in one case with a peritoneal swab. In at least one of these localizations cultivation of the pathogen was successful. Detection rate was reduced to 94% (15 of 16 hares) considering only the results of the cultures of the lungs and spleens. For a sensitive and rapid detection of F. tularensis subsp. holarctica, the PCR is a suitable method thereby avoiding hazardous multiplying of the pathogen. However, cultivation of F. tularensis is often a prerequisite for further studies on antibiotic resistance patterns of the pathogen, molecular epidemiological and pathological analyses of tularaemia.

  9. A Francisella-like endosymbiont in the Gulf Coast tick evolved from a mammalian pathogen

    PubMed Central

    Gerhart, Jonathan G.; Moses, Abraham S.; Raghavan, Rahul

    2016-01-01

    Ticks (order Ixodida) vector pathogenic bacteria that cause diseases in humans and other mammals. They also contain bacteria that are closely related to pathogens but function as endosymbionts that provide nutrients that are missing from mammalian blood—their sole food source. For instance, mammalian pathogens such as Coxiella burnetii and Francisella tularensis, as well as Coxiella-like and Francisella-like endosymbionts (CLEs and FLEs, respectively) occur in ticks worldwide. However, it is not clear whether the pathogens evolved from symbionts or symbionts from pathogens. Recent studies have indicated that C. burnetii likely originated from a tick-associated ancestor, but the origins of FLEs are not clear. In this study, we sequenced the genome of an FLE, termed FLE-Am, present in the Gulf Coast tick, Amblyomma maculatum. We show that FLE-Am likely evolved from a pathogenic strain of Francisella, indicating that tick endosymbionts can evolve from mammalian pathogens. Although the genome of FLE-Am is almost the same size as the genomes of pathogenic Francisella strains, about one-third of its protein-coding genes contain inactivating mutations. The relatively low coding capacity and extensive metabolic capabilities indicate that FLE-Am transitioned recently to its current endosymbiotic lifestyle and likely replaced an ancient endosymbiont with degraded functionality. PMID:27645766

  10. A biolayer interferometry-based assay for rapid and highly sensitive detection of biowarfare agents.

    PubMed

    Mechaly, Adva; Cohen, Hila; Cohen, Ofer; Mazor, Ohad

    2016-08-01

    Biolayer interferometry (BLI) is an optical technique that uses fiber-optic biosensors for label-free real-time monitoring of protein-protein interactions. In this study, we coupled the advantages of the Octet Red BLI system (automation, fluidics-free, and on-line monitoring) with a signal enhancement step and developed a rapid and sensitive immunological-based method for detection of biowarfare agents. As a proof of concept, we chose to demonstrate the efficacy of this novel assay for the detection of agents representing two classes of biothreats, proteinaceous toxins, and bacterial pathogens: ricin, a lethal plant toxin, and the gram-negative bacterium Francisella tularensis, the causative agent of tularemia. The assay setup consisted of biotinylated antibodies immobilized to the biosensor coupled with alkaline phosphatase-labeled antibodies as the detection moiety to create nonsoluble substrate crystals that precipitate on the sensor surface, thereby inducing a significant wavelength interference. It was found that this BLI-based assay enables sensitive detection of these pathogens (detection limits of 10 pg/ml and 1 × 10(4) pfu/ml ricin and F. tularensis, respectively) within a very short time frame (17 min). Owing to its simplicity, this assay can be easily adapted to detect other analytes in general, and biowarfare agents in particular, in a rapid and sensitive manner. PMID:27156814

  11. Phylogenetic analysis of the Francisella-like endosymbionts of Dermacentor ticks.

    PubMed

    Scoles, Glen A

    2004-05-01

    Bacterial endosymbionts with significant homology to Francisella tularensis (gamma-proteobacteria) have been described from at least five species of ticks in three different genera, including two North American Dermacentor species [D. andersoni Stiles and D. variabilis (Say)]. The evolutionary relationships among the Francisella-like endosymbionts (FLE) from different hosts and between FLE and the arthropod-borne pathogen F. tularensis are not known. A 1,169-base fragment of the 16s rDNA and a 713-base fragment of the F. tularensis 17-kDa lipoprotein gene homolog of the FLE of six North American Dermacentor tick species [D. anderson, D. variabilis, D. albipictus (Packard), D. occidentalis Marx, D. hunteri Bishopp, and D. (Anocentor) nitens Neumann] and of Amblyomma maculatum Koch and Ornithodoros porcinus (Murry 1877, sensu Walton 1979) as outgroups, were subjected to phylogenetic analysis. These gene phylogenies were compared with a phylogeny of the same tick species constructed from a 435-base fragment of the tick mitochondrial 16s rDNA. Although the phylogenies of the FLE and their tick hosts are parallel at the genus level, the Dermacentor FLE are unresolved at the species level. The FLE and the Dermacentor ticks show little sign of co-speciation, possibly indicating that the association between these endosymbiont and the Dermacentor ticks is of a relatively recent origin. Several ticks were co-infected, either with two FLE with divergent 17-kDa lipoprotein gene sequences or with FLE and an unidentified species of spotted fever group rickettsia (alpha-proteobacteria). Infection with FLE does not seem to have precluded infection with either a second closely related gamma-proteobacterial symbiont or with a second less closely related alpha-proteobacterial symbiont.

  12. Identification of Genes Required for Secretion of the Francisella Oxidative Burst-Inhibiting Acid Phosphatase AcpA.

    PubMed

    Hoang, Ky Van; Chen, Carolyn G; Koopman, Jacob; Moshiri, Jasmine; Adcox, Haley E; Gunn, John S

    2016-01-01

    Francisella tularensis is a Tier 1 bioterror threat and the intracellular pathogen responsible for tularemia in humans and animals. Upon entry into the host, Francisella uses multiple mechanisms to evade killing. Our previous studies have shown that after entering its primary cellular host, the macrophage, Francisella immediately suppresses the oxidative burst by secreting a series of acid phosphatases including AcpA-B-C and HapA, thereby evading the innate immune response of the macrophage and enhancing survival and further infection. However, the mechanism of acid phosphatase secretion by Francisella is still unknown. In this study, we screened for genes required for AcpA secretion in Francisella. We initially demonstrated that the known secretion systems, the putative Francisella-pathogenicity island (FPI)-encoded Type VI secretion system and the Type IV pili, do not secrete AcpA. Using random transposon mutagenesis in conjunction with ELISA, Western blotting and acid phosphatase enzymatic assays, a transposon library of 5450 mutants was screened for strains with a minimum 1.5-fold decrease in secreted (culture supernatant) AcpA, but no defect in cytosolic AcpA. Three mutants with decreased supernatant AcpA were identified. The transposon insertion sites of these mutants were revealed by direct genomic sequencing or inverse-PCR and sequencing. One of these mutants has a severe defect in AcpA secretion (at least 85% decrease) and is a predicted hypothetical inner membrane protein. Interestingly, this mutant also affected the secretion of the FPI-encoded protein, VgrG. Thus, this screen identified novel protein secretion factors involved in the subversion of host defenses. PMID:27199935

  13. Identification of Genes Required for Secretion of the Francisella Oxidative Burst-Inhibiting Acid Phosphatase AcpA

    PubMed Central

    Hoang, Ky Van; Chen, Carolyn G.; Koopman, Jacob; Moshiri, Jasmine; Adcox, Haley E.; Gunn, John S.

    2016-01-01

    Francisella tularensis is a Tier 1 bioterror threat and the intracellular pathogen responsible for tularemia in humans and animals. Upon entry into the host, Francisella uses multiple mechanisms to evade killing. Our previous studies have shown that after entering its primary cellular host, the macrophage, Francisella immediately suppresses the oxidative burst by secreting a series of acid phosphatases including AcpA-B-C and HapA, thereby evading the innate immune response of the macrophage and enhancing survival and further infection. However, the mechanism of acid phosphatase secretion by Francisella is still unknown. In this study, we screened for genes required for AcpA secretion in Francisella. We initially demonstrated that the known secretion systems, the putative Francisella-pathogenicity island (FPI)-encoded Type VI secretion system and the Type IV pili, do not secrete AcpA. Using random transposon mutagenesis in conjunction with ELISA, Western blotting and acid phosphatase enzymatic assays, a transposon library of 5450 mutants was screened for strains with a minimum 1.5-fold decrease in secreted (culture supernatant) AcpA, but no defect in cytosolic AcpA. Three mutants with decreased supernatant AcpA were identified. The transposon insertion sites of these mutants were revealed by direct genomic sequencing or inverse-PCR and sequencing. One of these mutants has a severe defect in AcpA secretion (at least 85% decrease) and is a predicted hypothetical inner membrane protein. Interestingly, this mutant also affected the secretion of the FPI-encoded protein, VgrG. Thus, this screen identified novel protein secretion factors involved in the subversion of host defenses. PMID:27199935

  14. A Two-component Kdo Hydrolase in the Inner Membrane of Francisella novicida

    PubMed Central

    Zhao, Jinshi; Raetz, Christian R. H.

    2010-01-01

    Lipid A coats the outer surface of the outer membrane of Gram-negative bacteria. In Francisella tularensis subspecies novicida lipid A is present either as the covalently attached anchor of lipopolysaccharide (LPS) or as free lipid A. The lipid A moiety of Francisella LPS is linked to the core domain by a single 2-keto-3-deoxy-D-manno-octulosonic acid (Kdo) residue. F. novicida KdtA is bifunctional, but F. novicida contains a membrane-bound Kdo hydrolase that removes the outer Kdo unit. The hydrolase consists of two proteins (KdoH1 and KdoH2), which are expressed from adjacent, co-transcribed genes. KdoH1 (related to sialidases) has a single predicted N-terminal transmembrane segment. KdoH2 contains 7 putative transmembrane sequences. Neither protein alone catalyzes Kdo cleavage when expressed in E. coli. Activity requires simultaneous expression of both proteins or mixing of membranes from strains expressing the individual proteins under in vitro assay conditions in the presence of non-ionic detergent. In E. coli expressing KdoH1 and KdoH2, hydrolase activity is localized in the inner membrane. WBB06, a heptose-deficient E. coli mutant that makes Kdo2-lipid A as its sole LPS, accumulates Kdo-lipid A when expressing the both hydrolase components, and 1-dephospho-Kdo-lipid A when expressing both the hydrolase and the Francisella lipid A 1-phosphatase (LpxE). PMID:20662782

  15. Identification, Distribution and Population Dynamics of Francisella-like Endosymbiont in Haemaphysalis doenitzi (Acari: Ixodidae)

    PubMed Central

    Liu, Jian-Nan; Yu, Zhi-Jun; Liu, Li-Meng; Li, Ning-Xin; Wang, Rong-Rong; Zhang, Chun-Mian; Liu, Jing-Ze

    2016-01-01

    Francisella-like endosymbionts (FLEs) with significant homology to Francisella tularensis (γ-proteobacteria) have been characterized in several tick species, whereas knowledge on their distribution and population dynamics in ticks remains meager. Hence, in the current study, we identified a novel Francisella-like endosymbiont (FLEs-Hd) from the tick Haemaphysalis doenitzi and evaluated the putative functions of this symbiont. Results indicated that FLEs-Hd had 100% infection rate and a perfect vertical transmission in H. doenitzi, and that it is distributed in ovaries, malpighian tubules, salivary glands and midguts of the ticks, suggesting that FLEs-Hd presumably is a crucial symbiont of the host without specific tissue tropism. To further explore the function of the symbiont, the population dynamics of FLEs-Hd at each developmental stage of ticks and in tissues at different reproductive statuses were determined by real-time quantitative polymerase chain reaction (real-time qPCR). Results showed that the high density and regular population dynamics of FLEs-Hd appeared in female ovaries, suggesting that the symbiont may provide necessary nutrients or regulators to ensure normal ovary development of ticks. PMID:27731377

  16. Francisella sp., an emerging pathogen of tilapia, Oreochromis niloticus (L.), in Costa Rica.

    PubMed

    Soto, E; Hawke, J P; Fernandez, D; Morales, J A

    2009-08-01

    Francisella sp. is an emergent bacterial pathogen that causes acute to chronic disease in warm and cold water cultured and wild fish species. During the past 3 years, the bacterium has been detected in tilapia, Oreochromis niloticus, cultured in Costa Rica. Infected fish presented non-specific clinical signs, such as erratic swimming, anorexia, anaemia, exophthalmia and high mortality. Upon macroscopic and microscopic examination, several internal organs (mainly spleen and kidney) were enlarged and contained white nodules. Histological examination revealed the presence of multifocal granulomatous lesions, with the presence of numerous small, pleomorphic, cocco-bacilli. The bacteria were isolated from infected tilapia on selective media and grown on several media with and without antibiotics. Specific PCR primers to the Francisella genus were used to confirm the preliminary diagnoses. In comparison with several bacterial 16S rRNA sequences, our isolate was found to share 99% identity with other Fransicella spp. isolated from fish, and more than 97% identity to the human pathogen Francisella tularensis. Koch's postulates were fulfilled after experimental intraperitoneal and gill exposure challenges. PMID:19515205

  17. 42 CFR 73.5 - Exemptions for HHS select agents and toxins.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ..., Ebola viruses, Francisella tularensis, Lassa fever virus, Marburg virus, South American Haemorrhagic Fever viruses (Junin, Machupo, Sabia, Flexal, Guanarito), Variola major virus (Smallpox virus), Variola... as the Virus-Serum-Toxin Act (21 U.S.C. 151-159), or (4) The Federal Insecticide, Fungicide,...

  18. 42 CFR 73.5 - Exemptions for HHS select agents and toxins.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ..., Ebola viruses, Francisella tularensis, Lassa fever virus, Marburg virus, South American Haemorrhagic Fever viruses (Junin, Machupo, Sabia, Flexal, Guanarito), Variola major virus (Smallpox virus), Variola... as the Virus-Serum-Toxin Act (21 U.S.C. 151-159), or (4) The Federal Insecticide, Fungicide,...

  19. 42 CFR 73.5 - Exemptions for HHS select agents and toxins.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ..., Ebola viruses, Francisella tularensis, Lassa fever virus, Marburg virus, South American Haemorrhagic Fever viruses (Junin, Machupo, Sabia, Flexal, Guanarito), Variola major virus (Smallpox virus), Variola... as the Virus-Serum-Toxin Act (21 U.S.C. 151-159), or (4) The Federal Insecticide, Fungicide,...

  20. Molecular Survey of Zoonotic Agents in Rodents and Other Small Mammals in Croatia.

    PubMed

    Tadin, Ante; Tokarz, Rafal; Markotić, Alemka; Margaletić, Josip; Turk, Nenad; Habuš, Josipa; Svoboda, Petra; Vucelja, Marko; Desai, Aaloki; Jain, Komal; Lipkin, W Ian

    2016-02-01

    Croatia is a focus for many rodent-borne zoonosis. Here, we report a survey of 242 rodents and small mammals, including 43 Myodes glareolus, 131 Apodemus flavicollis, 53 Apodemus agrarius, three Apodemus sylvaticus, six Sorex araneus, four Microtus arvalis, one Microtus agrestis, and one Muscardinus avellanarius, collected at eight sites in Croatia over an 8-year period. Multiplex MassTag polymerase chain reaction (PCR) was used for detection of Borrelia, Rickettsia, Bartonella, Babesia, Ehrlichia, Anaplasma, Francisella tularensis, and Coxiella burnetii. Individual PCR assays were used for detection of Leptospira, lymphocytic choriomeningitis virus, orthopoxviruses, flaviviruses, hantaviruses, and Toxoplasma gondii. Of the rodents, 52 (21.5%) were infected with Leptospira, 9 (3.7%) with Borrelia miyamotoi, 5 (2%) with Borrelia afzelii, 29 (12.0%) with Bartonella, 8 (3.3%) with Babesia microti, 2 (0.8%) with Ehrlichia, 4 (1.7%) with Anaplasma, 2 (0.8%) with F. tularensis, 43 (17.8%) with hantaviruses, and 1 (0.4%) with an orthopoxvirus. Other agents were not detected. Multiple infections were found in 32 rodents (13.2%): dual infections in 26 rodents (10.7%), triple infections in four rodents (2.9%), and quadruple infections in two rodents (0.8%). Our findings indicate that rodents in Croatia harbor a wide range of bacteria and viruses that are pathogenic to humans.

  1. Nanolipoprotein Particles (NLPs) as Versatile Vaccine Platforms for Co-delivery of Multiple Adjuvants with Subunit Antigens from Burkholderia spp. and F. tularensis - Technical Report

    SciTech Connect

    Fischer, N. O.

    2015-01-13

    The goal of this proposal is to demonstrate that colocalization of protein subunit antigens and adjuvants on nanolipoprotein particles (NLPs) can increase the protective efficacy of subunit antigens from Burkholderia spp. and Francisella tularensis against an aerosol challenge. In the third quarter of the third year, F344 rats vaccinated with adjuvanted NLP formulations were challenged with F. tularensis SCHU S4 at Battelle. Preliminary data indicate that up to 65% of females vaccinated intranasally with an NLP-based formulation survived this challenge, compared to only 20% survival of naïve animals. In addition, NLPs were successfully formulated with Burkholderia protein antigens. IACUC approval for immunological assessments in BALB/c mice was received and we anticipate that these assessments will begin by March 2015, pending ACURO approval.

  2. Nanolipoprotein Particles (NLPs) as Versatile Vaccine Platforms for Co-delivery of Multiple Adjuvants with Subunit Antigens from Burkholderia spp. and F. tularensis - Technical Report

    SciTech Connect

    Fischer, N. O.

    2015-01-06

    The goal of this proposal is to demonstrate that colocalization of protein subunit antigens and adjuvants on nanolipoprotein particles (NLPs) can increase the protective efficacy of subunit antigens from Burkholderia spp. and Francisella tularensis against an aerosol challenge. In the second quarter of the third year, LLNL finalized all immunological assessments of NLP vaccine formulations in the F344 model. Battelle has immunized rats with three unique NLP formulations by either intramuscular or intranasal administration. All inoculations have been completed, and protective efficacy against an aerosolized challenge will begin at the end of October, 2014.

  3. Francisella philomiragia Infection and Lethality in Mammalian Tissue Culture Cell Models, Galleria mellonella, and BALB/c Mice

    PubMed Central

    Propst, Crystal N.; Pylypko, Stephanie L.; Blower, Ryan J.; Ahmad, Saira; Mansoor, Mohammad; van Hoek, Monique L.

    2016-01-01

    Francisella (F.) philomiragia is a Gram-negative bacterium with a preference for brackish environments that has been implicated in causing bacterial infections in near-drowning victims. The purpose of this study was to characterize the ability of F. philomiragia to infect cultured mammalian cells, a commonly used invertebrate model, and, finally, to characterize the ability of F. philomiragia to infect BALB/c mice via the pulmonary (intranasal) route of infection. This study shows that F. philomiragia infects J774A.1 murine macrophage cells, HepG2 cells and A549 human Type II alveolar epithelial cells. However, replication rates vary depending on strain at 24 h. F. philomiragia infection after 24 h was found to be cytotoxic in human U937 macrophage-like cells and J774A.1 cells. This is in contrast to the findings that F. philomiragia was non-cytotoxic to human hepatocellular carcinoma cells, HepG2 cells and A549 cells. Differential cytotoxicity is a point for further study. Here, it was demonstrated that F. philomiragia grown in host-adapted conditions (BHI, pH 6.8) is sensitive to levofloxacin but shows increased resistance to the human cathelicidin LL-37 and murine cathelicidin mCRAMP when compared to related the Francisella species, F. tularensis subsp. novicida and F. tularensis subsp. LVS. Previous findings that LL-37 is strongly upregulated in A549 cells following F. tularensis subsp. novicida infection suggest that the level of antimicrobial peptide expression is not sufficient in cells to eradicate the intracellular bacteria. Finally, this study demonstrates that F. philomiragia is lethal in two in vivo models; Galleria mellonella via hemocoel injection, with a LD50 of 1.8 × 103, and BALB/c mice by intranasal infection, with a LD50 of 3.45 × 103. In conclusion, F. philomiragia may be a useful model organism to study the genus Francisella, particularly for those researchers with interest in studying microbial ecology or environmental strains of Francisella

  4. Discovery of a novel and potent class of F. tularensis enoyl-reductase (FabI) inhibitors by molecular shape and electrostatic matching

    PubMed Central

    Hevener, Kirk E.; Mehboob, Shahila; Su, Pin-Chih; Truong, Kent; Boci, Teuta; Deng, Jiangping; Ghassemi, Mahmood; Cook, James L.; Johnson, Michael E.

    2011-01-01

    Enoyl-acyl carrier protein (ACP) reductase, FabI, is a key enzyme in the bacterial fatty acid biosynthesis pathway (FAS II). FabI is an NADH-dependent oxidoreductase that acts to reduce enoyl-ACP substrates in a final step of the pathway. The absence of this enzyme in humans makes it an attractive target for the development of new antibacterial agents. FabI is known to be unresponsive to structure-based design efforts due to a high degree of induced fit and a mobile flexible loop encompassing the active site. Here we discuss the development, validation, and careful application of a ligand-based virtual screen used for the identification of novel inhibitors of the Francisella tularensis FabI target. In this study, four known classes of FabI inhibitors were used as templates for virtual screens that involved molecular shape and electrostatic matching. The program ROCS was used to search a high-throughput screening library for compounds that matched any of the four molecular shape queries. Matching compounds were further refined using the program EON, which compares and scores compounds by matching electrostatic properties. Using these techniques, 50 compounds were selected, ordered, and tested. The tested compounds possessed novel chemical scaffolds when compared to the input query compounds. Several hits with low micromolar activity were identified and follow-up scaffold-based searches resulted in the identification of a lead series with sub-micromolar enzyme inhibition, high ligand efficiency, and a novel scaffold. Additionally, one of the most active compounds showed promising whole-cell antibacterial activity against several Gram-positive and Gram-negative species, including the target pathogen. The results of a preliminary structure-activity relationship analysis are presented. PMID:22098466

  5. Comparative phosphoproteomics reveals components of host cell invasion and post-transcriptional regulation during Francisella infection

    SciTech Connect

    Nakayasu, Ernesto S.; Tempel, Rebecca; Cambronne, Xiaolu A.; Petyuk, Vladislav A.; Jones, Marcus B.; Gritsenko, Marina A.; Monroe, Matthew E.; Yang, Feng; Smith, Richard D.; Adkins, Joshua N.; Heffron, Fred

    2013-09-22

    Francisella tularensis is a facultative intracellular bacterium that causes the deadly disease tularemia. Most evidence suggests that Francisella is not well recognized by the innate immune system that normally leads to cytokine expression and cell death. In previous work, we identified new bacterial factors that were hyper-cytotoxic to macrophages. Four of the identified hyper-cytotoxic strains (lpcC, manB, manC and kdtA) had an impaired lipopolysaccharide (LPS) synthesis and produced an exposed lipid A lacking the O-antigen. These mutants were not only hyper-cytotoxic but also were phagocytosed at much higher rates compared to the wild type parent strain. To elucidate the cellular signaling underlying this enhanced phagocytosis and cell death, we performed a large-scale comparative phosphoproteomic analysis of cells infected with wild-type and delta-lpcC F. novicida. Our data suggest that not only actin but also intermediate filaments and microtubules are important for F. novicida entry into the host cells. In addition, we observed differential phosphorylation of tristetraprolin (TTP), a key component of the mRNA-degrading machinery that controls the expression of a variety of genes including many cytokines. Infection with the delta-lpcC mutant induced the hyper-phosphorylation and inhibition of TTP, leading to the production of cytokines such as IL-1beta and TNF-alpha which may kill the host cells by triggering apoptosis. Together, our data provide new insights for Francisella invasion and a post-transcriptional mechanism that prevents the expression of host immune response factors that controls infection by this pathogen.

  6. Receptor mimicry as novel therapeutic treatment for biothreat agents.

    PubMed

    Thomas, Richard J

    2010-01-01

    The specter of intentional release of pathogenic microbes and their toxins is a real threat. This article reviews the literature on adhesins of biothreat agents, their interactions with oligosaccharides and the potential for anti-adhesion compounds as an alternative to conventional therapeutics. The minimal binding structure of ricin has been well characterised and offers the best candidate for successful anti-adhesion therapy based on the Galβ1-4GlcNAc structure. The botulinum toxin serotypes A-F bind to a low number of gangliosides (GT1b, GQ1b, GD1a and GD1b) hence it should be possible to determine the minimal structure for binding. The minimal disaccharide sequence of GalNAcβ1-4Gal found in the gangliosides asialo-GM1 and asialo-GM2 is required for adhesion for many respiratory pathogens. Although a number of adhesins have been identified in bacterial biothreat agents such as Yersinia pestis, Bacillus anthracis, Francisella tularensis, Brucella species and Burkholderia pseudomallei, specific information regarding their in vivo expression during pneumonic infection is lacking. Limited oligosaccharide inhibition studies indicate the potential of GalNAcβ1-4Gal, GalNAcβ-3Gal and the hydrophobic compound, para-nitrophenol as starting points for the rational design of generic anti-adhesion compounds. A cocktail of multivalent oligosaccharides based on the minimal binding structures of identified adhesins would offer the best candidates for anti-adhesion therapy. PMID:21327124

  7. Detection of Lyme Disease and Q Fever Agents in Wild Rodents in Central Italy

    PubMed Central

    Di Domenico, Marco; Dall'Acqua, Francesca; Sozio, Giulia; Cammà, Cesare

    2015-01-01

    Abstract The maintenance of tick-borne disease agents in the environment strictly depends on the relationship between tick vectors and their hosts, which act as reservoirs for these pathogens. A pilot study aimed to investigate wild rodents as reservoirs for zoonotic tick-borne pathogens (Borrelia burgdorferi sensu lato (s.l.), Coxiella burnetii, Francisella tularensis, and Anaplasma phagocytophilum) was carried out in an area of Gran Sasso e Monti della Laga National Park (Abruzzi Region, central Italy), a wide protected area where, despite sporadic reports of infection in humans and animals, eco-epidemiological data on these diseases are still not available. Rodents were trapped and released at the capture site after the collection of feeding ticks and blood samples. In all, 172 ticks were collected; the most frequent species was Ixodes acuminatus (53%). Out of 88 tick pools, 11 resulted positive for C. burnetii and 13 for B. burgdorferi s.l.; the Borrelia afzelii genospecies was identified in one Ixodes ricinus tick collected from one Apodemus sp. rodent. Out of 143 blood samples, seven Apodemus spp. and five Myodes glareolus were positive for B. burgdorferi s.l. and two Apodemus spp. were positive for C. burnetii. All samples (ticks and blood) were negative for F. tularensis and A. phagocytophilum. This is the first report of B. burgdorferi s.l. in the environment for Abruzzi Region. Data on the presence of B. burgdorferi s.l. are similar to that observed in other Mediterranean countries. The present work is also the first report of C. burnetii in wild rodents in Italy. C. burnetii infection has been largely investigated in Italy in ruminant farms by serology and molecular methods, but information on ecology and on the wild cycle are still lacking. Further studies including genotyping should be performed and species-specific differences between wild rodent reservoirs of Q fever and Lyme disease agents should be investigated. PMID:26134933

  8. Combination of biobarcode assay with on-chip capillary electrophoresis for ultrasensitive and multiplex biological agent detection.

