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Sample records for francisella tularensis genes

  1. Targeted gene disruption in Francisella tularensis by group II introns.

    PubMed

    Rodriguez, Stephen A; Davis, Greg; Klose, Karl E

    2009-11-01

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

  2. Targeted inactivation of francisella tularensis genes by group II introns.

    PubMed

    Rodriguez, Stephen A; Yu, Jieh-Juen; Davis, Greg; Arulanandam, Bernard P; Klose, Karl E

    2008-05-01

    Studies of the molecular mechanisms of pathogenesis of Francisella tularensis, the causative agent of tularemia, have been hampered by a lack of genetic techniques for rapid targeted gene disruption in the most virulent subspecies. Here we describe efficient targeted gene disruption in F. tularensis utilizing mobile group II introns (targetrons) specifically optimized for F. tularensis. Utilizing a targetron targeted to blaB, which encodes ampicillin resistance, we showed that the system works at high efficiency in three different subspecies: F. tularensis subsp. tularensis, F. tularensis subsp. holarctica, and "F. tularensis subsp. novicida." A targetron was also utilized to inactivate F. tularensis subsp. holarctica iglC, a gene required for virulence. The iglC gene is located within the Francisella pathogenicity island (FPI), which has been duplicated in the most virulent subspecies. Importantly, the iglC targetron targeted both copies simultaneously, resulting in a strain mutated in both iglC genes in a single step. This system will help illuminate the contributions of specific genes, and especially those within the FPI, to the pathogenesis of this poorly studied organism.

  3. 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...

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

    PubMed

    Brunton, J; Steele, S; Miller, C; Lovullo, E; Taft-Benz, S; Kawula, T

    2015-08-01

    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 cells, including, but not limited to, macrophages and epithelial cells. We developed a luminescence reporter system to facilitate a large-scale transposon mutagenesis screen to identify genes required for growth in macrophage and epithelial cell lines. We screened 7,454 individual mutants, 269 of which exhibited reduced intracellular growth. Transposon insertions in the 269 growth-defective strains mapped to 68 different genes. FTT_0924, a gene of unknown function but highly conserved among Francisella species, was identified in this screen to be defective for intracellular growth within both macrophage and epithelial cell lines. FTT_0924 was required for full Schu S4 virulence in a murine pulmonary infection model. The ΔFTT_0924 mutant bacterial membrane is permeable when replicating in hypotonic solution and within macrophages, resulting in strongly reduced viability. The permeability and reduced viability were rescued when the mutant was grown in a hypertonic solution, indicating that FTT_0924 is required for resisting osmotic stress. The ΔFTT_0924 mutant was also significantly more sensitive to β-lactam antibiotics than Schu S4. Taken together, the data strongly suggest that FTT_0924 is required for maintaining peptidoglycan integrity and virulence.

  5. Identifying Francisella tularensis Genes Required for Growth in Host Cells

    PubMed Central

    Brunton, J.; Steele, S.; Miller, C.; Lovullo, E.; Taft-Benz, S.

    2015-01-01

    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 cells, including, but not limited to, macrophages and epithelial cells. We developed a luminescence reporter system to facilitate a large-scale transposon mutagenesis screen to identify genes required for growth in macrophage and epithelial cell lines. We screened 7,454 individual mutants, 269 of which exhibited reduced intracellular growth. Transposon insertions in the 269 growth-defective strains mapped to 68 different genes. FTT_0924, a gene of unknown function but highly conserved among Francisella species, was identified in this screen to be defective for intracellular growth within both macrophage and epithelial cell lines. FTT_0924 was required for full Schu S4 virulence in a murine pulmonary infection model. The ΔFTT_0924 mutant bacterial membrane is permeable when replicating in hypotonic solution and within macrophages, resulting in strongly reduced viability. The permeability and reduced viability were rescued when the mutant was grown in a hypertonic solution, indicating that FTT_0924 is required for resisting osmotic stress. The ΔFTT_0924 mutant was also significantly more sensitive to β-lactam antibiotics than Schu S4. Taken together, the data strongly suggest that FTT_0924 is required for maintaining peptidoglycan integrity and virulence. PMID:25987704

  6. 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

  7. Francisella tularensis Subspecies holarctica, Tasmania, Australia, 2011

    PubMed Central

    Jackson, Justin; McGregor, Alistair; Cooley, Louise; Ng, Jimmy; Brown, Mitchell; Ong, Chong Wei; Darcy, Catharine

    2012-01-01

    We report a case of ulceroglandular tularemia that developed in a woman after she was bitten by a ringtail possum (Pseudocheirus peregrinus) in a forest in Tasmania, Australia. Francisella tularensis subspecies holarctica was identified. This case indicates the emergence of F. tularensis type B in the Southern Hemisphere. PMID:22931809

  8. Detection of Francisella tularensis in voles in Finland.

    PubMed

    Rossow, Heidi; Sissonen, Susanna; Koskela, Katja A; Kinnunen, Paula M; Hemmilä, Heidi; Niemimaa, Jukka; Huitu, Otso; Kuusi, Markku; Vapalahti, Olli; Henttonen, Heikki; Nikkari, Simo

    2014-03-01

    Francisella tularensis is a highly virulent intracellular bacterium causing the zoonotic disease tularemia. It recurrently causes human and animal outbreaks in northern Europe, including Finland. Although F. tularensis infects several mammal species, only rodents and lagomorphs seem to have importance in its ecology. Peak densities of rodent populations may trigger tularemia outbreaks in humans; however, it is still unclear to which extent rodents or other small mammals maintain F. tularensis in nature. The main objective of this study was to obtain information about the occurrence of F. tularensis in small mammals in Finland. We snap-trapped 547 wild small mammals representing 11 species at 14 locations around Finland during 6 years and screened them for the presence of F. tularensis DNA using PCR analysis. High copy number of F. tularensis-specific DNA was detected in tissue samples of five field voles (Microtus agrestis) originating from one location and 2 years. According to DNA sequences of the bacterial 23S ribosomal RNA gene amplified from F. tularensis-infected voles, the infecting agent belongs to the subspecies holarctica. To find out the optimal tissue for tularemia screening in voles, we compared the amounts of F. tularensis DNA in lungs, liver, spleen, and kidney of the infected animals. F. tularensis DNA was detectable in high levels in all four organs except for one animal, whose kidney was F. tularensis DNA-negative. Thus, at least liver, lung, and spleen seem suitable for F. tularensis screening in voles. Thus, liver, lung, and spleen all seem suitable for F. tularensis screening in voles. In conclusion, field voles can be heavily infected with F. tularensis subsp. holarctica and thus potentially serve as the source of infection in humans and other mammals.

  9. Detection of Francisella tularensis in Voles in Finland

    PubMed Central

    Sissonen, Susanna; Koskela, Katja A.; Kinnunen, Paula M.; Hemmilä, Heidi; Niemimaa, Jukka; Huitu, Otso; Kuusi, Markku; Vapalahti, Olli; Henttonen, Heikki; Nikkari, Simo

    2014-01-01

    Abstract Francisella tularensis is a highly virulent intracellular bacterium causing the zoonotic disease tularemia. It recurrently causes human and animal outbreaks in northern Europe, including Finland. Although F. tularensis infects several mammal species, only rodents and lagomorphs seem to have importance in its ecology. Peak densities of rodent populations may trigger tularemia outbreaks in humans; however, it is still unclear to which extent rodents or other small mammals maintain F. tularensis in nature. The main objective of this study was to obtain information about the occurrence of F. tularensis in small mammals in Finland. We snap-trapped 547 wild small mammals representing 11 species at 14 locations around Finland during 6 years and screened them for the presence of F. tularensis DNA using PCR analysis. High copy number of F. tularensis-specific DNA was detected in tissue samples of five field voles (Microtus agrestis) originating from one location and 2 years. According to DNA sequences of the bacterial 23S ribosomal RNA gene amplified from F. tularensis–infected voles, the infecting agent belongs to the subspecies holarctica. To find out the optimal tissue for tularemia screening in voles, we compared the amounts of F. tularensis DNA in lungs, liver, spleen, and kidney of the infected animals. F. tularensis DNA was detectable in high levels in all four organs except for one animal, whose kidney was F. tularensis DNA-negative. Thus, at least liver, lung, and spleen seem suitable for F. tularensis screening in voles. Thus, liver, lung, and spleen all seem suitable for F. tularensis screening in voles. In conclusion, field voles can be heavily infected with F. tularensis subsp. holarctica and thus potentially serve as the source of infection in humans and other mammals. PMID:24575824

  10. Role of Glycosylation/Deglycolysation Processes in Francisella tularensis Pathogenesis.

    PubMed

    Barel, Monique; Charbit, Alain

    2017-01-01

    Francisella tularensis is able to invade, survive and replicate inside a variety of cell types. However, in vivo F. tularensis preferentially enters host macrophages where it rapidly escapes to the cytosol to avoid phagosomal stresses and to multiply to high numbers. We previously showed that human monocyte infection by F. tularensis LVS triggered deglycosylation of the glutamine transporter SLC1A5. However, this deglycosylation, specifically induced by Francisella infection, was not restricted to SLC1A5, suggesting that host protein deglycosylation processes in general might contribute to intracellular bacterial adaptation. Indeed, we later found that Francisella infection modulated the transcription of numerous glycosidase and glycosyltransferase genes in human macrophages and analysis of cell extracts revealed an important increase of N and O-protein glycosylation. In eukaryotic cells, glycosylation has significant effects on protein folding, conformation, distribution, stability, and activity and dysfunction of protein glycosylation may lead to development of diseases like cancer and pathogenesis of infectious diseases. Pathogenic bacteria have also evolved dedicated glycosylation machineries and have notably been shown to use these glycoconjugates as ligands to specifically interact with the host. In this review, we will focus on Francisella and summarize our current understanding of the importance of these post-translational modifications on its intracellular niche adaptation.

  11. Role of Glycosylation/Deglycolysation Processes in Francisella tularensis Pathogenesis

    PubMed Central

    Barel, Monique; Charbit, Alain

    2017-01-01

    Francisella tularensis is able to invade, survive and replicate inside a variety of cell types. However, in vivo F. tularensis preferentially enters host macrophages where it rapidly escapes to the cytosol to avoid phagosomal stresses and to multiply to high numbers. We previously showed that human monocyte infection by F. tularensis LVS triggered deglycosylation of the glutamine transporter SLC1A5. However, this deglycosylation, specifically induced by Francisella infection, was not restricted to SLC1A5, suggesting that host protein deglycosylation processes in general might contribute to intracellular bacterial adaptation. Indeed, we later found that Francisella infection modulated the transcription of numerous glycosidase and glycosyltransferase genes in human macrophages and analysis of cell extracts revealed an important increase of N and O-protein glycosylation. In eukaryotic cells, glycosylation has significant effects on protein folding, conformation, distribution, stability, and activity and dysfunction of protein glycosylation may lead to development of diseases like cancer and pathogenesis of infectious diseases. Pathogenic bacteria have also evolved dedicated glycosylation machineries and have notably been shown to use these glycoconjugates as ligands to specifically interact with the host. In this review, we will focus on Francisella and summarize our current understanding of the importance of these post-translational modifications on its intracellular niche adaptation. PMID:28377902

  12. Genome-Wide Identification of Francisella tularensis Virulence Determinants▿

    PubMed Central

    Su, Jingliang; Yang, Jun; Zhao, Daimin; Kawula, Thomas H.; Banas, Jeffrey A.; Zhang, Jing-Ren

    2007-01-01

    Francisella tularensis is a gram-negative pathogen that causes life-threatening infections in humans and has potential for use as a biological weapon. The genetic basis of the F. tularensis virulence is poorly understood. This study screened a total of 3,936 transposon mutants of the live vaccine strain for infection in a mouse model of respiratory tularemia by signature-tagged mutagenesis. We identified 341 mutants attenuated for infection in the lungs. The transposon disruptions were mapped to 95 different genes, virtually all of which are also present in the genomes of other F. tularensis strains, including human pathogenic F. tularensis strain Schu S4. A small subset of these attenuated mutants carried insertions in the genes encoding previously known virulence factors, but the majority of the identified genes have not been previously linked to F. tularensis virulence. Among these are genes encoding putative membrane proteins, proteins associated with stress responses, metabolic proteins, transporter proteins, and proteins with unknown functions. Several attenuated mutants contained disruptions in a putative capsule locus which partially resembles the poly-γ-glutamate capsule biosynthesis locus of Bacillus anthracis, the anthrax agent. Deletional mutation analysis confirmed that this locus is essential for F. tularensis virulence. PMID:17420240

  13. 21 CFR 866.3280 - Francisella tularensis serological reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Francisella tularensis in serum or to identify Francisella tularensis in cultured isolates derived from... provides epidemiological information on this disease. Tularemia is a desease principally of rodents, but... fleas and ticks. The disease takes on several forms depending upon the site of infection, such as...

  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. 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.

  16. Molecular method for discrimination between Francisella tularensis and Francisella-like endosymbionts.

    PubMed

    Escudero, Raquel; Toledo, A; Gil, Horacio; Kovácsová, Katarina; Rodríguez-Vargas, Manuela; Jado, Isabel; García-Amil, Cristina; Lobo, Bruno; Bhide, Mangesh; Anda, Pedro

    2008-09-01

    Environmental studies on the distribution of Francisella spp. are hampered by the frequency of Francisella-like endosymbionts that can produce a misleading positive result. A new, efficient molecular method for detection of Francisella tularensis and its discrimination from Francisella-like endosymbionts, as well as two variants associated with human disease (unusual F. tularensis strain FnSp1 and F. tularensis subsp. novicida-like strain 3523), is described. The method is highly specific and sensitive, detecting up to one plasmid copy or 10 genome equivalents.

  17. Paired-End Sequence Mapping Detects Extensive Genomic Rearrangement and Translocation During Divergence of Francisella tularensis Subspecies Tularensis and Francisella tularensis Subspecies holarctica Populations

    DTIC Science & Technology

    2006-03-30

    such as Acanthamoeba castellanii, may also serve as a host for maintenance ofF. tularensis in the aquatic cycle [10]. The species F. tularensis is...and growth of Francisella tularensis in Acanthamoeba castellanii. Appl Environ Microbiol, 2003. 69(1): p. 600-6. 11. Chu, M.C. and R.S. Weyant

  18. Multiple Francisella tularensis Subspecies and Clades, Tularemia Outbreak, Utah

    PubMed Central

    Petersen, Jeannine M.; Carlson, Jennifer K.; Dietrich, Gabrielle; Eisen, Rebecca J.; Coombs, Jana; Janusz, Aimee M.; Summers, JoDee; Ben Beard, C.

    2008-01-01

    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

  19. Comparison of Francisella tularensis genomes reveals evolutionary events associated with the emergence of human pathogenic strains

    PubMed Central

    Rohmer, Laurence; Fong, Christine; Abmayr, Simone; Wasnick, Michael; Larson Freeman, Theodore J; Radey, Matthew; Guina, Tina; Svensson, Kerstin; Hayden, Hillary S; Jacobs, Michael; Gallagher, Larry A; Manoil, Colin; Ernst, Robert K; Drees, Becky; Buckley, Danielle; Haugen, Eric; Bovee, Donald; Zhou, Yang; Chang, Jean; Levy, Ruth; Lim, Regina; Gillett, Will; Guenthener, Don; Kang, Allison; Shaffer, Scott A; Taylor, Greg; Chen, Jinzhi; Gallis, Byron; D'Argenio, David A; Forsman, Mats; Olson, Maynard V; Goodlett, David R; Kaul, Rajinder; Miller, Samuel I; Brittnacher, Mitchell J

    2007-01-01

    Background Francisella tularensis subspecies tularensis and holarctica are pathogenic to humans, whereas the two other subspecies, novicida and mediasiatica, rarely cause disease. To uncover the factors that allow subspecies tularensis and holarctica to be pathogenic to humans, we compared their genome sequences with the genome sequence of Francisella tularensis subspecies novicida U112, which is nonpathogenic to humans. Results Comparison of the genomes of human pathogenic Francisella strains with the genome of U112 identifies genes specific to the human pathogenic strains and reveals pseudogenes that previously were unidentified. In addition, this analysis provides a coarse chronology of the evolutionary events that took place during the emergence of the human pathogenic strains. Genomic rearrangements at the level of insertion sequences (IS elements), point mutations, and small indels took place in the human pathogenic strains during and after differentiation from the nonpathogenic strain, resulting in gene inactivation. Conclusion The chronology of events suggests a substantial role for genetic drift in the formation of pseudogenes in Francisella genomes. Mutations that occurred early in the evolution, however, might have been fixed in the population either because of evolutionary bottlenecks or because they were pathoadaptive (beneficial in the context of infection). Because the structure of Francisella genomes is similar to that of the genomes of other emerging or highly pathogenic bacteria, this evolutionary scenario may be shared by pathogens from other species. PMID:17550600

  20. Methods for Enhanced Culture Recovery of Francisella tularensis

    PubMed Central

    Petersen, Jeannine M.; Schriefer, Martin E.; Gage, Kenneth L.; Montenieri, John A.; Carter, Leon G.; Stanley, Miles; Chu, May C.

    2004-01-01

    Francisella tularensis is found in a wide variety of hosts and extrahost environments, making culture recovery a diagnostic challenge. Here we demonstrate improved recovery times and good sensitivity (90%) when cultures were inoculated on the site of an investigation using fresh tissues. For contaminated specimens, antibiotic supplementation of enriched cysteine heart agar blood culture medium improved recovery of F. tularensis by 81.1%. For transport of tissues, immediate freezing yielded culture recovery rates as high as 94%. PMID:15184180

  1. Genetic identification of unique immunological responses in mice infected with virulent and attenuated Francisella tularensis

    PubMed Central

    Kingry, Luke C.; Troyer, Ryan M.; Marlenee, Nicole L.; Bielefeldt-Ohmann, Helle; Bowen, Richard A.; Schenkel, Alan R.; Dow, Steven W.; Slayden, Richard A.

    2010-01-01

    Francisella tularensis is a category A select agent based on its infectivity and virulence but disease mechanisms in Francisella tularensis infection remain poorly understood. Murine pulmonary models of infection were therefore employed to assess and compare dissemination and pathology and to elucidate the host immune response to infection with the highly virulent Type A F. tularensis strain Schu4 versus the less virulent Type B live vaccine strain (LVS). We found that dissemination and pathology in the spleen was significantly greater in mice infected with F. tularensis Schu4 compared to mice infected with F. tularensis LVS. Using gene expression profiling to compare the response to infection with the two F. tularensis strains, we found that there were significant differences in the expression of genes involved in the apoptosis pathway, antigen processing and presentation pathways, and inflammatory response pathways in mice infected with Schu4 when compared to LVS. These transcriptional differences coincided with marked differences in dissemination and severity of organ lesions in mice infected with the Schu4 and LVS strains. Therefore, these findings indicate that altered apoptosis, antigen presentation and production of inflammatory mediators explain the differences in pathogenicity of F. tularensis Schu4 and LVS. PMID:21070859

  2. Respiratory Tularemia: Francisella Tularensis and Microarray Probe Designing

    PubMed Central

    Ranjbar, Reza; Behzadi, Payam; Mammina, Caterina

    2016-01-01

    Background: Francisella tularensis (F. tularensis) is the etiological microorganism for tularemia. There are different forms of tularemia such as respiratory tularemia. Respiratory tularemia is the most severe form of tularemia with a high rate of mortality; if not treated. Therefore, traditional microbiological tools and Polymerase Chain Reaction (PCR) are not useful for a rapid, reliable, accurate, sensitive and specific diagnosis. But, DNA microarray technology does. DNA microarray technology needs to appropriate microarray probe designing. Objective: The main goal of this original article was to design suitable long oligo microarray probes for detection and identification of F. tularensis. Method: For performing this research, the complete genomes of F. tularensis subsp. tularensis FSC198, F. tularensis subsp. holarctica LVS, F. tularensis subsp. mediasiatica, F. tularensis subsp. novicida (F. novicida U112), and F. philomiragia subsp. philomiragia ATCC 25017 were studied via NCBI BLAST tool, GView and PanSeq Servers and finally the microarray probes were produced and processed via AlleleID 7.7 software and Oligoanalyzer tool, respectively. Results: In this in silico investigation, a number of long oligo microarray probes were designed for detecting and identifying F. tularensis. Among these probes, 15 probes were recognized as the best candidates for microarray chip designing. Conclusion: Calibrated microarray probes reduce the biasis of DNA microarray technology as an advanced, rapid, accurate and cost-effective molecular diagnostic tool with high specificity and sensitivity. Professional microarray probe designing provides us with much more facility and flexibility regarding preparation of a microarray diagnostic chip. PMID:28077973

  3. 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 §...

  4. 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 §...

  5. 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 §...

  6. 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 §...

  7. Francisella tularensis endocarditis: two case reports and a literature review.

    PubMed

    Gaci, Rostane; Alauzet, Corentine; Selton-Suty, Christine; Lozniewski, Alain; Pulcini, Céline; May, Thierry; Goehringer, François

    2017-02-01

    We report the first two cases of infective endocarditis caused by Francisella tularensis in Europe (two cases have previously been reported outside Europe). We suggest clinicians should consider tularemia as a possible diagnosis in endemic regions in cases of culture-negative endocarditis.

  8. Survival and Growth of Francisella tularensis in Acanthamoeba castellanii

    PubMed Central

    Abd, Hadi; Johansson, Thorsten; Golovliov, Igor; Sandström, Gunnar; Forsman, Mats

    2003-01-01

    Francisella tularensis is a highly infectious, facultative intracellular bacterium which causes epidemics of tularemia in both humans and mammals at regular intervals. The natural reservoir of the bacterium is largely unknown, although it has been speculated that protozoa may harbor it. To test this hypothesis, Acanthamoeba castellanii was cocultured with a strain of F. tularensis engineered to produce green fluorescent protein (GFP) in a nutrient-rich medium. GFP fluorescence within A. castellanii was then monitored by flow cytometry and fluorescence microscopy. In addition, extracellular bacteria were distinguished from intracellular bacteria by targeting with monoclonal antibodies. Electron microscopy was used to determine the intracellular location of F. tularensis in A. castellanii, and viable counts were obtained for both extracellular and intracellular bacteria. The results showed that many F. tularensis cells were located intracellularly in A. castellanii cells. The bacteria multiplied within intracellular vacuoles and eventually killed many of the host cells. F. tularensis was found in intact trophozoites, excreted vesicles, and cysts. Furthermore, F. tularensis grew faster in cocultures with A. castellanii than it did when grown alone in the same medium. This increase in growth was accompanied by a decrease in the number of A. castellanii cells. The interaction between F. tularensis and amoebae demonstrated in this study indicates that ubiquitous protozoa might be an important environmental reservoir for F. tularensis. PMID:12514047

  9. A novel nanoprobe for the sensitive detection of Francisella tularensis.

    PubMed

    Kim, Ji-eun; Seo, Youngmin; Jeong, Yoon; Hwang, Mintai P; Hwang, Jangsun; Choo, Jaebum; Hong, Jong Wook; Jeon, Jun Ho; Rhie, Gi-eun; Choi, Jonghoon

    2015-11-15

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

  10. 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.

  11. Description of two new plasmids isolated from Francisella philomiragia strains and construction of shuttle vectors for the study of Francisella tularensis.

    PubMed

    Le Pihive, E; Blaha, D; Chenavas, S; Thibault, F; Vidal, D; Valade, E

    2009-11-01

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

  12. [CITRULLINUREIDASE GENE DIVERSITY IN THE GENUS FRANCISELLA].

    PubMed

    Timofeev, V S; Bakhteeva, I V; Pavlov, V M; Mokrievich, A N

    2015-01-01

    This work describes the results, of the in silico analysis of the genetic diversity of the citrullinureidase gene (ctu) in two species of bacteria of the genus Francisella: tularensis (ssp. tularensis, holarctica, mediasiatica, novicida) and philomiragia. The strains of the Central Asiatic subspecies possessing the citrullinureidase activity differ in the gene ctu from the ssp tularensis Schu by three nucleotide substitutions leading to two insignificant amino acid substitutions in the encoded polypeptide. In the strain F. tularensis of the ssp. holarctica the gene ctu encodes inactive enzyme, which is probably due to amino acid substitutions: 151 Gly --> Asp, 183 Pro --> Leu, 222 Asp --> Asn. Except for the Japan biovar bacteria, in all strains of the Holarctic subspecies there are two stop codons in the gene ctu. The bacteria of the subspecies novicida contain the ctu gene only in the strain 3523, whereas the other strains contain the gene FTN_0827 encoding the C-N hydrolase, which probably provides the citrullinureidase activity.

  13. [Real time PCR hybridization for the rapid and specific identification of Francisella tularensis].

    PubMed

    Bielawska-Drózd, Agata; Niemcewicz, Marcin; Gaweł, Jerzy; Bartoszcze, Michał; Graniak, Grzegorz; Joniec, Justyna; Kołodziej, Marcin

    2010-01-01

    Tularemia is highly infectious and fatal zoonotic disease caused by Gram negative bacteria Francisella tularensis. The necessity to undergo medical treatment in early phase of illness in humans and possibility of making use of bacterial aerosol by terrorists in an attack create an urgent need to implement a rapid and effective method which enables to identify the agent. In our study two primers FopA F/R and hybridization probes FopA S1/S2 designed from fopA gene sequence, were tested for their potential applicability to identify F. tularensis. In this research 50 strains of F. tularensis were used and the test gave positive results. Reaction specificity was confirmed by using of non-Francisella tularensis bacterial species. The results obtained in the real-time PCR reaction with primers Tul4 F/R and hybridization probes Tul4 S1/S2, designed from tul4 gene, were comparable to the results from previous experiment with fopA - primers set. Investigation of fopA and tul4 primers and hybridization probes properties revealed characteristic Tm (melting temperature) value of the products--61 degrees C and 60 degrees C, respectively. Detection sensitivity was remarkably higher when fopA primers set was used 1 fg/microl, and for tul4 primers set, minimal detectable concentration is 10 fg/microl.

  14. Identification of two substrates of FTS_1067 protein - An essential virulence factor of Francisella tularensis.

    PubMed

    Spidlova, Petra; Senitkova, Iva; Link, Marek; Stulik, Jiri

    2016-11-15

    Francisella tularensis is a highly virulent intracellular pathogen with the capacity to infect a variety of hosts including humans. One of the most important proteins involved in F. tularensis virulence and pathogenesis is the protein DsbA. This protein is annotated as a lipoprotein with disulfide oxidoreductase/isomerase activity. Therefore, its interactions with different substrates, including probable virulence factors, to assist in their proper folding are anticipated. We aimed to use the immunopurification approach to find DsbA (gene locus FTS_1067) interacting partners in F. tularensis subsp. holarctica strain FSC200 and compare the identified substrates with proteins which were found in our previous comparative proteome analysis. As a result of our work two FTS_1067 substrates, D-alanyl-D-alanine carboxypeptidase family protein and HlyD family secretion protein, were identified. Bacterial two-hybrid systems were further used to test their relevance in confirming FTS_1067 protein interactions.

  15. Characterization of Francisella tularensis Outer Membrane Proteins▿ †

    PubMed Central

    Huntley, Jason F.; Conley, Patrick G.; Hagman, Kayla E.; Norgard, Michael V.

    2007-01-01

    Francisella tularensis is a gram-negative coccobacillus that is capable of causing severe, fatal disease in a number of mammalian species, including humans. Little is known about the proteins that are surface exposed on the outer membrane (OM) of F. tularensis, yet identification of such proteins is potentially fundamental to understanding the initial infection process, intracellular survival, virulence, immune evasion and, ultimately, vaccine development. To facilitate the identification of putative F. tularensis outer membrane proteins (OMPs), the genomes of both the type A strain (Schu S4) and type B strain (LVS) were subjected to six bioinformatic analyses for OMP signatures. Compilation of the bioinformatic predictions highlighted 16 putative OMPs, which were cloned and expressed for the generation of polyclonal antisera. Total membranes were extracted from both Schu S4 and LVS by spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation, which separated OMs from cytoplasmic (inner) membrane and other cellular compartments. Validation of OM separation and enrichment was confirmed by probing sucrose gradient fractions with antibodies to putative OMPs and inner membrane proteins. F. tularensis OMs typically migrated in sucrose gradients between densities of 1.17 and 1.20 g/ml, which differed from densities typically observed for other gram-negative bacteria (1.21 to 1.24 g/ml). Finally, the identities of immunogenic proteins were determined by separation on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analysis. This is the first report of a direct method for F. tularensis OM isolation that, in combination with computational predictions, offers a more comprehensive approach for the characterization of F. tularensis OMPs. PMID:17114266

  16. 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.

  17. 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

  18. 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.

  19. Francisella philomiragia comb. nov. (formerly Yersinia philomiragia) and Francisella tularensis biogroup novicida (formerly Francisella novicida) associated with human disease.

    PubMed Central

    Hollis, D G; Weaver, R E; Steigerwalt, A G; Wenger, J D; Moss, C W; Brenner, D J

    1989-01-01

    Over a 12-year period, 16 human strains of a gram-negative, catalase-positive, halophilic, aerobic, nonmotile, small coccoid bacterium were received for identification. On the bases of biochemical characteristics and cellular fatty acid profiles, 14 of these strains were similar to the "Philomiragia" bacterium (Yersinia philomiragia, species incertae sedis). Additional characteristics were growth on Thayer-Martin agar but no growth or sparse, delayed growth on MacConkey agar; oxidase positive; acid production, often weak and delayed, from D-glucose, sucrose, and maltose; urease negative; no reduction of nitrates; and H2S produced but often delayed in triple sugar iron agar. Both the human isolates and the "Philomiragia" bacterium contained C10:0, C14:0, C16:0, C18:1 omega 9c, C18:0, 3-OH C18:0, C22:0, and C24:1 as major cellular fatty acids and ubiquinone eight (Q8) as the major isoprenoid quinone. These cellular acids in these relative amounts have been found previously only in Francisella tularensis and Francisella novicida, suggesting a relationship between the "Philomiragia" bacterium and Francisella species. Of the 14 human "Philomiragia"-like isolates, 9 were from blood, 3 were from lung biopsies or pleural fluid, and one each was from peritoneal fluid and cerebrospinal fluid. DNA relatedness studies (hydroxyapatite method, 50 and 65 degrees C) showed that these 14 strains were a single group that was the same species as the "Philomiragia" bacterium. Two other human strains were oxidase negative and H2S negative. They formed a single DNA relatedness group that was indistinguishable from the type strains of both F. tularensis and F. novicida. DNA relatedness of "Philomiragia" bacterium type and other strains to strains of F. novicida and F. tularensis, including the type strains, was 35 to 46%. One of the two F. novicida- and F. tularensis-like strains was isolated from blood, and the other was isolated from a cervical lymph node. On the basis of these

  20. Systems approach to investigating host-pathogen interactions in infections with the biothreat agent Francisella. Constraints-based model of Francisella tularensis

    PubMed Central

    2010-01-01

    Background Francisella tularensis is a prototypic example of a pathogen for which few experimental datasets exist, but for which copious high-throughout data are becoming available because of its re-emerging significance as biothreat agent. The virulence of Francisella tularensis depends on its growth capabilities within a defined environmental niche of the host cell. Results We reconstructed the metabolism of Francisella as a stoichiometric matrix. This systems biology approach demonstrated that changes in carbohydrate utilization and amino acid metabolism play a pivotal role in growth, acid resistance, and energy homeostasis during infection with Francisella. We also show how varying the expression of certain metabolic genes in different environments efficiently controls the metabolic capacity of F. tularensis. Selective gene-expression analysis showed modulation of sugar catabolism by switching from oxidative metabolism (TCA cycle) in the initial stages of infection to fatty acid oxidation and gluconeogenesis later on. Computational analysis with constraints derived from experimental data revealed a limited set of metabolic genes that are operational during infection. Conclusions This integrated systems approach provides an important tool to understand the pathogenesis of an ill-characterized biothreat agent and to identify potential novel drug targets when rapid target identification is required should such microbes be intentionally released or become epidemic. PMID:20731870

  1. Seroprevalence study of Francisella tularensis among hunters in Germany.

    PubMed

    Jenzora, Andrea; Jansen, Andreas; Ranisch, Heidrun; Lierz, Michael; Wichmann, Ole; Grunow, Roland

    2008-07-01

    In 2005 and 2006, Francisella tularensis unexpectedly reemerged in western Germany, when several semi-free-living marmosets (Callithrix jacchus) in a research facility died from tularemia and a group of hare hunters became infected. It is believed that hunters may have an elevated risk to be exposed to zoonotic pathogens, including F. tularensis. A previous cross-sectional study of the German population (n=6883) revealed a prevalence of 0.2%. Here, we investigated 286 sera from individuals mainly hunting in districts with emerging tularemia cases (group 1) and 84 sera from a region currently not conspicuous for tularemia (group 2). Methods included standard enzyme-linked immunosorbent assay (ELISA), Western blot analysis and indirect immunofluorescence assay. We found five out of the 286 hunters (1.7%; 95% CI 0.6-4.0%) in group 1 positive with standard ELISA and Western blot, but none in the Berlin area (group 2; 95% CI 0-0.04%). Group 1 showed an elevated risk for hunters to be seropositive for F. tularensis compared with the cross-sectional study (OR=7.7; P<0.001). This indicates a higher prevalence for tularemia in hunters of a suspected endemic region of Germany.