    PubMed

    Cho, Minkyung; Chung, Soyi; Jung, Jae Hwan; Rhie, Gi-eun; Jeon, Jun Ho; Seo, Tae Seok

    2014-11-15

    Early diagnosis of biological agents is of paramount importance to prevent the casualties and fatal disease in human during bioterrorism or biological warfare. In this study, we reported an efficient and sensitive multiplex biological agent detection method based on the DNA biobarcode assay and the micro-capillary electrophoresis (μCE) technology. Monoplex as well as multiplex pathogen identification was performed using five targets including Bacillus anthracis, Francisella tularensis, Yersinia pestis, Vaccinia virus and Botulinum toxin A. Through the DNA biobarcode assay process, the magnetic microparticle-pathogen-polystyrene microbead complexes were formed, and the FAM labeled single stranded barcode DNA could be released from the complexes upon denaturation. Different lengths of a barcode DNA were designed to designate each pathogen, so that the specific peak elution time in the capillary electrophoresis on a chip allows us to distinguish the target with high accuracy within 3 min. We improved the assignment accuracy of the peak in the electropherogram by adding two bracket ladders. Owing to the abundant amount of barcode DNAs, the presence of B. anthracis, F. tularensis, Y. pestis, Vaccinia virus was confirmed with a limit of detection of 50CFU/mL, while Botulinum toxin A was analyzed even at a concentration of 12.5 ag/mL. Multiple pathogen detection was also successfully conducted in a phosphate buffered saline (PBS) as well as a serum medium with background of other pathogens. Thus, our analytical platform based on the biobarcode assay and on-chip CE analysis provides rapid, sensitive, multiplex, and accurate biological agent identification.

  9. Performance of Traditional and Molecular Methods for Detecting Biological Agents in Drinking Water

    USGS Publications Warehouse

    Francy, Donna S.; Bushon, Rebecca N.; Brady, Amie M.G.; Bertke, Erin E.; Kephart, Christopher M.; Likirdopulos, Christina A.; Mailot, Brian E.; Schaefer, Frank W.; Lindquist, H.D. Alan

    2009-01-01

    To reduce the impact from a possible bioterrorist attack on drinking-water supplies, analytical methods are needed to rapidly detect the presence of biological agents in water. To this end, 13 drinking-water samples were collected at 9 water-treatment plants in Ohio to assess the performance of a molecular method in comparison to traditional analytical methods that take longer to perform. Two 100-liter samples were collected at each site during each sampling event; one was seeded in the laboratory with six biological agents - Bacillus anthracis (B. anthracis), Burkholderia cepacia (as a surrogate for Bu. pseudomallei), Francisella tularensis (F. tularensis), Salmonella Typhi (S. Typhi), Vibrio cholerae (V. cholerae), and Cryptospordium parvum (C. parvum). The seeded and unseeded samples were processed by ultrafiltration and analyzed by use of quantiative polymerase chain reaction (qPCR), a molecular method, and culture methods for bacterial agents or the immunomagnetic separation/fluorescent antibody (IMS/FA) method for C. parvum as traditional methods. Six replicate seeded samples were also processed and analyzed. For traditional methods, recoveries were highly variable between samples and even between some replicate samples, ranging from below detection to greater than 100 percent. Recoveries were significantly related to water pH, specific conductance, and dissolved organic carbon (DOC) for all bacteria combined by culture methods, but none of the water-quality characteristics tested were related to recoveries of C. parvum by IMS/FA. Recoveries were not determined by qPCR because of problems in quantifying organisms by qPCR in the composite seed. Instead, qPCR results were reported as detected, not detected (no qPCR signal), or +/- detected (Cycle Threshold or 'Ct' values were greater than 40). Several sample results by qPCR were omitted from the dataset because of possible problems with qPCR reagents, primers, and probes. For the remaining 14 qPCR results

  10. Development of a real-time PCR assay for identification and quantification of the fish pathogen Francisella noatunensis subsp. orientalis.

    PubMed

    Soto, Esteban; Bowles, Kimberly; Fernandez, Denise; Hawke, John P

    2010-04-01

    Members of the genus Francisella are small Gram-negative facultative intracellular bacteria that cause francisellosis in a wide variety of fish species worldwide. F. noatunensis subsp. orientalis has been recently described as a warm-water pathogen of tilapia Oreochromis spp. In this study, a quantitative real-time polymerase chain reaction (qPCR) TaqMan probe assay was developed to rapidly and accurately detect and quantify F. noatunensis subsp. orientalis from fish tissue. The target region of the assay was the F. tularensis iglC gene homologue previously found in F. noatunensis subsp. orientalis. Probe specificity was confirmed by the lack of signal and cross-reactivity with 12 common fish pathogens, 2 subspecies of F. tularensis, F. noatunensis subsp. noatunensis, and tilapia tissue. The range of linearity was determined to be 50 fg to 1.4 mg, and the lower limit of detection was 50 fg of DNA (equivalent to approximately 25 genome equivalents) per reaction. A similar sensitivity was observed with DNA extracted from a mixture of F. noatunensis subsp. orientalis and fish tissue. The assay was also able to detect and quantify F. noatunensis subsp. orientalis from the spleens of experimentally infected tilapia. No signal was observed in the control groups. In conclusion, we have developed a highly sensitive and specific assay that can be used for the specific identification and quantification of F. noatunensis subsp. orientalis. PMID:20481087

  11. Fiber-optic microsphere-based arrays for multiplexed biological warfare agent detection.

    PubMed

    Song, Linan; Ahn, Soohyoun; Walt, David R

    2006-02-15

    We report a multiplexed high-density DNA array capable of rapid, sensitive, and reliable identification of potential biological warfare agents. An optical fiber bundle containing 6000 individual 3.1-mum-diameter fibers was chemically etched to yield microwells and used as the substrate for the array. Eighteen different 50-mer single-stranded DNA probes were covalently attached to 3.1-mum microspheres. Probe sequences were designed for Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella melitensis, Clostridium botulinum, Vaccinia virus, and one biological warfare agent (BWA) simulant, Bacillus thuringiensis kurstaki. The microspheres were distributed into the microwells to form a randomized multiplexed high-density DNA array. A detection limit of 10 fM in a 50-microL sample volume was achieved within 30 min of hybridization for B. anthracis, Y. pestis, Vaccinia virus, and B. thuringiensis kurstaki. We used both specific responses of probes upon hybridization to complementary targets as well as response patterns of the multiplexed array to identify BWAs with high accuracy. We demonstrated the application of this multiplexed high-density DNA array for parallel identification of target BWAs in spiked sewage samples after PCR amplification. The array's miniaturized feature size, fabrication flexibility, reusability, and high reproducibility may enable this array platform to be integrated into a highly sensitive, specific, and reliable portable instrument for in situ BWA detection.

  12. Nanoparticle-labeled DNA capture elements for detection and identification of biological agents

    NASA Astrophysics Data System (ADS)

    Kiel, Johnathan L.; Holwitt, Eric A.; Parker, Jill E.; Vivekananda, Jeevalatha; Franz, Veronica

    2004-12-01

    Aptamers, synthetic DNA capture elements (DCEs), can be made chemically or in genetically engineered bacteria. DNA capture elements are artificial DNA sequences, from a random pool of sequences, selected for their specific binding to potential biological warfare or terrorism agents. These sequences were selected by an affinity method using filters to which the target agent was attached and the DNA isolated and amplified by polymerase chain reaction (PCR) in an iterative, increasingly stringent, process. The probes can then be conjugated to Quantum Dots and super paramagnetic nanoparticles. The former provide intense, bleach-resistant fluorescent detection of bioagent and the latter provide a means to collect the bioagents with a magnet. The fluorescence can be detected in a flow cytometer, in a fluorescence plate reader, or with a fluorescence microscope. To date, we have made DCEs to Bacillus anthracis spores, Shiga toxin, Venezuelan Equine Encephalitis (VEE) virus, and Francisella tularensis. DCEs can easily distinguish Bacillus anthracis from its nearest relatives, Bacillus cereus and Bacillus thuringiensis. Development of a high through-put process is currently being investigated.

  13. Neither Neoplasia Nor Tuberculosis, but Francisella

    PubMed Central

    Mambie, Adeline; Wallet, Frédéric; Scherman, Laurine; Armand, Sylvie; Vervelle, Christine; Faure, Karine; Guery, Benoit; Titécat, Marie; Loïez, Caroline

    2016-01-01

    Tularaemia is an emerging anthropozoonosis transmitted by contact with infected animals and through arthropod bites, inhalation, or ingestion. We describe a pulmonary nodule suggesting cancer in a 70-year-old man. Histological analysis excluded neoplasia, and bacteriological culture excluded tuberculosis. Serological testing and PCR Francisella were positive for this hunter patient, then treated by ciprofloxacin with a favourable outcome. PMID:27419157

  14. Considerations in detecting CDC select agents under field conditions

    NASA Astrophysics Data System (ADS)

    Spinelli, Charles; Soelberg, Scott; Swanson, Nathaneal; Furlong, Clement; Baker, Paul

    2008-04-01

    Surface Plasmon Resonance (SPR) has become a widely accepted technique for real-time detection of interactions between receptor molecules and ligands. Antibody may serve as receptor and can be attached to the gold surface of the SPR device, while candidate analyte fluids contact the detecting antibody. Minute, but detectable, changes in refractive indices (RI) indicate that analyte has bound to the antibody. A decade ago, an inexpensive, robust, miniature and fully integrated SPR chip, called SPREETA, was developed. University of Washington (UW) researchers subsequently developed a portable, temperature-regulated instrument, called SPIRIT, to simultaneously use eight of these three-channel SPREETA chips. A SPIRIT prototype instrument was tested in the field, coupled to a remote reporting system on a surrogate unmanned aerial vehicle (UAV). Two target protein analytes were released sequentially as aerosols with low analyte concentration during each of three flights and were successfully detected and verified. Laboratory experimentation with a more advanced SPIRIT instrument demonstrated detection of very low levels of several select biological agents that might be employed by bioterrorists. Agent detection under field-like conditions is more challenging, especially as analyte concentrations are reduced and complex matricies are introduced. Two different sample preconditioning protocols have been developed for select agents in complex matrices. Use of these preconditioning techniques has allowed laboratory detection in spiked heavy mud of Francisella tularensis at 10 3 CFU/ml, Bacillus anthracis spores at 10 3 CFU/ml, Staphylococcal enterotoxin B (SEB) at 1 ng/ml, and Vaccinia virus (a smallpox simulant) at 10 5 PFU/ml. Ongoing experiments are aimed at simultaneous detection of multiple agents in spiked heavy mud, using a multiplex preconditioning protocol.

  15. Detection of Microbial Agents in Ticks Collected from Migratory Birds in Central Italy

    PubMed Central

    Toma, Luciano; Mancini, Fabiola; Di Luca, Marco; Cecere, Jacopo G.; Bianchi, Riccardo; Khoury, Cristina; Quarchioni, Elisa; Manzia, Francesca; Rezza, Giovanni

    2014-01-01

    Abstract Tick species characterization and molecular studies were performed within ornithological surveys conducted during 2010 and 2011 in the Lazio Region of central Italy. A total of 137 ticks were collected from 41 migratory birds belonging to 17 species (four partial migrants and 13 long-distance migrants). Most ticks were nymphs, with a predominance of Hyalomma marginatum marginatum and H. m. rufipes, and a small portion of Ixodes and Amblyomma species. All tick species analyzed were infected, and the molecular pathogen recognition revealed the presence of Rickettsia aeschlimannii, Rickettsia africae, Erlichia spp., Coxiella burnetii, Borrelia burgdorferi sensu lato group, and Babesia microti, whereas no genomic DNA of Bartonella spp. or Francisella tularensis was detected. The results of the survey show that H. marginatum ticks appear to be a vector of microbial agents that may affect human and animal health and that migratory birds may be an important carrier of these ticks. Additional studies are needed to better investigate the role of migratory birds in the epidemiology of these pathogens. PMID:24576218

  16. Development of a panel of recombinase polymerase amplification assays for detection of biothreat agents.

    PubMed

    Euler, Milena; Wang, Yongjie; Heidenreich, Doris; Patel, Pranav; Strohmeier, Oliver; Hakenberg, Sydney; Niedrig, Matthias; Hufert, Frank T; Weidmann, Manfred

    2013-04-01

    Syndromic panels for infectious disease have been suggested to be of value in point-of-care diagnostics for developing countries and for biodefense. To test the performance of isothermal recombinase polymerase amplification (RPA) assays, we developed a panel of 10 RPAs for biothreat agents. The panel included RPAs for Francisella tularensis, Yersinia pestis, Bacillus anthracis, variola virus, and reverse transcriptase RPA (RT-RPA) assays for Rift Valley fever virus, Ebola virus, Sudan virus, and Marburg virus. Their analytical sensitivities ranged from 16 to 21 molecules detected (probit analysis) for the majority of RPA and RT-RPA assays. A magnetic bead-based total nucleic acid extraction method was combined with the RPAs and tested using inactivated whole organisms spiked into plasma. The RPA showed comparable sensitivities to real-time RCR assays in these extracts. The run times of the assays at 42°C ranged from 6 to 10 min, and they showed no cross-detection of any of the target genomes of the panel nor of the human genome. The RPAs therefore seem suitable for the implementation of syndromic panels onto microfluidic platforms.

  17. Development of a Panel of Recombinase Polymerase Amplification Assays for Detection of Biothreat Agents

    PubMed Central

    Euler, Milena; Wang, Yongjie; Heidenreich, Doris; Patel, Pranav; Strohmeier, Oliver; Hakenberg, Sydney; Niedrig, Matthias; Hufert, Frank T.

    2013-01-01

    Syndromic panels for infectious disease have been suggested to be of value in point-of-care diagnostics for developing countries and for biodefense. To test the performance of isothermal recombinase polymerase amplification (RPA) assays, we developed a panel of 10 RPAs for biothreat agents. The panel included RPAs for Francisella tularensis, Yersinia pestis, Bacillus anthracis, variola virus, and reverse transcriptase RPA (RT-RPA) assays for Rift Valley fever virus, Ebola virus, Sudan virus, and Marburg virus. Their analytical sensitivities ranged from 16 to 21 molecules detected (probit analysis) for the majority of RPA and RT-RPA assays. A magnetic bead-based total nucleic acid extraction method was combined with the RPAs and tested using inactivated whole organisms spiked into plasma. The RPA showed comparable sensitivities to real-time RCR assays in these extracts. The run times of the assays at 42°C ranged from 6 to 10 min, and they showed no cross-detection of any of the target genomes of the panel nor of the human genome. The RPAs therefore seem suitable for the implementation of syndromic panels onto microfluidic platforms. PMID:23345286

  18. Exploitation of complement regulatory proteins by Borrelia and Francisella.

    PubMed

    Madar, Marian; Bencurova, Elena; Mlynarcik, Patrik; Almeida, André M; Soares, Renata; Bhide, Katarina; Pulzova, Lucia; Kovac, Andrej; Coelho, Ana V; Bhide, Mangesh

    2015-06-01

    Pathogens have developed sophisticated mechanisms of complement evasion such as binding to the host complement regulatory proteins (CRPs) on their surface or expression of CRP mimicking molecules. The ability of pathogens to evade the complement system has been correlated with pathogenesis and host selectivity. Hitherto, little work has been undertaken to determine whether Borrelia and Francisella exploit various CRPs to block complement attack. Seventeen Borrelia (twelve species) and six Francisella (three subspecies) strains were used to assess their ability to bind human, sheep and cattle CRPs or mimic membrane associated complement regulators. A series of experiments including affinity ligand binding experiments, pull-down assays and mass spectrometry based protein identification, revealed an array of CRP binding proteins of Borrelia and Francisella. Unlike Francisella, Borrelia strains were able to bind multiple human CRPs. Three strains of Borrelia (SKT-4, SKT-2 and HO14) showed the presence of a human CD46-homologous motif, indicating their ability to possess putative human CD46 mimicking molecules. Similarly, five strains of Borrelia and two strains of Francisella may have surface proteins with human CD59-homologous motifs. Among ovine and bovine CRPs, the only CRP bound by Francisella (LVS, Tul4 strain) was vitronectin, while ovine C4BP, ovine factor H and bovine factor H were bound to Borrelia strains SKT-2, DN127 and Co53. This study presents an array of proteins of Borrelia and Francisella that bind CRPs or may mimic membrane-CRPs, thus enabling multiphasic complement evasion strategies of these pathogens.

  19. DESTRUCTION OF FRANCISELLA TULARENSIS AND YERSINIA PESTIS PERSISTENCE OF BACILLUS ANTHRACIS SPORES AND CLOSTRIDIUM BOTULINUM IN MUNICIPAL SOLID LANDFILL LEACHATES

    EPA Science Inventory

    The United States Environmental Protection Agency Office of Research and Development National Homeland Security Research Center (NHSRC) in collaboration with the Department of Defense Edgewood Chemical Biological Center (ECBC) are evaluating the permanence of biological and chemi...

  20. Dissection of Francisella-Host Cell Interactions in Dictyostelium discoideum.

    PubMed

    Lampe, Elisabeth O; Brenz, Yannick; Herrmann, Lydia; Repnik, Urska; Griffiths, Gareth; Zingmark, Carl; Sjöstedt, Anders; Winther-Larsen, Hanne C; Hagedorn, Monica

    2016-03-01

    Francisella bacteria cause severe disease in both vertebrates and invertebrates and include one of the most infectious human pathogens. Mammalian cell lines have mainly been used to study the mechanisms by which Francisella manipulates its host to replicate within a large variety of hosts and cell types, including macrophages. Here, we describe the establishment of a genetically and biochemically tractable infection model: the amoeba Dictyostelium discoideum combined with the fish pathogen Francisella noatunensis subsp. noatunensis. Phagocytosed F. noatunensis subsp. noatunensis interacts with the endosomal pathway and escapes further phagosomal maturation by translocating into the host cell cytosol. F. noatunensis subsp. noatunensis lacking IglC, a known virulence determinant required for Francisella intracellular replication, follows the normal phagosomal maturation and does not grow in Dictyostelium. The attenuation of the F. noatunensis subsp. noatunensis ΔiglC mutant was confirmed in a zebrafish embryo model, where growth of F. noatunensis subsp. noatunensis ΔiglC was restricted. In Dictyostelium, F. noatunensis subsp. noatunensis interacts with the autophagic machinery. The intracellular bacteria colocalize with autophagic markers, and when autophagy is impaired (Dictyostelium Δatg1), F. noatunensis subsp. noatunensis accumulates within Dictyostelium cells. Altogether, the Dictyostelium-F. noatunensis subsp. noatunensis infection model recapitulates the course of infection described in other host systems. The genetic and biochemical tractability of the system allows new approaches to elucidate the dynamic interactions between pathogenic Francisella and its host organism. PMID:26712555

  1. Dissection of Francisella-Host Cell Interactions in Dictyostelium discoideum

    PubMed Central

    Lampe, Elisabeth O.; Brenz, Yannick; Herrmann, Lydia; Repnik, Urska; Griffiths, Gareth; Zingmark, Carl; Sjöstedt, Anders; Winther-Larsen, Hanne C.

    2015-01-01

    Francisella bacteria cause severe disease in both vertebrates and invertebrates and include one of the most infectious human pathogens. Mammalian cell lines have mainly been used to study the mechanisms by which Francisella manipulates its host to replicate within a large variety of hosts and cell types, including macrophages. Here, we describe the establishment of a genetically and biochemically tractable infection model: the amoeba Dictyostelium discoideum combined with the fish pathogen Francisella noatunensis subsp. noatunensis. Phagocytosed F. noatunensis subsp. noatunensis interacts with the endosomal pathway and escapes further phagosomal maturation by translocating into the host cell cytosol. F. noatunensis subsp. noatunensis lacking IglC, a known virulence determinant required for Francisella intracellular replication, follows the normal phagosomal maturation and does not grow in Dictyostelium. The attenuation of the F. noatunensis subsp. noatunensis ΔiglC mutant was confirmed in a zebrafish embryo model, where growth of F. noatunensis subsp. noatunensis ΔiglC was restricted. In Dictyostelium, F. noatunensis subsp. noatunensis interacts with the autophagic machinery. The intracellular bacteria colocalize with autophagic markers, and when autophagy is impaired (Dictyostelium Δatg1), F. noatunensis subsp. noatunensis accumulates within Dictyostelium cells. Altogether, the Dictyostelium-F. noatunensis subsp. noatunensis infection model recapitulates the course of infection described in other host systems. The genetic and biochemical tractability of the system allows new approaches to elucidate the dynamic interactions between pathogenic Francisella and its host organism. PMID:26712555

  2. MsbA Transporter-dependent Lipid A 1-Dephosphorylation on the Periplasmic Surface of the Inner Membrane: TOPOGRAPHY OF FRANCISELLA NOVICIDA LpxE EXPRESSED IN ESCHERICHIA COLI*

    PubMed Central

    Wang, Xiaoyuan; Karbarz, Mark J.; McGrath, Sara C.; Cotter, Robert J.; Raetz, Christian R. H.

    2008-01-01

    The lipid A anchor of Francisella tularensis lipopolysaccharide (LPS) lacks both phosphate groups present in Escherichia coli lipid A. Membranes of Francisella novicida (an environmental strain related to F. tularensis) contain enzymes that dephosphorylate lipid A and its precursors at the 1- and 4′-positions. We now report the cloning and characterization of a membrane-bound phosphatase of F. novicida that selectively dephosphorylates the 1-position. By transferring an F. novicida genomic DNA library into E. coli and selecting for low level polymyxin resistance, we isolated FnlpxE as the structural gene for the 1-phosphatase, an inner membrane enzyme of 239 amino acid residues. Expression of FnlpxE in a heptose-deficient mutant of E. coli caused massive accumulation of a previously uncharacterized LPS molecule, identified by mass spectrometry as 1-dephospho-Kdo2-lipid A. The predicted periplasmic orientation of the FnLpxE active site suggested that LPS export might be required for 1-dephosphorylation of lipid A. LPS and phospholipid export depend on the activity of MsbA, an essential inner membrane ABC transporter. Expression of FnlpxE in the msbA temperature-sensitive E. coli mutant WD2 resulted in 90% 1-dephosphorylation of lipid A at the permissive temperature (30 °C). However, the 1-phosphate group of newly synthesized lipid A was not cleaved at the nonpermissive temperature (44 °C). Our findings provide the first direct evidence that lipid A 1-dephosphorylation catalyzed by LpxE occurs on the periplasmic surface of the inner membrane. PMID:15339914

  3. A procedure for differentiating between the intentional release of biological warfare agents and natural outbreaks of disease: its use in analyzing the tularemia outbreak in Kosovo in 1999 and 2000.

    PubMed

    Grunow, R; Finke, E-J

    2002-08-01

    The events of 11 September and the subsequent anthrax outbreaks in the USA have opened the world's eyes to the threat posed by terrorist groups, criminal organizations and lone operators who will stop at nothing to achieve their goals. The open or covert use of pathogens and toxins as biological warfare agents can no longer be ruled out. Against this background, the appearance of an unusual disease must be studied in order to clarify whether it is a natural or artificially caused occurrence. This issue was recently raised in discussions with local representatives and relief organizations during a tularemia epidemic in Kosovo from October 1999 to May 2000. This paper will present a procedure which attempts to use certain criteria to identify or rule out the use of biological warfare agents in the event of an unusual outbreak of disease. Data and findings gathered by routine epidemiologic and microbiological studies often provide only an indirect answer to this problem. For this reason, various criteria were formulated and points allocated to represent their importance, allowing us to deduce in a semiquantitative manner the degree of possibility of an artificial genesis of outbreaks. The significance and characterization of each criterion are discussed. An analysis of the tularemia epidemic in Kosovo based on the procedure described here indicates that a deliberate release of the causative agent of tularemia, Francisella tularensis, as a biological warfare agent is doubtful. In this paper, an approach is described to discriminate between the intentional use of biological warfare agents and natural outbreaks of infectious diseases. The developed model is flexible and considers the political, military and social analysis of the crisis-afflicted region, the specific features of the pathogen, and the epidemiologic and clinical characteristics of the epidemic.

  4. Biofilm formation of Francisella noatunensis subsp. orientalis.

    PubMed

    Soto, Esteban; Halliday-Simmonds, Iona; Francis, Stewart; Kearney, Michael T; Hansen, John D

    2015-12-31

    Francisella noatunensis subsp. orientalis (Fno) is an emergent fish pathogen in both marine and fresh water environments. The bacterium is suspected to persist in the environment even without the presence of a suitable fish host. In the present study, the influence of different abiotic factors such as salinity and temperature were used to study the biofilm formation of different isolates of Fno including intracellular growth loci C (iglC) and pathogenicity determinant protein A (pdpA) knockout strains. Finally, we compared the susceptibility of planktonic and biofilm to three disinfectants used in the aquaculture and ornamental fish industry, namely Virkon(®), bleach and hydrogen peroxide. The data indicates that Fno is capable of producing biofilms within 24 h where both salinity as well as temperature plays a role in the growth and biofilm formation of Fno. Mutations in the iglC or pdpA, both known virulence factors, do not appear to affect the capacity of Fno to produce biofilms, and the minimum inhibitory concentration, and minimum biocidal concentration for the three disinfectants were lower than the minimum biofilm eradication concentration values. This information needs to be taken into account if trying to eradicate the pathogen from aquaculture facilities or aquariums.

  5. Biofilm formation of Francisella noatunensis subsp. orientalis

    USGS Publications Warehouse

    Soto, Esteban; Halliday-Wimmonds, Iona; Kearney, Michael T; Hansen, John D.

    2015-01-01

    Francisella noatunensis subsp. orientalis (Fno) is an emergent fish pathogen in both marine and fresh water environments. The bacterium is suspected to persist in the environment even without the presence of a suitable fish host. In the present study, the influence of different abiotic factors such as salinity and temperature were used to study the biofilm formation of different isolates of Fno including intracellular growth loci C (iglC)and pathogenicity determinant protein A (pdpA) knockout strains. Finally, we compared the susceptibility of planktonic and biofilm to three disinfectants used in the aquaculture and ornamental fish industry, namely Virkon®, bleach and hydrogen peroxide. The data indicates that Fno is capable of producing biofilms within 24 h where both salinity as well as temperature plays a role in the growth and biofilm formation of Fno. Mutations in theiglC or pdpA, both known virulence factors, do not appear to affect the capacity of Fno to produce biofilms, and the minimum inhibitory concentration, and minimum biocidal concentration for the three disinfectants were lower than the minimum biofilm eradication concentration values. This information needs to be taken into account if trying to eradicate the pathogen from aquaculture facilities or aquariums.