  2. 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

  3. Glutathione provides a source of cysteine essential for intracellular multiplication of Francisella tularensis.

    PubMed

    Alkhuder, Khaled; Meibom, Karin L; Dubail, Iharilalao; Dupuis, Marion; Charbit, Alain

    2009-01-01

    Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularemia. Its ability to multiply and survive in macrophages is critical for its virulence. By screening a bank of HimarFT transposon mutants of the F. tularensis live vaccine strain (LVS) to isolate intracellular growth-deficient mutants, we selected one mutant in a gene encoding a putative gamma-glutamyl transpeptidase (GGT). This gene (FTL_0766) was hence designated ggt. The mutant strain showed impaired intracellular multiplication and was strongly attenuated for virulence in mice. Here we present evidence that the GGT activity of F. tularensis allows utilization of glutathione (GSH, gamma-glutamyl-cysteinyl-glycine) and gamma-glutamyl-cysteine dipeptide as cysteine sources to ensure intracellular growth. This is the first demonstration of the essential role of a nutrient acquisition system in the intracellular multiplication of F. tularensis. GSH is the most abundant source of cysteine in the host cytosol. Thus, the capacity this intracellular bacterial pathogen has evolved to utilize the available GSH, as a source of cysteine in the host cytosol, constitutes a paradigm of bacteria-host adaptation.

  4. 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....

  5. Performance of a Handheld PCR Instrument in the Detection of Bacillus anthracis, Francisella tularensis, and Yersinia pestis: Sensitivity, Specificity, and Effect of Interferents on Assay Results

    DTIC Science & Technology

    2004-12-01

    1 PERFORMANCE OF A HANDHELD PCR INSTRUMENT IN THE DETECTION OF BACILLUS ANTHRACIS, FRANCISELLA TULARENSIS, AND YERSINIA PESTIS: SENSITIVITY...fluorogenic PCR assay reagents for the detection of three biological threat agents, Bacillus anthracis (BA), Francisella tularensis (FT), and Yersinia...TITLE AND SUBTITLE Performance Of A Handheld Pcr Instrument In The Detection Of Bacillus Anthracis, Francisella Tularensis, And Yersinia Pestis

  6. IglE Is an Outer Membrane-Associated Lipoprotein Essential for Intracellular Survival and Murine Virulence of Type A Francisella tularensis

    PubMed Central

    Robertson, Gregory T.; Child, Robert; Ingle, Christine; Celli, Jean

    2013-01-01

    IglE is a small, hypothetical protein encoded by the duplicated Francisella pathogenicity island (FPI). Inactivation of both copies of iglE rendered Francisella tularensis subsp. tularensis Schu S4 avirulent and incapable of intracellular replication, owing to an inability to escape the phagosome. This defect was fully reversed following single-copy expression of iglE in trans from attTn7 under the control of the Francisella rpsL promoter, thereby establishing that the loss of iglE, and not polar effects on downstream vgrG gene expression, was responsible for the defect. IglE is exported to the Francisella outer membrane as an ∼13.9-kDa lipoprotein, determined on the basis of a combination of selective Triton X-114 solubilization, radiolabeling with [3H]palmitic acid, and sucrose density gradient membrane partitioning studies. Lastly, a genetic screen using the iglE-null live vaccine strain resulted in the identification of key regions in the carboxyl terminus of IglE that are required for intracellular replication of Francisella tularensis in J774A.1 macrophages. Thus, IglE is essential for Francisella tularensis virulence. Our data support a model that likely includes protein-protein interactions at or near the bacterial cell surface that are unknown at present. PMID:23959721

  7. A method for functional trans-complementation of intracellular Francisella tularensis.

    PubMed

    Steele, Shaun; Taft-Benz, Sharon; Kawula, Thomas

    2014-01-01

    -complementation using bead-bound bacteria co-infections is a method to rapidly identify mutants that fail to modify a host response. Francisella tularensis is a facultative intracellular bacterial pathogen and is the causative agent of the disease tularemia. F. tularensis enters host cells through phagocytosis, escapes the phagosome, and replicates in the host cell cytosol while suppressing cytokine secretion [1]-[4]. Although substantial progress has been made in understanding the intracellular life cycle of F. tularensis, the F. tularensis proteins responsible for manipulating many host cell pathways are unknown. Identifying novel host-pathogen effector proteins is difficult because there is no rapid method to reliably distinguish between bacterial proteins that modify host processes and proteins that are involved in bacterial processes that are required for the bacteria to survive or replicate in the intracellular environment. The ability to identify mutants that are deficient for host-pathogen interactions is important because it can aid in prioritizing the investigation of genes of interest and in downstream experimental design. Moreover, certain mutant phenotypes, such as decreased phagosomal escape, hinder investigation of other potential phenotypes. A method to specifically complement these phenotypes would allow for further characterizations of certain F. tularensis mutants. Thus we sought to develop a method to easily identify and functionally complement mutants that are deficient for interactions with the host.

  8. 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

  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. 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.

  11. 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

  12. Open and compressed conformations of Francisella tularensis ClpP

    PubMed Central

    Díaz‐Sáez, Laura; Pankov, Genady

    2016-01-01

    ABSTRACT Caseinolytic proteases are large oligomeric assemblies responsible for maintaining protein homeostasis in bacteria and in so doing influence a wide range of biological processes. The functional assembly involves three chaperones together with the oligomeric caseinolytic protease catalytic subunit P (ClpP). This protease represents a potential target for therapeutic intervention in pathogenic bacteria. Here, we detail an efficient protocol for production of recombinant ClpP from Francisella tularensis, and the structural characterization of three crystal forms which grow under similar conditions. One crystal form reveals a compressed state of the ClpP tetradecamer and two forms an open state. A comparison of the two types of structure infers that differences at the enzyme active site result from a conformational change involving a highly localized disorder‐order transition of a β‐strand α‐helix combination. This transition occurs at a subunit‐subunit interface. Our study may now underpin future efforts in a structure‐based approach to target ClpP for inhibitor or activator development. Proteins 2016; 85:188–194. © 2016 Wiley Periodicals, Inc. PMID:27802578

  13. Amblyomma americanum as a Bridging Vector for Human Infection with Francisella tularensis

    PubMed Central

    2015-01-01

    The γ-proteobacterium Francisella tularensis causes seasonal tick-transmitted tularemia outbreaks in natural rabbit hosts and incidental infections in humans in the south-central United States. Although Dermacentor variabilis is considered a primary vector for F. tularensis, Amblyomma americanum is the most abundant tick species in this endemic region. A systematic study of F. tularensis colonization of A. americanum was undertaken to better understand its potential to serve as an overwintering reservoir for F. tularensis and as a bridging vector for human infections. Colony-reared A. americanum were artificially fed F. tularensis subspecies holarctica strain LVS via glass capillaries and colonization levels determined. Capillary-fed larva and nymph were initially infected with 104 CFU/tick which declined prior to molting for both stages, but rebounded post-molting in nymphs and persisted in 53% at 103 to 108 CFU/nymph at 168 days post-capillary feeding (longest sampling time in the study). In contrast, only 18% of adults molted from colonized nymphs maintained LVS colonization at 101 to 105 CFU/adult at 168 days post-capillary feeding (longest sampling time). For adults, LVS initially colonized the gut and disseminated to salivary glands by 24 h and had an ID50 of <5CFU in mice. Francisella tularensis infected the ovaries of gravid females, but transmission to eggs was infrequent and transovarial transmission to hatched larvae was not observed. The prolonged persistence of F. tularensis in A. americanum nymphs supports A. americanum as an overwintering reservoir for F. tularensis from which seasonal epizootics may originate; however, although the rapid dissemination of F. tularensis from gut to salivary glands in adults A. americanum is compatible with intermittent feeding adult males acting as bridging vectors for incidental F. tularensis infections of humans, acquisition of F. tularensis by adults may be unlikely based on adult feeding preference for larger

  14. 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.

  15. Detection of Francisella tularensis within infected mouse tissues by using a hand-held PCR thermocycler.

    PubMed

    Emanuel, Peter A; Bell, Ryan; Dang, Jessica L; McClanahan, Rebecca; David, John C; Burgess, Robert J; Thompson, Joseph; Collins, Lisa; Hadfield, Ted

    2003-02-01

    The diagnosis of human cases of tularemia often relies upon the demonstration of an antibody response to Francisella tularensis or the direct culturing of the bacteria from the patient. Antibody response is not detectable until 2 weeks or more after infection, and culturing requires special media and suspicion of tularemia. In addition, handling live Francisella poses a risk to laboratory personnel due to the highly infectious nature of this pathogen. In an effort to develop a rapid diagnostic assay for tularemia, we investigated the use of TaqMan 5' hydrolysis fluorogenic PCR to detect the organism in tissues of infected mice. Mice were infected to produce respiratory tularemia. The fopA and tul4 genes of F. tularensis were amplified from infected spleen, lung, liver, and kidney tissues sampled over a 5-day period. The samples were analyzed using the laboratory-based Applied Biosystems International 7900 and the Smiths Detection-Edgewood BioSeeq, a hand-held portable fluorescence thermocycler designed for use in the field. A comparison of culturing and PCR for detection of bacteria in infected tissues shows that culturing was more sensitive than PCR. However, the results for culture take 72 h, whereas PCR results were available within 4 h. PCR was able to detect infection in all the tissues tested. Lung tissue showed the earliest response at 2 days when tested with the ABI 7900 and in 3 days when tested with the BioSeeq. The results were in agreement between the ABI 7900 and the BioSeeq when presented with the same sample. Template preparation may account for the loss of sensitivity compared to culturing techniques. The hand-held BioSeeq thermocycler shows promise as an expedient means of forward diagnosis of infection in the field.

  16. Genome sequence and phenotypic analysis of a first German Francisella sp. isolate (W12-1067) not belonging to the species Francisella tularensis

    PubMed Central

    2014-01-01

    Background Francisella isolates from patients suffering from tularemia in Germany are generally strains of the species F. tularensis subsp. holarctica. To our knowledge, no other Francisella species are known for Germany. Recently, a new Francisella species could be isolated from a water reservoir of a cooling tower in Germany. Results We identified a Francisella sp. (isolate W12-1067) whose 16S rDNA is 99% identical to the respective nucleotide sequence of the recently published strain F. guangzhouensis. The overall sequence identity of the fopA, gyrA, rpoA, groEL, sdhA and dnaK genes is only 89%, indicating that strain W12-1067 is not identical to F. guangzhouensis. W12-1067 was isolated from a water reservoir of a cooling tower of a hospital in Germany. The growth optimum of the isolate is approximately 30°C, it can grow in the presence of 4–5% NaCl (halotolerant) and is able to grow without additional cysteine within the medium. The strain was able to replicate within a mouse-derived macrophage-like cell line. The whole genome of the strain was sequenced (~1.7 mbp, 32.2% G + C content) and the draft genome was annotated. Various virulence genes common to the genus Francisella are present, but the Francisella pathogenicity island (FPI) is missing. However, another putative type-VI secretion system is present within the genome of strain W12-1067. Conclusions Isolate W12-1067 is closely related to the recently described F. guangzhouensis species and it replicates within eukaryotic host cells. Since W12-1067 exhibits a putative new type-VI secretion system and F. tularensis subsp. holarctica was found not to be the sole species in Germany, the new isolate is an interesting species to be analyzed in more detail. Further research is needed to investigate the epidemiology, ecology and pathogenicity of Francisella species present in Germany. PMID:24961323

  17. 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

    2016-03-04

    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.

  18. 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.

  19. A multiplex real-time PCR assay for the detection and differentiation of Francisella tularensis subspecies.

    PubMed

    Gunnell, Mark K; Lovelace, Charity D; Satterfield, Benjamin A; Moore, Emily A; O'Neill, Kim L; Robison, Richard A

    2012-11-01

    Francisella tularensis is the aetiological agent of tularaemia, a zoonotic disease with worldwide prevalence. F. tularensis is a highly pathogenic organism and has been designated a category A biothreat agent by the Centers for Disease Control and Prevention. Tularaemia is endemic in much of the USA, Europe and parts of Asia. It is transmitted by numerous vectors and vehicles such as deer flies, ticks and rabbits. Currently, there are four recognized subspecies of F. tularensis: tularensis (type A), holarctica (type B), mediasiatica and novicida. Within the type A classification there are two subclassifications, type A.I and A.II, each with a specific geographical distribution across the USA. F. tularensis subsp. holartica (type B) is found in both the USA and Europe. Because of virulence differences among subtypes, it is important that health departments, hospitals and other government agencies are able to quickly identify each subtype. The purpose of this study was to develop a multiplex real-time PCR assay for the identification and discrimination of type A.I, type A.II, type B and novicida subspecies of F. tularensis. The assay was validated using 119 isolates of F. tularensis, three of its nearest neighbours and 14 other bacterial pathogens. This assay proved to be ~98 % successful at identifying the known subspecies of F. tularensis and could prove to be a useful tool in the characterization of this important pathogen.

  20. Geographic Differentiation of Francisella Tularensis using Molecular Methods

    DTIC Science & Technology

    2006-05-01

    For his extensive global F. tularensis DNA collection, I am grateful to Dr. Anders Johansson from the Division of Infectious Diseases and Clinical...dissemination, its extremely low infectious dose of only ten to fifty organisms when acquired through the inhalation route in humans, and the potential...in this chapter. 14 1.1.3 Ecology of F. tularensis F. tularensis is a widely infectious zoonotic pathogen and has been isolated from as many as 250

  1. Cytosolic clearance of replication-deficient mutants reveals Francisella tularensis interactions with the autophagic pathway.

    PubMed

    Chong, Audrey; Wehrly, Tara D; Child, Robert; Hansen, Bryan; Hwang, Seungmin; Virgin, Herbert W; Celli, Jean

    2012-09-01

    Cytosolic bacterial pathogens must evade intracellular innate immune recognition and clearance systems such as autophagy to ensure their survival and proliferation. The intracellular cycle of the bacterium Francisella tularensis is characterized by rapid phagosomal escape followed by extensive proliferation in the macrophage cytoplasm. Cytosolic replication, but not phagosomal escape, requires the locus FTT0369c, which encodes the dipA gene (deficient in intracellular replication A). Here, we show that a replication-deficient, ∆dipA mutant of the prototypical SchuS4 strain is eventually captured from the cytosol of murine and human macrophages into double-membrane vacuoles displaying the late endosomal marker, LAMP1, and the autophagy-associated protein, LC3, coinciding with a reduction in viable intracellular bacteria. Capture of SchuS4ΔdipA was not dipA-specific as other replication-deficient bacteria, such as chloramphenicol-treated SchuS4 and a purine auxotroph mutant SchuS4ΔpurMCD, were similarly targeted to autophagic vacuoles. Vacuoles containing replication-deficient bacteria were labeled with ubiquitin and the autophagy receptors SQSTM1/p62 and NBR1, and their formation was decreased in macrophages from either ATG5-, LC3B- or SQSTM1-deficient mice, indicating recognition by the ubiquitin-SQSTM1-LC3 pathway. While a fraction of both the wild-type and the replication-impaired strains were ubiquitinated and recruited SQSTM1, only the replication-defective strains progressed to autophagic capture, suggesting that wild-type Francisella interferes with the autophagic cascade. Survival of replication-deficient strains was not restored in autophagy-deficient macrophages, as these bacteria died in the cytosol prior to autophagic capture. Collectively, our results demonstrate that replication-impaired strains of Francisella are cleared by autophagy, while replication-competent bacteria seem to interfere with autophagic recognition, therefore ensuring survival

  2. 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.

  3. 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

  4. First indication for a functional CRISPR/Cas system in Francisella tularensis.

    PubMed

    Schunder, Eva; Rydzewski, Kerstin; Grunow, Roland; Heuner, Klaus

    2013-03-01

    Francisella tularensis is a zoonotic agent and the subspecies novicida is proposed to be a water-associated bacterium. The intracellular pathogen F. tularensis causes tularemia in humans and is known for its potential to be used as a biological threat. We analyzed the genome sequence of F. tularensis subsp. novicida U112 in silico for the presence of a putative functional CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system. CRISPR/Cas systems are known to encode an RNA-guided adaptive immunity-like system to protect bacteria against invading genetic elements like bacteriophages and plasmids. In this work, we present a first indication that F. tularensis subsp. novicida encodes a functional CRISPR/Cas defence system. Additionally, we identified various spacer DNAs homologous to a putative phage present within the genome of F. tularensis subsp. novicida-like strain 3523. CRISPR/Cas is also present in F. tularensis subsp. tularensis, holarctica, and mediasiatica, but these systems seem to be non-functional.