  6. Biofilm formation of Francisella noatunensis subsp. orientalis.

    PubMed

    Soto, Esteban; Halliday-Simmonds, Iona; Francis, Stewart; Kearney, Michael T; Hansen, John D

    2015-12-31

    Francisella noatunensis subsp. orientalis (Fno) is an emergent fish pathogen in both marine and fresh water environments. The bacterium is suspected to persist in the environment even without the presence of a suitable fish host. In the present study, the influence of different abiotic factors such as salinity and temperature were used to study the biofilm formation of different isolates of Fno including intracellular growth loci C (iglC) and pathogenicity determinant protein A (pdpA) knockout strains. Finally, we compared the susceptibility of planktonic and biofilm to three disinfectants used in the aquaculture and ornamental fish industry, namely Virkon(®), bleach and hydrogen peroxide. The data indicates that Fno is capable of producing biofilms within 24 h where both salinity as well as temperature plays a role in the growth and biofilm formation of Fno. Mutations in the iglC or pdpA, both known virulence factors, do not appear to affect the capacity of Fno to produce biofilms, and the minimum inhibitory concentration, and minimum biocidal concentration for the three disinfectants were lower than the minimum biofilm eradication concentration values. This information needs to be taken into account if trying to eradicate the pathogen from aquaculture facilities or aquariums. PMID:26507830

  7. Francisella guangzhouensis sp. nov., isolated from air-conditioning systems.

    PubMed

    Qu, Ping-Hua; Chen, Shou-Yi; Scholz, Holger C; Busse, Hans-Jürgen; Gu, Quan; Kämpfer, Peter; Foster, Jeffrey T; Glaeser, Stefanie P; Chen, Cha; Yang, Zhi-Chong

    2013-10-01

    Four strains (08HL01032(T), 09HG994, 10HP82-6 and 10HL1960) were isolated from water of air-conditioning systems of various cooling towers in Guangzhou city, China. Cells were Gram-stain-negative coccobacilli without flagella, catalase-positive and oxidase-negative, showing no reduction of nitrate, no hydrolysis of urea and no production of H2S. Growth was characteristically enhanced in the presence of l-cysteine, which was consistent with the properties of members of the genus Francisella. The quinone system was composed of ubiquinone Q-8 with minor amounts of Q-9. The polar lipid profile consisted of the predominant lipids phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, two unidentified phospholipids (PL2, PL3), an unidentified aminophospholipid and an unidentified glycolipid (GL2). The polyamine pattern consisted of the major compounds spermidine, cadaverine and spermine. The major cellular fatty acids were C10 : 0, C14 : 0, C16 : 0, C18 : 1ω9c and C18 : 1 3-OH. A draft whole-genome sequence of the proposed type strain 08HL01032(T) was generated. Comparative sequence analysis of the complete 16S and 23S rRNA genes confirmed affiliation to the genus Francisella, with 95 % sequence identity to the closest relatives in the database, the type strains of Francisella philomiragia and Francisella noatunensis subsp. orientalis. Full-length deduced amino acid sequences of various housekeeping genes, recA, gyrB, groEL, dnaK, rpoA, rpoB, rpoD, rpoH, fopA and sdhA, exhibited similarities of 67-92 % to strains of other species of the genus Francisella. Strains 08HL01032(T), 09HG994, 10HP82-6 and 10HL1960 exhibited highly similar pan-genome PCR profiles. Both the phenotypic and molecular data support the conclusion that the four strains belong to the genus Francisella but exhibit considerable divergence from all recognized Francisella species. Therefore, we propose the name Francisella guangzhouensis sp

  8. Multi-platform comparison of ten commercial master mixes for probe-based real-time polymerase chain reaction detection of bioterrorism threat agents for surge preparedness.

    PubMed

    Buzard, Gregory S; Baker, Daniel; Wolcott, Mark J; Norwood, David A; Dauphin, Leslie A

    2012-11-30

    The Centers for Disease Control and Prevention and United States Army Research Institute for Infectious Diseases have developed real-time PCR assays for the detection of bioterrorism threat agents. These assays all rely on a limited number of approved real-time PCR master mixes. Because the availability of these reagents is a critical element of bioterrorism preparedness, we undertook a joint national preparedness exercise to address the potential surge needs resulting from a large-scale bio-emergency. We identified 9 commercially-available potential alternatives to an existing approved master mix (LightCycler FastStart DNA Master HybProbes): the TaqMan Fast Universal PCR master mix, OmniMix HS, FAST qPCR master mix, EXPRESS qPCR SuperMix kit, QuantiFast Probe PCR kit, LightCycler FastStart DNA Master(PLUS) HybProbe, Brilliant II FAST qPCR master mix, ABsolute Fast QPCR Mix and the HotStart IT Taq master mix. The performances of these kits were evaluated by the use of real-time PCR assays for four bioterrorism threat agents: Bacillus anthracis, Brucella melitensis, Burkholderia mallei and Francisella tularensis. The master mixes were compared for target-specific detection levels, as well as consistency of results among three different real-time PCR platforms (LightCycler, SmartCycler and 7500 Fast Dx). Real-time PCR analysis revealed that all ten kits performed well for agent detection on the 7500 Fast Dx instrument; however, the QuantiFast Probe PCR kit yielded the most consistently positive results across multiple real-time PCR platforms. We report that certain combinations of commonly used master mixes and instruments are not as reliable as others at detecting low concentrations of target DNA. Furthermore, our study provides laboratories the option to select from the commercial kits we evaluated to suit their preparedness needs. PMID:23107058

  9. Multi-platform comparison of ten commercial master mixes for probe-based real-time polymerase chain reaction detection of bioterrorism threat agents for surge preparedness.

    PubMed

    Buzard, Gregory S; Baker, Daniel; Wolcott, Mark J; Norwood, David A; Dauphin, Leslie A

    2012-11-30

    The Centers for Disease Control and Prevention and United States Army Research Institute for Infectious Diseases have developed real-time PCR assays for the detection of bioterrorism threat agents. These assays all rely on a limited number of approved real-time PCR master mixes. Because the availability of these reagents is a critical element of bioterrorism preparedness, we undertook a joint national preparedness exercise to address the potential surge needs resulting from a large-scale bio-emergency. We identified 9 commercially-available potential alternatives to an existing approved master mix (LightCycler FastStart DNA Master HybProbes): the TaqMan Fast Universal PCR master mix, OmniMix HS, FAST qPCR master mix, EXPRESS qPCR SuperMix kit, QuantiFast Probe PCR kit, LightCycler FastStart DNA Master(PLUS) HybProbe, Brilliant II FAST qPCR master mix, ABsolute Fast QPCR Mix and the HotStart IT Taq master mix. The performances of these kits were evaluated by the use of real-time PCR assays for four bioterrorism threat agents: Bacillus anthracis, Brucella melitensis, Burkholderia mallei and Francisella tularensis. The master mixes were compared for target-specific detection levels, as well as consistency of results among three different real-time PCR platforms (LightCycler, SmartCycler and 7500 Fast Dx). Real-time PCR analysis revealed that all ten kits performed well for agent detection on the 7500 Fast Dx instrument; however, the QuantiFast Probe PCR kit yielded the most consistently positive results across multiple real-time PCR platforms. We report that certain combinations of commonly used master mixes and instruments are not as reliable as others at detecting low concentrations of target DNA. Furthermore, our study provides laboratories the option to select from the commercial kits we evaluated to suit their preparedness needs.

  10. Cutaneous Infection Caused by a Novel Francisella sp.

    PubMed Central

    Respicio-Kingry, Laurel B.; Byrd, Lori; Allison, Ashley; Brett, Meghan; Scott-Waldron, Christine; Galliher, Karen; Hannah, Paul; Mead, Paul

    2013-01-01

    A 69-year-old patient presented with a tender, thickly crusted skin lesion of 1 week's duration. A bacterial culture swab taken from the underlying granular tissue yielded a pure isolate of a Gram-negative coccobacillus, presumptively identified as a novel Francisella species via 16S rRNA and multilocus gene sequence analysis. PMID:23903547

  11. Molecular cloning of the recA gene and construction of a recA strain of Francisella novicida.

    PubMed Central

    Berg, J M; Mdluli, K E; Nano, F E

    1992-01-01

    A gene locus that is functionally analogous to the recA gene of Escherichia coli was molecularly cloned from Francisella novicida. The cloned gene was found to suppress the sensitivity of an E. coli strain to DNA-damaging agents and to support genetic recombination in E. coli. After transposon mutagenesis, the recA-like gene locus was returned to F. novicida and a UV-sensitive F. novicida strain was isolated. In contrast to the wild-type strain, this UV-sensitive strain could not be transformed with chromosomal DNA. Images PMID:1309722

  12. Screen of FDA-approved drug library identifies maprotiline, an antibiofilm and antivirulence compound with QseC sensor-kinase dependent activity in Francisella novicida.

    PubMed

    Dean, Scott N; van Hoek, Monique L

    2015-01-01

    Development of new therapeutics against Select Agents such as Francisella is critical preparation in the event of bioterrorism. Testing FDA-approved drugs for this purpose may yield new activities unrelated to their intended purpose and may hasten the discovery of new therapeutics. A library of 420 FDA-approved drugs was screened for antibiofilm activity against a model organism for human tularemia, Francisella (F.) novicida, excluding drugs that significantly inhibited growth. The initial screen was based on the 2-component system (TCS) dependent biofilm effect, thus, the QseC dependence of maprotiline anti-biofilm action was demonstrated. By comparing their FDA-approved uses, chemical structures, and other properties of active drugs, toremifene and polycyclic antidepressants maprotiline and chlorpromazine were identified as being highly active against F. novicida biofilm formation. Further down-selection excluded toremifene for its membrane active activity and chlorpromazine for its high antimicrobial activity. The mode of action of maprotiline against F. novicida was sought. It was demonstrated that maprotiline was able to significantly down-regulate the expression of the virulence factor IglC, encoded on the Francisella Pathogenicity Island (FPI), suggesting that maprotiline is exerting an effect on bacterial virulence. Further studies showed that maprotiline significantly rescued F. novicida infected wax worm larvae. In vivo studies demonstrated that maprotiline treatment could prolong time to disease onset and survival in F. novicida infected mice. These results suggest that an FDA-approved drug such as maprotiline has the potential to combat Francisella infection as an antivirulence agent, and may have utility in combination with antibiotics. PMID:26155740

  13. Screen of FDA-approved drug library identifies maprotiline, an antibiofilm and antivirulence compound with QseC sensor-kinase dependent activity in Francisella novicida

    PubMed Central

    Dean, Scott N; van Hoek, Monique L

    2015-01-01

    Development of new therapeutics against Select Agents such as Francisella is critical preparation in the event of bioterrorism. Testing FDA-approved drugs for this purpose may yield new activities unrelated to their intended purpose and may hasten the discovery of new therapeutics. A library of 420 FDA-approved drugs was screened for antibiofilm activity against a model organism for human tularemia, Francisella (F.) novicida, excluding drugs that significantly inhibited growth. The initial screen was based on the 2-component system (TCS) dependent biofilm effect, thus, the QseC dependence of maprotiline anti-biofilm action was demonstrated. By comparing their FDA-approved uses, chemical structures, and other properties of active drugs, toremifene and polycyclic antidepressants maprotiline and chlorpromazine were identified as being highly active against F. novicida biofilm formation. Further down-selection excluded toremifene for its membrane active activity and chlorpromazine for its high antimicrobial activity. The mode of action of maprotiline against F. novicida was sought. It was demonstrated that maprotiline was able to significantly down-regulate the expression of the virulence factor IglC, encoded on the Francisella Pathogenicity Island (FPI), suggesting that maprotiline is exerting an effect on bacterial virulence. Further studies showed that maprotiline significantly rescued F. novicida infected wax worm larvae. In vivo studies demonstrated that maprotiline treatment could prolong time to disease onset and survival in F. novicida infected mice. These results suggest that an FDA-approved drug such as maprotiline has the potential to combat Francisella infection as an antivirulence agent, and may have utility in combination with antibiotics. PMID:26155740

  14. Characterization of Francisella species isolated from the cooling water of an air conditioning system.

    PubMed

    Gu, Quan; Li, Xunde; Qu, Pinghua; Hou, Shuiping; Li, Juntao; Atwill, Edward R; Chen, Shouyi

    2015-01-01

    Strains of Francisella spp. were isolated from cooling water from an air conditioning system in Guangzhou, China. These strains are Gram negative, coccobacilli, non-motile, oxidase negative, catalase negative, esterase and lipid esterase positive. In addition, these bacteria grow on cysteine-supplemented media at 20 °C to 40 °C with an optimal growth temperature of 30 °C. Analysis of 16S rRNA gene sequences revealed that these strains belong to the genus Francisella. Biochemical tests and phylogenetic and BLAST analyses of 16S rRNA, rpoB and sdhA genes indicated that one strain was very similar to Francisella philomiragia and that the other strains were identical or highly similar to the Francisella guangzhouensis sp. nov. strain 08HL01032 we previously described. Biochemical and molecular characteristics of these strains demonstrated that multiple Francisella species exist in air conditioning systems. PMID:26413079

  15. Characterization of Francisella species isolated from the cooling water of an air conditioning system.

    PubMed

    Gu, Quan; Li, Xunde; Qu, Pinghua; Hou, Shuiping; Li, Juntao; Atwill, Edward R; Chen, Shouyi

    2015-01-01

    Strains of Francisella spp. were isolated from cooling water from an air conditioning system in Guangzhou, China. These strains are Gram negative, coccobacilli, non-motile, oxidase negative, catalase negative, esterase and lipid esterase positive. In addition, these bacteria grow on cysteine-supplemented media at 20 °C to 40 °C with an optimal growth temperature of 30 °C. Analysis of 16S rRNA gene sequences revealed that these strains belong to the genus Francisella. Biochemical tests and phylogenetic and BLAST analyses of 16S rRNA, rpoB and sdhA genes indicated that one strain was very similar to Francisella philomiragia and that the other strains were identical or highly similar to the Francisella guangzhouensis sp. nov. strain 08HL01032 we previously described. Biochemical and molecular characteristics of these strains demonstrated that multiple Francisella species exist in air conditioning systems.

  16. Characterization of Francisella species isolated from the cooling water of an air conditioning system

    PubMed Central

    Gu, Quan; Li, Xunde; Qu, Pinghua; Hou, Shuiping; Li, Juntao; Atwill, Edward R.; Chen, Shouyi

    2015-01-01

    Strains of Francisella spp. were isolated from cooling water from an air conditioning system in Guangzhou, China. These strains are Gram negative, coccobacilli, non-motile, oxidase negative, catalase negative, esterase and lipid esterase positive. In addition, these bacteria grow on cysteine-supplemented media at 20 °C to 40 °C with an optimal growth temperature of 30 °C. Analysis of 16S rRNA gene sequences revealed that these strains belong to the genus Francisella. Biochemical tests and phylogenetic and BLAST analyses of 16S rRNA, rpoB and sdhA genes indicated that one strain was very similar to Francisella philomiragia and that the other strains were identical or highly similar to the Francisella guangzhouensis sp. nov. strain 08HL01032 we previously described. Biochemical and molecular characteristics of these strains demonstrated that multiple Francisella species exist in air conditioning systems. PMID:26413079

  17. Biocidal and Sporicidal Efficacy of Pathoster® 0.35% and Pathoster® 0.50% Against Bacterial Agents in Potential Bioterrorism Use

    PubMed Central

    Candeliere, Antonio; Donatiello, Adelia; Pagano, Stefania; Iatarola, Michela; Tolve, Francesco; Antonino, Leonardo; Fasanella, Antonio

    2016-01-01

    The use of products that can neutralize or significantly reduce the microbial load and that are not harmful to human health and the environment represents a milestone in the fight against the spread of infectious diseases. Peracetic acid, besides being an excellent sterilizing and sporicidal agent, is harmless to humans and the environment when it is used in a common dosage. However, the high costs and loss of efficacy of the product very quickly after its reconstitution limit its use. We evaluated the efficacy and stability of 2 commercial products, based on stabilized peracetic acid (Pathoster® 0.35% and Pathoster® 0.50%) used against spores of Bacillus anthracis and spores of Bacillus cereus and vegetative forms of Yersinia pestis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Brucella abortus, and Brucella melitensis. The efficacy tests were based on the direct contact of the products with a standard suspension of the bacteria. The stability of the products was defined as the period of time during which the biocidal and sporicidal properties remained unchanged. The limit of effectiveness was the period after which the product was unable to exert a complete sterilization after a contact of 5 minutes with at least 1 of the 8 bacteria used in this work. Both formulations showed good efficacy against the microorganisms used in the study, confirming the utility of peracetic acid as a sterilizing product. After the reconstitution, Pathoster® 0.35% was stable until 16±1 days, while Pathoster® 0.50% was stable until 24±1 days. The formulations used in this study showed good performance and a significant stability of peracetic acid. PMID:27482880

  18. Biocidal and Sporicidal Efficacy of Pathoster(®) 0.35% and Pathoster(®) 0.50% Against Bacterial Agents in Potential Bioterrorism Use.

    PubMed

    Candeliere, Antonio; Campese, Emanuele; Donatiello, Adelia; Pagano, Stefania; Iatarola, Michela; Tolve, Francesco; Antonino, Leonardo; Fasanella, Antonio

    2016-01-01

    The use of products that can neutralize or significantly reduce the microbial load and that are not harmful to human health and the environment represents a milestone in the fight against the spread of infectious diseases. Peracetic acid, besides being an excellent sterilizing and sporicidal agent, is harmless to humans and the environment when it is used in a common dosage. However, the high costs and loss of efficacy of the product very quickly after its reconstitution limit its use. We evaluated the efficacy and stability of 2 commercial products, based on stabilized peracetic acid (Pathoster(®) 0.35% and Pathoster(®) 0.50%) used against spores of Bacillus anthracis and spores of Bacillus cereus and vegetative forms of Yersinia pestis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Brucella abortus, and Brucella melitensis. The efficacy tests were based on the direct contact of the products with a standard suspension of the bacteria. The stability of the products was defined as the period of time during which the biocidal and sporicidal properties remained unchanged. The limit of effectiveness was the period after which the product was unable to exert a complete sterilization after a contact of 5 minutes with at least 1 of the 8 bacteria used in this work. Both formulations showed good efficacy against the microorganisms used in the study, confirming the utility of peracetic acid as a sterilizing product. After the reconstitution, Pathoster(®) 0.35% was stable until 16±1 days, while Pathoster(®) 0.50% was stable until 24±1 days. The formulations used in this study showed good performance and a significant stability of peracetic acid. PMID:27482880

  19. Serology for tularemia

    MedlinePlus

    Tularemia test; Serology for Francisella tularensis ... This blood test is done when tularemia is suspected. ... Saunders; 2011:chap 44. Penn RL. Francisella tularensis (Tularemia). In: Bennett JE, Dolin R, Blaser MJ, eds. ...

  20. Duplex PCR assay and in situ hybridization for detection of Francisella spp. and Francisella noatunensis subsp. orientalis in red tilapia.

    PubMed

    Dong, Ha T; Gangnonngiw, Warachin; Phiwsaiya, Kornsunee; Charoensapsri, Walaiporn; Nguyen, Vuong V; Nilsen, Pål; Pradeep, Padmaja J; Withyachumnarnkul, Boonsirm; Senapin, Saengchan; Rodkhum, Channarong

    2016-06-15

    Conventional isolation and identification based on phenotypic characteristics is challenging with the highly fastidious, intracellular bacterium Francisella noatunensis subsp. orientalis (Fno). Here, we developed a duplex PCR method for simultaneous detection of the Francisella genus and Fno in one PCR reaction and an in situ hybridization method for paraffin section based diagnosis of Fno. The PCR results showed genus- and species-specific bands (1140 and 203 bp) from Fno but only one genus-specific band (1140 bp) from F. noatunensis subsp. noatunensis. Sensitivity of the duplex PCR assay revealed a detection limit of 20 to 200 fg genomic DNA (~10 to 100 genome equivalents) depending on DNA template extraction methods. The newly developed duplex PCR assay could be used to detect Fno from clinically sick fish exhibiting signs of visceral granulomas and would also be able to detect Fno infection in naturally diseased fish without symptoms of francisellosis, indicating potential application for diagnosis of field samples. The in situ hybridization assay using Fno species-specific probe revealed positive signals in multiple organs including the spleen, liver, kidney, gills and intestine of infected fish. PMID:27304869

  1. Host immune response and acute disease in a zebrafish model of francisella pathogenesis

    USGS Publications Warehouse

    Vojtech, L.N.; Sanders, G.E.; Conway, C.; Ostland, V.; Hansen, J.D.

    2009-01-01

    Members of the bacterial genus Francisella are highly virulent and infectious pathogens. New models to study Francisella pathogenesis in evolutionarily distinct species are needed to provide comparative insight, as the mechanisms of host resistance and pathogen virulence are not well understood. We took advantage of the recent discovery of a novel species of Francisella to establish a zebrafish/Francisella comparative model of pathogenesis and host immune response. Adult zebraflsh were susceptible to acute Francisella-induced disease and suffered mortality in a dose-dependent manner. Using immunohistochemical analysis, we localized bacterial antigens primarily to lymphoid tissues and livers of zebraflsh following infection by intraperitoneal injection, which corresponded to regions of local cellular necrosis. Francisella sp. bacteria replicated rapidly in these tissues beginning 12 h postinfection, and bacterial titers rose steadily, leveled off, and then decreased by 7 days postinfection. Zebraflsh mounted a significant tissue-specific proinflammatory response to infection as measured by the upregulation of interleukin-l?? (IL-1??), gamma interferon, and tumor necrosis factor alpha mRNA beginning by 6 h postinfection and persisting for up to 7 days postinfection. In addition, exposure of zebraflsh to heat-killed bacteria demonstrated that the significant induction of IL-?? was highly specific to live bacteria. Taken together, the pathology and immune response to acute Francisella infection in zebraflsh share many features with those in mammals, highlighting the usefulness of this new model system for addressing both general and specific questions about Francisella host-pathogen interactions via an evolutionary approach. Copyright ?? 2009, American Society for Microbiology. All Rights Reserved.

  2. Transfer of immunity against lethal murine Francisella infection by specific antibody depends on host gamma interferon and T cells.

    PubMed Central

    Rhinehart-Jones, T R; Fortier, A H; Elkins, K L

    1994-01-01

    Both serum and spleen cells from mice immune to Francisella tularensis transfer protection to naive recipients. Here we characterize the mechanism of protection induced by transfer of immune mouse serum (IMS). IMS obtained 4 weeks after intradermal infection with 10(3) bacteria of the live vaccine strain (LVS) contained high levels of immunoglobulin G2 (IgG2a) and IgM (end point titers, 1:16,600 and 1:7,200, respectively) and little IgG1, IgG2b, or IgG3. LVS-specific antibodies were detected 5 days after intradermal infection, and reached peak levels by 2 weeks postinfection. Only sera obtained 10 days or more after sublethal infection, when IgG titers peaked, transferred protection against a challenge of 100 50% lethal doses (LD50s). Purified high-titer IgG anti-LVS antibody but not IgM anti-LVS antibody was responsible for transfer of protection against an intraperitoneal challenge of up to 3,000 LD50s. IMS had no direct toxic effects on LVS and did not affect uptake or growth of bacteria in association with peritoneal cells. One day after LVS infection, liver, spleen, and lung tissue from mice treated with IMS contained 1 to 2 log units fewer bacteria than did tissue from mice treated with normal mouse serum or phosphate-buffered saline. Between 2 and 4 days after infection, however, bacterial growth rates in tissues were similar in both serum-protected mice and unprotected mice. Bacterial burdens in IMS-treated, LVS-infected mice declined in infected tissues after day 5, whereas control animals died. This lag phase suggested that development of a host response was involved in complete bacterial clearance. In fact, transfer of IMS into normal recipients that were simultaneously treated with anti-gamma interferon and challenged with LVS did not protect mice from death. Further, transfer of IMS into athymic nu/nu mice did not protect against LVS challenge; protection was, however, reconstituted by transfer of normal T cells into nu/nu mice. Thus, "passive

  3. First identification of Francisella noatunensis subsp. orientalis causing mortality in Mexican tilapia Oreochromis spp.

    PubMed

    Ortega, Cesar; Mancera, Gerardo; Enríquez, Ricardo; Vargas, Augusto; Martínez, Simón; Fajardo, Raúl; Avendaño-Herrera, Ruben; Navarrete, María José; Romero, Alex

    2016-08-01

    Francisellosis, an emerging disease in tilapia Oreochromis spp., is caused by the facultative, intracellular bacterium Francisella noatunensis subsp. orientalis, which is present in various countries where tilapia farming is commercially important. We confirmed the presence of francisellosis in Mexican tilapia cultures in association with an outbreak during the second semester of 2012. Broodstock fish presented a mortality rate of approximately 40%, and disease was characterized by histologically classified granulomas, or whitish nodules, in different organs, mainly the spleen and kidney. Through DNA obtained from infected tissue and pure cultures in a cysteine heart medium supplemented with hemoglobin, F. noatunensis subsp. orientalis was initially confirmed through the amplification and analysis of the 16S rRNA gene and the internal transcribed spacer region. Phylogenetic analysis of these genes demonstrated close similarity with previously reported F. noatunensis subsp. orientalis sequences obtained from infected tilapia from various countries. The identification of this subspecies as the causative agent of the outbreak was confirmed using the iglC gene as a target sequence, which showed 99.5% identity to 2 F. noatunensis subsp. orientalis strains (Ethime-1 and Toba04). These findings represent the first documented occurrence of francisellosis in Mexican tilapia cultures, which highlights the importance of establishing preventative measures to minimize the spread of this disease within the Mexican aquaculture industry. PMID:27503916

  4. First identification of Francisella noatunensis subsp. orientalis causing mortality in Mexican tilapia Oreochromis spp.

    PubMed

    Ortega, Cesar; Mancera, Gerardo; Enríquez, Ricardo; Vargas, Augusto; Martínez, Simón; Fajardo, Raúl; Avendaño-Herrera, Ruben; Navarrete, María José; Romero, Alex

    2016-08-01

    Francisellosis, an emerging disease in tilapia Oreochromis spp., is caused by the facultative, intracellular bacterium Francisella noatunensis subsp. orientalis, which is present in various countries where tilapia farming is commercially important. We confirmed the presence of francisellosis in Mexican tilapia cultures in association with an outbreak during the second semester of 2012. Broodstock fish presented a mortality rate of approximately 40%, and disease was characterized by histologically classified granulomas, or whitish nodules, in different organs, mainly the spleen and kidney. Through DNA obtained from infected tissue and pure cultures in a cysteine heart medium supplemented with hemoglobin, F. noatunensis subsp. orientalis was initially confirmed through the amplification and analysis of the 16S rRNA gene and the internal transcribed spacer region. Phylogenetic analysis of these genes demonstrated close similarity with previously reported F. noatunensis subsp. orientalis sequences obtained from infected tilapia from various countries. The identification of this subspecies as the causative agent of the outbreak was confirmed using the iglC gene as a target sequence, which showed 99.5% identity to 2 F. noatunensis subsp. orientalis strains (Ethime-1 and Toba04). These findings represent the first documented occurrence of francisellosis in Mexican tilapia cultures, which highlights the importance of establishing preventative measures to minimize the spread of this disease within the Mexican aquaculture industry.

  5. [Tularaemia - an overview of the current knowledge].

    PubMed

    Lukásová, Eva; Cermák, Pavel; Smelá, Gabriela; Jedlicková, Anna

    2010-02-01

    Francisella tularensis belongs to the family Francisellaceae. It is the aetiological agent of a zoonosis called tularaemia, spread throughout the northern hemisphere. Currently, several subspecies of F. tularensis may be distinguished with various pathogenicity and geographical distribution. In human medicine, only sporadic infections or local epidemics are reported. Given the fact that F. tularensis is highly pathogenic for humans and is easily spread by aerosol, water or food, it may be exploited as a biological weapon. It belongs to fastidious strains requiring specially prepared culture media.