  5. 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.

  6. Mast cell TLR2 signaling is crucial for effective killing of Francisella tularensis.

    PubMed

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

    2012-06-01

    TLR signaling is critical for early host defense against pathogens, but the contributions of mast cell TLR-mediated mechanisms and subsequent effector functions during pulmonary infection are 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 were used 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 MHC class II and lysosomal-associated membrane protein 2. Infected TLR2(-/-) mast cells, in contrast to wild-type and TLR4(-/-) cells, lacked detectable IL-4 and displayed increased cell death with a 2-3 log increase of F. tularensis replication, but could be rescued with rIL-4 treatment. Importantly, MHC class II and lysosomal-associated membrane protein 2 localization with labeled F. tularensis in the lungs was greater in wild-type 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.

  7. 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.

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

    PubMed

    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.

  9. 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

  10. Genome-Wide DNA Microarray Analysis of Francisella tularensis Strains Demonstrates Extensive Genetic Conservation within the Species but Identifies Regions That Are Unique to the Highly Virulent F. tularensis subsp. tularensis

    PubMed Central

    Broekhuijsen, Martien; Larsson, Pär; Johansson, Anders; Byström, Mona; Eriksson, Ulla; Larsson, Eva; Prior, Richard G.; Sjöstedt, Anders; Titball, Richard W.; Forsman, Mats

    2003-01-01

    Francisella tularensis is a potent pathogen and a possible bioterrorism agent. Little is known, however, to explain the molecular basis for its virulence and the distinct differences in virulence found between the four recognized subspecies, F. tularensis subsp. tularensis, F. tularensis subsp. mediasiatica, F. tularensis subsp. holarctica, and F. tularensis subsp. novicida. We developed a DNA microarray based on 1,832 clones from a shotgun library used for sequencing of the highly virulent strain F. tularensis subsp. tularensis Schu S4. This allowed a genome-wide analysis of 27 strains representing all four subspecies. Overall, the microarray analysis confirmed a limited genetic variation within the species F. tularensis, and when the strains were compared, at most 3.7% of the probes showed differential hybridization. Cluster analysis of the hybridization data revealed that the causative agents of type A and type B tularemia, i.e., F. tularensis subsp. tularensis and F. tularensis subsp. holarctica, respectively, formed distinct clusters. Despite marked differences in their virulence and geographical origin, a high degree of genomic similarity between strains of F. tularensis subsp. tularensis and F. tularensis subsp. mediasiatica was apparent. Strains from Japan clustered separately, as did strains of F. tularensis subsp. novicida. Eight regions of difference (RD) 0.6 to 11.5 kb in size, altogether comprising 21 open reading frames, were identified that distinguished strains of the moderately virulent subspecies F. tularensis subsp. holarctica and the highly virulent subspecies F. tularensis subsp. tularensis. One of these regions, RD1, allowed for the first time the development of an F. tularensis-specific PCR assay that discriminates each of the four subspecies. PMID:12843022

  11. Purification and biophysical characterization of the CapA membrane protein FTT0807 from Francisella tularensis.

    PubMed

    Martin-Garcia, Jose M; Hansen, Debra T; Zook, James; Loskutov, Andrey V; Robida, Mark D; Craciunescu, Felicia M; Sykes, Kathryn F; Wachter, Rebekka M; Fromme, Petra; Allen, James P

    2014-04-01

    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.

  12. 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

  13. Structure and Function of REP34 Implicates Carboxypeptidase Activity in Francisella tularensis Host Cell Invasion*

    PubMed Central

    Feld, Geoffrey K.; El-Etr, Sahar; Corzett, Michele H.; Hunter, Mark S.; Belhocine, Kamila; Monack, Denise M.; Frank, Matthias; Segelke, Brent W.; Rasley, Amy

    2014-01-01

    Francisella tularensis is the etiological agent of tularemia, or rabbit fever. Although F. tularensis is a recognized biothreat agent with broad and expanding geographical range, its mechanism of infection and environmental persistence remain poorly understood. Previously, we identified seven F. tularensis proteins that induce a rapid encystment phenotype (REP) in the free-living amoeba, Acanthamoeba castellanii. Encystment is essential to the pathogen's long term intracellular survival in the amoeba. Here, we characterize the cellular and molecular function of REP34, a REP protein with a mass of 34 kDa. A REP34 knock-out strain of F. tularensis has a reduced ability to both induce encystment in A. castellanii and invade human macrophages. We determined the crystal structure of REP34 to 2.05-Å resolution and demonstrate robust carboxypeptidase B-like activity for the enzyme. REP34 is a zinc-containing monomeric protein with close structural homology to the metallocarboxypeptidase family of peptidases. REP34 possesses a novel topology and substrate binding pocket that deviates from the canonical funnelin structure of carboxypeptidases, putatively resulting in a catalytic role for a conserved tyrosine and distinct S1′ recognition site. Taken together, these results identify REP34 as an active carboxypeptidase, implicate the enzyme as a potential key F. tularensis effector protein, and may help elucidate a mechanistic understanding of F. tularensis infection of phagocytic cells. PMID:25231992

  14. 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

  15. A spontaneous mutation in kdsD, a biosynthesis gene for 3 Deoxy-D-manno-Octulosonic Acid, occurred in a ciprofloxacin resistant strain of Francisella tularensis and caused a high level of attenuation in murine models of tularemia.

    PubMed

    Chance, Taylor; Chua, Jennifer; Toothman, Ronald G; Ladner, Jason T; Nuss, Jonathan E; Raymond, Jo Lynne; Biot, Fabrice V; Demons, Samandra; Miller, Lynda; Halasohoris, Stephanie; Mou, Sherry; Koroleva, Galina; Lovett, Sean; Palacios, Gustavo; Vietri, Nicholas J; Worsham, Patricia L; Cote, Christopher K; Kijek, Todd M; Bozue, Joel A

    2017-01-01

    Francisella tularensis, a gram-negative facultative intracellular bacterial pathogen, is the causative agent of tularemia and able to infect many mammalian species, including humans. Because of its ability to cause a lethal infection, low infectious dose, and aerosolizable nature, F. tularensis subspecies tularensis is considered a potential biowarfare agent. Due to its in vitro efficacy, ciprofloxacin is one of the antibiotics recommended for post-exposure prophylaxis of tularemia. In order to identify therapeutics that will be efficacious against infections caused by drug resistant select-agents and to better understand the threat, we sought to characterize an existing ciprofloxacin resistant (CipR) mutant in the Schu S4 strain of F. tularensis by determining its phenotypic characteristics and sequencing the chromosome to identify additional genetic alterations that may have occurred during the selection process. In addition to the previously described genetic alterations, the sequence of the CipR mutant strain revealed several additional mutations. Of particular interest was a frameshift mutation within kdsD which encodes for an enzyme necessary for the production of 3-Deoxy-D-manno-Octulosonic Acid (KDO), an integral component of the lipopolysaccharide (LPS). A kdsD mutant was constructed in the Schu S4 strain. Although it was not resistant to ciprofloxacin, the kdsD mutant shared many phenotypic characteristics with the CipR mutant, including growth defects under different conditions, sensitivity to hydrophobic agents, altered LPS profiles, and attenuation in multiple models of murine tularemia. This study demonstrates that the KdsD enzyme is essential for Francisella virulence and may be an attractive therapeutic target for developing novel medical countermeasures.

  16. A spontaneous mutation in kdsD, a biosynthesis gene for 3 Deoxy-D-manno-Octulosonic Acid, occurred in a ciprofloxacin resistant strain of Francisella tularensis and caused a high level of attenuation in murine models of tularemia

    PubMed Central

    Chance, Taylor; Toothman, Ronald G.; Nuss, Jonathan E.; Raymond, Jo Lynne; Biot, Fabrice V.; Demons, Samandra; Miller, Lynda; Halasohoris, Stephanie; Mou, Sherry; Koroleva, Galina; Lovett, Sean; Palacios, Gustavo; Vietri, Nicholas J.; Worsham, Patricia L.; Cote, Christopher K.; Kijek, Todd M.; Bozue, Joel A.

    2017-01-01

    Francisella tularensis, a gram–negative facultative intracellular bacterial pathogen, is the causative agent of tularemia and able to infect many mammalian species, including humans. Because of its ability to cause a lethal infection, low infectious dose, and aerosolizable nature, F. tularensis subspecies tularensis is considered a potential biowarfare agent. Due to its in vitro efficacy, ciprofloxacin is one of the antibiotics recommended for post-exposure prophylaxis of tularemia. In order to identify therapeutics that will be efficacious against infections caused by drug resistant select-agents and to better understand the threat, we sought to characterize an existing ciprofloxacin resistant (CipR) mutant in the Schu S4 strain of F. tularensis by determining its phenotypic characteristics and sequencing the chromosome to identify additional genetic alterations that may have occurred during the selection process. In addition to the previously described genetic alterations, the sequence of the CipR mutant strain revealed several additional mutations. Of particular interest was a frameshift mutation within kdsD which encodes for an enzyme necessary for the production of 3-Deoxy-D-manno-Octulosonic Acid (KDO), an integral component of the lipopolysaccharide (LPS). A kdsD mutant was constructed in the Schu S4 strain. Although it was not resistant to ciprofloxacin, the kdsD mutant shared many phenotypic characteristics with the CipR mutant, including growth defects under different conditions, sensitivity to hydrophobic agents, altered LPS profiles, and attenuation in multiple models of murine tularemia. This study demonstrates that the KdsD enzyme is essential for Francisella virulence and may be an attractive therapeutic target for developing novel medical countermeasures. PMID:28328947

  17. Experimental Infection of voles with Francisella tularensis indicates their amplification role in tularemia outbreaks.

    PubMed

    Rossow, Heidi; Forbes, Kristian M; Tarkka, Eveliina; Kinnunen, Paula M; Hemmilä, Heidi; Huitu, Otso; Nikkari, Simo; Henttonen, Heikki; Kipar, Anja; Vapalahti, Olli

    2014-01-01

    Tularemia outbreaks in humans have been linked to fluctuations in rodent population density, but the mode of bacterial maintenance in nature is unclear. Here we report on an experiment to investigate the pathogenesis of Francisella tularensis infection in wild rodents, and thereby assess their potential to spread the bacterium. We infected 20 field voles (Microtus agrestis) and 12 bank voles (Myodes glareolus) with a strain of F. tularensis ssp. holarctica isolated from a human patient. Upon euthanasia or death, voles were necropsied and specimens collected for histological assessment and identification of bacteria by immunohistology and PCR. Bacterial excretion and a rapid lethal clinical course with pathological changes consistent with bacteremia and tissue necrosis were observed in infected animals. The results support a role for voles as an amplification host of F. tularensis, as excreta and, in particular, carcasses with high bacterial burden could serve as a source for environmental contamination.

  18. Delayed presence of alternatively activated macrophages during a Francisella tularensis infection.

    PubMed

    D'Elia, Riccardo V; Laws, Thomas R; Núñez, Alejandro; Taylor, Christopher; Clark, Graeme C

    2015-01-01

    Francisella tularensis is an intracellular bacterium that has the ability to multiply within the macrophage. The phenotype of a macrophage can determine whether the infection is cleared or the host succumbs to disease. Previously published data has suggested that F. tularensis LVS actively induces the alternative phenotype as a survival mechanism. In these studies we demonstrate that this is not the case for the more virulent strain of F. tularensis SCHU-S4. During an intranasal mouse model of infection, immuno-histochemistry identified that iNOS positive ("classical") macrophages are present at 72 h post-infection and remain high (supported by CCL-5 release) in numbers. In contrast, arginase/FIZZ-1 positive ("alternative") cells appear later and in low numbers during the development of the disease tularemia.

  19. Experimental Infection of Voles with Francisella tularensis Indicates Their Amplification Role in Tularemia Outbreaks

    PubMed Central

    Rossow, Heidi; Forbes, Kristian M.; Tarkka, Eveliina; Kinnunen, Paula M.; Hemmilä, Heidi; Huitu, Otso; Nikkari, Simo; Henttonen, Heikki; Kipar, Anja; Vapalahti, Olli

    2014-01-01

    Tularemia outbreaks in humans have been linked to fluctuations in rodent population density, but the mode of bacterial maintenance in nature is unclear. Here we report on an experiment to investigate the pathogenesis of Francisella tularensis infection in wild rodents, and thereby assess their potential to spread the bacterium. We infected 20 field voles (Microtus agrestis) and 12 bank voles (Myodes glareolus) with a strain of F. tularensis ssp. holarctica isolated from a human patient. Upon euthanasia or death, voles were necropsied and specimens collected for histological assessment and identification of bacteria by immunohistology and PCR. Bacterial excretion and a rapid lethal clinical course with pathological changes consistent with bacteremia and tissue necrosis were observed in infected animals. The results support a role for voles as an amplification host of F. tularensis, as excreta and, in particular, carcasses with high bacterial burden could serve as a source for environmental contamination. PMID:25271640

  20. 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

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

    2014-04-08

    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 identifying and

  1. 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

  2. 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

    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.

  3. Nasal Acai Polysaccharides Potentiate Innate Immunity to Protect against Pulmonary Francisella tularensis and Burkholderia pseudomallei Infections

    PubMed Central

    Skyberg, Jerod A.; Rollins, MaryClare F.; Holderness, Jeff S.; Marlenee, Nicole L.; Schepetkin, Igor A.; Goodyear, Andrew; Dow, Steven W.; Jutila, Mark A.; Pascual, David W.

    2012-01-01

    Pulmonary Francisella tularensis and Burkholderia pseudomallei infections are highly lethal in untreated patients, and current antibiotic regimens are not always effective. Activating the innate immune system provides an alternative means of treating infection and can also complement antibiotic therapies. Several natural agonists were screened for their ability to enhance host resistance to infection, and polysaccharides derived from the Acai berry (Acai PS) were found to have potent abilities as an immunotherapeutic to treat F. tularensis and B. pseudomallei infections. In vitro, Acai PS impaired replication of Francisella in primary human macrophages co-cultured with autologous NK cells via augmentation of NK cell IFN-γ. Furthermore, Acai PS administered nasally before or after infection protected mice against type A F. tularensis aerosol challenge with survival rates up to 80%, and protection was still observed, albeit reduced, when mice were treated two days post-infection. Nasal Acai PS administration augmented intracellular expression of IFN-γ by NK cells in the lungs of F. tularensis-infected mice, and neutralization of IFN-γ ablated the protective effect of Acai PS. Likewise, nasal Acai PS treatment conferred protection against pulmonary infection with B. pseudomallei strain 1026b. Acai PS dramatically reduced the replication of B. pseudomallei in the lung and blocked bacterial dissemination to the spleen and liver. Nasal administration of Acai PS enhanced IFN-γ responses by NK and γδ T cells in the lungs, while neutralization of IFN-γ totally abrogated the protective effect of Acai PS against pulmonary B. pseudomallei infection. Collectively, these results demonstrate Acai PS is a potent innate immune agonist that can resolve F. tularensis and B. pseudomallei infections, suggesting this innate immune agonist has broad-spectrum activity against virulent intracellular pathogens. PMID:22438809

  4. Decontamination of a hospital room using gaseous chlorine dioxide: Bacillus anthracis, Francisella tularensis, and Yersinia pestis.

    PubMed

    Lowe, John J; Gibbs, Shawn G; Iwen, Peter C; Smith, Philip W; Hewlett, Angela L

    2013-01-01

    This study assessed the efficacy of gaseous chlorine dioxide for inactivation of Bacillus anthracis, Francisella tularensis, and Yersinia pestis in a hospital patient care suite. Spore and vegetative cells of Bacillus anthracis Sterne 34F2, spores of Bacillus atrophaeus ATCC 9372 and vegetative cells of both Francisella tularensis ATCC 6223 and Yersinia pestis A1122 were exposed to gaseous chlorine dioxide in a patient care suite. Organism inactivation was then assessed by log reduction in viable organisms postexposure to chlorine dioxide gas compared to non-exposed control organism. Hospital room decontamination protocols utilizing chlorine dioxide gas concentrations of 377 to 385 ppm maintained to exposures of 767 ppm-hours with 65% relative humidity consistently achieved complete inactivation of B. anthracis and B. atrophaeus spores, as well as vegetative cells of B. anthracis, F. tularensis, and Y. pestis. Decrease in exposure (ppm-hours) and relative humidity (<65%) or restricting airflow reduced inactivation but achieved >8 log reductions in organisms. Up to 10-log reductions were achieved in a hospital room with limited impact on adjacent areas, indicating chlorine dioxide concentrations needed for decontamination of highly concentrated (>6 logs) organisms can be achieved throughout a hospital room. This study translates laboratory chlorine dioxide fumigation studies applied in a complex clinical environment.