  6. Effect of Aerosol Age on the Infectivity of Airborne Pasteurella tularensis for Macaca mulatta and Man

    PubMed Central

    Sawyer, William D.; Jemski, Joseph V.; Hogge, Arthur L.; Eigelsbach, Henry T.; Wolfe, Elwood K.; Dangerfield, Harry G.; Gochenour, William S.; Crozier, Dan

    1966-01-01

    Sawyer, William D. (U.S. Army Medical Unit, Fort Detrick, Frederick, Md.), Joseph V. Jemski, Arthur L. Hogge, Jr., Henry T. Eigelsbach, Elwood K. Wolfe, Harry G. Dangerfield, William S. Gochenour, Jr., and Dan Crozier. Effect of aerosol age on the infectivity of airborne Pasteurella tularensis for Macaca mulatta and man. J. Bacteriol. 91:2180–2184. 1966.—In aging aerosols of Pasteurella tularensis SCHU-S4, the respiratory infectivity for man and Macaca mulatta decreased more rapidly than the viability of the organisms. Infectivity was diminished after 120 min, and was reduced 10-fold after 180 min. These findings confirmed previous observations made in mice and guinea pigs, and also revealed that smaller losses of infectivity were detectable in the primate hosts. PMID:4957611

  7. Finished genome assembly of warm spring isolate Francisella novicida DPG 3A-IS

    DOE PAGES

    Johnson, Shannon L.; Minogue, Timothy D.; Daligault, Hajnalka E.; Wolcott, Mark J.; Teshima, Hazuki; Coyne, Susan R.; Davenport, Karen W.; Jaissle, James G.; Chain, Patrick S.

    2015-09-17

    We sequenced the complete genome of Francisella novicida DPG 3A-IS to closed and finished status. This is a warm spring isolate recovered from Hobo Warm Spring (Utah, USA). The last assembly is available in NCBI under accession number CP012037.

  8. Francisella Infection in Cultured Tilapia in Thailand and the Inflammatory Cytokine Response.

    PubMed

    Jantrakajorn, Sasibha; Wongtavatchai, Janenuj

    2016-06-01

    Francisella infections developed in freshwater Nile Tilapia Oreochromis niloticus and red tilapia Oreochromis spp. farms in Thailand during 2012-2014. The diseased fish were lethargic and pale in color and showed numerous white nodules in their enlarged spleens. Histopathological examination and electron microscopy suggested that the white nodules were multifocal granulomas consisting of coccobacilli within vacuolated cells. Isolation of Francisella-like bacteria was achieved from 42 of 100 samples, while polymerase chain reaction confirmed Francisella infections in all samples. Analysis of the 16S rRNA gene from samples obtained from three different geographical culture areas revealed more than 99% similarity with F. noatunensis subsp. orientalis. The influence of Francisella infection on inflammatory cytokines was determined on splenic cells of fish intraperitoneally injected with the bacteria (0.8 × 10(5) colony-forming units per fish). Infected tilapia showed significantly greater expression of the pro-inflammatory genes interleukin-1β (IL-1β) and tumor necrotic factor-α (TNF-α) within 24 h postinjection (hpi) and for up to 96 hpi. However, down-regulation of an anti-inflammatory gene, transforming growth factor-β (TGF-β) was observed as early as 24 hpi. This investigation demonstrates that an imbalance between pro- and anti-inflammatory cytokines in response to the infection may account for the substantial number of granulomas in fish hematopoietic tissues that was found in the later stage of the disease. Received September 9, 2015; accepted December 13, 2015. PMID:27196982

  9. Molecular detection of Rickettsia, Anaplasma, Coxiella and Francisella bacteria in ticks collected from Artiodactyla in Thailand.

    PubMed

    Sumrandee, Chalao; Baimai, Visut; Trinachartvanit, Wachareeporn; Ahantarig, Arunee

    2016-07-01

    A total of 79 ticks collected from Sambar deer (Cervus unicolor), Barking deer (Muntiacus muntjak) and Wild boar (Sus scrofa) were examined by PCR for the presence of Rickettsia, Anaplasma, Coxiella, and Francisella bacteria. Of the 79 ticks, 13% tested positive for Rickettsia, 15% tested positive for Anaplasma, 4% tested positive for Coxiella, and 3% tested positive for Francisella. Interestingly, triple infection with Anaplasma, Rickettsia and Francisella was determined in a Dermacentor auratus tick. Moreover, another triple infection with Rickettsia, Anaplasma, and Coxiella was found in a Haemaphysalis lagrangei tick. Double infection of Rickettsia with Coxiella was also detected in another H. lagrangei tick. From the phylogenetic analyses, we found a Rickettsia sp. with a close evolutionary relationship to Rickettsia bellii in the H. lagrangei tick. We also found the first evidence of a Rickettsia sp. that is closely related to Rickettsia tamurae in Rhipicephalus (Boophilus) microplus ticks from Thailand. H. lagrangei and Haemaphysalis obesa ticks collected from Sambar deer tested positive for Anaplasma species form the same clade with Anaplasma bovis. In contrast, other H. lagrangei ticks collected from Sambar deer and D. auratus ticks collected from Wild boar were also reported for the first time to be infected with an Anaplasma species that is closely related to Anaplasma platys. The phylogenetic analysis of the 16S rRNA gene of Coxiella bacteria revealed that Coxiella symbionts from H. lagrangei formed a distinctly different lineage from Coxiella burnetii (a human pathogen). Additionally, Francisella bacteria identified in D. auratus ticks were found to be distantly related to a group of pathogenic Francisella species. The identification of these bacteria in several feeding ticks suggests the risk of various emerging tick-borne diseases and endosymbionts in humans, wildlife, and domestic animals in Thailand. PMID:26934997

  10. Antimicrobial and antibiofilm activity of cathelicidins and short, synthetic peptides against Francisella.

    PubMed

    Amer, Lilian S; Bishop, Barney M; van Hoek, Monique L

    2010-05-28

    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 (microM), 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=50 nM). 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. PMID:20399752

  11. Glutamate Utilization Couples Oxidative Stress Defense and the Tricarboxylic Acid Cycle in Francisella Phagosomal Escape

    PubMed Central

    Ramond, Elodie; Gesbert, Gael; Rigard, Mélanie; Dairou, Julien; Dupuis, Marion; Dubail, Iharilalao; Meibom, Karin; Henry, Thomas; Barel, Monique; Charbit, Alain

    2014-01-01

    Intracellular bacterial pathogens have developed a variety of strategies to avoid degradation by the host innate immune defense mechanisms triggered upon phagocytocis. Upon infection of mammalian host cells, the intracellular pathogen Francisella replicates exclusively in the cytosolic compartment. Hence, its ability to escape rapidly from the phagosomal compartment is critical for its pathogenicity. Here, we show for the first time that a glutamate transporter of Francisella (here designated GadC) is critical for oxidative stress defense in the phagosome, thus impairing intra-macrophage multiplication and virulence in the mouse model. The gadC mutant failed to efficiently neutralize the production of reactive oxygen species. Remarkably, virulence of the gadC mutant was partially restored in mice defective in NADPH oxidase activity. The data presented highlight links between glutamate uptake, oxidative stress defense, the tricarboxylic acid cycle and phagosomal escape. This is the first report establishing the role of an amino acid transporter in the early stage of the Francisella intracellular lifecycle. PMID:24453979

  12. [Microbiological and clinical aspects of tularaemia].

    PubMed

    Pavliš, Oto; Pohanka, Miroslav

    2011-10-01

    Francisella tularensis belongs to the most important biological agents potentially applicable in biological warfare and bioterrorism. High virulence, easy and rapid spread among individual vectors, stability of the cells in aerosol and good penetration into the lungs make F. tularensis one of the most important biological warfare agents in both human and veterinary medicine. The text provides comprehensive data about tularaemia and outlines the fate of the pathogen in the host. Special attention is paid to immunological aspects of the disease, therapy, and diagnostic methods.

  13. Novel genomic tools for specific and real-time detection of biothreat and frequently encountered foodborne pathogens.

    PubMed

    Woubit, Abdela; Yehualaeshet, Teshome; Habtemariam, Tsegaye; Samuel, Temesgen

    2012-04-01

    The bacterial genera Escherichia, Salmonella, Shigella, Vibrio, Yersinia, and Francisella include important food safety and biothreat agents. By extensive mining of the whole genome and protein databases of diverse, closely and distantly related bacterial species and strains, we have identified novel genome regions, which we utilized to develop a rapid detection platform for these pathogens. The specific genomic targets we have identified to design the primers in Francisella tularensis subsp. tularensis, F. tularensis subsp. novicida, Shigella dysenteriae, Salmonella enterica serovar Typhimurium, Vibrio cholerae, Yersinia pestis, and Yersinia pseudotuberculosis contained either known genes or putative proteins. Primer sets were designed from the target regions for use in real-time PCR assays to detect specific biothreat pathogens at species or strain levels. The primer sets were first tested by in silico PCR against whole-genome sequences of different species, subspecies, or strains and then by in vitro PCR against genomic DNA preparations from 23 strains representing six biothreat agents (Escherichia coli O157:H7 strain EDL 933, Shigella dysenteriae, S. enterica serovar Typhi, F. tularensis subsp. tularensis, V. cholerae, and Y. pestis) and six foodborne pathogens (Salmonella Typhimurium, Salmonella Saintpaul, Shigella sonnei, F. tularensis subsp. novicida, Vibrio parahaemolyticus, and Y. pseudotuberculosis). Each pathogen was specifically identifiable at the genus and species levels. Sensitivity assays performed with purified DNA showed the lowest detection limit of 128 fg of DNA/μl for F. tularensis subsp. tularensis. A preliminary test to detect Shigella organisms in a milk matrix also enabled the detection of 6 to 60 CFU/ml. These new tools could ultimately be used to develop platforms to simultaneously detect these pathogens. PMID:22488053

  14. Complete Genome Sequence of Francisella guangzhouensis Strain 08HL01032T, Isolated from Air-Conditioning Systems in China.

    PubMed

    Svensson, Daniel; Öhrman, Caroline; Bäckman, Stina; Karlsson, Edvin; Nilsson, Elin; Byström, Mona; Lärkeryd, Adrian; Myrtennäs, Kerstin; Stenberg, Per; Qu, Ping-Hua; Trygg, Johan; Scholz, Holger C; Forsman, Mats; Sjödin, Andreas

    2015-03-19

    We present the complete genome sequence of Francisella guangzhouensis strain 08HL01032(T), which consists of one chromosome (1,658,482 bp) and one plasmid (3,045 bp) with G+C contents of 32.0% and 28.7%, respectively.

  15. Efficacy of florfenicol for control of mortality with Francisella noatunensis subsp. orientalis in Nile tilapia, oreochromis niloticus (L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Francisella noatunensis subsp. orientalis (Fno) (syn. F. asiatica) is an emergent Gram-negative facultative intracellular bacterium. Although it is considered one of the most pathogenic bacteria in fish, there are no commercially available treatments of vaccines. The objective of this project was ...

  16. Finished genome assembly of warm spring isolate Francisella novicida DPG 3A-IS

    SciTech Connect

    Johnson, Shannon L.; Minogue, Timothy D.; Daligault, Hajnalka E.; Wolcott, Mark J.; Teshima, Hazuki; Coyne, Susan R.; Davenport, Karen W.; Jaissle, James G.; Chain, Patrick S.

    2015-09-17

    We sequenced the complete genome of Francisella novicida DPG 3A-IS to closed and finished status. This is a warm spring isolate recovered from Hobo Warm Spring (Utah, USA). The last assembly is available in NCBI under accession number CP012037.

  17. Francisella philomiragia Bacteremia in a Patient with Acute Respiratory Insufficiency and Acute-on-Chronic Kidney Disease

    PubMed Central

    Humphries, Romney M.; Mattison, H. Reid; Miles, Jessica E.; Simpson, Edward R.; Corbett, Ian J.; Schmitt, Bryan H.; May, M.

    2015-01-01

    Francisella philomiragia is a very uncommon pathogen of humans. Diseases caused by it are protean and have been reported largely in near-drowning victims and those with chronic granulomatous disease. We present a case of F. philomiragia pneumonia with peripheral edema and bacteremia in a renal transplant patient and review the diverse reports of F. philomiragia infections. PMID:26400786

  18. Francisella noatunensis subsp. noatunensis replicates within Atlantic cod (Gadus morhua L.) leucocytes and inhibits respiratory burst activity.

    PubMed

    Vestvik, Nils; Rønneseth, Anita; Kalgraff, Cathrine A K; Winther-Larsen, Hanne C; Wergeland, Heidrun I; Haugland, Gyri T

    2013-09-01

    Francisella noatunensis subsp. noatunensis, causing granulomatosis in cod, has been shown to reside within cod immune cells, mainly within monocytes and macrophages. In the present study, we analysed the ability of the bacterium to replicate within adherent cells isolated from head kidney by in vitro infection of leucocytes. Two different technical approaches for flow cytometry analyses were performed for detection of intracellular bacteria. The presence of the wild type was assessed after identification by intracellular binding of specific antibodies to the pathogen. The other way was to use green fluorescent protein (GFP) transformed bacterium for infection studies allowing direct measurements of fluorescence from infected cells. By both methods we found an increase in fluorescence in infected cells, verifying bacterial replication, both after 4 and 28 h post infection in leucocytes isolated from head kidney (HKL). The GFP transformed bacterium was similar to the wild type in growth and infectivity pattern, showing that it can be a valuable tool for further studies of infection routes and pathology. Further, F. noatunensis subsp. noatunensis was found to inhibit respiratory burst activity, a potent pathogen killing mechanism, in cod leucocytes, but not in such cells from salmon. Our findings may indicate that inhibition of respiratory burst during Francisella infection is a key to its intracellular existence. This strategy seems to be conserved through evolution as it is also observed during infections in higher vertebrates caused by bacteria within the Francisella genus. The results presented here, showing the intracellular existence of Francisella, its replication within leucocytes and the inhibitory effect on respiratory burst, strongly support that these factors contribute to disease and pathology in infected cod. The intracellular replication shown in the present study might contribute to explain the problems of obtaining protective vaccines against

  19. An outbreak of disease caused by Francisella sp. in Nile tilapia Oreochromis niloticus at a recirculation fish farm in the UK.

    PubMed

    Jeffery, Keith R; Stone, David; Feist, Stephen W; Verner-Jeffreys, David W

    2010-09-01

    This study details the first diagnosis of Francisella sp. in tilapia in the United Kingdom. Losses of tilapia fry at a recirculation fish farm in England were investigated, giving a presumptive positive diagnosis of infection with Francisella sp. by histopathological examination. Most fish sampled showed moderate to marked pathology of the major organs, with lesions being present in most tissues. The most obvious host response was granuloma formulation. A subsequent follow-up visit provided further evidence for the presence of a Francisella species. PCR amplicons were obtained using Francisella spp.-specific primers that shared 100% sequence identity with the 16S rRNA gene of the type strain of the species F. asiatica previously described as the cause of disease in tilapia in Southeast Asia and Central America. This outbreak and the subsequent investigation emphasise the importance of strict biosecurity at fish farms and the care that needs to be taken when using a new supplier of fish. PMID:21387995

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

    PubMed

    Banik, Sukalyani; Mansour, Ahd Ahmed; Suresh, Ragavan Varadharajan; Wykoff-Clary, Sherri; Malik, Meenakshi; McCormick, Alison A; Bakshi, Chandra Shekhar

    2015-01-01

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

  1. Diagnostic procedures in tularaemia with special focus on molecular and immunological techniques.

    PubMed

    Splettstoesser, W D; Tomaso, H; Al Dahouk, S; Neubauer, H; Schuff-Werner, P

    2005-08-01

    Tularaemia is a severe bacterial zoonosis caused by the highly infectious agent Francisella tularensis. It is endemic in countries of the northern hemisphere ranging from North America to Europe, Asia and Japan. Very recently, Francisella-like strains causing disease in humans were described from tropical northern Australia. In the last decade, efforts have been made to develop sensitive and specific immunological and molecular techniques for the laboratory diagnosis of tularaemia and also for the definite identification of members of the species F. tularensis and its four subspecies. Screening for the keyword 'Francisella' a Medline search over the last decade was performed and articles describing diagnostic methods for tularaemia and its causative agent were selected. Besides classical microbiological techniques (cultivation, biochemical profiling, susceptibility testing) several new immunological and molecular approaches to identify F. tularensis have been introduced employing highly specific antibodies and various polymerase chain reaction (PCR)-based methods. Whereas direct antigen detection by enzyme-linked immunosorbent assay (ELISA) or immunofluorescence might allow early presumptive diagnosis of tularaemia, these methods--like all PCR techniques--still await further evaluation. Therefore, diagnosis of tularaemia still relies mainly on the demonstration of specific antibodies in the host. ELISA and immunoblot methods started to replace the standard tube or micro-agglutination assays. However, the diagnostic value of antibody detection in the very early clinical phase of tularaemia is limited. Francisella tularensis is regarded as a 'highest priority' biological agent (category 'A' according to the CDC, Atlanta, GA, USA), thus rapid and reliable diagnosis of tularaemia is required not only for a timely onset of therapy, the handling of outbreak investigations but also for the surveillance of endemic foci. Only very recently, evaluated test kits for

  2. Transovarial Transmission of Francisella-Like Endosymbionts and Anaplasma phagocytophilum Variants in Dermacentor albipictus (Acari: Ixodidae)

    PubMed Central

    BALDRIDGE, GERALD D.; SCOLES, GLEN. A.; BURKHARDT, NICOLE Y.; SCHLOEDER, BRIAN; KURTTI, TIMOTHY J.; MUNDERLOH, ULRIKE G.

    2009-01-01

    Dermacentor albipictus (Packard) is a North American tick that feeds on cervids and livestock. It is a suspected vector of anaplasmosis in cattle, but its microbial flora and vector potential remain underevaluated. We screened D. albipictus ticks collected from Minnesota white-tailed deer (Odocoileus virginianus) for bacteria of the genera Anaplasma, Ehrlichia, Francisella, and Rickettsia using polymerase chain reaction (PCR) gene amplification and sequence analyses. We detected Anaplasma phagocytophilum and Francisella-like endosymbionts (FLEs) in nymphal and adult ticks of both sexes at 45 and 94% prevalences, respectively. The A. phagocytophilum and FLEs were transovarially transmitted to F1 larvae by individual ticks at efficiencies of 10–40 and 95–100%, respectively. The FLEs were transovarially transmitted to F2 larvae obtained as progeny of adults from F1 larval ticks reared to maturity on a calf, but A. phagocytophilum were not. Based on PCR and tissue culture inoculation assays, A. phagocytophilum and FLEs were not transmitted to the calf. The amplified FLE 16S rRNA gene sequences were identical to that of an FLE detected in a D. albipictus from Texas, whereas those of the A. phagocytophilum were nearly identical to those of probable human-nonpathogenic A. phagocytophilum WI-1 and WI-2 variants detected in white-tailed deer from central Wisconsin. However, the D. albipictus A. phagocytophilum sequences differed from that of the nonpathogenic A. phagocytophilum variant-1 associated with Ixodes scapularis ticks and white-tailed deer as well as that of the human-pathogenic A. phagocytophilum ha variant associated with I. scapularis and the white-footed mouse, Peromyscus leucopus. The transovarial transmission of A. phagocytophilum variants in Dermacentor ticks suggests that maintenance of A. phagocytophilum in nature may not be solely dependent on horizontal transmission. PMID:19496436

  3. Restriction of Francisella novicida Genetic Diversity during Infection of the Vector Midgut

    PubMed Central

    Reif, Kathryn E.; Palmer, Guy H.; Crowder, David W.; Ueti, Massaro W.; Noh, Susan M.

    2014-01-01

    The genetic diversity of pathogens, and interactions between genotypes, can strongly influence pathogen phenotypes such as transmissibility and virulence. For vector-borne pathogens, both mammalian hosts and arthropod vectors may limit pathogen genotypic diversity (number of unique genotypes circulating in an area) by preventing infection or transmission of particular genotypes. Mammalian hosts often act as “ecological filters” for pathogen diversity, where novel variants are frequently eliminated because of stochastic events or fitness costs. However, whether vectors can serve a similar role in limiting pathogen diversity is less clear. Here we show using Francisella novicida and a natural tick vector of Francisella spp. (Dermacentor andersoni), that the tick vector acted as a stronger ecological filter for pathogen diversity compared to the mammalian host. When both mice and ticks were exposed to mixtures of F. novicida genotypes, significantly fewer genotypes co-colonized ticks compared to mice. In both ticks and mice, increased genotypic diversity negatively affected the recovery of available genotypes. Competition among genotypes contributed to the reduction of diversity during infection of the tick midgut, as genotypes not recovered from tick midguts during mixed genotype infections were recovered from tick midguts during individual genotype infection. Mediated by stochastic and selective forces, pathogen genotype diversity was markedly reduced in the tick. We incorporated our experimental results into a model to demonstrate how vector population dynamics, especially vector-to-host ratio, strongly affected pathogen genotypic diversity in a population over time. Understanding pathogen genotypic population dynamics will aid in identification of the variables that most strongly affect pathogen transmission and disease ecology. PMID:25392914

  4. Aquatic Francisella-like bacterium associated with mortality of intensively cultured hybrid striped bass Morone chrysops x M. saxatilis.

    PubMed

    Ostland, V E; Stannard, J A; Creek, J J; Hedrick, R P; Ferguson, H W; Carlberg, J M; Westerman, M E

    2006-10-17

    The present study identifies an emerging disease associated with an aquatic Francisella-like bacterium that can cause mortality in hybrid striped bass Morone chrysops x M. saxatilis reared intensively in freshwater. Clinically affected fish were lethargic, had scattered haemorrhagic cutaneous lesions and diffuse gill pallor. The head kidney and spleen were markedly swollen and contained numerous interstitial granulomas; histological examination revealed small, pleomorphic Gram-negative coccobacilli within vacuolated cells. The bacterium could not be cultured from head kidney homogenates either with standard or enriched microbiological media or following inoculation of a Chinook salmon embryo (CHSE)-214 cell line. No amplification product was obtained from head kidney DNA by polymerase chain reaction (PCR) assay using Piscirickettsia salmonis-specific primers. PCR analysis of infected head kidney homogenate with primers designed for the eubacterial 16S rRNA produced a single amplicon. Phylogenetic analysis of this DNA sequence demonstrated that the sequence aligned most closely with members of the genus Francisella, identified from tilapia Oreochromis spp. in Taiwan and an aquatic Francisella species that was recently isolated from the three-line grunt Parapristipoma trilineatum in Japan. This Francisella-like disease was transmitted to naive hybrid striped bass fingerlings by intraperitoneal injection of tissue homogenates prepared from a natural outbreak. All fish developed gross and histological lesions identical to those from natural outbreaks. Intracellular Gram-negative bacteria were observed within the cytoplasm of cells (presumably macrophages) within the granulomas, but bacteria were not recovered. The 16S DNA sequence of the bacterium obtained from tissues of experimentally infected fish was identical to that obtained from the fish used as infected donor tissue. PMID:17140136

  5. Bis-indolic compounds as potential new therapeutic alternatives for tularaemia.

    PubMed

    Caspar, Yvan; Sutera, Vivien; Boisset, Sandrine; Denis, Jean-Noël; Maurin, Max

    2014-01-01

    Francisella tularensis is the etiological agent of tularaemia and a CDC class A biological threat agent. Few antibiotic classes are currently useful in treating tularaemia, including the aminoglycosides gentamicin and streptomycin, fluoroquinolones, and tetracyclines. However, treatment failures and relapses remain frequent and F. tularensis strains resistant to antibiotics have been easily selected in vitro. In this study, we evaluated the activity of new synthetic bis-indole derivatives against this pathogen. Minimum inhibitory concentrations (MICs) of four compounds (dcm01 to dcm04) were determined for the reference strains F. tularensis subsp. holarctica LVS NCTC10857, F. tularensis subsp. novicida CIP56.12 and F. philomiragia ATCC25015, and for 41 clinical strains of F. tularensis subsp. holarctica isolated in France. Minimal bactericidal concentrations (MBCs) were determined for the dcm02 and dcm04 compounds for the LVS and two clinical strains. Killing curves were also determined for the same three strains exposed to dcm04. All tested bis-indole compounds were bacteriostatic against F. tularensis subsp. holarctica strains, with a MIC90 of 8 μg/mL for dcm01, dcm02, and dcm03, and 2 μg/mL for dcm04. Only one strain was resistant to both dcm01 and dcm03, with MICs > 32 μg/mL. In contrast, F. tularensis subsp. novicida was resistant to all derivatives and F. philomiragia was only susceptible to dcm02 and dcm04, with MICs of 16 and 4 μg/mL, respectively. MBC and killing curve experiments revealed significant bactericidal activity (i.e., 3-log reduction of the bacterial inoculum) of the dcm02 and dcm04 compounds only for the LVS strain. In conclusion, we have identified novel synthetic bis-indole compounds that are active against F. tularensis subsp. holarctica. They may be drug candidates for the development of new therapeutic alternatives for tularaemia treatment. Their further characterization is needed, especially identification of their bacterial targets.

  6. A Francisella Virulence Factor Catalyzes an Essential Reaction of Biotin Synthesis

    PubMed Central

    Feng, Youjun; Napier, Brooke A.; Manandhar, Miglena; Henke, Sarah K; Weiss, David S.; Cronan, John E.

    2014-01-01

    Summary We recently identified a gene (FTN_0818) required for Francisella virulence that seemed likely involved in biotin metabolism. However, the molecular function of this virulence determinant was unclear. Here we show that this protein named BioJ is the enzyme of the biotin biosynthesis pathway that determines the chain length of the biotin valeryl side chain. Expression of bioJ allows growth of an E. coli bioH strain on biotin-free medium, indicating functional equivalence of BioJ to the paradigm pimeloyl-ACP methyl ester carboxyl-esterase, BioH. BioJ was purified to homogeneity, shown to be monomeric and capable of hydrolysis of its physiological substrate methyl pimeloyl-ACP to pimeloyl-ACP, the precursor required to begin formation of the fused heterocyclic rings of biotin. Phylogenetic analyses confirmed that distinct from BioH, BioJ represents a novel sub-clade of the α/β-hydrolase family. Structure-guided mapping combined with site-directed mutagenesis revealed that the BioJ catalytic triad consists of Ser151, Asp248 and His278, all of which are essential for activity and virulence. The biotin synthesis pathway was reconstituted in vitro and the physiological role of BioJ directly assayed. To the best of our knowledge, these data represent further evidence linking biotin synthesis to bacterial virulence. PMID:24313380

  7. Improved Broth Microdilution Method for Antimicrobial Susceptibility Testing of Francisella Noatunensis Orientalis.

    PubMed

    Soto, Esteban; Halliday-Simmonds, Iona; Francis, Stewart; Fraites, Trellor; Martínez-López, Beatriz; Wiles, Judy; Hawke, John P; Endris, Richard D

    2016-09-01

    In this project we optimized a minimal inhibitory concentration testing protocol for Francisella noatunensis orientalis. Thirty-three F. noatunensis orientalis isolates recovered from different fish species and locations were tested, and Escherichia coli ATCC 25922 was used as a quality control reference strain. A modified cation-adjusted Mueller Hinton broth supplemented with 2% IsoVitalex and 0.1% glucose (MMH) was tested at a pH of 6.4 ± 0.1, 7.1 ± 0.1, and 7.3 ± 0.1. Growth curves generated for F. noatunensis orientalis indicated that MMH at a pH of 6.4 ± 0.1 provided optimal growth. There were no significant differences in the growth curves obtained from isolates recovered from different fish species or from fresh or marine water. The pH of 6.4 ± 0.1 in the MMH media interfered with the inhibitory properties of the potentiated sulfonamides (ormetoprim-sulfadimethoxine and trimethoprim-sulfamethoxazole) when using the E. coli ATCC reference strain. Minimal inhibitory concentrations of eight antimicrobials (gentamicin, enrofloxacin, ampicillin, oxytetracycline, erythromycin, florfenicol, flumequine, and oxolinic acid) were similar for all F. noatunensis orientalis isolates. The in vitro susceptibility data provided here can provide a baseline for monitoring the development of antimicrobial resistance among F. noatunensis orientalis isolates, as well as provide valuable data in the development of potential therapeutics. Received October 27, 2015; accepted April 13, 2016. PMID:27484609

  8. Roles of inflammatory caspases during processing of zebrafish interleukin-1β in Francisella noatunensis infection

    USGS Publications Warehouse

    Vojtech, Lucia N.; Scharping, Nichole; Woodson, James C.; Hansen, John D.