  5. [Surveillance of Francisella tularensis infection in dogs in Bratislava].

    PubMed

    Gurycová, D; Kopcok, M

    1992-03-01

    Out of 548 serologically investigated dogs from Bratislava and other regions of Slovakia and Moravia, antibodies to F. tularensis were found in 16.4% (Tabs. I, II). In all the investigated groups of dogs from the region of Bratislava the highest seroprevalence by F. tularensis was recorded in watch dogs kept on farms and in cooperatives--37.5% and in rambling dogs--20.7% (Tab. I). The highest seropositivity was found in one to three year old dogs--22.2% (Tab. III). A similar degree of seroprevalence was also observed in one to three years old police dogs which came from the endemic region of tularemia--West Slovakia (19.3%) and East Slovakia (25.6%)--Tab. IV. These facts indicate the persistence of active natural foci in these regions. Serological investigations of the relatively great number of dogs from different regions of Slovakia showed that the presence of F. tularensis antibodies in this animal species, mainly in the watch dogs group, can be taken as a convenient marker or indicator of the existence of active natural foci of tularemia and as a suitable component for surveillance of this diseases.

  6. Francisella Tularensis Blue–Gray Phase Variation Involves Structural Modifications of Lipopolysaccharide O-Antigen, Core and Lipid A and Affects Intramacrophage Survival and Vaccine Efficacy

    PubMed Central

    Soni, Shilpa; Ernst, Robert K.; Muszyński, Artur; Mohapatra, Nrusingh P.; Perry, Malcolm B.; Vinogradov, Evgeny; Carlson, Russell W.; Gunn, John S.

    2010-01-01

    Francisella tularensis is a CDC Category A biological agent and a potential bioterrorist threat. There is no licensed vaccine against tularemia in the United States. A long-standing issue with potential Francisella vaccines is strain phase variation to a gray form that lacks protective capability in animal models. Comparisons of the parental strain (LVS) and a gray variant (LVSG) have identified lipopolysaccharide (LPS) alterations as a primary change. The LPS of the F. tularensis variant strain gains reactivity to F. novicida anti-LPS antibodies, suggesting structural alterations to the O-antigen. However, biochemical and structural analysis of the F. tularensis LVSG and LVS LPS demonstrated that LVSG has less O-antigen but no major O-antigen structural alterations. Additionally, LVSG possesses structural differences in both the core and lipid A regions, the latter being decreased galactosamine modification. Recent work has identified two genes important in adding galactosamine (flmF2 and flmK) to the lipid A. Quantitative real-time PCR showed reduced transcripts of both of these genes in the gray variant when compared to LVS. Loss of flmF2 or flmK caused less frequent phase conversion but did not alter intramacrophage survival or colony morphology. The LVSG strain demonstrated an intramacrophage survival defect in human and rat but not mouse macrophages. Consistent with this result, the LVSG variant demonstrated little change in LD50 in the mouse model of infection. Furthermore, the LVSG strain lacks the protective capacity of F. tularensis LVS against virulent Type A challenge. These data suggest that the LPS of the F. tularensis LVSG phase variant is dramatically altered. Understanding the mechanism of blue to gray phase variation may lead to a way to inhibit this variation, thus making future F. tularensis vaccines more stable and efficacious. PMID:21687776

  7. 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

  8. Comparison of Five Commercial DNA Extraction Kits for the Recovery of Francisella Tularensis DNA from Spiked Soil Samples

    DTIC Science & Technology

    2007-01-01

    protozoa. The fact that F. tularensis has been shown to survive in amoebae in the laboratory [8], and has been isolated from natural water sources [9,10...play a role in the maintenance of this bacterium in the environment. Francisella strains are extremely difficult to culture from environmental...sources [12]; however, recent molecular surveys have identified Francisella-like bacteria in soil samples from Houston, TX, Denver, Co [13], and from

  9. 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

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

    PubMed Central

    Goralski, Tyler D. P.; Dewan, Kalyan K.; Alumasa, John N.; Avanzato, Victoria; Place, David E.; Markley, Rachel L.; Katkere, Bhuvana; Rabadi, Seham M.; Bakshi, Chandra Shekhar

    2016-01-01

    Bacteria require at least one pathway to rescue ribosomes stalled at the ends of mRNAs. The primary pathway for ribosome rescue is trans-translation, which is conserved in >99% of sequenced bacterial genomes. Some species also have backup systems, such as ArfA or ArfB, which can rescue ribosomes in the absence of sufficient trans-translation activity. Small-molecule inhibitors of ribosome rescue have broad-spectrum antimicrobial activity against bacteria grown in liquid culture. These compounds were tested against the tier 1 select agent Francisella tularensis to determine if they can limit bacterial proliferation during infection of eukaryotic cells. The inhibitors KKL-10 and KKL-40 exhibited exceptional antimicrobial activity against both attenuated and fully virulent strains of F. tularensis in vitro and during ex vivo infection. Addition of KKL-10 or KKL-40 to macrophages or liver cells at any time after infection by F. tularensis prevented further bacterial proliferation. When macrophages were stimulated with the proinflammatory cytokine gamma interferon before being infected by F. tularensis, addition of KKL-10 or KKL-40 reduced intracellular bacteria by >99%, indicating that the combination of cytokine-induced stress and a nonfunctional ribosome rescue pathway is fatal to F. tularensis. Neither KKL-10 nor KKL-40 was cytotoxic to eukaryotic cells in culture. These results demonstrate that ribosome rescue is required for F. tularensis growth at all stages of its infection cycle and suggest that KKL-10 and KKL-40 are good lead compounds for antibiotic development. PMID:26953190

  11. Biology of Francisella tularensis Subspecies holarctica Live Vaccine Strain in the Tick Vector Dermacentor variabilis

    PubMed Central

    Mani, Rinosh J.; Reichard, Mason V.; Morton, Rebecca J.; Kocan, Katherine M.; Clinkenbeard, Kenneth D.

    2012-01-01

    Background The γ-proteobacterium Francisella tularensis is the etiologic agent of seasonal tick-transmitted tularemia epizootics in rodents and rabbits and of incidental infections in humans. The biology of F. tularensis in its tick vectors has not been fully described, particularly with respect to its quanta and duration of colonization, tissue dissemination, and transovarial transmission. A systematic study of the colonization of Dermacentor variabilis by the F. tularensis subsp. holarctica live vaccine strain (LVS) was undertaken to better understand whether D. variabilis may serve as an inter-epizootic reservoir for F. tularensis. Methodology/Principal Findings Colony-reared larva, nymph, and adult D. variabilis were artificially fed LVS via glass capillary tubes fitted over the tick mouthparts, and the level of colonization determined by microbial culture. Larvae and nymphs were initially colonized with 8.8±0.8×101 and 1.1±0.03×103 CFU/tick, respectively. Post-molting, a significant increase in colonization of both molted nymphs and adults occurred, and LVS persisted in 42% of molted adult ticks at 126 days post-capillary tube feeding. In adult ticks, LVS initially colonized the gut, disseminated to hemolymph and salivary glands by 21 days, and persisted up to 165 days. LVS was detected in the salivary secretions of adult ticks after four days post intra-hemocoelic inoculation, and LVS recovered from salivary gland was infectious to mice with an infectious dose 50% of 3 CFU. LVS in gravid female ticks colonized via the intra-hemocoelic route disseminated to the ovaries and then to the oocytes, but the pathogen was not recovered from the subsequently-hatched larvae. Conclusions/Significance This study demonstrates that D. variabilis can be efficiently colonized with F. tularensis using artificial methods. The persistence of F. tularensis in D. variabilis suggests that this tick species may be involved in the maintenance of enzootic foci of tularemia in the

  12. 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.

  13. Modulation of virulence factors in Francisella tularensis determines human macrophage responses

    PubMed Central

    Carlson, Paul E.; Carroll, James A.; O’Dee, Dawn M.; Nau, Gerard J.

    2009-01-01

    Francisella tularensis, the causative agent of tularemia and Category A biodefense agent, is known to replicate within host macrophages, though the pathogenesis of this organism is incompletely understood. We have isolated a variant of F. tularensis Live Vaccine Strain (LVS) based on colony morphology and its effect on macrophages. Human monocyte-derived macrophages produced more tumor necrosis factor α (TNFα), interleukin (IL)-1β, IL-6, and IL-12 p40 following exposure to the variant, designated the activating variant (ACV). The immunoreactivity of the lipopolysaccharide (LPS) from both LVS and ACV was comparable to the previously described blue variant and was distinct from the gray variant of LVS. We found, however, the soluble protein fractions of LVS and ACV differed. Further investigation using two-dimensional gel electrophoresis demonstrated higher levels of several proteins in the parental LVS isolate. The differentially-expressed proteins featured several associated with virulence in F. tularensis and other pathogens, including intracellular growth locus C (IglC), a σ54 modulation protein family member (YhbH), and aconitase. ACV reverted to the LVS phenotype, indicated by low cytokine induction and high IglC expression, after growth in a chemically-defined media. These data provide evidence that the levels of virulence factors in F. tularensis are modulated based on culture conditions and that this modulation impacts host responses. This work provides a basis for investigation of Francisella virulence factor regulation and the identification of additional factors, co-regulated with IglC, that affect macrophage responses. PMID:17369012

  14. Sensitivity of Francisella tularensis to ultrapure water and deoxycholate: implications for bacterial intracellular growth assay in macrophages

    PubMed Central

    Chalabaev, Sabina; Anderson, Christine A.; Onderdonk, Andrew B.; Kasper, Dennis L.

    2011-01-01

    The ability of Francisella tularensis to replicate in macrophages is critical for its pathogenesis, therefore intracellular growth assays are important tools for assessing virulence. We show that two lysis solutions commonly used in these assays, deionized water and deoxycholate in PBS, lead to highly inaccurate measurements of intracellular bacterial survival. PMID:21420447

  15. 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.

  16. Nucleolin, a Shuttle Protein Promoting Infection of Human Monocytes by Francisella tularensis

    PubMed Central

    Barel, Monique; Meibom, Karin; Charbit, Alain

    2010-01-01

    Background Francisella tularensis is a highly virulent facultative intracellular bacterium, disseminating in vivo mainly within host mononuclear phagocytes. After entry into macrophages, F. tularensis initially resides in a phagosomal compartment, whose maturation is then arrested. Bacteria escape rapidly into the cytoplasm, where they replicate freely. We recently demonstrated that nucleolin, an eukaryotic protein able to traffic from the nucleus to the cell surface, acted as a surface receptor for F. tularensis LVS on human monocyte-like THP-1 cells. Methodology/Principal Findings Here, we followed the fate of nucleolin once F. tularensis has been endocytosed. We first confirmed by siRNA silencing experiments that expression of nucleolin protein was essential for binding of LVS on human macrophage-type THP-1 cells. We then showed that nucleolin co-localized with intracellular bacteria in the phagosomal compartment. Strikingly, in that compartment, nucleolin also co-localized with LAMP-1, a late endosomal marker. Co-immunoprecipation assays further demonstrated an interaction of nucleolin with LAMP-1. Co-localization of nucleolin with LVS was no longer detectable at 24 h when bacteria were multiplying in the cytoplasm. In contrast, with an iglC mutant of LVS, which remains trapped into the phagosomal compartment, or with inert particles, nucleolin/bacteria co-localization remained almost constant. Conclusions/Significance We herein confirm the importance of nucleolin expression for LVS binding and its specificity as nucleolin is not involved in binding of another intracellular pathogen as L. monocytogenes or an inert particle. Association of nucleolin with F. tularensis during infection continues intracellularly after endocytosis of the bacteria. The present work therefore unravels for the first time the presence of nucleolin in the phagosomal compartment of macrophages. PMID:21152024

  17. 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.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27098957

  19. Characterization of the O-antigen polymerase (Wzy) of Francisella tularensis.

    PubMed

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

    2010-09-03

    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 (Gly(176), Asp(177), Gly(323), and Tyr(324)) 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.

  20. 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

  1. 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

  2. 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

    2015-06-02

    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.

  3. 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

  4. 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.

  5. Aged mice display an altered pulmonary host response to Francisella tularensis live vaccine strain (LVS) infections

    PubMed Central

    CA, Mares; SS, Ojeda; Q, Li; EG, Morris; JJ, Coalson; JM, Teale

    2012-01-01

    Aging is a complex phenomenon that has been shown to affect many organ systems including the innate and adaptive immune systems. The current study was designed to examine the potential effect of immunosenescence on the pulmonary immune response using a Francisella tularensis live vaccine strain (LVS) inhalation infection model. F. tularensis is a gram-negative intracellular pathogen that can cause a severe pneumonia.In this study both young (8-12 week old) and aged (20-24 month old) mice were infected intranasally with LVS. Lung tissues from young and aged mice were used to assess pathology, recruitment of immune cell types and cytokine expression levels at various times post infection. Bacterial burdens were also assessed. Interestingly, the lungs of aged animals harbored fewer organisms at early time points of infection (day 1, day 3) compared with their younger counterparts. In addition, only aged animals displayed small perivascular aggregates at these early time points that appeared mostly mononuclear in nature. However, the kinetics of infiltrating polymorphonuclear neutrophils (PMNs) and increased cytokine levels measured in the bronchial alveolar lavage fluid (BALF) were delayed in infected aged animals relative to young infected animals with neutrophils appearing at day 5 post infection (PI) in the aged animals as opposed to day 3 PI in the young infected animals. Also evident were alterations in the ratios of mononuclear to PMNs at distinct post infection times. The above evidence indicates that aged mice elicit an altered immune response in the lung to respiratory Francisella tularensis LVS infections compared to their younger counterparts. PMID:19825409

  6. Lack of OxyR and KatG Results in Extreme Susceptibility of Francisella tularensis LVS to Oxidative Stress and Marked Attenuation In vivo

    PubMed Central

    Honn, Marie; Lindgren, Helena; Bharath, Gurram K.; Sjöstedt, Anders

    2017-01-01

    Francisella tularensis is an intracellular bacterium and as such is expected to encounter a continuous attack by reactive oxygen species (ROS) in its intracellular habitat and efficiently coping with oxidative stress is therefore essential for its survival. The oxidative stress response system of F. tularensis is complex and includes multiple antioxidant enzymes and pathways, including the transcriptional regulator OxyR and the H2O2-decomposing enzyme catalase, encoded by katG. The latter is regulated by OxyR. A deletion of either of these genes, however, does not severely compromise the virulence of F. tularensis and we hypothesized that if the bacterium would be deficient of both catalase and OxyR, then the oxidative defense and virulence of F. tularensis would become severely hampered. To test this hypothesis, we generated a double deletion mutant, ΔoxyR/ΔkatG, of F. tularensis LVS and compared its phenotype to the parental LVS strain and the corresponding single deletion mutants. In accordance with the hypothesis, ΔoxyR/ΔkatG was distinctly more susceptible than ΔoxyR and ΔkatG to H2O2, ONOO−, and O2-, moreover, it hardly grew in mouse-derived BMDM or in mice, whereas ΔkatG and ΔoxyR grew as well as F. tularensis LVS in BMDM and exhibited only slight attenuation in mice. Altogether, the results demonstrate the importance of catalase and OxyR for a robust oxidative stress defense system and that they act cooperatively. The lack of both functions render F. tularensis severely crippled to handle oxidative stress and also much attenuated for intracellular growth and virulence. PMID:28174696

  7. Francisella tularensis Antioxidants Harness Reactive Oxygen Species to Restrict Macrophage Signaling and Cytokine Production*

    PubMed Central

    Melillo, Amanda A.; Bakshi, Chandra Shekhar; Melendez, J. Andrés

    2010-01-01

    Francisella tularensis is the etiologic agent of the highly infectious animal and human disease tularemia. Its extreme infectivity and virulence are associated with its ability to evade immune detection, which we now link to its robust reactive oxygen species-scavenging capacity. Infection of primary human monocyte-derived macrophages with virulent F. tularensis SchuS4 prevented proinflammatory cytokine production in the presence or absence of IFN-γ compared with infection with the attenuated live vaccine strain. SchuS4 infection also blocked signals required for macrophage cytokine production, including Akt phosphorylation, IκBα degradation, and NF-κB nuclear localization and activation. Concomitant with SchuS4-mediated suppression of Akt phosphorylation was an increase in the levels of the Akt antagonist PTEN. Moreover, SchuS4 prevented the H2O2-dependent oxidative inactivation of PTEN compared with a virulent live vaccine strain. Mutation of catalase (katG) sensitized F. tularensis to H2O2 and enhanced PTEN oxidation, Akt phosphorylation, NF-κB activation, and inflammatory cytokine production. Together, these findings suggest a novel role for bacterial antioxidants in restricting macrophage activation through their ability to preserve phosphatases that temper kinase signaling and proinflammatory cytokine production. PMID:20558723

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

    PubMed

    Heine, Henry S; Chuvala, Lara; Riggins, Renaldo; Cirz, Ryan; Cass, Robert; Louie, Arnold; Drusano, G L

    2016-01-11

    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.

  10. 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.

  11. 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.

  12. GroEL and Lipopolysaccharide from Francisella tularensis Live Vaccine Strain Synergistically Activate Human Macrophages ▿

    PubMed Central

    Noah, Courtney E.; Malik, Meenakshi; Bublitz, DeAnna C.; Camenares, Devin; Sellati, Timothy J.; Benach, Jorge L.; Furie, Martha B.

    2010-01-01

    Francisella tularensis, the causative agent of tularemia, interacts with host cells of innate immunity in an atypical manner. For most Gram-negative bacteria, the release of lipopolysaccharide (LPS) from their outer membranes stimulates an inflammatory response. When LPS from the attenuated live vaccine strain (LVS) or the highly virulent Schu S4 strain of F. tularensis was incubated with human umbilical vein endothelial cells, neither species of LPS induced expression of the adhesion molecule E-selectin or secretion of the chemokine CCL2. Moreover, a high concentration (10 μg/ml) of LVS or Schu S4 LPS was required to stimulate production of CCL2 by human monocyte-derived macrophages (huMDM). A screen for alternative proinflammatory factors of F. tularensis LVS identified the heat shock protein GroEL as a potential candidate. Recombinant LVS GroEL at a concentration of 10 μg/ml elicited secretion of CXCL8 and CCL2 by huMDM through a TLR4-dependent mechanism. When 1 μg of LVS GroEL/ml was added to an equivalent amount of LVS LPS, the two components synergistically activated the huMDM to produce CXCL8. Schu S4 GroEL was less stimulatory than LVS GroEL and showed a lesser degree of synergy when combined with Schu S4 LPS. These findings suggest that the intrinsically low proinflammatory activity of F. tularensis LPS may be increased in the infected human host through interactions with other components of the bacterium. PMID:20123721

  13. Structure-Function Analysis of DipA, a Francisella tularensis Virulence Factor Required for Intracellular Replication

    PubMed Central

    Chong, Audrey; Child, Robert; Wehrly, Tara D.; Rockx-Brouwer, Dedeke; Qin, Aiping; Mann, Barbara J.; Celli, Jean

    2013-01-01

    Francisella tularensis is a highly infectious bacterium whose virulence relies on its ability to rapidly reach the macrophage cytosol and extensively replicate in this compartment. We previously identified a novel Francisella virulence factor, DipA (FTT0369c), which is required for intramacrophage proliferation and survival, and virulence in mice. DipA is a 353 amino acid protein with a Sec-dependent signal peptide, four Sel1-like repeats (SLR), and a C-terminal coiled-coil (CC) domain. Here, we determined through biochemical and localization studies that DipA is a membrane-associated protein exposed on the surface of the prototypical F. tularensis subsp. tularensis strain SchuS4 during macrophage infection. Deletion and substitution mutagenesis showed that the CC domain, but not the SLR motifs, of DipA is required for surface exposure on SchuS4. Complementation of the dipA mutant with either DipA CC or SLR domain mutants did not restore intracellular growth of Francisella, indicating that proper localization and the SLR domains are required for DipA function. Co-immunoprecipitation studies revealed interactions with the Francisella outer membrane protein FopA, suggesting that DipA is part of a membrane-associated complex. Altogether, our findings indicate that DipA is positioned at the host–pathogen interface to influence the intracellular fate of this pathogen. PMID:23840797

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

    PubMed

    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

    2008-10-01

    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.

  15. German Francisella tularensis isolates from European brown hares (Lepus europaeus) reveal genetic and phenotypic diversity

    PubMed Central

    2013-01-01

    Background Tularemia is a zoonotic disease caused by Francisella tularensis that has been found in many different vertebrates. In Germany most human infections are caused by contact with infected European brown hares (Lepus europaeus). The aim of this study was to elucidate the epidemiology of tularemia in hares using phenotypic and genotypic characteristics of F. tularensis. Results Cultivation of F. tularensis subsp. holarctica bacteria from organ material was successful in 31 of 52 hares that had a positive PCR result targeting the Ft-M19 locus. 17 isolates were sensitive to erythromycin and 14 were resistant. Analysis of VNTR loci (Ft-M3, Ft-M6 and Ft-M24), INDELs (Ftind33, Ftind38, Ftind49, RD23) and SNPs (B.17, B.18, B.19, and B.20) was shown to be useful to investigate the genetic relatedness of Francisella strains in this set of strains. The 14 erythromycin resistant isolates were assigned to clade B.I, and 16 erythromycin sensitive isolates to clade B.IV and one isolate was found to belong to clade B.II. MALDI-TOF mass spectrometry (MS) was useful to discriminate strains to the subspecies level. Conclusions F. tularensis seems to be a re-emerging pathogen in Germany. The pathogen can easily be identified using PCR assays. Isolates can also be identified within one hour using MALDI-TOF MS in laboratories where specific PCR assays are not established. Further analysis of strains requires genotyping tools. The results from this study indicate a geographical segregation of the phylogenetic clade B.I and B.IV, where B.I strains localize primarily within eastern Germany and B.IV strains within western Germany. This phylogeographical pattern coincides with the distribution of biovar I (erythromycin sensitive) and biovar II (erythromycin resistance) strains. When time and costs are limiting parameters small numbers of isolates can be analysed using PCR assays combined with DNA sequencing with a focus on genetic loci that are most likely discriminatory among

  16. 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.

  17. Francisella tularensis Schu S4 lipopolysaccharide core sugar and O-antigen mutants are attenuated in a mouse model of tularemia.

    PubMed

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

    2014-04-01

    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.

  18. 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

  19. 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.

  20. Genomic Deletion Marking an Emerging Subclone of Francisella tularensis subsp. holarctica in France and the Iberian Peninsula▿ †

    PubMed Central

    Dempsey, M. P.; Dobson, M.; Zhang, C.; Zhang, M.; Lion, C.; Gutiérrez-Martín, C. B.; Iwen, P. C.; Fey, P. D.; Olson, M. E.; Niemeyer, D.; Francesconi, S.; Crawford, R.; Stanley, M.; Rhodes, J.; Wagner, D. M.; Vogler, A. J.; Birdsell, D.; Keim, P.; Johansson, A.; Hinrichs, S. H.; Benson, A. K.

    2007-01-01

    Francisella tularensis subsp. holarctica is widely disseminated in North America and the boreal and temperate regions of the Eurasian continent. Comparative genomic analyses identified a 1.59-kb genomic deletion specific to F. tularensis subsp. holarctica isolates from Spain and France. Phylogenetic analysis of strains carrying this deletion by multiple-locus variable-number tandem repeat analysis showed that the strains comprise a highly related set of genotypes, implying that these strains were recently introduced or recently emerged by clonal expansion in France and the Iberian Peninsula. PMID:17890329

  1. Long-range dispersal moved Francisella tularensis into Western Europe from the East.

    PubMed

    Dwibedi, Chinmay; Birdsell, Dawn; Lärkeryd, Adrian; Myrtennäs, Kerstin; Öhrman, Caroline; Nilsson, Elin; Karlsson, Edvin; Hochhalter, Christian; Rivera, Andrew; Maltinsky, Sara; Bayer, Brittany; Keim, Paul; Scholz, Holger C; Tomaso, Herbert; Wittwer, Matthias; Beuret, Christian; Schuerch, Nadia; Pilo, Paola; Hernández Pérez, Marta; Rodriguez-Lazaro, David; Escudero, Raquel; Anda, Pedro; Forsman, Mats; Wagner, David M; Larsson, Pär; Johansson, Anders

    2016-12-01

    For many infections transmitting to humans from reservoirs in nature, disease dispersal patterns over space and time are largely unknown. Here, a reversed genomics approach helped us understand disease dispersal and yielded insight into evolution and biological properties of Francisella tularensis, the bacterium causing tularemia. We whole-genome sequenced 67 strains and characterized by single-nucleotide polymorphism assays 138 strains, collected from individuals infected 1947-2012 across Western Europe. We used the data for phylogenetic, population genetic and geographical network analyses. All strains (n=205) belonged to a monophyletic population of recent ancestry not found outside Western Europe. Most strains (n=195) throughout the study area were assigned to a star-like phylogenetic pattern indicating that colonization of Western Europe occurred via clonal expansion. In the East of the study area, strains were more diverse, consistent with a founder population spreading from east to west. The relationship of genetic and geographic distance within the F. tularensis population was complex and indicated multiple long-distance dispersal events. Mutation rate estimates based on year of isolation indicated null rates; in outbreak hotspots only, there was a rate of 0.4 mutations/genome/year. Patterns of nucleotide substitution showed marked AT mutational bias suggestive of genetic drift. These results demonstrate that tularemia has moved from east to west in Europe and that F. tularensis has a biology characterized by long-range geographical dispersal events and mostly slow, but variable, replication rates. The results indicate that mutation-driven evolution, a resting survival phase, genetic drift and long-distance geographical dispersal events have interacted to generate genetic diversity within this species.

  2. Long-range dispersal moved Francisella tularensis into Western Europe from the East

    PubMed Central

    Dwibedi, Chinmay; Birdsell, Dawn; Lärkeryd, Adrian; Myrtennäs, Kerstin; Öhrman, Caroline; Nilsson, Elin; Karlsson, Edvin; Hochhalter, Christian; Rivera, Andrew; Maltinsky, Sara; Bayer, Brittany; Keim, Paul; Scholz, Holger C.; Tomaso, Herbert; Wittwer, Matthias; Beuret, Christian; Schuerch, Nadia; Pilo, Paola; Hernández Pérez, Marta; Rodriguez-Lazaro, David; Escudero, Raquel; Anda, Pedro; Forsman, Mats; Wagner, David M.; Larsson, Pär

    2016-01-01

    For many infections transmitting to humans from reservoirs in nature, disease dispersal patterns over space and time are largely unknown. Here, a reversed genomics approach helped us understand disease dispersal and yielded insight into evolution and biological properties of Francisella tularensis, the bacterium causing tularemia. We whole-genome sequenced 67 strains and characterized by single-nucleotide polymorphism assays 138 strains, collected from individuals infected 1947-2012 across Western Europe. We used the data for phylogenetic, population genetic and geographical network analyses. All strains (n=205) belonged to a monophyletic population of recent ancestry not found outside Western Europe. Most strains (n=195) throughout the study area were assigned to a star-like phylogenetic pattern indicating that colonization of Western Europe occurred via clonal expansion. In the East of the study area, strains were more diverse, consistent with a founder population spreading from east to west. The relationship of genetic and geographic distance within the F. tularensis population was complex and indicated multiple long-distance dispersal events. Mutation rate estimates based on year of isolation indicated null rates; in outbreak hotspots only, there was a rate of 0.4 mutations/genome/year. Patterns of nucleotide substitution showed marked AT mutational bias suggestive of genetic drift. These results demonstrate that tularemia has moved from east to west in Europe and that F. tularensis has a biology characterized by long-range geographical dispersal events and mostly slow, but variable, replication rates. The results indicate that mutation-driven evolution, a resting survival phase, genetic drift and long-distance geographical dispersal events have interacted to generate genetic diversity within this species. PMID:28348839

  3. Nonrandom Distribution of Vector Ticks (Dermacentor variabilis) Infected by Francisella tularensis

    PubMed Central

    Goethert, Heidi K.; Telford, Sam R.

    2009-01-01

    The island of Martha's Vineyard, Massachusetts, is the site of a sustained outbreak of tularemia due to Francisella tularensis tularensis. Dog ticks, Dermacentor variabilis, appear to be critical in the perpetuation of the agent there. Tularemia has long been characterized as an agent of natural focality, stably persisting in characteristic sites of transmission, but this suggestion has never been rigorously tested. Accordingly, we sought to identify a natural focus of transmission of the agent of tularemia by mapping the distribution of PCR-positive ticks. From 2004 to 2007, questing D. variabilis were collected from 85 individual waypoints along a 1.5 km transect in a field site on Martha's Vineyard. The positions of PCR-positive ticks were then mapped using ArcGIS. Cluster analysis identified an area approximately 290 meters in diameter, 9 waypoints, that was significantly more likely to yield PCR-positive ticks (relative risk 3.3, P = 0.001) than the rest of the field site. Genotyping of F. tularensis using variable number tandem repeat (VNTR) analysis on PCR-positive ticks yielded 13 different haplotypes, the vast majority of which was one dominant haplotype. Positive ticks collected in the cluster were 3.4 times (relative risk = 3.4, P<0.0001) more likely to have an uncommon haplotype than those collected elsewhere from the transect. We conclude that we have identified a microfocus where the agent of tularemia stably perpetuates and that this area is where genetic diversity is generated. PMID:19247435

  4. Modulation of hepatic PPAR expression during Ft LVS LPS-induced protection from Francisella tularensis LVS infection

    PubMed Central

    2010-01-01

    Background It has been shown previously that administration of Francisella tularensis (Ft) Live Vaccine Strain (LVS) lipopolysaccharide (LPS) protects mice against subsequent challenge with Ft LVS and blunts the pro-inflammatory cytokine response. Methods To further investigate the molecular mechanisms that underlie Ft LVS LPS-mediated protection, we profiled global hepatic gene expression following Ft LVS LPS or saline pre-treatment and subsequent Ft LVS challenge using Affymetrix arrays. Results A large number of genes (> 3,000) were differentially expressed at 48 hours post-infection. The degree of modulation of inflammatory genes by infection was clearly attenuated by pre-treatment with Ft LVS LPS in the surviving mice. However, Ft LVS LPS alone had a subtle effect on the gene expression profile of the uninfected mice. By employing gene set enrichment analysis, we discovered significant up-regulation of the fatty acid metabolism pathway, which is regulated by peroxisome proliferator activated receptors (PPARs). Conclusions We hypothesize that the LPS-induced blunting of pro-inflammatory response in mouse is, in part, mediated by PPARs (α and γ). PMID:20082697

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

    PubMed Central

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

    2012-01-01

    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. PMID:22486311

  6. 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.