    2012-01-01

    The interleukin-1 family of cytokines are essential for the control of pathogenic microbes but are also responsible for devastating autoimmune pathologies. Consequently, tight regulation of inflammatory processes is essential for maintaining homeostasis. In mammals, interleukin-1 beta (IL-1β) is primarily regulated at two levels, transcription and processing. The main pathway for processing IL-1β is the inflammasome, a multiprotein complex that forms in the cytosol and which results in the activation of inflammatory caspase (caspase 1) and the subsequent cleavage and secretion of active IL-1β. Although zebrafish encode orthologs of IL-1β and inflammatory caspases, the processing of IL-1β by activated caspase(s) has never been examined. Here, we demonstrate that in response to infection with the fish-specific bacterial pathogen Francisella noatunensis, primary leukocytes from adult zebrafish display caspase-1-like activity that results in IL-1β processing. Addition of caspase 1 or pancaspase inhibitors considerably abrogates IL-1β processing. As in mammals, this processing event is concurrent with the secretion of cleaved IL-1β into the culture medium. Furthermore, two putative zebrafish inflammatory caspase orthologs, caspase A and caspase B, are both able to cleave IL-1β, but with different specificities. These results represent the first demonstration of processing and secretion of zebrafish IL-1β in response to a pathogen, contributing to our understanding of the evolutionary processes governing the regulation of inflammation.                   

  9. A Francisella virulence factor catalyses an essential reaction of biotin synthesis.

    PubMed

    Feng, Youjun; Napier, Brooke A; Manandhar, Miglena; Henke, Sarah K; Weiss, David S; Cronan, John E

    2014-01-01

    We recently identified a gene (FTN_0818) required for Francisella virulence that seemed likely involved in biotin metabolism. However, the molecular function of this virulence determinant was unclear. Here we show that this protein named BioJ is the enzyme of the biotin biosynthesis pathway that determines the chain length of the biotin valeryl side-chain. Expression of bioJ allows growth of an Escherichia coli bioH strain on biotin-free medium, indicating functional equivalence of BioJ to the paradigm pimeloyl-ACP methyl ester carboxyl-esterase, BioH. BioJ was purified to homogeneity, shown to be monomeric and capable of hydrolysis of its physiological substrate methyl pimeloyl-ACP to pimeloyl-ACP, the precursor required to begin formation of the fused heterocyclic rings of biotin. Phylogenetic analyses confirmed that distinct from BioH, BioJ represents a novel subclade of the α/β-hydrolase family. Structure-guided mapping combined with site-directed mutagenesis revealed that the BioJ catalytic triad consists of Ser151, Asp248 and His278, all of which are essential for activity and virulence. The biotin synthesis pathway was reconstituted reaction in vitro and the physiological role of BioJ directly assayed. To the best of our knowledge, these data represent further evidence linking biotin synthesis to bacterial virulence. PMID:24313380

  10. Agent Orange

    MedlinePlus

    ... Index Agent Orange Agent Orange Home Facts about Herbicides Veterans' Diseases Birth Defects Benefits Exposure Locations Provider ... millions of gallons of Agent Orange and other herbicides on trees and vegetation during the Vietnam War. ...

  11. Efficacy of florfenicol for control of mortality associated with Francisella noatunensis subsp. orientalis in Nile tilapia, Oreochromis niloticus (L.).

    PubMed

    Soto, E; Kidd, S; Gaunt, P S; Endris, R

    2013-04-01

    Francisella noatunensis subsp. orientalis (Fno) (syn. F. asiatica) is an emergent Gram-negative facultative intracellular bacterium. Although it is considered one of the most pathogenic bacteria in fish, there are no commercially available treatments or vaccines. The objective of this project was to determine the most efficacious concentration of florfenicol (FFC) [10, 15 or 20 mg FFC kg(-1) body weight (bw) per days for 10 days] administered in feed to control experimentally induced infections of Fno in Nile tilapia, Oreochromis niloticus (L.), reared in a recirculating aquaculture system. The cumulative mortality of fish that received 0, 10, 15 or 20 mg FFC kg(-1)  bw per day was 60, 37, 14 and 16%, respectively. Francisella noatunensis subsp. orientalis genome equivalents were detected in water from all challenged groups with slight reduction in the concentration in the florfenicol-treated groups 4 days after treatment. The mean LOG of CFU Fno mg(-1) spleen was 3-5 and was present in all challenged groups at necropsy 11 days after treatment (21 days after challenge). Results show that florfenicol administered at doses of 15 and 20 mg FFC kg(-1)  bw per days for 10 days significantly reduced mortality associated with francisellosis in Nile tilapia. PMID:23134104

  12. Simultaneous, specific and real-time detection of biothreat and frequently encountered food-borne pathogens

    PubMed Central

    Woubit, Abdela Salah; Yehualaeshet, Teshome; Habtemariam, Tsegaye; Samuel, Temesgen

    2012-01-01

    The bacterial genera Escherichia, Salmonella, Shigella, Vibrio, Yersinia and Francisella include important food safety and biothreat agents causing food-related and other human illnesses worldwide. We aimed to develop rapid methods with the capability to simultaneously and differentially detect all six pathogens in one run. Our initial experiments to use previously reported sets of primers revealed non-specificity of some of the sequences when tested against a broader array of pathogens, or proved not optimal for simultaneous detection parameters. By extensive mining of the whole genome and protein databases of diverse closely and distantly related bacterial species and strains, we have identified unique genome regions, which we utilized to develop a detection platform. Twelve of the specific genomic targets we have identified to design the primers in F. tularensis ssp. tularensis, F. tularensis ssp. novicida, S. dysentriae, S. typhimurium, V. cholera, Y. pestis, and Y. pseudotuberculosis contained either hypothetical or putative proteins, the functions of which have not been clearly defined. Corresponding primer sets were designed from the target regions for use in real-time PCR assays to detect specific biothreat pathogens at species or strain levels. The primer sets were first tested by in-silico PCR against whole genome sequences of different species, sub-species, or strains and then by in vitro PCR against genomic DNA preparations from 23 strains representing six biothreat agents (E.coli O157:H7 strain EDL 933, Shigella dysentriae, Salmonella typhi, Francisella tularensis ssp. tularensis, Vibrio cholera, and Yersinia pestis) and six foodborne pathogens (Salmonella typhimurium, Salmonella saintpaul, Shigella sonnei, Francisella novicida, Vibrio parahemolytica and Yersinia pseudotuberculosis). Each pathogen was specifically identifiable at the genus and species levels. Sensitivity assays performed using purified DNA showed the lowest detection limit of 640 fg

  13. Louse-borne bacterial pathogens in lice (Phthiraptera) of rodents and cattle from Egypt.

    PubMed

    Reeves, Will K; Szumlas, Daniel E; Moriarity, John R; Loftis, Amanda D; Abbassy, Magda M; Helmy, Ibrahim M; Dasch, Gregory A

    2006-04-01

    We collected 1,023 lice, representing 5 species, from rats and domestic cattle throughout 13 governorates in Egypt and tested these lice for Anaplasma marginale, Bartonella spp., Brucella spp., Borrelia recurrentis, Coxiella burnetii, Francisella tularensis, and Rickettsia spp. by PCR amplification and sequencing. Five different louse-borne bacterial agents were detected in lice from rodents or cattle, including "Bartonella rattimassiliensis", "B. phoceensis", and Bartonella sp. near Bartonella tribocorum, Coxiella burnetii, and Rickettsia typhi. More lice from governorates bordering the Mediterranean and Red Seas contained pathogens. Our data indicate that lice of urban and domestic animals harbor pathogenic or potentially pathogenic bacterial agents throughout Egypt.

  14. Biological Agents

    MedlinePlus

    ... to Z Index Contact Us FAQs What's New Biological Agents This page requires that javascript be enabled ... and Health Topics A-Z Index What's New Biological agents include bacteria, viruses, fungi, other microorganisms and ...

  15. Current status of vaccine development for tularemia preparedness

    PubMed Central

    Hong, Kee-Jong; Park, Pil-Gu; Seo, Sang-Hwan; Rhie, Gi-eun

    2013-01-01

    Tularemia is a high-risk infectious disease caused by Gram-negative bacterium Francisella tularensis. Due to its high fatality at very low colony-forming units (less than 10), F. tularensis is considered as a powerful potential bioterrorism agent. Vaccine could be the most efficient way to prevent the citizen from infection of F. tularensis when the bioterrorism happens, but officially approved vaccine with both efficacy and safety is not developed yet. Research for the development of tularemia vaccine has been focusing on the live attenuated vaccine strain (LVS) for long history, still there are no LVS confirmed for the safety which should be an essential factor for general vaccination program. Furthermore the LVS did not show protection efficacy against high-risk subspecies tularensis (type A) as high as the level against subspecies holarctica (type B) in human. Though the subunit or recombinant vaccine candidates have been considered for better safety, any results did not show better prevention efficacy than the LVS candidate against F. tularensis infection. Currently there are some more trials to develop vaccine using mutant strains or nonpathogenic F. novicida strain, but it did not reveal effective candidates overwhelming the LVS either. Difference in the protection efficacy of LVS against type A strain in human and the low level protection of many subunit or recombinant vaccine candidates lead the scientists to consider the live vaccine development using type A strain could be ultimate answer for the tularemia vaccine development. PMID:23596588

  16. Detection of Multiple Waterborne Pathogens Using Microsequencing Arrays

    EPA Science Inventory

    Aims: A microarray was developed to simultaneously detect Cryptosporidium parvum, Cryptosporidium hominis, Enterococcus faecium, Bacillus anthracis and Francisella tularensis in water. Methods and Results: A DNA microarray was designed to contain probes that specifically dete...

  17. 42 CFR 73.9 - Responsible Official.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... neurotoxins, Brucella melitensis, Francisella tularensis, Ebola viruses, Hendra virus, Marburg virus, Lassa fever virus, Nipah virus, Rift Valley fever virus, South American Haemorrhagic Fever viruses (Junin, Machupo, Sabia, Flexal, Guanarito), Variola major virus (Smallpox virus), Variola minor...

  18. 42 CFR 73.9 - Responsible Official.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... neurotoxins, Brucella melitensis, Francisella tularensis, Ebola viruses, Hendra virus, Marburg virus, Lassa fever virus, Nipah virus, Rift Valley fever virus, South American Haemorrhagic Fever viruses (Junin, Machupo, Sabia, Flexal, Guanarito), Variola major virus (Smallpox virus), Variola minor...

  19. 42 CFR 73.9 - Responsible Official.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... neurotoxins, Brucella melitensis, Francisella tularensis, Ebola viruses, Hendra virus, Marburg virus, Lassa fever virus, Nipah virus, Rift Valley fever virus, South American Haemorrhagic Fever viruses (Junin, Machupo, Sabia, Flexal, Guanarito), Variola major virus (Smallpox virus), Variola minor...

  20. Serologic Survey of Snowshoe Hares (Lepus americanus) in the Greater Yellowstone Area for Brucellosis, Tularemia, and Snowshoe Hare Virus.

    PubMed

    Tyers, Dan; Zimmer, Jeremy; Lewandowski, Kristen; Hennager, Steve; Young, John; Pappert, Ryan; Panella, Amanda; Kosoy, Olga

    2015-07-01

    We examined sera from snowshoe hares (Lepus americanus) livetrapped in the northern Greater Yellowstone Area (GYA), US, for antibodies to Brucella abortus, Francisella tularensis, and snowshoe hare virus (SSHV). Zero of 90, 0 of 67, and 40 of 100 samples were antibody positive for B. abortus, F. tularensis, and SSHV, respectively. Hares were trapped from 2009 to 2012, and of the six animals that were captured twice with at least 1 yr between captures, four developed antibody to SSHV, indicating active exposure to the agent. These findings suggest snowshoe hares in the GYA do not play a significant role as a reservoir of B. abortus, but do maintain the zoonotic, encephalitic SSHV in the population.

  1. Bacterial biofilms of importance to medicine and bioterrorism: proteomic techniques to identify novel vaccine components and drug targets.

    PubMed

    Hassett, Daniel J; Limbach, Patrick A; Hennigan, Robert F; Klose, Karl E; Hancock, Robert E W; Platt, Mark D; Hunt, Donald F

    2003-12-01

    Biofilms are highly ordered microbial communities enmeshed in a carefully sculpted matrix designed for survival of organisms either in multi- or mono-genus/species in a specific microniche. In human disease, biofilm infections are some of the most recalcitrant to treat. Even with rigorous antibiotic regimens, some biofilms, such as those within the thick airway mucus of cystic fibrosis (CF) patients, persist throughout the course of the disease process. In this editorial, discussion will cover the utility of using advanced proteomic techniques to help identify potential weaknesses in the already impressive defensive armamentarium of biofilm bacteria. Two biofilm systems will be discussed herein, one of which is that of Pseudomonas aeruginosa biofilms within CF airway biofilms. The other is referred to as persistent 'bioterrorist agent biofilms' in which Francisella tularensis can grow on surfaces where environmental amoeba can phagocytose them, allowing for growth of F. tularensis within the amoebae. PMID:14640945

  2. Serologic Survey of Snowshoe Hares (Lepus americanus) in the Greater Yellowstone Area for Brucellosis, Tularemia, and Snowshoe Hare Virus.

    PubMed

    Tyers, Dan; Zimmer, Jeremy; Lewandowski, Kristen; Hennager, Steve; Young, John; Pappert, Ryan; Panella, Amanda; Kosoy, Olga

    2015-07-01

    We examined sera from snowshoe hares (Lepus americanus) livetrapped in the northern Greater Yellowstone Area (GYA), US, for antibodies to Brucella abortus, Francisella tularensis, and snowshoe hare virus (SSHV). Zero of 90, 0 of 67, and 40 of 100 samples were antibody positive for B. abortus, F. tularensis, and SSHV, respectively. Hares were trapped from 2009 to 2012, and of the six animals that were captured twice with at least 1 yr between captures, four developed antibody to SSHV, indicating active exposure to the agent. These findings suggest snowshoe hares in the GYA do not play a significant role as a reservoir of B. abortus, but do maintain the zoonotic, encephalitic SSHV in the population. PMID:26161724

  3. Tularaemia: a challenging zoonosis.

    PubMed

    Carvalho, C L; Lopes de Carvalho, I; Zé-Zé, L; Núncio, M S; Duarte, E L

    2014-03-01

    In recent years, several emerging zoonotic vector-borne infections with potential impact on human health have been identified in Europe, including tularaemia, caused by Francisella tularensis. This remarkable pathogen, one of the most virulent microorganisms currently known, has been detected in increasingly new settings and in a wide range of wild species, including lagomorphs, rodents, carnivores, fish and invertebrate arthropods. Also, a renewed concern has arisen with regard to F. tularensis: its potential use by bioterrorists. Based on the information published concerning the latest outbreaks, the aim of this paper is to review the main features of the agent, its biology, immunology and epidemiology. Moreover, special focus will be given to zoonotic aspects of the disease, as tularaemia outbreaks in human populations have been frequently associated with disease in animals.

  4. Sunscreening Agents

    PubMed Central

    Martis, Jacintha; Shobha, V; Sham Shinde, Rutuja; Bangera, Sudhakar; Krishnankutty, Binny; Bellary, Shantala; Varughese, Sunoj; Rao, Prabhakar; Naveen Kumar, B.R.

    2013-01-01

    The increasing incidence of skin cancers and photodamaging effects caused by ultraviolet radiation has increased the use of sunscreening agents, which have shown beneficial effects in reducing the symptoms and reoccurrence of these problems. Many sunscreen compounds are in use, but their safety and efficacy are still in question. Efficacy is measured through indices, such as sun protection factor, persistent pigment darkening protection factor, and COLIPA guidelines. The United States Food and Drug Administration and European Union have incorporated changes in their guidelines to help consumers select products based on their sun protection factor and protection against ultraviolet radiation, whereas the Indian regulatory agency has not yet issued any special guidance on sunscreening agents, as they are classified under cosmetics. In this article, the authors discuss the pharmacological actions of sunscreening agents as well as the available formulations, their benefits, possible health hazards, safety, challenges, and proper application technique. New technologies and scope for the development of sunscreening agents are also discussed as well as the role of the physician in patient education about the use of these agents. PMID:23320122

  5. Antiparasitic agents.

    PubMed

    Rosenblatt, J E

    1992-03-01

    In recent years, introduction of new and more effective agents has improved the overall therapy for parasitic infections. This field, however, is still plagued by numerous problems, including the development of resistance to antimicrobial agents (especially with malaria), unavailability of agents in the United States or lack of approval by the Food and Drug Administration, and major toxicities or lack of experience in pregnant women and children, which limits use in these groups of patients. Widespread resistance of Plasmodium falciparum to chloroquine and other agents has complicated the treatment and prophylaxis of this type of malaria. A combination of quinine and Fansidar is usually effective oral therapy for falciparum malaria; quinidine may be administered if intravenous therapy is needed. Mefloquine, which is currently recommended for prophylaxis against chloroquine-resistant P. falciparum, is also effective for single-dose oral treatment, although this regimen has not yet been approved by the Food and Drug Administration. Metronidazole has been widely used for treatment of gastroenteritis due to Entamoeba histolytica and Giardia lamblia (not approved by the Food and Drug Administration for the latter) and is considered safe and effective. A new macrolide, azithromycin, has been reported to be effective for cryptosporidiosis in experimental animals; currently, no effective therapy is available for human infections. Combinations of sulfonamides with other antifolates, trimethoprim or pyrimethamine, are recommended therapy for Pneumocystis carinii pneumonia or toxoplasmosis, respectively. Therapies for the various types of leishmaniasis and trypanosomiasis are complex, often toxic, and often of limited efficacy. The benzimidazoles are effective for roundworm infections, although thiabendazole has severe toxic effects. The recent introduction of ivermectin has revolutionized the treatment and control of onchocerciasis. Another relatively new agent, praziquantel

  6. Elevated AIM2-mediated pyroptosis triggered by hypercytotoxic Francisella mutant strains is attributed to increased intracellular bacteriolysis

    PubMed Central

    Peng, Kaitian; Broz, Petr; Jones, Jonathan; Joubert, Lydia-Marie; Monack, Denise

    2011-01-01

    Intracellular bacterial pathogens Francisella novicida and the Live Vaccine Strain (LVS) are recognized in the macrophage cytosol by the AIM2 inflammasome, which leads to the activation of caspase-1 and the processing and secretion of active IL-1β, IL-18 and pyroptosis. Previous studies have reported that F. novicida and LVS mutants in specific genes (e.g., FTT0584, mviN and ripA) induce elevated inflammasome activation and hypercytotoxicity in host cells, leading to the proposal that F. novicida and LVS may have proteins that actively modulate inflammasome activation. However, there has been no direct evidence of such inflammasome evasion mechanisms. Here, we demonstrate for the first time that the above mutants, along with a wide range of F. novicida hypercytotoxic mutants that are deficient for membrane-associated proteins (ΔFTT0584, ΔmviN, ΔripA, ΔfopA and ΔFTN1217) or deficient for genes involved in O-antigen or LPS biosynthesis (ΔwbtA and ΔlpxH) lyse more intracellularly, thus activating increased levels of AIM2-dependent pyroptosis and other innate immune signaling pathways. This suggests that an inflammasome-specific evasion mechanism may not be present in F. novicida and LVS. Furthermore, future studies may need to consider increased bacterial lysis as a possible cause of elevated stimulation of multiple innate immune pathways when the protein composition or surface carbohydrates of the bacterial membrane is altered. PMID:21883803

  7. Antidiabetic Agents.

    ERIC Educational Resources Information Center

    Plummer, Nancy; Michael, Nancy, Ed.

    This module on antidiabetic agents is intended for use in inservice or continuing education programs for persons who administer medications in long-term care facilities. Instructor information, including teaching suggestions, and a listing of recommended audiovisual materials and their sources appear first. The module goal and objectives are then…

  8. Tularemia in Alaska, 1938 - 2010

    PubMed Central

    2011-01-01

    Tularemia is a serious, potentially life threatening zoonotic disease. The causative agent, Francisella tularensis, is ubiquitous in the Northern hemisphere, including Alaska, where it was first isolated from a rabbit tick (Haemophysalis leporis-palustris) in 1938. Since then, F. tularensis has been isolated from wildlife and humans throughout the state. Serologic surveys have found measurable antibodies with prevalence ranging from < 1% to 50% and 4% to 18% for selected populations of wildlife species and humans, respectively. We reviewed and summarized known literature on tularemia surveillance in Alaska and summarized the epidemiological information on human cases reported to public health officials. Additionally, available F. tularensis isolates from Alaska were analyzed using canonical SNPs and a multi-locus variable-number tandem repeats (VNTR) analysis (MLVA) system. The results show that both F. t. tularensis and F. t. holarctica are present in Alaska and that subtype A.I, the most virulent type, is responsible for most recently reported human clinical cases in the state. PMID:22099502

  9. Tularemia in Alaska, 1938 - 2010.

    PubMed

    Hansen, Cristina M; Vogler, Amy J; Keim, Paul; Wagner, David M; Hueffer, Karsten

    2011-01-01

    Tularemia is a serious, potentially life threatening zoonotic disease. The causative agent, Francisella tularensis, is ubiquitous in the Northern hemisphere, including Alaska, where it was first isolated from a rabbit tick (Haemophysalis leporis-palustris) in 1938. Since then, F. tularensis has been isolated from wildlife and humans throughout the state. Serologic surveys have found measurable antibodies with prevalence ranging from < 1% to 50% and 4% to 18% for selected populations of wildlife species and humans, respectively. We reviewed and summarized known literature on tularemia surveillance in Alaska and summarized the epidemiological information on human cases reported to public health officials. Additionally, available F. tularensis isolates from Alaska were analyzed using canonical SNPs and a multi-locus variable-number tandem repeats (VNTR) analysis (MLVA) system. The results show that both F. t. tularensis and F. t. holarctica are present in Alaska and that subtype A.I, the most virulent type, is responsible for most recently reported human clinical cases in the state. PMID:22099502

  10. Type I Interferon signaling constrains IL-17A/F secretion by γδ T cells during bacterial infections

    PubMed Central

    Henry, Thomas; Kirimanjeswara, Girish S.; Ruby, Thomas; Jones, Jonathan W.; Peng, Kaitian; Perret, Magali; Ho, Lena; Sauer, John-Demian; Iwakura, Yoichiro; Metzger, Dennis W.; Monack, Denise M.

    2010-01-01

    Recognition of intracellular bacteria by macrophages leads to secretion of type I Interferons. However, the role of type I IFN during bacterial infection is still poorly understood. Francisella tularensis, the causative agent of tularemia, is a pathogenic bacterium that replicates in the cytosol of macrophages leading to secretion of type I IFN. Here, we investigated the role of type I IFN in a mouse model of tularemia. Mice deficient for type I IFN receptor (IFNAR1−/−) are more resistant to intradermal infection with F. tularensis subspecies novicida (F. novicida). Increased resistance to infection was associated with a specific increase in IL-17A/F and a corresponding expansion of an IL-17A+ γδ T cell population, indicating that type I IFN negatively regulate the number of IL-17A+ γδ T cells during infection. Furthermore, IL-17A-deficient mice contained fewer neutrophils compared to WT mice upon infection, indicating that IL-17A contributes to neutrophil expansion during F. novicida infection. Accordingly, an increase in IL-17A in IFNAR1−/− mice correlated with an increase in splenic neutrophil numbers. Similar results were obtained in a mouse model of pneumonic tularemia using the highly virulent Francisella tularensis subspecies tularensis SchuS4 strain and in a mouse model of systemic Listeria monocytogenes infection. Our results indicate that the type I IFN-mediated negative regulation of IL-17A+ γδ T cell expansion is conserved during bacterial infections. We propose that this newly described activity of type I IFN signaling might participate in the resistance of the IFNAR1−/− mice to infection with F. novicida and other intracellular bacteria. PMID:20176744

  11. cGAS and Ifi204 Cooperate To Produce Type I IFNs in Response to Francisella Infection

    PubMed Central

    Storek, Kelly M.; Gertsvolf, Nina A.; Ohlson, Maikke B.

    2015-01-01

    Type I IFN production is an important host immune response against viral and bacterial infections. However, little is known about the ligands and corresponding host receptors that trigger type I IFN production during bacterial infections. We used a model intracellular pathogen, Francisella novicida, to begin characterizing the type I IFN response to bacterial pathogens. F. novicida replicates in the cytosol of host cells and elicits a robust type I IFN response that is largely TLR independent, but is dependent on the adapter molecule STING, suggesting that the type I IFN stimulus during F. novicida infection is cytosolic. In this study, we report that the cytosolic DNA sensors, cyclic GMP-AMP synthase (cGAS) and Ifi204, are both required for the STING-dependent type I IFN response to F. novicida infection in both primary and immortalized murine macrophages. We created cGAS, Ifi204, and Sting functional knockouts in RAW264.7 macrophages and demonstrated that cGAS and Ifi204 cooperate to sense dsDNA and activate the STING-dependent type I IFN pathway. In addition, we show that dsDNA from F. novicida is an important type I IFN stimulating ligand. One outcome of cGAS–STING signaling is the activation of the absent in melanoma 2 inflammasome in response to F. novicida infection. Whereas the absent in melanoma 2 inflammasome is beneficial to the host during F. novicida infection, type I IFN signaling by STING and IFN regulatory factor 3 is detrimental to the host during F. novicida infection. Collectively, our studies indicate that cGAS and Ifi204 cooperate to sense cytosolic dsDNA and F. novicida infection to produce a strong type I IFN response. PMID:25710914

  12. KGB agents

    NASA Astrophysics Data System (ADS)

    Gaina, Alex

    A short story is reported in which the activity of Communist Party of the USSR and secret KGB agents, which were payed by the State, in view of controlling of the conscience of population. The story reffers to the Physics Department of the Moscow University, Planing Institute of the Gosplan of Moldavian S.S.R. and Chishinau Technical University (actually: Technical University of Moldova), where the author has worked during Soviet times. Almost every 6-th citizen in the USSR was engaged in this activity, while actually the former communists rule in the Republic of Moldova.