  7. Reproducible and Quantitative Model of Infection of Dermacentor variabilis with the Live Vaccine Strain of Francisella tularensis

    PubMed Central

    Coburn, Jenifer; Maier, Tamara; Casey, Monika; Padmore, Lavinia; Sato, Hiromi

    2014-01-01

    Pathogen life cycles in mammalian hosts have been studied extensively, but studies with arthropod vectors represent considerable challenges. In part this is due to the difficulty of delivering a reproducible dose of bacteria to follow arthropod-associated replication. We have established reproducible techniques to introduce known numbers of Francisella tularensis strain LVS from mice into Dermacentor variabilis nymphs. Using this model infection system, we performed dose-response infection experiments and followed bacterial replication through the molt to adults and at later time points. During development to adults, bacteria replicate to high numbers and can be found associated with the gut tissues, salivary glands, and hemolymph of adult ticks. Further, we can transmit a mutant of LVS (LVS ΔpurMCD) that cannot replicate in macrophages in vitro or in mice to nymphs. Our data show that the LVS ΔpurMCD mutant cannot be transstadially transmitted from nymphs to adult ticks. We then show that a plasmid-complemented strain of this mutant is recoverable in adult ticks and necessary for bacterial replication during the molt. In a mixed-infection assay (ΔpurMCD mutant versus ΔpurMCD complement), 98% of the recovered bacteria retained the plasmid marker. These data suggest that the ΔpurMCD mutation cannot be rescued by the presence a complemented strain in a mixed infection. Importantly, our infection model provides a platform to test specific mutants for their replication in ticks, perform competition studies, and use other genetic techniques to identify F. tularensis genes that are expressed or required in this unique environment. PMID:25362054

  8. [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

  9. Large Scale Structural Rearrangement of a Serine Hydrolase from Francisella tularensis Facilitates Catalysis*

    PubMed Central

    Filippova, Ekaterina V.; Weston, Leigh A.; Kuhn, Misty L.; Geissler, Brett; Gehring, Alexandra M.; Armoush, Nicola; Adkins, Chinessa T.; Minasov, George; Dubrovska, Ievgeniia; Shuvalova, Ludmilla; Winsor, James R.; Lavis, Luke D.; Satchell, Karla J. F.; Becker, Daniel P.; Anderson, Wayne F.; Johnson, R. Jeremy

    2013-01-01

    Tularemia is a deadly, febrile disease caused by infection by the Gram-negative bacterium, Francisella tularensis. Members of the ubiquitous serine hydrolase protein family are among current targets to treat diverse bacterial infections. Herein we present a structural and functional study of a novel bacterial carboxylesterase (FTT258) from F. tularensis, a homologue of human acyl protein thioesterase (hAPT1). The structure of FTT258 has been determined in multiple forms, and unexpectedly large conformational changes of a peripheral flexible loop occur in the presence of a mechanistic cyclobutanone ligand. The concomitant changes in this hydrophobic loop and the newly exposed hydrophobic substrate binding pocket suggest that the observed structural changes are essential to the biological function and catalytic activity of FTT258. Using diverse substrate libraries, site-directed mutagenesis, and liposome binding assays, we determined the importance of these structural changes to the catalytic activity and membrane binding activity of FTT258. Residues within the newly exposed hydrophobic binding pocket and within the peripheral flexible loop proved essential to the hydrolytic activity of FTT258, indicating that structural rearrangement is required for catalytic activity. Both FTT258 and hAPT1 also showed significant association with liposomes designed to mimic bacterial or human membranes, respectively, even though similar structural rearrangements for hAPT1 have not been reported. The necessity for acyl protein thioesterases to have maximal catalytic activity near the membrane surface suggests that these conformational changes in the protein may dually regulate catalytic activity and membrane association in bacterial and human homologues. PMID:23430251

  10. Isolation of Francisella tularensis and Yersinia pestis from Blood Cultures by Plasma Purification and Immunomagnetic Separation Accelerates Antibiotic Susceptibility Determination

    PubMed Central

    Aloni-Grinstein, Ronit; Schuster, Ofir; Yitzhaki, Shmuel; Aftalion, Moshe; Maoz, Sharon; Steinberger-Levy, Ida; Ber, Raphael

    2017-01-01

    The early symptoms of tularemia and plague, which are caused by Francisella tularensis and Yersinia pestis infection, respectively, are common to other illnesses, resulting in a low index of suspicion among clinicians. Moreover, because these diseases can be treated only with antibiotics, rapid isolation of the bacteria and antibiotic susceptibility testing (AST) are preferable. Blood cultures of patients may serve as a source for bacteria isolation. However, due to the slow growth rates of F. tularensis and Y. pestis on solid media, isolation by plating blood culture samples on proper agar plates may require several days. Thus, improving the isolation procedure prior to antibiotic susceptibility determination is a major clinically relevant need. In this study, we developed a rapid, selective procedure for the isolation of F. tularensis and Y. pestis from blood cultures. We examined drop-plating and plasma purification followed by immunomagnetic separation (IMS) as alternative isolation methods. We determined that replacing the classical isolation method with drop-plating is advantageous with respect to time at the expense of specificity. Hence, we also examined isolation by IMS. Sub-localization of F. tularensis within blood cultures of infected mice has revealed that the majority of the bacteria are located within the extracellular fraction, in the plasma. Y. pestis also resides within the plasma. Therefore, the plasma fraction was isolated from blood cultures and subjected to an IMS procedure using polyclonal anti-F. tularensis live vaccine strain (LVS) or anti-Y. pestis antibodies conjugated to 50-nm nano-beads. The time required to reach an inoculum of sufficient bacteria for AST was shortest when using the plasma and IMSs for both bacteria, saving up to 2 days of incubation for F. tularensis and 1 day for Y. pestis. Our isolation procedure provides a proof of concept for the clinical relevance of rapid isolation for AST from F. tularensis- and Y. pestis

  11. Bioavailability and efficacy of levofloxacin against Francisella tularensis in the common marmoset (Callithrix jacchus).

    PubMed

    Nelson, Michelle; Lever, Mark S; Dean, Rachel E; Pearce, Peter C; Stevens, Daniel J; Simpson, Andrew J H

    2010-09-01

    Pharmacokinetic and efficacy studies with levofloxacin were performed in the common marmoset (Callithrix jacchus) model of inhalational tularemia. Plasma levofloxacin pharmacokinetics were determined in six animals in separate single-dose and multidose studies. Plasma drug concentrations were analyzed using liquid chromatography-tandem mass spectrometry-electrospray ionization. On day 7 of a twice-daily dosing regimen of 40 mg/kg, the levofloxacin half-life, maximum concentration, and area under the curve in marmoset plasma were 2.3 h, 20.9 microg/ml, and 81.4 microg/liter/h, respectively. An efficacy study was undertaken using eight treated and two untreated control animals. Marmosets were challenged with a mean of 1.5 x 10(2) CFU of Francisella tularensis by the airborne route. Treated animals were administered 16.5 mg/kg levofloxacin by mouth twice daily, based on the pharmacokinetic parameters, beginning 24 h after challenge. Control animals had a raised core body temperature by 57 h postchallenge and died from infection by day 5. All of the other animals survived, remained afebrile, and lacked overt clinical signs. No bacteria were recovered from the organs of these animals at postmortem after culling at day 24 postchallenge. In conclusion, postexposure prophylaxis with orally administered levofloxacin was efficacious against acute inhalational tularemia in the common marmoset. The marmoset appears to be an appropriate animal model for the evaluation of postexposure therapies.

  12. Characterization of Monoclonal Antibodies to Terminal and Internal O-Antigen Epitopes of Francisella tularensis Lipopolysaccharide

    PubMed Central

    Roche, Marly I.; Lu, Zhaohua; Hui, Julia H.

    2011-01-01

    The lipopolysaccharide (LPS) of Francisella tularensis (Ft), the Gram negative bacterium that causes tularemia, has been shown to be a main protective antigen in mice and humans; we have previously demonstrated that murine anti-Ft LPS IgG2a monoclonal antibodies (MAbs) can protect mice against otherwise lethal intranasal infection with the Ft live vaccine strain (LVS). Here we show that four IgG2a anti-LPS MAbs are specific for the O-polysaccharide (O-antigen [OAg]) of Ft LPS. But whereas three of the MAbs bind to immunodominant repeating internal epitopes, one binds to a unique terminal epitope of Ft OAg. This was deduced from its even binding to both long and short chains of the LPS ladder in Western blots, its rapid decrease in ELISA binding to decreasing solid-phase LPS concentrations, its inability to compete for LPS binding with a representative of the other three MAbs, and its inability to immunoprecipitate OAg despite its superior agglutination titer. Biacore analysis showed the end-binding MAb to have higher bivalent avidity for Ft OAg than the internal-binding MAbs and provided an immunogenicity explanation for the predominance of internal-binding anti-Ft OAg MAbs. These findings demonstrate that non-overlapping epitopes can be targeted by antibodies to Ft OAg, which may inform the design of vaccines and immunotherapies against tularemia. PMID:21466282

  13. Exposure of laboratory workers to Francisella tularensis despite a bioterrorism procedure.

    PubMed

    Shapiro, Daniel S; Schwartz, Donald R

    2002-06-01

    A rapidly fatal case of pulmonary tularemia in a 43-year-old man who was transferred to a tertiary care facility is presented. The microbiology laboratory and autopsy services were not notified of the clinical suspicion of tularemia by the service caring for the patient. Despite having a laboratory bioterrorism procedure in place and adhering to established laboratory protocol, 12 microbiology laboratory employees were exposed to Francisella tularensis and the identification of the organism was delayed due to lack of notification of the laboratory of the clinical suspicion of tularemia. A total of 11 microbiology employees and two persons involved in performing the patient's autopsy received prophylactic doxycycline due to concerns of transmission. None of them developed signs or symptoms of tularemia. One microbiology laboratory employee was pregnant and declined prophylactic antibiotics. As a result of this event, the microbiology laboratory has incorporated flow charts directly into the bench procedures for several highly infectious agents that may be agents of bioterrorism. This should permit more rapid recognition of an isolate for referral to a Level B laboratory for definitive identification and should improve laboratory safety.

  14. 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.

  15. Immunization with heat-killed Francisella tularensis LVS elicits protective antibody-mediated immunity.

    PubMed

    Lavine, Christy L; Clinton, Shawn R; Angelova-Fischer, Irena; Marion, Tony N; Bina, Xiaowen R; Bina, James E; Whitt, Michael A; Miller, Mark A

    2007-11-01

    Francisella tularensis (FT) has been classified by the CDC as a category A pathogen because of its high virulence and the high mortality rate associated with infection via the aerosol route. Because there is no licensed vaccine available for FT, development of prophylactic and therapeutic regimens for the prevention/treatment of infection is a high priority. In this report, heat-killed FT live vaccine strain (HKLVS) was employed as a vaccine immunogen, either alone or in combination with an adjuvant, and was found to elicit protective immunity against high-dose FT live vaccine strain (FTLVS) challenge. FT-specific antibodies produced in response to immunization with HKLVS alone were subsequently found to completely protect naive mice against high-dose FT challenge in both infection-interference and passive immunization experiments. Additional passive immunization trials employing serum collected from mice immunized with a heat-killed preparation of an O-antigen-deficient transposon mutant of FTLVS (HKLVS-OAg(neg)) yielded similar results. These findings demonstrated that FT-specific antibodies alone can confer immunity against high-dose FTLVS challenge, and they reveal that antibody-mediated protection is not dependent upon production of LPS-specific antibodies.

  16. 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

  17. Francisella tularensis type A strains cause the rapid encystment of Acanthamoeba castellanii and survive in amoebal cysts for three weeks postinfection.

    PubMed

    El-Etr, Sahar H; Margolis, Jeffrey J; Monack, Denise; Robison, Richard A; Cohen, Marissa; Moore, Emily; Rasley, Amy

    2009-12-01

    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 to 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 is caused by factor(s) secreted by amoebae and/or F. tularensis into the coculture medium. Further, our results indicate that in contrast to the live vaccine strain LVS, virulent strains of F. tularensis can survive in A. castellanii cysts for at least 3 weeks postinfection and that the 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 interactions between F. tularensis strains and amoebae may play a role in the environmental persistence of F. tularensis.

  18. 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.

  19. IL-12Rβ2 is critical for survival of primary Francisella tularensis LVS infection

    PubMed Central

    Melillo, Amanda A.; Foreman, Oded; Elkins, Karen L.

    2013-01-01

    Using a panel of vaccines that provided different degrees of protection, we previously identified the IL-12 receptor subunit β2 as a mediator, whose relative expression correlated with strength of protection against secondary lethal challenge of vaccinated mice with an intracellular bacterium, the LVS of Francisella tularensis. The present study therefore tested the hypothesis that IL-12Rβ2 is an important mediator in resistance to LVS by directly examining its role during infections. IL-12Rβ2 KO mice were highly susceptible to LVS primary infection, administered i.d. or i.n. The LD50 of LVS infection of KO mice were 2 logs lower than those of WT mice, regardless of route. Five days after infection with LVS, bacterial organ burdens were significantly higher in IL-12Rβ2 KO mice. IL-12Rβ2 KO mice infected with lethal doses of LVS had more severe liver pathology, including significant increases in the liver enzymes ALT and AST. Despite decreased levels of IFN-γ, LVS-vaccinated IL-12Rβ2 KO mice survived large lethal LVS secondary challenge. Consistent with in vivo protection, in vitro intramacrophage LVS growth was well-controlled in cocultures containing WT or IL-12Rβ2 KO LVS-immune splenocytes. Thus, survival of secondary LVS challenge was not strictly dependent on IL-12Rβ2. However, IL-12Rβ2 is important in parenteral and mucosal host resistance to primary LVS infection and in the ability of WT mice to clear LVS infection and serves to restrict liver damage. PMID:23440500

  20. 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

  1. Structural Analysis of a Protective Epitope of the Francisella tularensis O-Polysaccharide†

    PubMed Central

    Rynkiewicz, Michael J.; Lu, Zhaohua; Hui, Julia H.; Sharon, Jacqueline; Seaton, Barbara A.

    2012-01-01

    Francisella tularensis (Ft), the Gram negative facultative intracellular bacterium that causes tularemia, is considered a biothreat due to its high infectivity and the high mortality rate of respiratory disease. The Ft lipopolysaccharide (Ft LPS) is thought to be a main protective antigen in mice and humans, and we have previously demonstrated the protective effect of the Ft LPS-specific monoclonal antibody Ab52 in a mouse model of respiratory tularemia. Immunochemical characterization has shown that the epitope recognized by Ab52 is contained within two internal repeat units of the O-polysaccharide [O-antigen (OAg)] of Ft LPS. To further localize the Ab52 epitope and understand the molecular interactions between the antibody and the saccharide, we now solved the X-ray crystal structure of the Fab fragment of Ab52 and derived an antibody-antigen complex using molecular docking. The docked complex, refined through energy minimization, reveals an antigen binding site in the shape of a large canyon with a central pocket that accommodates a V-shaped epitope consisting of six sugar residues, α-D-GalpNAcAN(1→4)-α-D-GalpNAcAN(1→3)-β-D-QuipNAc(1→2)-β-D-Quip4NFm(1→4)-α-D-GalpNAcAN(1→4)-α-D-GalpNAcAN. These results inform the development of vaccines and immunotherapeutic/immunoprophylactic antibodies against Ft by suggesting a desired topology for antibody binding to internal epitopes of Ft LPS. This is the first report of an X-ray crystal structure of a monoclonal antibody that targets a protective Ft B cell epitope. PMID:22747335

  2. Utilization of an unstable plasmid and the I-SceI endonuclease to generate routine markerless deletion mutants in Francisella tularensis

    PubMed Central

    Horzempa, Joseph; Shanks, Robert M.Q.; Brown, Matthew J.; Russo, Brian C.; O’Dee, Dawn M.; Nau, Gerard J.

    2011-01-01

    We engineered an efficient system to make Francisella tularensis deletion mutations using an unstable, poorly maintained plasmid to enhance the likelihood of homologous recombination. For counterselection, we adapted a strategy using I-SceI, which causes a double-stranded break in the integrated suicide vector, forcing a second recombination to mediate allelic replacement. PMID:19879904

  3. High-Quality Draft Genome Sequence of Francisella tularensis subsp. holarctica Strain 08T0073 Isolated from a Wild European Hare

    PubMed Central

    Thomas, Prasad; Myrtennäs, Kerstin; Forsman, Mats; Braune, Silke; Runge, Martin; Tomaso, Herbert

    2017-01-01

    ABSTRACT Here, we report a high-quality draft genome sequence of Francisella tularensis subsp. holarctica strain 08T0073, isolated from the cadaver of a wild European hare (Lepus europaeus) found near Helmstedt, Lower Saxony, Germany, in 2007. In Germany, infected hares are a major source of tularemia in humans. PMID:28336603

  4. 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.

  5. 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

  6. 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

  7. Monitoring biothreat agents (Francisella tularensis, Bacillus anthracis and Yersinia pestis) with a portable real-time PCR instrument.

    PubMed

    Mölsä, Markos; Hemmilä, Heidi; Katz, Anna; Niemimaa, Jukka; Forbes, Kristian M; Huitu, Otso; Stuart, Peter; Henttonen, Heikki; Nikkari, Simo

    2015-08-01

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

  8. Genetic engineering of Francisella tularensis LVS for use as a novel live vaccine platform against Pseudomonas aeruginosa infections.

    PubMed

    Robinson, Cory M; Kobe, Brianna N; Schmitt, Deanna M; Phair, Brian; Gilson, Tricia; Jung, Joo-Yong; Roberts, Lawton; Liao, Jialin; Camerlengo, Chelsea; Chang, Brandon; Davis, Mackenzie; Figurski, Leah; Sindeldecker, Devin; Horzempa, Joseph

    2015-01-01

    Francisella tularensis LVS (Live Vaccine Strain) is an attenuated bacterium that has been used as a live vaccine. Patients immunized with this organism show a very long-term memory response (over 30 years post vaccination) evidenced by the presence of indicators of robust cell-mediated immunity. Because F. tularensis LVS is such a potent vaccine, we hypothesized that this organism would be an effective vaccine platform. First, we sought to determine if we could genetically modify this strain to produce protective antigens of a heterologous pathogen. Currently, there is not a licensed vaccine against the important opportunistic bacterial pathogen, Pseudomonas aeruginosa. Because many P. aeruginosa strains are also drug resistant, the need for effective vaccines is magnified. Here, F. tularensis LVS was genetically modified to express surface proteins PilAPa, OprFPa, and FliCPa of P. aeruginosa. Immunization of mice with LVS expressing the recombinant FliCPa led to a significant production of antibodies specific for P. aeruginosa. However, mice that had been immunized with LVS expressing PilAPa or OprFPa did not produce high levels of antibodies specific for P. aerugionsa. Therefore, the recombinant LVS strain engineered to produce FliCPa may be able to provide immune protection against a P. aeruginosa challenge. However for future use of this vaccine platform, selection of the appropriate recombinant antigen is critical as not all recombinant antigens expressed in this strain were immunogenic.