  13. Filling agents.

    PubMed

    Glavas, Ioannis P

    2005-06-01

    Injectable fillers have become an important component of minimally invasive facial rejuvenation modalities. Their ease of use, effectiveness, low morbidity, and fast results with minimal downtime are factors that have made them popular among patients. Soft tissue augmentation has evolved to a unique combination of medicine and art. A wide selection of available agents and new products, each one with unique properties, may be used alone or in combination. The physician acquires the tools to rebalance facial characteristics not only by filling wrinkles but also by having the ability to shape the face and restore bony contours and lines. Careful selection of candidates, realistic expectations, and an understanding of the limitations of fillers are crucial for a successful result.

  14. Health care agents

    MedlinePlus

    Durable power of attorney for health care; Health care proxy; End-of-life - health care agent; Life support treatment - ... Respirator - health care agent; Ventilator - health care agent; Power of attorney - health care agent; POA - health care ...

  15. Detecting agents.

    PubMed Central

    Johnson, Susan C

    2003-01-01

    This paper reviews a recent set of behavioural studies that examine the scope and nature of the representational system underlying theory-of-mind development. Studies with typically developing infants, adults and children with autism all converge on the claim that there is a specialized input system that uses not only morphological cues, but also behavioural cues to categorize novel objects as agents. Evidence is reviewed in which 12- to 15-month-old infants treat certain non-human objects as if they have perceptual/attentional abilities, communicative abilities and goal-directed behaviour. They will follow the attentional orientation of an amorphously shaped novel object if it interacts contingently with them or with another person. They also seem to use a novel object's environmentally directed behaviour to determine its perceptual/attentional orientation and object-oriented goals. Results from adults and children with autism are strikingly similar, despite adults' contradictory beliefs about the objects in question and the failure of children with autism to ultimately develop more advanced theory-of-mind reasoning. The implications for a general theory-of-mind development are discussed. PMID:12689380

  16. Being prepared: bioterrorism and mass prophylaxis: part II.

    PubMed

    Weant, Kyle A; Bailey, Abby M; Fleishaker, Elise L; Justice, Stephanie B

    2014-01-01

    Although several biological agents have been recognized as presenting a significant threat to public health if used in a bioterrorist attack, those that are of greatest importance are known as the Category A agents: Bacillus anthracis (anthrax); variola major (smallpox); Yersinia pestis (plague); Francisella tularensis (tularemia); ribonucleic acid viruses (hemorrhagic fevers); and Clostridium botulinum (botulism toxin). In the previous issue, Part I of this review focused on the clinical presentation and treatment of anthrax, plague, and tularemia. In this second part of this 2-part review of these agents, the focus is on the clinical presentation and treatment of smallpox, viral hemorrhagic fevers, and botulism toxin. The utilization of mass prophylaxis to limit the morbidity and mortality associated with all these agents is also discussed along with the role emergency care personnel play in its implementation.

  17. An outbreak of granulomatous inflammation associated with Francisella noatunensis subsp. orientalis in farmed tilapia ( Oreochromis niloticus × O. aureus) in China

    NASA Astrophysics Data System (ADS)

    Lin, Qiang; Li, Ningqiu; Fu, Xiaozhe; Hu, Qiandong; Chang, Ouqin; Liu, Lihui; Zhang, Defeng; Wang, Guangjun; San, Guibao; Wu, Shuqin

    2016-05-01

    In 2013, a novel disease was detected in tilapia ( Oreochromis niloticus × O. aureus) in Guangzhou, South China. To identify the causative pathogen, we conducted histological examination, bacteria isolation, and purification, and sequenced the 16S rRNA gene of isolates. Infected fish had a large number of white granulomas in the kidney, liver, heart, and spleen. The head kidney and spleen were markedly swollen. A bacterium strain designated as gz201301 was recovered from the spleen of infected tilapia. The 16S rRNA sequence of gz201301 revealed that it was highly similar to F. noatunensis subsp. orientalis. This is the first report of a Francisella-like infection in farmed tilapia in China.

  18. Preparing Change Agents for Change Agent Roles.

    ERIC Educational Resources Information Center

    Sedlacek, James R.

    Seventy-seven Spanish- and Portuguese-speaking agricultural change agents from developing Central and South American countries responded to a questionnaire which sought perceptions of the roles in which the change agents felt they were involved and the roles for which they felt they were being trained. The agents were participating in training…

  19. Genetic diversity of bacterial agents detected in ticks removed from asymptomatic patients in northeastern Italy.

    PubMed

    Sanogo, Y O; Parola, P; Shpynov, S; Camicas, J L; Brouqui, P; Caruso, G; Raoult, D

    2003-06-01

    A total of 360 ticks were removed from 353 asymptomatic subjects in Belluno Province, Italy and surrounding areas, from 1998 to 2001. Ticks were identified as Ixodes ricinus (357), Ixodes hexagonus (1), Rhipicephalus sanguineus (1), and Ixodes ventalloi (1). Tick DNA was investigated by PCR and subsequent sequencing of amplified products to identity associated bacterial agents. Primers targeting different genes of Rickettsia (gltA and OmpA), Borrelia (16S rDNA, rpoB), Francisella (16S rDNA), and all genera members of the Anaplasmataceae (16S rDNA), were used. DNA of bacterial agents was identified in 28 Ixodes ricinus specimens (7.8%). Rickettsia helvetica was detected in 7 ticks. Rickettsia sp. IRS4 and Borrelia afzelii was detected in 4 ticks each. B. garinii and B. valaisiana were identified in one tick each. Anaplasma phagocytophilum, the agent of human granulocytic ehrlichiosis, was identified in 1 specimen of I. ricinus. A new Ehrlichia sp. ("Candidatus Ehrlichia walkerii", sp. nov.) was identified in 10 I. ricinus specimens.

  20. Remote Agent Demonstration

    NASA Technical Reports Server (NTRS)

    Dorais, Gregory A.; Kurien, James; Rajan, Kanna

    1999-01-01

    We describe the computer demonstration of the Remote Agent Experiment (RAX). The Remote Agent is a high-level, model-based, autonomous control agent being validated on the NASA Deep Space 1 spacecraft.

  1. Zoonotic vector-borne bacterial pathogens in California mountain lions (Puma concolor), 1987-2010.

    PubMed

    Girard, Yvette A; Swift, Pamela; Chomel, Bruno B; Kasten, Rickie W; Fleer, Katryna; Foley, Janet E; Torres, Steven G; Johnson, Christine K

    2012-11-01

    Sera collected from 442 mountain lions in 48 California counties between the years of 1987 and 2010 were tested using immunofluorescence assays and agglutination tests for the presence of antibodies reactive to Yersinia pestis, Francisella tularensis, Bartonella henselae, Borrelia burgdorferi, and Anaplasma phagocytophilum antigens. Data were analyzed for spatial and temporal trends in seropositivity. Seroprevalences for B. burgdorferi (19.9%) and B. henselae (37.1%) were relatively high, with the highest exposure in the Central Coast region for B. henselae. B. henselae DNA amplified in mountain lion samples was genetically similar to human-derived Houston-1 and domestic cat-derived U4 B. henselae strains at the gltA and ftsZ loci. The statewide seroprevalences of Y. pestis (1.4%), F. tularensis (1.4%), and A. phagocytophilum (5.9%), were comparatively low. Sera from Y. pestis- and F. tularensis-seropositive mountain lions were primarily collected in the Eastern and Western Sierra Nevada, and samples reactive to Y. pestis antigen were collected exclusively from adult females. Adult age (≥ 2 years) was a risk factor for B. burgdorferi exposure. Over 70% of tested animals were killed on depredation permits, and therefore were active near areas with livestock and human residential communities. Surveillance of mountain lions for these bacterial vector-borne and zoonotic agents may be informative to public health authorities, and the data are useful for detecting enzootic and peridomestic pathogen transmission patterns, particularly in combination with molecular characterization of the infecting organisms. PMID:22925024

  2. Zoonotic vector-borne bacterial pathogens in California mountain lions (Puma concolor), 1987-2010.

    PubMed

    Girard, Yvette A; Swift, Pamela; Chomel, Bruno B; Kasten, Rickie W; Fleer, Katryna; Foley, Janet E; Torres, Steven G; Johnson, Christine K

    2012-11-01

    Sera collected from 442 mountain lions in 48 California counties between the years of 1987 and 2010 were tested using immunofluorescence assays and agglutination tests for the presence of antibodies reactive to Yersinia pestis, Francisella tularensis, Bartonella henselae, Borrelia burgdorferi, and Anaplasma phagocytophilum antigens. Data were analyzed for spatial and temporal trends in seropositivity. Seroprevalences for B. burgdorferi (19.9%) and B. henselae (37.1%) were relatively high, with the highest exposure in the Central Coast region for B. henselae. B. henselae DNA amplified in mountain lion samples was genetically similar to human-derived Houston-1 and domestic cat-derived U4 B. henselae strains at the gltA and ftsZ loci. The statewide seroprevalences of Y. pestis (1.4%), F. tularensis (1.4%), and A. phagocytophilum (5.9%), were comparatively low. Sera from Y. pestis- and F. tularensis-seropositive mountain lions were primarily collected in the Eastern and Western Sierra Nevada, and samples reactive to Y. pestis antigen were collected exclusively from adult females. Adult age (≥ 2 years) was a risk factor for B. burgdorferi exposure. Over 70% of tested animals were killed on depredation permits, and therefore were active near areas with livestock and human residential communities. Surveillance of mountain lions for these bacterial vector-borne and zoonotic agents may be informative to public health authorities, and the data are useful for detecting enzootic and peridomestic pathogen transmission patterns, particularly in combination with molecular characterization of the infecting organisms.

  3. Biological warfare agents.

    PubMed

    Pohanka, Miroslav; Kuca, Kamil

    2010-01-01

    Biological warfare agents are a group of pathogens and toxins of biological origin that can be potentially misused for military or criminal purposes. The present review attempts to summarize necessary knowledge about biological warfare agents. The historical aspects, examples of applications of these agents such as anthrax letters, biological weapons impact, a summary of biological warfare agents and epidemiology of infections are described. The last section tries to estimate future trends in research on biological warfare agents.

  4. Spacecraft sanitation agent development

    NASA Technical Reports Server (NTRS)

    1972-01-01

    The development of an effective sanitizing agent that is compatible with the spacecraft environment and the human occupant is discussed. Experimental results show that two sanitation agents must be used to satisfy mission requirements: one agent for personal hygiene and one for equipment maintenance. It was also recommended that a water rinse be used with the agents for best results, and that consideration be given to using the agents pressure packed or in aerosol formulations.

  5. Chemical warfare agents.

    PubMed

    Chauhan, S; Chauhan, S; D'Cruz, R; Faruqi, S; Singh, K K; Varma, S; Singh, M; Karthik, V

    2008-09-01

    Chemical warfare agents (CWA's) are defined as any chemical substance whose toxic properties are utilised to kill, injure or incapacitate an enemy in warfare and associated military operations. Chemical agents have been used in war since times immemorial, but their use reached a peak during World War I. During World War II only the Germans used them in the infamous gas chambers. Since then these have been intermittently used both in war and acts of terrorisms. Many countries have stockpiles of these agents. There has been a legislative effort worldwide to ban the use of CWA's under the chemical weapons convention which came into force in 1997. However the manufacture of these agents cannot be completely prohibited as some of them have potential industrial uses. Moreover despite the remedial measures taken so far and worldwide condemnation, the ease of manufacturing these agents and effectiveness during combat or small scale terrorist operations still make them a powerful weapon to reckon with. These agents are classified according to mechanism of toxicity in humans into blister agents, nerve agents, asphyxiants, choking agents and incapacitating/behavior altering agents. Some of these agents can be as devastating as a nuclear bomb. In addition to immediate injuries caused by chemical agents, some of them are associated with long term morbidities and psychological problems. In this review we will discuss briefly about the historical background, properties, manufacture techniques and industrial uses, mechanism of toxicity, clinical features of exposure and pharmacological management of casualties caused by chemical agents. PMID:21783898

  6. Being prepared: bioterrorism and mass prophylaxis: part I.

    PubMed

    Weant, Kyle A; Bailey, Abby M; Fleishaker, Elise L; Justice, Stephanie B

    2014-01-01

    Bioterrorism presents a real and omnipresent risk to public health throughout the world. More than 30 biological agents have been identified as possessing the potential to be deployed in a bioterrorist attack. Those that have been determined to be of the greatest concern and possess the greatest potential of use in this arena are known as the Category A agents: Bacillus anthracis (anthrax); Variola major (smallpox); Yersinia pestis (plague); Francisella tularensis (tularemia); viral hemorrhagic fevers; and Clostridium botulinum toxin (botulism toxin). Although the Centers for Disease Control and Prevention utilizes surveillance systems to identify illnesses, the weight of diagnosing, effectively treating, and notifying the appropriate public health officials lies squarely on the shoulders of emergency care personnel. Part I of this two-part review will focus on the clinical presentation and treatment of anthrax, plague, and tularemia. The subsequent Part II of this review will discuss smallpox, viral hemorrhagic fevers, botulism toxin, and the provision of mass prophylaxis.

  7. Mobile Agents Applications.

    ERIC Educational Resources Information Center

    Martins, Rosane Maria; Chaves, Magali Ribeiro; Pirmez, Luci; Rust da Costa Carmo, Luiz Fernando

    2001-01-01

    Discussion of the need to filter and retrieval relevant information from the Internet focuses on the use of mobile agents, specific software components which are based on distributed artificial intelligence and integrated systems. Surveys agent technology and discusses the agent building package used to develop two applications using IBM's Aglet…

  8. Hydroxypyridonate chelating agents

    DOEpatents

    Raymond, Kenneth N.; Scarrow, Robert C.; White, David L.

    1987-01-01

    Chelating agents having 1-hydroxy-2-pyridinone (HOPO) and related moieties incorporated within their structures, including polydentate HOPO-substituted polyamines such as spermidine and spermine, and HOPO-substituted desferrioxamine. The chelating agents are useful in selectively removing certain cations from solution, and are particularly useful as ferric ion and actinide chelators. Novel syntheses of the chelating agents are provided.

  9. Standard Agent Framework 1

    SciTech Connect

    Goldsmith, Steven Y.

    1999-04-06

    The Standard Agent framework provides an extensible object-oriented development environment suitable for use in both research and applications projects. The SAF provides a means for constructing and customizing multi-agent systems through specialization of standard base classes (architecture-driven framework) and by composition of component classes (data driven framework). The standard agent system is implemented as an extensible object-centerd framework. Four concrete base classes are developed: (1) Standard Agency; (2) Standard Agent; (3) Human Factor, and (4) Resources. The object-centered framework developed and utilized provides the best comprimise between generality and flexibility available in agent development systems today.

  10. Immunotherapy for tularemia.

    PubMed

    Skyberg, Jerod A

    2013-11-15

    Francisella tularensis is a gram-negative bacterium that causes the zoonotic disease tularemia. Francisella is highly infectious via the respiratory route (~10 CFUs) and pulmonary infections due to type A strains of F. tularensis are highly lethal in untreated patients (> 30%). In addition, no vaccines are licensed to prevent tularemia in humans. Due to the high infectivity and mortality of pulmonary tularemia, F. tularensis has been weaponized, including via the introduction of antibiotic resistance, by several countries. Because of the lack of efficacious vaccines, and concerns about F. tularensis acquiring resistance to antibiotics via natural or illicit means, augmentation of host immunity, and humoral immunotherapy have been investigated as countermeasures against tularemia. This manuscript will review advances made and challenges in the field of immunotherapy against tularemia.

  11. [Investigation of tularemia seroprevalence in the rural area of Thrace region in Turkey].

    PubMed

    Dedeoğlu Kilinç, Günay; Gürcan, Saban; Eskiocak, Muzaffer; Kiliç, Haluk; Kunduracilar, Hakan

    2007-07-01

    The first published tularemia epidemic in Turkey had been reported in 1936 from Luleburgaz (located in European part-Thrace region-of Turkey), and the second was in 1945 again in the same province. Following a long period of time without any tularemia report from Thrace region, in 2005 another epidemic occurred in a village of Edirne, another province located in the same region. Since there is presumptive evidence of circulation of the infectious agent, Francisella tularensis in Thrace region of Turkey, a large scale seroepidemiological study is needed. In this study, the presence of antibodies against F. tularensis in 1782 subjects, choosen by "thirty cluster" method, inhabiting in 90 different villages of Edirne, Kirklareli, and Tekirdag provinces in Thrace Region, were investigated. The subjects were included to the study on the basis of volunteering (74.3% were male; mean age: 46 years; age range: 6-92 years) and demographical characteristics and their possible risky behaviours were recorded in a questionnaire form. Antibodies specific for F. tularensis were screened by microagglutination test, and were found positive in five (0.3%) of the subjects between the titers of 1/20- 1/160. All of the seropositive subjects were adult males (ages between 22-74 years); three were living in the two villages of Kirklareli, while the others were from the villages of Tekirdag and Edirne. Rose Bengal test was also found positive in three of the seropositive subjects, and with the thought of a probable cross reaction they were taken into an advanced investigation for brucellosis. The risk evaluation revealed that male gender, being together with livestock and exposure to ticks were the major risk factors. Since the data of this study indicated that F. tularensis is in circulation in Thrace Region, the educational programmes for both the healthcare workers and inhabitants of this region should be attempted for the prevention of a possible epidemic.

  12. Agent Architectures for Compliance

    NASA Astrophysics Data System (ADS)

    Burgemeestre, Brigitte; Hulstijn, Joris; Tan, Yao-Hua

    A Normative Multi-Agent System consists of autonomous agents who must comply with social norms. Different kinds of norms make different assumptions about the cognitive architecture of the agents. For example, a principle-based norm assumes that agents can reflect upon the consequences of their actions; a rule-based formulation only assumes that agents can avoid violations. In this paper we present several cognitive agent architectures for self-monitoring and compliance. We show how different assumptions about the cognitive architecture lead to different information needs when assessing compliance. The approach is validated with a case study of horizontal monitoring, an approach to corporate tax auditing recently introduced by the Dutch Customs and Tax Authority.

  13. Effect of size and temperature at vaccination on immunization and protection conferred by a live attenuated Francisella noatunensis immersion vaccine in red hybrid tilapia.

    PubMed

    Soto, Esteban; Brown, Nicholas; Gardenfors, Zackarias O; Yount, Shaun; Revan, Floyd; Francis, Stewart; Kearney, Michael T; Camus, Alvin

    2014-12-01

    Francisella noatunensis subsp. orientalis (Fno) is a pleomorphic, facultative intracellular, Gram-negative, emerging bacterial pathogen of marine and fresh water fish with worldwide distribution. In this study, the efficacy of an attenuated Fno intracellular growth locus C (iglC) mutant was evaluated for use as a live immersion vaccine, when administered to hybrid tilapia at two different stages of growth (5 g fry and 10 g fingerlings) and at two temperatures (25 °C and 30 °C). To determine vaccine efficacy, mortality, days to first death, and Fno genome equivalents (GE) in the spleens of survivors, as well as serum and mucus antibody levels, were evaluated after 30 d in fish challenged with a wild type virulent strain. Both size and temperature at vaccination played an important role in immunization and protection. Fry vaccinated at 25 °C were not protected when compared to non-vaccinated fry at 25 °C (p = 0.870). In contrast, 5 g fry vaccinated at 30 °C were significantly protected compared to non-vaccinated fry at 30 °C (p = 0.038). Although lower mortalities occurred, 10 g fingerlings vaccinated at 25 °C were not protected, compared to non-vaccinated fingerlings at 25 °C (p = 0.328), while, 10 g fingerlings vaccinated at 30 °C were significantly protected, compared to non-vaccinated fingerlings at 30 °C (p = 0.038). Additionally, overall mortality of 5 g fish was significantly higher than in 10 g fish. Mortality was also significantly higher in fish subjected to a 30 to 25 °C temperature change one week prior to challenge, than in fish maintained at the same temperature during vaccination and challenge. This information demonstrates that both temperature and size at vaccination are important factors when implementing immunization prophylaxis in cultured tilapia.

  14. 13 CFR 107.1620 - Functions of agents, including Central Registration Agent, Selling Agent and Fiscal Agent.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 13 Business Credit and Assistance 1 2010-01-01 2010-01-01 false Functions of agents, including... Assistance SMALL BUSINESS ADMINISTRATION SMALL BUSINESS INVESTMENT COMPANIES SBA Financial Assistance for... Functions of agents, including Central Registration Agent, Selling Agent and Fiscal Agent. (a) Agents....

  15. Detecting biological warfare agents.

    PubMed

    Song, Linan; Ahn, Soohyoun; Walt, David R

    2005-10-01

    We developed a fiber-optic, microsphere-based, high-density array composed of 18 species-specific probe microsensors to identify biological warfare agents. We simultaneously identified multiple biological warfare agents in environmental samples by looking at specific probe responses after hybridization and response patterns of the multiplexed array.

  16. Travel Agent Course Outline.

    ERIC Educational Resources Information Center

    British Columbia Dept. of Education, Victoria.

    Written for college entry-level travel agent training courses, this course outline can also be used for inservice training programs offered by travel agencies. The outline provides information on the work of a travel agent and gives clear statements on what learners must be able to do by the end of their training. Material is divided into eight…

  17. Change Agent Survival Guide

    ERIC Educational Resources Information Center

    Dunbar, Folwell L.

    2011-01-01

    Consulting is a rough racket. Only a tarantula hair above IRS agents, meter maids and used car sales people, the profession is a prickly burr for slings and arrows. Throw in education, focus on dysfunctional schools and call oneself a "change agent," and this bad rap all but disappears. Unfortunately, though, consulting/coaching/mentoring in…

  18. The 'Hittite plague', an epidemic of tularemia and the first record of biological warfare.

    PubMed

    Trevisanato, Siro Igino

    2007-01-01

    A long-lasting epidemic that plagued the Eastern Mediterranean in the 14th century BC was traced back to a focus in Canaan along the Arwad-Euphrates trading route. The symptoms, mode of infection, and geographical area, identified the agent as Francisella tularensis, which is also credited for outbreaks in Canaan around 1715 BC and 1075 BC. At first, the 14th century epidemic contaminated an area stretching from Cyprus to Iraq, and from Israel to Syria, sparing Egypt and Anatolia due to quarantine and political boundaries, respectively. Subsequently, wars spread the disease to central Anatolia, from where it was deliberately brought to Western Anatolia, in what constitutes the first known record of biological warfare. Finally, Aegean soldiers fighting in western Anatolia returned home to their islands, further spreading the epidemic.

  19. Development of Liposomal Ciprofloxacin to Treat Lung Infections.

    PubMed

    Cipolla, David; Blanchard, Jim; Gonda, Igor

    2016-01-01

    Except for management of Pseudomonas aeruginosa (PA) in cystic fibrosis, there are no approved inhaled antibiotic treatments for any other diseases or for infections from other pathogenic microorganisms such as tuberculosis, non-tuberculous mycobacteria, fungal infections or potential inhaled biowarfare agents including Francisella tularensis, Yersinia pestis and Coxiella burnetii (which cause pneumonic tularemia, plague and Q fever, respectively). Delivery of an antibiotic formulation via the inhalation route has the potential to provide high concentrations at the site of infection with reduced systemic exposure to limit side effects. A liposomal formulation may improve tolerability, increase compliance by reducing the dosing frequency, and enhance penetration of biofilms and treatment of intracellular infections. Two liposomal ciprofloxacin formulations (Lipoquin(®) and Pulmaquin(®)) that are in development by Aradigm Corporation are described here. PMID:26938551

  20. Tularemia vaccine development: paralysis or progress?

    PubMed Central

    Sunagar, Raju; Kumar, Sudeep; Franz, Brian J; Gosselin, Edmund J

    2016-01-01

    Francisella tularensis (Ft) is a gram-negative intercellular pathogen and category A biothreat agent. However, despite 15 years of strong government investment and intense research focused on the development of a US Food and Drug Administration-approved vaccine against Ft, the primary goal remains elusive. This article reviews research efforts focused on developing an Ft vaccine, as well as a number of important factors, some only recently recognized as such, which can significantly impact the development and evaluation of Ft vaccine efficacy. Finally, an assessment is provided as to whether a US Food and Drug Administration-approved Ft vaccine is likely to be forthcoming and the potential means by which this might be achieved. PMID:27200274

  1. Tularemia Outbreaks and Common Vole (Microtus arvalis) Irruptive Population Dynamics in Northwestern Spain, 1997-2014.

    PubMed

    Luque-Larena, Juan José; Mougeot, François; Roig, Dolors Vidal; Lambin, Xavier; Rodríguez-Pastor, Ruth; Rodríguez-Valín, Elena; Anda, Pedro; Escudero, Raquel

    2015-09-01

    During the last decades, large tularemia outbreaks in humans have coincided in time and space with population outbreaks of common voles in northwestern Spain, leading us to hypothesize that this rodent species acts as a key spillover agent of Francisella tularensis in the region. Here, we evaluate for the first time a potential link between irruptive vole numbers and human tularemia outbreaks in Spain. We compiled vole abundance estimates obtained through live-trapping monitoring studies and official reports of human tularemia cases during the period 1997-2014. We confirm a significant positive association between yearly cases of tularemia infection in humans and vole abundance. High vole densities during outbreaks (up to 1000 voles/hectare) may therefore enhance disease transmission and spillover contamination in the environment. If this ecological link is further confirmed, the apparent multiannual cyclicity of common vole outbreaks might provide a basis for forecasting the risk of tularemia outbreaks in northwestern Spain. PMID:26333034

  2. [Experimental study on the possibility of using live tularemia vaccine to increase resistance to heterologous infection disease].

    PubMed

    Iliukhin, V I; Plekhanova, N G; Senina, T V; Stanovaia, O V; Kislichkin, N N

    2004-01-01

    In experiments on guinea pigs immunized with Francisella tularensis 15, or live tularemia vaccine (LTV), the level of heterologous protective effect to dangerous infectious diseases caused by Yersinia pestis, Burkholderia pseudomallei, B. mallei, Mycobacterium tuberculosis was studied. The study revealed that during the first 4 weeks after the subcutaneous immunization with LTV the level of resistance of the immunized animals to heterologous infective agent reliably increased as indicated by the survival rate of the animals, as well as by the survival time of those killed by infection, in comparison with the controls. Later (on day 150 after immunization) differences in death rate between the groups perceptibly decreased. Nevertheless, the 1 1/2-fold increase of the survival time of the challenged immunized animals in comparison with the controls proved the possibility of using immunization with LTV for the urgent prophylaxis and treatment not only of tularemia, but also of plague, glanders, melioidosis and tuberculosis.

  3. First Pediatric Case of Tularemia after a Coyote Bite.

    PubMed

    Chomel, Bruno B; Morton, Jane A; Kasten, Rickie W; Chang, Chao-Chin

    2016-01-01

    Bite-transmitted tularemia is a rare event in humans and most of the cases have been associated with cat bites. We report the first pediatric case of tularemia caused by a coyote (Canis latrans) bite. Coyotes can be healthy carriers of Francisella tularensis and transmit this infectious agent through a bite. Pediatricians should be aware of this risk after a carnivore bite and implement appropriate antibiotic therapy, as amoxicillin/clavulanate potassium (Augmentin) may have prolonged the typical two to three days' incubation period commonly observed for tularemia after an animal bite and was not effective in preventing clinical signs in this child. Finally, it emphasizes again the importance of early and late serum samples for appropriate serodiagnostic.

  4. Polyclonal Antibody Production for Membrane Proteins via Genetic Immunization

    PubMed Central

    Hansen, Debra T.; Robida, Mark D.; Craciunescu, Felicia M.; Loskutov, Andrey V.; Dörner, Katerina; Rodenberry, John-Charles; Wang, Xiao; Olson, Tien L.; Patel, Hetal; Fromme, Petra; Sykes, Kathryn F.

    2016-01-01

    Antibodies are essential for structural determinations and functional studies of membrane proteins, but antibody generation is limited by the availability of properly-folded and purified antigen. We describe the first application of genetic immunization to a structurally diverse set of membrane proteins to show that immunization of mice with DNA alone produced antibodies against 71% (n = 17) of the bacterial and viral targets. Antibody production correlated with prior reports of target immunogenicity in host organisms, underscoring the efficiency of this DNA-gold micronanoplex approach. To generate each antigen for antibody characterization, we also developed a simple in vitro membrane protein expression and capture method. Antibody specificity was demonstrated upon identifying, for the first time, membrane-directed heterologous expression of the native sequences of the FopA and FTT1525 virulence determinants from the select agent Francisella tularensis SCHU S4. These approaches will accelerate future structural and functional investigations of therapeutically-relevant membrane proteins. PMID:26908053

  5. Tularemia Outbreaks and Common Vole (Microtus arvalis) Irruptive Population Dynamics in Northwestern Spain, 1997-2014.

    PubMed

    Luque-Larena, Juan José; Mougeot, François; Roig, Dolors Vidal; Lambin, Xavier; Rodríguez-Pastor, Ruth; Rodríguez-Valín, Elena; Anda, Pedro; Escudero, Raquel

    2015-09-01

    During the last decades, large tularemia outbreaks in humans have coincided in time and space with population outbreaks of common voles in northwestern Spain, leading us to hypothesize that this rodent species acts as a key spillover agent of Francisella tularensis in the region. Here, we evaluate for the first time a potential link between irruptive vole numbers and human tularemia outbreaks in Spain. We compiled vole abundance estimates obtained through live-trapping monitoring studies and official reports of human tularemia cases during the period 1997-2014. We confirm a significant positive association between yearly cases of tularemia infection in humans and vole abundance. High vole densities during outbreaks (up to 1000 voles/hectare) may therefore enhance disease transmission and spillover contamination in the environment. If this ecological link is further confirmed, the apparent multiannual cyclicity of common vole outbreaks might provide a basis for forecasting the risk of tularemia outbreaks in northwestern Spain.

  6. First Pediatric Case of Tularemia after a Coyote Bite.

    PubMed

    Chomel, Bruno B; Morton, Jane A; Kasten, Rickie W; Chang, Chao-Chin

    2016-01-01

    Bite-transmitted tularemia is a rare event in humans and most of the cases have been associated with cat bites. We report the first pediatric case of tularemia caused by a coyote (Canis latrans) bite. Coyotes can be healthy carriers of Francisella tularensis and transmit this infectious agent through a bite. Pediatricians should be aware of this risk after a carnivore bite and implement appropriate antibiotic therapy, as amoxicillin/clavulanate potassium (Augmentin) may have prolonged the typical two to three days' incubation period commonly observed for tularemia after an animal bite and was not effective in preventing clinical signs in this child. Finally, it emphasizes again the importance of early and late serum samples for appropriate serodiagnostic. PMID:26885419

  7. Development of Liposomal Ciprofloxacin to Treat Lung Infections

    PubMed Central

    Cipolla, David; Blanchard, Jim; Gonda, Igor

    2016-01-01

    Except for management of Pseudomonas aeruginosa (PA) in cystic fibrosis, there are no approved inhaled antibiotic treatments for any other diseases or for infections from other pathogenic microorganisms such as tuberculosis, non-tuberculous mycobacteria, fungal infections or potential inhaled biowarfare agents including Francisella tularensis, Yersinia pestis and Coxiella burnetii (which cause pneumonic tularemia, plague and Q fever, respectively). Delivery of an antibiotic formulation via the inhalation route has the potential to provide high concentrations at the site of infection with reduced systemic exposure to limit side effects. A liposomal formulation may improve tolerability, increase compliance by reducing the dosing frequency, and enhance penetration of biofilms and treatment of intracellular infections. Two liposomal ciprofloxacin formulations (Lipoquin® and Pulmaquin®) that are in development by Aradigm Corporation are described here. PMID:26938551

  8. First Pediatric Case of Tularemia after a Coyote Bite

    PubMed Central

    Chomel, Bruno B.; Morton, Jane A.; Kasten, Rickie W.; Chang, Chao-chin

    2016-01-01

    Bite-transmitted tularemia is a rare event in humans and most of the cases have been associated with cat bites. We report the first pediatric case of tularemia caused by a coyote (Canis latrans) bite. Coyotes can be healthy carriers of Francisella tularensis and transmit this infectious agent through a bite. Pediatricians should be aware of this risk after a carnivore bite and implement appropriate antibiotic therapy, as amoxicillin/clavulanate potassium (Augmentin) may have prolonged the typical two to three days' incubation period commonly observed for tularemia after an animal bite and was not effective in preventing clinical signs in this child. Finally, it emphasizes again the importance of early and late serum samples for appropriate serodiagnostic. PMID:26885419

  9. [Tularemia in Germany].

    PubMed

    Kohlmann, R; Geis, G; Gatermann, S G

    2014-07-01

    The bacterium Francisella tularensis is known for more than 100 years by now as the etiological agent of the disease tularemia, a zoonotic infection with a worldwide distribution in the Northern Hemisphere. The prevalence of tularemia shows a wide geographic variation, being comparably infrequent in Germany. Tularemia can present itself with multiple clinical manifestations including ulceroglandular, glandular, oropharyngeal, oculoglandular, respiratory and typhoidal forms. Due to the low prevalence and the unspecific symptomatology, a rapid diagnosis and early start of an effective therapy are rarely obtained. Thus, in this article we summarize important aspects concerning etiology, ecology and routes of transmission, recent epidemiologic situation, clinical picture, diagnostics and treatment of tularemia, focusing on the situation in Germany.

  10. Ferrimagnetic susceptibility contrast agents.

    PubMed

    Bach-Gansmo, T

    1993-01-01

    Contrast agents based on superparamagnetic particles have been in clinical development for more than 5 years, and the complexity of their effects is still not elucidated. The relaxivities are frequently used to give an idea of their efficacy, but these parameters can only be used if they are concentration independent. For large superparamagnetic systems, the evolution of the transverse magnetization is biexponential, after an initial loss of magnetization. Both these characteristics of large superparamagnetic systems should lead to prudence in using the relaxivities as indicators of contrast medium efficacy. Susceptibility induced artefacts have been associated with the use of superparamagnetic contrast agents since the first imaging evaluation took place. The range of concentrations where good contrast effect was achieved without inducing artefacts, as well as blurring and metal artefacts were evaluated. The influence of motion on the induction of artefacts was studied, and compared to the artefacts induced by a paramagnetic agent subject to motion. With a suitable concentration of a negative contrast agent, a signal void could be achieved in the region prone to motion, and no artefacts were induced. If the concentration was too high, a displacement of the region close to the contrast agent was observed. The artefacts occurred in a volume surrounding the contrast agent, i.e., also outside the imaging plane. In comparison a positive, paramagnetic contrast agent induced heavy artefacts in the phase encoding direction, appearing as both high intensity regions and black holes, in a mosaic pattern. Clinical trials of the oral contrast agent OMP for abdominal MR imaging showed this agent to be safe and efficacious. OMP increased the diagnostic efficacy of abdominal MR imaging in 2 of 3 cases examined, with a significant decrease in motion artefacts. Susceptibility contrast agents may also be of use in the evaluation of small lesions in the liver. Particulate material

  11. Standard Agent Framework 1

    1999-04-06

    The Standard Agent framework provides an extensible object-oriented development environment suitable for use in both research and applications projects. The SAF provides a means for constructing and customizing multi-agent systems through specialization of standard base classes (architecture-driven framework) and by composition of component classes (data driven framework). The standard agent system is implemented as an extensible object-centerd framework. Four concrete base classes are developed: (1) Standard Agency; (2) Standard Agent; (3) Human Factor, and (4)more » Resources. The object-centered framework developed and utilized provides the best comprimise between generality and flexibility available in agent development systems today.« less

  12. How do agents represent?

    NASA Astrophysics Data System (ADS)

    Ryan, Alex

    Representation is inherent to the concept of an agent, but its importance in complex systems has not yet been widely recognised. In this paper I introduce Peirce's theory of signs, which facilitates a definition of representation in general. In summary, representation means that for some agent, a model is used to stand in for another entity in a way that shapes the behaviour of the agent with respect to that entity. Representation in general is then related to the theories of representation that have developed within different disciplines. I compare theories of representation from metaphysics, military theory and systems theory. Additional complications arise in explaining the special case of mental representations, which is the focus of cognitive science. I consider the dominant theory of cognition — that the brain is a representational device — as well as the sceptical anti-representational response. Finally, I argue that representation distinguishes agents from non-representational objects: agents are objects capable of representation.

  13. Biological warfare agents.

    PubMed

    Thavaselvam, Duraipandian; Vijayaraghavan, Rajagopalan

    2010-07-01

    The recent bioterrorist attacks using anthrax spores have emphasized the need to detect and decontaminate critical facilities in the shortest possible time. There has been a remarkable progress in the detection, protection and decontamination of biological warfare agents as many instrumentation platforms and detection methodologies are developed and commissioned. Even then the threat of biological warfare agents and their use in bioterrorist attacks still remain a leading cause of global concern. Furthermore in the past decade there have been threats due to the emerging new diseases and also the re-emergence of old diseases and development of antimicrobial resistance and spread to new geographical regions. The preparedness against these agents need complete knowledge about the disease, better research and training facilities, diagnostic facilities and improved public health system. This review on the biological warfare agents will provide information on the biological warfare agents, their mode of transmission and spread and also the detection systems available to detect them. In addition the current information on the availability of commercially available and developing technologies against biological warfare agents has also been discussed. The risk that arise due to the use of these agents in warfare or bioterrorism related scenario can be mitigated with the availability of improved detection technologies.

  14. Biological warfare agents

    PubMed Central

    Thavaselvam, Duraipandian; Vijayaraghavan, Rajagopalan

    2010-01-01

    The recent bioterrorist attacks using anthrax spores have emphasized the need to detect and decontaminate critical facilities in the shortest possible time. There has been a remarkable progress in the detection, protection and decontamination of biological warfare agents as many instrumentation platforms and detection methodologies are developed and commissioned. Even then the threat of biological warfare agents and their use in bioterrorist attacks still remain a leading cause of global concern. Furthermore in the past decade there have been threats due to the emerging new diseases and also the re-emergence of old diseases and development of antimicrobial resistance and spread to new geographical regions. The preparedness against these agents need complete knowledge about the disease, better research and training facilities, diagnostic facilities and improved public health system. This review on the biological warfare agents will provide information on the biological warfare agents, their mode of transmission and spread and also the detection systems available to detect them. In addition the current information on the availability of commercially available and developing technologies against biological warfare agents has also been discussed. The risk that arise due to the use of these agents in warfare or bioterrorism related scenario can be mitigated with the availability of improved detection technologies. PMID:21829313

  15. Dioxin, agent orange

    SciTech Connect

    Gough, M.

    1986-01-01

    This book presents information on the following topics: dioxin, a prevalent problem; nobody wanted dioxin; agent organe and Vietnam; what we know about and may learn about agent orange and Veterans' health; agent organe and birth defects; dioxin in Missouri; 2, 4, 5-T: the U.S.' disappearing herbicide; Seveso: high-level environmental exposure; the nitro explosion; industrial exposures to dioxin; company behavior in the face of dioxin exposures; dioxin and specific cancers; animal tests of dioxin toxicity; dioxin decions; the present and the future.

  16. Riot Control Agents

    MedlinePlus

    ... your clothing, rapidly wash your entire body with soap and water, and get medical care as quickly ... agent from your skin with large amounts of soap and water. Washing with soap and water will ...

  17. Radioactive diagnostic agent

    SciTech Connect

    Shigematsu, A.; Aihara, M.; Matsuda, M.; Suzuki, A.; Tsuya, A.

    1984-02-07

    A radioactive diagnostic agent for renal cortex, adrenal cortex, myocardium, brain stem, spinal nerve, etc., which comprises as an essential component monoiodoacetic acid wherein the iodine atom is radioactive.

  18. Agent oriented programming

    NASA Technical Reports Server (NTRS)

    Shoham, Yoav

    1994-01-01

    The goal of our research is a methodology for creating robust software in distributed and dynamic environments. The approach taken is to endow software objects with explicit information about one another, to have them interact through a commitment mechanism, and to equip them with a speech-acty communication language. System-level applications include software interoperation and compositionality. A government application of specific interest is an infrastructure for coordination among multiple planners. Daily activity applications include personal software assistants, such as programmable email, scheduling, and new group agents. Research topics include definition of mental state of agents, design of agent languages as well as interpreters for those languages, and mechanisms for coordination within agent societies such as artificial social laws and conventions.

  19. Agent amplified communication

    SciTech Connect

    Kautz, H.; Selman, B.; Milewski, A.

    1996-12-31

    We propose an agent-based framework for assisting and simplifying person-to-person communication for information gathering tasks. As an example, we focus on locating experts for any specified topic. In our approach, the informal person-to-person networks that exist within an organization are used to {open_quotes}referral chain{close_quotes} requests for expertise. User-agents help automate this process. The agents generate referrals by analyzing records of e-mail communication patterns. Simulation results show that the higher responsiveness of an agent-based system can be effectively traded for the higher accuracy of a completely manual approach. Furthermore, preliminary experience with a group of users on a prototype system has shown that useful automatic referrals can be found in practice. Our experience with actual users has also shown that privacy concerns are central to the successful deployment of personal agents: an advanced agent-based system will therefore need to reason about issues involving trust and authority.

  20. Tularaemia: clinical aspects in Europe.

    PubMed

    Maurin, Max; Gyuranecz, Miklós

    2016-01-01

    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.

  1. Agent independent task planning

    NASA Technical Reports Server (NTRS)

    Davis, William S.

    1990-01-01

    Agent-Independent Planning is a technique that allows the construction of activity plans without regard to the agent that will perform them. Once generated, a plan is then validated and translated into instructions for a particular agent, whether a robot, crewmember, or software-based control system. Because Space Station Freedom (SSF) is planned for orbital operations for approximately thirty years, it will almost certainly experience numerous enhancements and upgrades, including upgrades in robotic manipulators. Agent-Independent Planning provides the capability to construct plans for SSF operations, independent of specific robotic systems, by combining techniques of object oriented modeling, nonlinear planning and temporal logic. Since a plan is validated using the physical and functional models of a particular agent, new robotic systems can be developed and integrated with existing operations in a robust manner. This technique also provides the capability to generate plans for crewmembers with varying skill levels, and later apply these same plans to more sophisticated robotic manipulators made available by evolutions in technology.

  2. Sunscreening agents: a review.

    PubMed

    Latha, M S; Martis, Jacintha; Shobha, V; Sham Shinde, Rutuja; Bangera, Sudhakar; Krishnankutty, Binny; Bellary, Shantala; Varughese, Sunoj; Rao, Prabhakar; Naveen Kumar, B R

    2013-01-01

    The increasing incidence of skin cancers and photodamaging effects caused by ultraviolet radiation has increased the use of sunscreening agents, which have shown beneficial effects in reducing the symptoms and reoccurrence of these problems. Many sunscreen compounds are in use, but their safety and efficacy are still in question. Efficacy is measured through indices, such as sun protection factor, persistent pigment darkening protection factor, and COLIPA guidelines. The United States Food and Drug Administration and European Union have incorporated changes in their guidelines to help consumers select products based on their sun protection factor and protection against ultraviolet radiation, whereas the Indian regulatory agency has not yet issued any special guidance on sunscreening agents, as they are classified under cosmetics. In this article, the authors discuss the pharmacological actions of sunscreening agents as well as the available formulations, their benefits, possible health hazards, safety, challenges, and proper application technique. New technologies and scope for the development of sunscreening agents are also discussed as well as the role of the physician in patient education about the use of these agents.

  3. MpcAgent

    2013-11-29

    MpcAgent software is a module for the VolltronLite platform from PNNL that regulates the operation of rooftop air conditioning units in small to medium commercial buildings for the purpose of reducing peak power consumption. The MpcAgent accomplishes this by restricting the number of units that may operate simultaneously and using a model predictive control strategy to select which units to operate in each control period. The outcome of this control is effective control of themore » building air temperature at the user specified set point while avoiding expensive peak demand charges that result from running all HVAC units simultaneously.« less

  4. MpcAgent

    SciTech Connect

    Nutaro, James

    2013-11-29

    MpcAgent software is a module for the VolltronLite platform from PNNL that regulates the operation of rooftop air conditioning units in small to medium commercial buildings for the purpose of reducing peak power consumption. The MpcAgent accomplishes this by restricting the number of units that may operate simultaneously and using a model predictive control strategy to select which units to operate in each control period. The outcome of this control is effective control of the building air temperature at the user specified set point while avoiding expensive peak demand charges that result from running all HVAC units simultaneously.

  5. Tularemia in Canada with a focus on Saskatchewan

    PubMed Central

    Martin, Tom; Holmes, Ian H.; Wobeser, Gary A.; Anthony, Rene F.; Greefkes, Ineke

    1982-01-01

    Although rare among humans in Canada, tularemia is often endemic in wildlife. The inhabitants of rural areas are especially likely to be exposed to the causative bacterium, Francisella tularensis, through trapping or through the bites of arthropods. Muskrats have replaced rabbits as the principal source of infection, as illustrated by a familial outbreak of oropharyngeal tularemia in Saskatchewan. In humans the disease has six distinct forms and can be asymptomatic, but it generally comes to medical attention as fever, persistent ulcers and enlarged lymph nodes. Serologic tests will confirm the diagnosis. Bien que la tularémie soit rare chez l'homme au Canada, elle existe souvent à l'état endémique parmi les animaux sauvages. Les habitants des régions rurales sont particuliérement susceptibles d'être exposés à l'agent étiologique, Francisella tularensis, lors du trappage ou par les morsures d'arthropodes. Le rat musqué a maintenant remplacé le lapin comme principale source d'infection, tel que l'illustre une poussée de tularémie oropharyngienne chez une famille de Saskatchewan. Chez l'humain la maladie prend six formes distinctes, et elle peut être asymptomatique, mais elle se présente généralement à l'attention du médecin comme une fièvre accompagnée d'ulcères persistants et d'une tuméfaction ganglionnaire. Les épreuves sérologiques confirment le diagnostic. PMID:7046896

  6. Raccoons and Skunks as Sentinels for Enzootic Tularemia

    PubMed Central

    Berrada, Zenda L.; Goethert, Heidi K.

    2006-01-01

    We analyzed sera from diverse mammals of Martha's Vineyard, Massachusetts, for evidence of Francisella tularensis exposure. Skunks and raccoons were frequently seroreactive, whereas white-footed mice, cottontail rabbits, deer, rats, and dogs were not. Tularemia surveillance may be facilitated by focusing on skunks and raccoons. PMID:16707067

  7. Treatment of Tularemia in Patient with Chronic Graft-versus-Host Disease

    PubMed Central

    Seibold, Erik; Knabbe, Cornelius; Kaufmann, Martin; Splettstoesser, Wolf

    2013-01-01

    We describe a case of human tularemia caused by Francisella tularensis subsp. holarctica in a stem cell transplant recipient with chronic graft-versus-host disease who was receiving levofloxacin prophylaxis. The infection was characterized by pneumonia with septic complications. The patient was successfully treated with doxycycline. PMID:23647853

  8. Tularemia among Free-Ranging Mice without Infection of Exposed Humans, Switzerland, 2012

    PubMed Central

    Origgi, Francesco C.; König, Barbara; Lindholm, Anna K.; Mayor, Désirée

    2015-01-01

    The animals primarily infected by Francisella tularensis are rapidly consumed by scavengers, hindering ecologic investigation of the bacterium. We describe a 2012 natural tularemia epizootic among house mice in Switzerland and the assessment of infection of exposed humans. The humans were not infected, but the epizootic coincided with increased reports of human cases in the area. PMID:25531919

  9. Development and evaluation of a latex agglutination test for the rapid serodiagnosis of tularemia.

    PubMed

    Rastawicki, Waldemar; Rokosz-Chudziak, Natalia; Chróst, Anna; Gierczyński, Rafał

    2015-05-01

    A latex agglutination test (LAT) was developed for a rapid detection of antibodies against Francisella tularensis. The assay is performed by mixing serum with antigen-coated latex beads and read within 5 min. Developed LAT has been proved to be a specific, sensitive, fast, easy-to-perform and cost-efficient tool for the routine diagnosis of tularemia.

  10. Tularemia among free-ranging mice without infection of exposed humans, Switzerland, 2012.

    PubMed

    Origgi, Francesco C; König, Barbara; Lindholm, Anna K; Mayor, Désirée; Pilo, Paola

    2015-01-01

    The animals primarily infected by Francisella tularensis are rapidly consumed by scavengers, hindering ecologic investigation of the bacterium. We describe a 2012 natural tularemia epizootic among house mice in Switzerland and the assessment of infection of exposed humans. The humans were not infected, but the epizootic coincided with increased reports of human cases in the area.

  11. Waterborne outbreak of tularemia associated with crayfish fishing.

    PubMed Central

    Anda, P.; Segura del Pozo, J.; Díaz García, J. M.; Escudero, R.; García Peña, F. J.; López Velasco, M. C.; Sellek, R. E.; Jiménez Chillarón, M. R.; Sánchez Serrano, L. P.; Martínez Navarro, J. F.

    2001-01-01

    In 1997, an outbreak of human tularemia associated with hare-hunting in central Spain affected 585 patients. We describe the identification of Francisella tularensis biovar palaearctica in a second outbreak of ulceroglandular tularemia associated with crayfish (Procambarus clarkii) fishing in a contaminated freshwater stream distant from the hare-associated outbreak. The second outbreak occurred 1 year after the first. PMID:11485678

  12. Agent Persuasion Mechanism of Acquaintance

    NASA Astrophysics Data System (ADS)

    Jinghua, Wu; Wenguang, Lu; Hailiang, Meng

    Agent persuasion can improve negotiation efficiency in dynamic environment based on its initiative and autonomy, and etc., which is being affected much more by acquaintance. Classification of acquaintance on agent persuasion is illustrated, and the agent persuasion model of acquaintance is also illustrated. Then the concept of agent persuasion degree of acquaintance is given. Finally, relative interactive mechanism is elaborated.

  13. 13 CFR 108.1620 - Functions of agents, including Central Registration Agent, Selling Agent and Fiscal Agent.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 13 Business Credit and Assistance 1 2010-01-01 2010-01-01 false Functions of agents, including Central Registration Agent, Selling Agent and Fiscal Agent. 108.1620 Section 108.1620 Business Credit and Assistance SMALL BUSINESS ADMINISTRATION NEW MARKETS VENTURE CAPITAL (âNMVCâ) PROGRAM SBA...

  14. Battlefield agent collaboration

    NASA Astrophysics Data System (ADS)

    Budulas, Peter P.; Young, Stuart H.; Emmerman, Philip J.

    2001-09-01

    Small air and ground physical agents (robots) will be ubiquitous on the battlefield of the 21st century, principally to lower the exposure to harm of our ground forces in urban and open terrain scenarios. Teams of small collaborating physical agents conducting tasks such as Reconnaissance, Surveillance, and Target Acquisition (RSTA), intelligence, chemical and biological agent detection, logistics, decoy, sentry; and communications relay will have advanced sensors, communications, and mobility characteristics. It is anticipated that there will be many levels of individual and team collaboration between the soldier and robot, robot to robot, and robot to mother ship. This paper presents applications and infrastructure components that illustrate each of these levels. As an example, consider the application where a team of twenty small robots must rapidly explore and define a building complex. Local interactions and decisions require peer to peer collaboration. Global direction and information fusion warrant a central team control provided by a mother ship. The mother ship must effectively deliver/retrieve, service, and control these robots as well as fuse the information gathered by these highly mobile robot teams. Any level of collaboration requires robust communications, specifically a mobile ad hoc network. The application of fixed ground sensors and mobile robots is also included in this paper. This paper discusses on going research at the U.S. Army Research Laboratory that supports the development of multi-robot collaboration. This research includes battlefield visualization, intelligent software agents, adaptive communications, sensor and information fusion, and multi-modal human computer interaction.

  15. Mobility control agent

    SciTech Connect

    Argabright, P.A.; Phillips, B.L.; Rhudy, J.S.

    1983-05-17

    Polymer mobility control agents useful in supplemental oil recovery processes, which give improved reciprocal relative mobilities, are prepared by initiating the polymerization of a monomer containing a vinyl group with a catalyst comprising a persulfate and ferrous ammonium sulfate. The vinyl monomer is an acrylyl, a vinyl cyanide, a styryl and water soluble salts thereof.

  16. E-Learning Agents

    ERIC Educational Resources Information Center

    Gregg, Dawn G.

    2007-01-01

    Purpose: The purpose of this paper is to illustrate the advantages of using intelligent agents to facilitate the location and customization of appropriate e-learning resources and to foster collaboration in e-learning environments. Design/methodology/approach: This paper proposes an e-learning environment that can be used to provide customized…

  17. Remote Agent Experiment

    NASA Technical Reports Server (NTRS)

    Benard, Doug; Dorais, Gregory A.; Gamble, Ed; Kanefsky, Bob; Kurien, James; Millar, William; Muscettola, Nicola; Nayak, Pandu; Rouquette, Nicolas; Rajan, Kanna; Norvig, Peter (Technical Monitor)

    2000-01-01

    Remote Agent (RA) is a model-based, reusable artificial intelligence (At) software system that enables goal-based spacecraft commanding and robust fault recovery. RA was flight validated during an experiment on board of DS1 between May 17th and May 21th, 1999.

  18. Can Subscription Agents Survive?

    ERIC Educational Resources Information Center

    Tuttle, Marcia

    1985-01-01

    With the saturation of traditional markets for their services, subscription agents have evolved from orders and invoices to serving customers by communicating with librarians and publishers and making automated and paper products available. Magazine fulfillment centers, publisher discounts, and electronic publishing will influence the subscription…

  19. Zoonotic infections in communities of the James Bay Cree territory: An overview of seroprevalence

    PubMed Central

    Sampasa-Kanyinga, Hugues; Lévesque, Benoit; Anassour-Laouan-Sidi, Elhadji; Côté, Suzanne; Serhir, Bouchra; Ward, Brian J; Libman, Michael D; Drebot, Michael A; Makowski, Kai; Dimitrova, Kristina; Ndao, Momar; Dewailly, Éric

    2013-01-01

    The Cree communities of James Bay are at risk for contracting infectious diseases transmitted by wildlife. Data from serological testing for a range of zoonotic infections performed in the general population (six communities), or trappers and their spouses (one community), were abstracted from four population-based studies conducted in Cree territory (Quebec) between 2005 and 2009. Evidence of exposure to Trichinella species, Toxoplasma gondii, Toxocara canis, Echinococcus granulosus, Leptospira species, Coxiella burnetii and Francisella tularensis was verified in all communities, whereas antibodies against Sin Nombre virus and California serogroup viruses (Jamestown Canyon and snowshoe hare viruses) were evaluated in three and six communities, respectively. Seroprevalence varied widely among communities: snowshoe hare virus (1% to 42%), F tularensis (14% to 37%), Leptospira species (10% to 27%), Jamestown Canyon virus (9% to 24%), C burnetii (0% to 18%), T gondii (4% to 12%), T canis (0% to 10%), E granulosus (0% to 4%) and Trichinella species (0% to 1%). No subject had serological evidence of Sin Nombre virus exposure. These data suggest that large proportions of the Cree population have been exposed to at least one of the targeted zoonotic agents. The Cree population, particularly those most heavily exposed to fauna, as well as the medical staff living in these regions, should be aware of these diseases. Greater awareness would not only help to decrease exposures but would also increase the chance of appropriate diagnostic testing. PMID:24421806

  20. Emulsified blasting agents

    SciTech Connect

    Chironis, N.P.

    1985-01-01

    This article describes an improved blasting agent which is being tailor-blended with bulk ANFO to provide more explosive energy and better water resistance when the blasting conditions call for it. The proportions of the emulsion/ANFO mix are easily changed at the blasthole site because both materials can be selectively mixed in modified bulk-explosive trucks before loading the blasting agents into the holes. Such blends are helping speed stripping at a number of surface mines and are leading to cost savings in production, ranging from 10% to 30%, depending upon application, even though the actual cost of a blend will be higher than if bulk ANFO is used alone.

  1. Rigid bifunctional chelating agents

    DOEpatents

    Sweet, Mark P.; Mease, Ronnie C.; Srivastava, Suresh C.

    2000-02-08

    Bicyclo[2.2.2]octane-2,3 diamine-N,N,N',N'-tetraacetic acids (BODTA) and bicyclo[2.2.1]heptane-2,3 diamine-N,N,N',N'-tetraacetic acid (BHDTA) are chelating agents useful in forming detectably labeled bioconjugate compounds for diagnostic and therapeutic purposes. New compounds and processes of forming BODTA and BHDTA are disclosed. Radioimmunoconjugates of the present invention show high and prolonged tumor uptake with low normal tissue uptakes.

  2. Rigid bifunctional chelating agents

    DOEpatents

    Sweet, M.P.; Mease, R.C.; Srivastava, S.C.

    1998-07-21

    Bicyclo[2.2.2] octane-2,3 diamine-N,N,N`,N`-tetraacetic acids (BODTA) and bicyclo[2.2.1] heptane-2,3 diamine-N,N,N`,N`-tetraacetic acid (BHDTA) are chelating agents useful in forming detectably labeled bioconjugate compounds for diagnostic and therapeutic purposes. New compounds and processes of forming BODTA and BHDTA are disclosed. Radioimmunoconjugates of the present invention show high and prolonged tumor uptake with low normal tissue uptakes.

  3. Rigid bifunctional chelating agents

    DOEpatents

    Sweet, Mark P.; Mease, Ronnie C.; Srivastava, Suresh C.

    1998-07-21

    Bicyclo›2.2.2! octane-2,3 diamine-N,N,N',N'-tetraacetic acids (BODTA) and bicyclo›2.2.1! heptane-2,3 diamine-N,N,N',N'-tetraacetic acid (BHDTA) are chelating agents useful in forming detectably labeled bioconjugate compounds for diagnostic and therapeutic purposes. New compounds and processes of forming BODTA and BHDTA are disclosed. Radioimmunoconjugates of the present invention show high and prolonged tumor uptake with low normal tissue uptakes.

  4. Surface polymerization agents

    SciTech Connect

    Taylor, C.; Wilkerson, C.

    1996-12-01

    This is the final report of a 1-year, Laboratory-Directed R&D project at LANL. A joint technical demonstration was proposed between US Army Missile Command (Redstone Arsenal) and LANL. Objective was to demonstrate that an unmanned vehicle or missile could be used as a platform to deliver a surface polymerization agent in such a manner as to obstruct the filters of an air-breathing mechanism, resulting in operational failure.

  5. [Two cases of tick-borne tularemia in Yozgat province, Turkey].

    PubMed

    Yeşilyurt, Murat; Kılıç, Selçuk; Cağaşar, Ozlem; Celebi, Bekir; Gül, Serdar

    2011-10-01

    Tularemia which has a worldwide distribution, is a zoonotic infection caused by Francisella tularensis. F.tularensis can infect a wide range of animals and can be transmitted to humans in a variety of ways, the most common being by the bite of an infected arthropod vector (usually tick) in the USA and Europe. The clinical presentations have been classically divided into ulceroglandular, glandular, oculoglandular, pharyngeal, respiratory, and typhoidal tularemia depending on the route of transmission. Arthropod-borne infection generally leads to the ulceroglandular form of tularemia. In Turkey, oropharyngeal form which is related to the consumption of contaminated water, is the most common presentation of tularemia. In this report, two cases of ulceroglandular tularemia which developed as a consequence of tick bite in Yozgat province have been presented. A 33-year-old female patient was admitted to the hospital with a tender lump on the right axilla. Empiric antibiotic treatment with amoxicillin clavulanate did not lead to an improvement in the painful axillary mass. She reported a tick bite on her right shoulder before development of fever, chills and regional tender lump. On physical examination, hyperemia was seen on the shoulder, with enlarged tender right axillary lymph node. The clinical diagnosis of suspected ulceroglandular tularemia was confirmed by the seroconversion (1/160 and 1/1280 titers in acute and convelescent sera, respectively) with microagglutination test (MAT) and F.tularensis DNA positivity in lymph node aspirate by polymerase chain reaction. The agent was identified as F.tularensis subsp. holarctica based on the results of amplification of target RD1 gene. Second case, a 18-year-old male, was admitted to our hospital with a-week history of sudden onset of fever, headache, generalized aches, vomiting, nause, and tender lump on the left axilla. On physical examination, an inflammatory eschar was seen on his scalp with enlarged cervical lymph

  6. Collaborating with Autonomous Agents

    NASA Technical Reports Server (NTRS)

    Trujillo, Anna C.; Cross, Charles D.; Fan, Henry; Hempley, Lucas E.; Motter, Mark A.; Neilan, James H.; Qualls, Garry D.; Rothhaar, Paul M.; Tran, Loc D.; Allen, B. Danette

    2015-01-01

    With the anticipated increase of small unmanned aircraft systems (sUAS) entering into the National Airspace System, it is highly likely that vehicle operators will be teaming with fleets of small autonomous vehicles. The small vehicles may consist of sUAS, which are 55 pounds or less that typically will y at altitudes 400 feet and below, and small ground vehicles typically operating in buildings or defined small campuses. Typically, the vehicle operators are not concerned with manual control of the vehicle; instead they are concerned with the overall mission. In order for this vision of high-level mission operators working with fleets of vehicles to come to fruition, many human factors related challenges must be investigated and solved. First, the interface between the human operator and the autonomous agent must be at a level that the operator needs and the agents can understand. This paper details the natural language human factors e orts that NASA Langley's Autonomy Incubator is focusing on. In particular these e orts focus on allowing the operator to interact with the system using speech and gestures rather than a mouse and keyboard. With this ability of the system to understand both speech and gestures, operators not familiar with the vehicle dynamics will be able to easily plan, initiate, and change missions using a language familiar to them rather than having to learn and converse in the vehicle's language. This will foster better teaming between the operator and the autonomous agent which will help lower workload, increase situation awareness, and improve performance of the system as a whole.

  7. Hydroxypyridonate and hydroxypyrimidinone chelating agents

    DOEpatents

    Raymond, Kenneth N.; Doble, Daniel M.; Sunderland, Christopher J.; Thompson, Marlon

    2005-01-25

    The present invention provides hydroxypyridinone and hydroxypyrimidone chelating agents. Also provides are Gd(III) complexes of these agents, which are useful as contrast enhancing agents for magnetic resonance imaging. The invention also provides methods of preparing the compounds of the invention, as well as methods of using the compounds in magnetic resonance imaging applications.

  8. Chemical warfare agents

    PubMed Central

    Ganesan, K.; Raza, S. K.; Vijayaraghavan, R.

    2010-01-01

    Among the Weapons of Mass Destruction, chemical warfare (CW) is probably one of the most brutal created by mankind in comparison with biological and nuclear warfare. Chemical weapons are inexpensive and are relatively easy to produce, even by small terrorist groups, to create mass casualties with small quantities. The characteristics of various CW agents, general information relevant to current physical as well as medical protection methods, detection equipment available and decontamination techniques are discussed in this review article. A brief note on Chemical Weapons Convention is also provided. PMID:21829312

  9. Holograms as Teaching Agents

    NASA Astrophysics Data System (ADS)

    Walker, Robin A.

    2013-02-01

    Hungarian physicist Dennis Gabor won the Pulitzer Prize for his 1947 introduction of basic holographic principles, but it was not until the invention of the laser in 1960 that research scientists, physicians, technologists and the general public began to seriously consider the interdisciplinary potentiality of holography. Questions around whether and when Three-Dimensional (3-D) images and systems would impact American entertainment and the arts would be answered before educators, instructional designers and students would discover how much Three-Dimensional Hologram Technology (3DHT) would affect teaching practices and learning environments. In the following International Symposium on Display Holograms (ISDH) poster presentation, the author features a traditional board game as well as a reflection hologram to illustrate conventional and evolving Three-Dimensional representations and technology for education. Using elements from the American children's toy Operation® (Hasbro, 2005) as well as a reflection hologram of a human brain (Ko, 1998), this poster design highlights the pedagogical effects of 3-D images, games and systems on learning science. As teaching agents, holograms can be considered substitutes for real objects, (human beings, organs, and animated characters) as well as agents (pedagogical, avatars, reflective) in various learning environments using many systems (direct, emergent, augmented reality) and electronic tools (cellphones, computers, tablets, television). In order to understand the particular importance of utilizing holography in school, clinical and public settings, the author identifies advantages and benefits of using 3-D images and technology as instructional tools.

  10. Lipid-lowering agents.

    PubMed

    Ewang-Emukowhate, Mfon; Wierzbicki, Anthony S

    2013-09-01

    The role of lipid lowering in reducing the risk of mortality and morbidity from cardiovascular disease (CVD) is well established. Treatment particularly aimed at decreasing low-density lipoprotein cholesterol (LDL-C) is effective in reducing the risk of death from coronary heart disease and stroke. Statins form the cornerstone of treatment. However, in some individuals with a high risk of CVD who are unable to achieve their target LDL-C due to either intolerance or lack of efficacy, there is the need for alternative therapies. This review provides an overview of the different classes of currently available lipid-lowering medications including statins, fibrates, bile acid sequestrants (resins), and omega-3 fatty acids. Data are presented on their indications, pharmacology, and the relevant end point clinical trial data with these drugs. It also discusses the human trial data on some novel therapeutic agents that are being developed including those for homozygous familial hypercholesterolemia--the antisense oligonucleotide mipomersen and the microsomal transfer protein inhibitor lomitapide. Data are presented on phase II and III trials on agents with potentially wider applications, cholesterol ester transfer protein inhibitors and proprotein convertase subtilisin kexin 9 inhibitors. The data on a licensed gene therapy for lipoprotein lipase deficiency are also presented. PMID:23811423

  11. [Bacteriophages as antibacterial agents].

    PubMed

    Shasha, Shaul M; Sharon, Nehama; Inbar, Michael

    2004-02-01

    Bacteriophages are viruses that only infect bacteria. They have played an important role in the development of molecular biology and have been used as anti-bacterial agents. Since their independent discovery by Twort and d'Herelle, they have been extensively used to prevent and treat bacterial infections, mainly in Eastern Europe and the former Soviet Union. In western countries this method has been sporadically employed on humans and domesticated animals. However, the discovery and widespread use of antibiotics, coupled with doubts about the efficacy of phage therapy, led to an eclipse in the use of phage in medicine. The emergence of antibiotic resistant bacteria, especially strains that are multiply resistant, has resulted in a renewed interest in alternatives to conventional drugs. One of the possible replacements for antibiotics is the use of bacteriophages as antimicrobial agents. This brief review aims to describe the history of bacteriophage and early clinical studies on their use in bacterial disease prophylaxis and therapy, and discuss the advantages and disadvantages of bacteriophage in this regard.

  12. Lipid-lowering agents.

    PubMed

    Ewang-Emukowhate, Mfon; Wierzbicki, Anthony S

    2013-09-01

    The role of lipid lowering in reducing the risk of mortality and morbidity from cardiovascular disease (CVD) is well established. Treatment particularly aimed at decreasing low-density lipoprotein cholesterol (LDL-C) is effective in reducing the risk of death from coronary heart disease and stroke. Statins form the cornerstone of treatment. However, in some individuals with a high risk of CVD who are unable to achieve their target LDL-C due to either intolerance or lack of efficacy, there is the need for alternative therapies. This review provides an overview of the different classes of currently available lipid-lowering medications including statins, fibrates, bile acid sequestrants (resins), and omega-3 fatty acids. Data are presented on their indications, pharmacology, and the relevant end point clinical trial data with these drugs. It also discusses the human trial data on some novel therapeutic agents that are being developed including those for homozygous familial hypercholesterolemia--the antisense oligonucleotide mipomersen and the microsomal transfer protein inhibitor lomitapide. Data are presented on phase II and III trials on agents with potentially wider applications, cholesterol ester transfer protein inhibitors and proprotein convertase subtilisin kexin 9 inhibitors. The data on a licensed gene therapy for lipoprotein lipase deficiency are also presented.

  13. [New agents for hypercholesterolemia].

    PubMed

    Pintó, Xavier; García Gómez, María Carmen

    2016-02-19

    An elevated proportion of high cardiovascular risk patients do not achieve the therapeutic c-LDL goals. This owes to physicians' inappropriate or insufficient use of cholesterol lowering medications or to patients' bad tolerance or therapeutic compliance. Another cause is an insufficient efficacy of current cholesterol lowering drugs including statins and ezetimibe. In addition, proprotein convertase subtilisin kexin type 9 inhibitors are a new cholesterol lowering medications showing safety and high efficacy to reduce c-LDL in numerous already performed or underway clinical trials, potentially allowing an optimal control of hypercholesterolemia in most patients. Agents inhibiting apolipoprotein B synthesis and microsomal transfer protein are also providing a new potential to decrease cholesterol in patients with severe hypercholesterolemia and in particular in homozygote familial hypercholesterolemia. Last, cholesteryl ester transfer protein inhibitors have shown powerful effects on c-HDL and c-LDL, although their efficacy in cardiovascular prevention and safety has not been demonstrated yet. We provide in this article an overview of the main characteristics of therapeutic agents for hypercholesterolemia, which have been recently approved or in an advanced research stage.

  14. Advances in antithrombotic agents.

    PubMed

    Chakrabarti, Ranjan; Das, Saibal Kumar

    2007-07-01

    Thrombosis is the condition where an imbalance in the homeostatic mechanism results in unwanted intravascular thrombus formation. Imbalances in this highly regulated process of coagulation and anticoagulation can lead to a variety of pathophysiological conditions leading to stroke, pulmonary heart attack and other serious conditions. In the western world, thromboembolic diseases are the leading cause of morbidity and mortality. Remarkable progress has occurred over the last decade in the development of antithrombotic drugs, which can be classified into 3 major categories - Anticoagulants, Antiplatelets and thrombolytics. Increased understanding of the pathobiology of thrombotic and vascular disorders has helped researchers to target novel pathways involving the coagulation, thrombolytic, fibrinolytic and integrin systems. Traditionally aspirin and unfractionated heparin was used for myocardial infarction. Newer antiplatelet agents such as, clopidogrel, GP IIb/IIIa inhibitors, low molecular weight heparin, direct thrombin inhibitors and several improved thrombolytic agents have been introduced for clinical use. This review will discuss different important drugs, which have been launched in recent years and also some new targets pursued by different companies. PMID:17630943

  15. Identification of disulfide bond isomerase substrates reveals bacterial virulence factors

    PubMed Central

    Ren, Guoping; Champion, Matthew M.; Huntley, Jason F.

    2014-01-01

    Summary Bacterial pathogens are exposed to toxic molecules inside the host and require efficient systems to form and maintain correct disulfide bonds for protein stability and function. The intracellular pathogen Francisella tularensis encodes a disulfide bond formation protein ortholog, DsbA, which previously was reported to be required for infection of macrophages and mice. However, the molecular mechanisms by which F. tularensis DsbA contributes to virulence are unknown. Here, we demonstrate that F. tularensis DsbA is a bifunctional protein that oxidizes and, more importantly, isomerizes complex disulfide connectivity in substrates. A single amino acid in the conserved cis-proline loop of the DsbA thioredoxin domain was shown to modulate both isomerase activity and F. tularensis virulence. Trapping experiments in F. tularensis identified over 50 F. tularensis DsbA substrates, including outer membrane proteins, virulence factors, and many hypothetical proteins. Six of these hypothetical proteins were randomly selected and deleted, revealing two novel proteins, FTL_1548 and FTL_1709, which are required for F. tularensis virulence. We propose that the extreme virulence of F. tularensis is partially due to the bifunctional nature of DsbA, that many of the newly-identified substrates are required for virulence, and that the development of future DsbA inhibitors could have broad anti-bacterial implications. PMID:25257164

  16. Flexible, secure agent development framework

    DOEpatents

    Goldsmith; Steven Y.

    2009-04-07

    While an agent generator is generating an intelligent agent, it can also evaluate the data processing platform on which it is executing, in order to assess a risk factor associated with operation of the agent generator on the data processing platform. The agent generator can retrieve from a location external to the data processing platform an open site that is configurable by the user, and load the open site into an agent substrate, thereby creating a development agent with code development capabilities. While an intelligent agent is executing a functional program on a data processing platform, it can also evaluate the data processing platform to assess a risk factor associated with performing the data processing function on the data processing platform.

  17. Fluoroquinolone antimicrobial agents.

    PubMed Central

    Wolfson, J S; Hooper, D C

    1989-01-01

    The fluoroquinolones, a new class of potent orally absorbed antimicrobial agents, are reviewed, considering structure, mechanisms of action and resistance, spectrum, variables affecting activity in vitro, pharmacokinetic properties, clinical efficacy, emergence of resistance, and tolerability. The primary bacterial target is the enzyme deoxyribonucleic acid gyrase. Bacterial resistance occurs by chromosomal mutations altering deoxyribonucleic acid gyrase and decreasing drug permeation. The drugs are bactericidal and potent in vitro against members of the family Enterobacteriaceae, Haemophilus spp., and Neisseria spp., have good activity against Pseudomonas aeruginosa and staphylococci, and (with several exceptions) are less potent against streptococci and have fair to poor activity against anaerobic species. Potency in vitro decreases in the presence of low pH, magnesium ions, or urine but is little affected by different media, increased inoculum, or serum. The effects of the drugs in combination with a beta-lactam or aminoglycoside are often additive, occasionally synergistic, and rarely antagonistic. The agents are orally absorbed, require at most twice-daily dosing, and achieve high concentrations in urine, feces, and kidney and good concentrations in lung, bone, prostate, and other tissues. The drugs are efficacious in treatment of a variety of bacterial infections, including uncomplicated and complicated urinary tract infections, bacterial gastroenteritis, and gonorrhea, and show promise for therapy of prostatitis, respiratory tract infections, osteomyelitis, and cutaneous infections, particularly when caused by aerobic gram-negative bacilli. Fluoroquinolones have also proved to be efficacious for prophylaxis against travelers' diarrhea and infection with gram-negative bacilli in neutropenic patients. The drugs are effective in eliminating carriage of Neisseria meningitidis. Patient tolerability appears acceptable, with gastrointestinal or central nervous

  18. Biological agents and pregnancy.

    PubMed

    Ekblad, U

    1995-08-01

    Pregnant women are exposed to many biological, eg microbial, agents, which are potentially harmful to the fetus. The reported rates of vertical transmission of hepatitis B and human immunodeficiency virus vary between 3 to 90% and 0 to 65%, respectively. The susceptibility to hepatitis B and human immunodeficiency infection is increased in pregnant physicians, midwives, and nurses because of the bloodborne nature of these viruses. Also, TORCH (toxoplasmosis-rubella-cytomegalovirus-herpes) infections, acquired during pregnancy, may result in congenital infection, and serious sequelae in the neonatal period or years after birth. Schoolteachers and daycare personnel have an increased risk of perinatal varicella, "fifth disease," and mumps. Perinatal listeriosis affects one in 20,000 births and may result in fetal wastage. Because of the risk of the possibility of vertical transmission, immunization during pregnancy with live virus vaccines is not recommended. PMID:8520961

  19. Arylthiosemicarbazones as antileishmanial agents.

    PubMed

    Manzano, José Ignacio; Cochet, Florent; Boucherle, Benjamin; Gómez-Pérez, Verónica; Boumendjel, Ahcène; Gamarro, Francisco; Peuchmaur, Marine

    2016-11-10

    Based on a screening process, we targeted substituted thiosemicarbazone as potential antileishmanial agents. Our objective was to identify the key structural elements contributing to the anti-parasite activity that might be used for development of effective drugs. A series of 32 compounds was synthesized and their efficacy was evaluated against the clinically relevant intracellular amastigotes of Leishmania donovani. From these, 22 compounds showed EC50 values below 10 μM with the most active derivative (compound 14) showing an EC50 of 0.8 μM with very low toxicity on two different mammalian cell lines. The most relevant structural elements required for higher activity indicate that the presence of a fused bicyclic aromatic ring such as a naphthalene bearing an alkyl or an alkoxy group substituent are prerequisites. Owing to the easy synthesis, high activity and low toxicity, the most active compounds could be considered as a lead for further development.

  20. Itch Management: Topical Agents.

    PubMed

    Metz, Martin; Staubach, Petra

    2016-01-01

    Chronic pruritus is a common problem in patients with inflammatory skin diseases as well as in subjects with dry or sensitive skin. Regardless of the underlying cause of the pruritus, a topical therapy is not only useful but most often necessary to achieve symptom control. A good topical therapy should fulfill different functions. An optimal basic therapy based on the condition of the skin is important to repair epithelial barrier defects and to rehydrate the skin. An adequate disease-specific topical therapy is crucial for inflamed skin, e.g. anti-inflammatory topical therapy is an important part in the treatment of atopic dermatitis. Finally, the use of specific antipruritic substances can help to improve pruritus in patients irrespective of the underlying disease. Here, we summarize topical agents used in the treatment of chronic pruritus. PMID:27578070

  1. [Ribonucleases as antiviral agents].

    PubMed

    Il'inskaia, O N; Shakh Makhmud, R

    2014-01-01

    Many ribonucleases (RNases) are able to inhibit the reproduction of viruses in infected cell cultures and laboratory animals, but molecular mechanisms of their antiviral activity remain unclear. The review observes the most known RNases which possess established antiviral effects, actually intracellular RNases (RNase L, MCPIPI protein, eosinophylic RNases) as well as exogenously applied ones (RNase A, BS-RNase, onconase, binase, synthetic RNases). Attention is given on two important but not always obligatory aspects in molecule of RNases, which have antiviral properties: catalytic activity and ability to the dimerization. The hypothetic scheme of virus elimination by exogenous RNases, that reflects possible types of interaction of viruses and RNases with a cell, is proposed. The evidence for RNases as classical components of immune defense which are perspective agents for development of new antiviral therapeutics is produced.

  2. Agent-based enterprise integration

    SciTech Connect

    N. M. Berry; C. M. Pancerella

    1998-12-01

    The authors are developing and deploying software agents in an enterprise information architecture such that the agents manage enterprise resources and facilitate user interaction with these resources. The enterprise agents are built on top of a robust software architecture for data exchange and tool integration across heterogeneous hardware and software. The resulting distributed multi-agent system serves as a method of enhancing enterprises in the following ways: providing users with knowledge about enterprise resources and applications; accessing the dynamically changing enterprise; locating enterprise applications and services; and improving search capabilities for applications and data. Furthermore, agents can access non-agents (i.e., databases and tools) through the enterprise framework. The ultimate target of the effort is the user; they are attempting to increase user productivity in the enterprise. This paper describes their design and early implementation and discusses the planned future work.

  3. Collaborating Fuzzy Reinforcement Learning Agents

    NASA Technical Reports Server (NTRS)

    Berenji, Hamid R.

    1997-01-01

    Earlier, we introduced GARIC-Q, a new method for doing incremental Dynamic Programming using a society of intelligent agents which are controlled at the top level by Fuzzy Relearning and at the local level, each agent learns and operates based on ANTARCTIC, a technique for fuzzy reinforcement learning. In this paper, we show that it is possible for these agents to compete in order to affect the selected control policy but at the same time, they can collaborate while investigating the state space. In this model, the evaluator or the critic learns by observing all the agents behaviors but the control policy changes only based on the behavior of the winning agent also known as the super agent.

  4. [Contact sensitization to external agents].

    PubMed

    Erdmann, S M; Merk, H-F

    2003-04-01

    The following review describes contact sensitization to topically applied medications--especially topical dermatological agents--and to external agents in the broadest sense. Particularly skin care products constitute a special source for sensitization due to their widespread use. Especially fragrances and preservatives in cosmetics play an important global role in eliciting contact allergies. Because of the extremely broad spectrum covered by the active and adjuvant ingredients contained in external agents, the following discussion focuses on specific substance groups.

  5. Broad-spectrum antiviral agents

    PubMed Central

    Zhu, Jun-Da; Meng, Wen; Wang, Xiao-Jia; Wang, Hwa-Chain R.

    2015-01-01

    Development of highly effective, broad-spectrum antiviral agents is the major objective shared by the fields of virology and pharmaceutics. Antiviral drug development has focused on targeting viral entry and replication, as well as modulating cellular defense system. High throughput screening of molecules, genetic engineering of peptides, and functional screening of agents have identified promising candidates for development of optimal broad-spectrum antiviral agents to intervene in viral infection and control viral epidemics. This review discusses current knowledge, prospective applications, opportunities, and challenges in the development of broad-spectrum antiviral agents. PMID:26052325

  6. Incorporating BDI Agents into Human-Agent Decision Making Research

    NASA Astrophysics Data System (ADS)

    Kamphorst, Bart; van Wissen, Arlette; Dignum, Virginia

    Artificial agents, people, institutes and societies all have the ability to make decisions. Decision making as a research area therefore involves a broad spectrum of sciences, ranging from Artificial Intelligence to economics to psychology. The Colored Trails (CT) framework is designed to aid researchers in all fields in examining decision making processes. It is developed both to study interaction between multiple actors (humans or software agents) in a dynamic environment, and to study and model the decision making of these actors. However, <