  9. Francisella tularensis modulates a distinct subset of regulatory factors and sustains mitochondrial integrity to impair human neutrophil apoptosis

    PubMed Central

    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. Herein 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 through 24–48 h of infection, and in this manner sustains mitochondrial integrity. Downstream of mitochondria, XIAP and 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 treatment of cancer that also induces neutrophil apoptosis and can promote resolution of several infectious and inflammatory disorders. Herein we confirm the ability of R-roscovitine to induce neutrophil apoptosis, yet also demonstrate that its efficacy is significantly impaired by F. tularensis. Collectively, our findings advance understanding of neutrophil apoptosis and its capacity to be manipulated by pathogenic bacteria. PMID:26906922

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

    PubMed Central

    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. PMID:26474413

  11. Genetic engineering of Francisella tularensis LVS for use as a novel live vaccine platform against Pseudomonas aeruginosa infections

    PubMed Central

    Robinson, Cory M; Kobe, Brianna N; Schmitt, Deanna M; Phair, Brian; Gilson, Tricia; Jung, Joo-Yong; Roberts, Lawton; Liao, Jialin; Camerlengo, Chelsea; Chang, Brandon; Davis, Mackenzie; Figurski, Leah; Sindeldecker, Devin; Horzempa, Joseph

    2015-01-01

    Francisella tularensis LVS (Live Vaccine Strain) is an attenuated bacterium that has been used as a live vaccine. Patients immunized with this organism show a very long-term memory response (over 30 years post vaccination) evidenced by the presence of indicators of robust cell-mediated immunity. Because F. tularensis LVS is such a potent vaccine, we hypothesized that this organism would be an effective vaccine platform. First, we sought to determine if we could genetically modify this strain to produce protective antigens of a heterologous pathogen. Currently, there is not a licensed vaccine against the important opportunistic bacterial pathogen, Pseudomonas aeruginosa. Because many P. aeruginosa strains are also drug resistant, the need for effective vaccines is magnified. Here, F. tularensis LVS was genetically modified to express surface proteins PilAPa, OprFPa, and FliCPa of P. aeruginosa. Immunization of mice with LVS expressing the recombinant FliCPa led to a significant production of antibodies specific for P. aeruginosa. However, mice that had been immunized with LVS expressing PilAPa or OprFPa did not produce high levels of antibodies specific for P. aerugionsa. Therefore, the recombinant LVS strain engineered to produce FliCPa may be able to provide immune protection against a P. aeruginosa challenge. However for future use of this vaccine platform, selection of the appropriate recombinant antigen is critical as not all recombinant antigens expressed in this strain were immunogenic. PMID:25617059

  12. 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

  13. 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.

  14. 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

  15. Monophosphoryl Lipid A Enhances Efficacy of a Francisella tularensis LVS-Catanionic Nanoparticle Subunit Vaccine against F. tularensis Schu S4 Challenge by Augmenting both Humoral and Cellular Immunity.

    PubMed

    Richard, Katharina; Mann, Barbara J; Qin, Aiping; Barry, Eileen M; Ernst, Robert K; Vogel, Stefanie N

    2017-03-01

    Francisella tularensis, a bacterial biothreat agent, has no approved vaccine in the United States. Previously, we showed that incorporating lysates from partially attenuated F. tularensis LVS or fully virulent F. tularensis Schu S4 strains into catanionic surfactant vesicle (V) nanoparticles (LVS-V and Schu S4-V, respectively) protected fully against F. tularensis LVS intraperitoneal (i.p.) challenge in mice. However, we achieved only partial protection against F. tularensis Schu S4 intranasal (i.n.) challenge, even when employing heterologous prime-boost immunization strategies. We now extend these findings to show that both LVS-V and Schu S4-V immunization (i.p./i.p.) elicited similarly high titers of anti-F. tularensis IgG and that the titers could be further increased by adding monophosphoryl lipid A (MPL), a nontoxic Toll-like receptor 4 (TLR4) adjuvant that is included in several U.S. FDA-approved vaccines. LVS-V+MPL immune sera also detected more F. tularensis antigens than LVS-V immune sera and, after passive transfer to naive mice, significantly delayed the time to death against F. tularensis Schu S4 subcutaneous (s.c.) but not i.n. challenge. Active immunization with LVS-V+MPL (i.p./i.p.) also increased the frequency of gamma interferon (IFN-γ)-secreting activated helper T cells, IFN-γ production, and the ability of splenocytes to control intramacrophage F. tularensis LVS replication ex vivo Active LVS-V+MPL immunization via heterologous routes (i.p./i.n.) significantly elevated IgA and IgG levels in bronchoalveolar lavage fluid and significantly enhanced protection against i.n. F. tularensis Schu S4 challenge (to ∼60%). These data represent a significant step in the development of a subunit vaccine against the highly virulent type A strains.

  16. [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

  17. 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.

  18. 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}.

  19. A Molecular Survey for Francisella tularensis and Rickettsia spp. in Haemaphysalis leporispalustris (Acari: Ixodidae) in Northern California.

    PubMed

    Roth, Tara; Lane, Robert S; Foley, Janet

    2016-12-28

    Francisella tularensis and Rickettsia spp. have been cultured from Haemaphysalis leporispalustris Packard, but their prevalence in this tick has not been determined using modern molecular methods. We collected H. leporispalustris by flagging vegetation and leaf litter and from lagomorphs (Lepus californicus Gray and Sylvilagus bachmani (Waterhouse)) in northern California. Francisella tularensis DNA was not detected in any of 1,030 ticks tested by polymerase chain reaction (PCR), whereas 0.4% of larvae tested in pools, 0 of 117 individual nymphs, and 2.3% of 164 adult ticks were PCR-positive for Rickettsia spp. Positive sites were Laurel Canyon Trail in Tilden Regional Park in Alameda Contra Costa County, with a Rickettsia spp. prevalence of 0.6% in 2009, and Hopland Research and Extension Center in Mendocino County, with a prevalence of 4.2% in 1988. DNA sequencing revealed R. felis, the agent of cat-flea typhus, in two larval pools from shaded California bay and live oak leaf litter in Contra Costa County and one adult tick from a L. californicus in chaparral in Mendocino County. The R. felis in unfed, questing larvae demonstrates that H. leporispalustris can transmit this rickettsia transovarially. Although R. felis is increasingly found in diverse arthropods and geographical regions, prior literature suggests a typical epidemiological cycle involving mesocarnivores and the cat flea, Ctenocephalides felis. To our knowledge, this is the first report of R. felis in H. leporispalustris. Natural infection and transovarial transmission of this pathogen in the tick indicate the existence of a previously undocumented wild-lands transmission cycle that may intersect mesocarnivore-reservoired cycles and collectively affect human health risk.

  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. Persistence of cell-mediated immunity three decades after vaccination with the live vaccine strain of Francisella tularensis

    PubMed Central

    Eneslätt, Kjell; Rietz, Cecilia; Rydén, Patrik; Stöven, Svenja; House, Robert V.; Wolfraim, Lawrence A.; Tärnvik, Arne; Sjöstedt, Anders

    2012-01-01

    Summary The efficacy of many vaccines against intracellular bacteria depends on the generation of cell-mediated immunity, but studies to determine the duration of immunity are usually confounded by re-exposure. The causative agent of tularemia, Francisella tularensis, is rare in most areas and, therefore, tularemia vaccination is an interesting model for studies of the longevity of vaccine-induced cell-mediated immunity. Here lymphocyte proliferation and cytokine production in response to F. tularensis were assayed in two groups of 16 individuals, vaccinated 1-3 or 27-34 years previously. As compared to naïve individuals, vaccinees of both groups showed higher proliferative responses and, out of 17 cytokines assayed, higher levels of MIP-1β, IFN-γ, IL-10, and IL-5 in response to recall stimulation. The responses were very similar in the two groups of vaccinees. A statistical model was developed to predict the immune status of the individuals and by use of two parameters, proliferative responses and levels of IFN-γ, 91.1% of the individuals were correctly classified. Using flow cytometry analysis, we demonstrated that during recall stimulation, expression of IFN-γ by CD4+CCR7+, CD4+CD62L+, CD8+CCR7+, and CD8+CD62L+ cells significantly increased in samples from vaccinated donors. In conclusion, cell-mediated immunity was found to persist three decades after tularemia vaccination without evidence of decline. PMID:21442618

  2. Cat-bite-induced Francisella tularensis infection with a false-positive serological reaction for Bartonella quintana

    PubMed Central

    Petersson, Evelina

    2017-01-01

    Introduction. Tularaemia is caused by infection with Francisella tularensistransmitted via direct contact with an infected hare carcass or indirectly through the bites of vectors, but may be cat-bite-associated as well. Medical history and reliable diagnostic analysis are important in order to differentiate it from other cat-associated infections, e.g. Bartonella spp. Case presentation. A healthy 56-year-old man was examined because of a cat-bite-associated ulceroglandular wound on his right thumb. Nineteen days after the cat bite occurred, a serology test was positive for anti-Bartonella quintana, but negative for anti-F. tularensis. Since Bartonella infections are rare in Sweden, another serology test was analysed 2 weeks later with a positive result for anti-F. tularensis. The patient was treated with doxycycline for 14 days and recovered. The patient was re-sampled after 18 months to obtain a convalescent sample. The acute and the convalescent samples were both analysed at a reference centre, with negative results for anti-Bartonella spp. this time. Conclusion. This case is enlightening about the importance of extending the medical history and re-sampling the patient for antibody detection when the clinical suspicion of cat-bite-associated tularaemia is high. The false-positive result for anti-B. quintana antibodies may have been due to technical issues with the assay, cross-reactivity or both. PMID:28348802

  3. Contribution of FcɛRI-associated vesicles to mast cell-macrophage communication following Francisella tularensis infection.

    PubMed

    Rodriguez, Annette R; Yu, Jieh-Juen; Navara, Christopher; Chambers, James P; Guentzel, M Neal; Arulanandam, Bernard P

    2016-10-01

    Understanding innate immune intercellular communication following microbial infection remains a key biological issue. Using live cell imaging, we demonstrate that mast cells actively extend cellular projections to sample the macrophage periphery during Francisella tularensis LVS infection. Mast cell MHCII(hi) expression was elevated from less than 1% to 13% during LVS infection. Direct contact during co-culture with macrophages further increased mast cell MHCII(hi) expression to approximately 87%. Confocal analyses of the cellular perimeter revealed mast cell caspase-1 was localized in close proximity with FcɛRI in uninfected mast cells, and repositioned to clustered regions upon LVS infection. Importantly, mast cell FcɛRI-encompassed vesicles are transferred to macrophages by trogocytosis, and macrophage caspase-1 expression is further up-regulated upon direct contact with mast cells. Our study reveals direct cellular interactions between innate cells that may impact the function of caspase-1, a known sensor of microbial danger and requirement for innate defense against many pathogenic microbes including F. tularensis.

  4. 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.

  5. Visualization of Murine Intranasal Dosing Efficiency Using Luminescent Francisella tularensis: Effect of Instillation Volume and Form of Anesthesia

    PubMed Central

    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

  6. Environmental Monitoring and Surveillance of Rodents and Vectors for Francisella tularensis Following Outbreaks of Human Tularemia in Georgia.

    PubMed

    Elashvili, Eka; Kracalik, Ian; Burjanadze, Irma; Datukishvili, Sophio; Chanturia, Gvantsa; Tsertsvadze, Nikoloz; Beridze, Levan; Shavishvili, Merab; Dzneladze, Archil; Grdzelidze, Marina; Imnadze, Paata; Pearson, Andrew; Blackburn, Jason K

    2015-10-01

    Tularemia is a re-emerging bacterial zoonosis, broadly distributed across the northern hemisphere. In Georgia, there is a history of human tularemia outbreaks dating back to the 1940s. In response to outbreaks, health officials initiated long-term field surveillance and environmental monitoring. The objective of our study was to obtain information from 57 years of field surveys to identify species that play a role in the occurrence Francisella tularensis subsp. holarctica in the environment in Georgia. We collected historical data on human outbreaks, field collections, population dynamics of the common vole (Microtus arvalis), and conducted surveys on small mammals and vectors from five regions in Georgia during 1956-2012. Bacterial isolation was conducted using standard culturing techniques, and isolation rates for species were obtained for a subset of years. We used a Spearman rank correlation to test for associations between the density of the common vole and isolation rates. From 1956 through 2012, there were four recorded outbreaks of human tularemia (362 cases). A total of 465 bacterial isolates of F. tularensis subsp. holarctica were obtained from 27 species and environmental samples. The number of isolations was highest in the common vole (M. arvalis; 149 isolates; 32%) and Dermacentor marginatus ticks (132 isolates; 28%); isolation rates ranged between 0-0.91% and 0-0.47%, respectively. Population dynamics of the common vole were not correlated with the isolation rate. Given the history of tularemia re-emergence in Georgia, continued field surveys and environmental monitoring may provide an early indication of outbreak risk in humans. In conclusion, our findings provide evidence of long-standing foci of F. tularensis subsp. holarctica that are likely maintained by the common vole-tick cycle.

  7. Live Attenuated Mutants of Francisella tularensis Protect Rabbits against Aerosol Challenge with a Virulent Type A Strain

    PubMed Central

    Smith, Le'Kneitah P.; Cole, Kelly Stefano; Santiago, Araceli E.; Mann, Barbara J.; Barry, Eileen M.

    2014-01-01

    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. PMID:24614653

  8. Environmental Monitoring and Surveillance of Rodents and Vectors for Francisella tularensis Following Outbreaks of Human Tularemia in Georgia

    PubMed Central

    Elashvili, Eka; Kracalik, Ian; Burjanadze, Irma; Datukishvili, Sophio; Chanturia, Gvantsa; Tsertsvadze, Nikoloz; Beridze, Levan; Shavishvili, Merab; Dzneladze, Archil; Grdzelidze, Marina; Imnadze, Paata; Pearson, Andrew

    2015-01-01

    Abstract Tularemia is a re-emerging bacterial zoonosis, broadly distributed across the northern hemisphere. In Georgia, there is a history of human tularemia outbreaks dating back to the 1940s. In response to outbreaks, health officials initiated long-term field surveillance and environmental monitoring. The objective of our study was to obtain information from 57 years of field surveys to identify species that play a role in the occurrence Francisella tularensis subsp. holarctica in the environment in Georgia. We collected historical data on human outbreaks, field collections, population dynamics of the common vole (Microtus arvalis), and conducted surveys on small mammals and vectors from five regions in Georgia during 1956–2012. Bacterial isolation was conducted using standard culturing techniques, and isolation rates for species were obtained for a subset of years. We used a Spearman rank correlation to test for associations between the density of the common vole and isolation rates. From 1956 through 2012, there were four recorded outbreaks of human tularemia (362 cases). A total of 465 bacterial isolates of F. tularensis subsp. holarctica were obtained from 27 species and environmental samples. The number of isolations was highest in the common vole (M. arvalis; 149 isolates; 32%) and Dermacentor marginatus ticks (132 isolates; 28%); isolation rates ranged between 0–0.91% and 0–0.47%, respectively. Population dynamics of the common vole were not correlated with the isolation rate. Given the history of tularemia re-emergence in Georgia, continued field surveys and environmental monitoring may provide an early indication of outbreak risk in humans. In conclusion, our findings provide evidence of long-standing foci of F. tularensis subsp. holarctica that are likely maintained by the common vole–tick cycle. PMID:26394283

  9. CpG oligodeoxyribonucleotides protect mice from Burholderia pseudomallei but not Francisella tularensis Schu 54 aersols

    DTIC Science & Technology

    2010-01-01

    live vaccine strain (LVS), when administered before parenteral challenge. Given the potential to develop CpG ODN as a pre-treatment for multiple...1] have been successfully developed as adjuvants for a broad array of bacterial subunit vaccines and are currently undergoing multiple clinical...tularensis live vaccine strain (LVS) [3] suggest that CpG ODN may also protect against human-virulent F. tularensis Schu S4 infection. Significantly

  10. 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

  11. 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.

  12. Host-Adaptation of Francisella tularensis Alters the Bacterium's Surface-Carbohydrates to Hinder Effectors of Innate and Adaptive Immunity

    PubMed Central

    Zarrella, Tiffany M.; Singh, Anju; Bitsaktsis, Constantine; Rahman, Tabassum; Sahay, Bikash; Feustel, Paul J.; Gosselin, Edmund J.; Sellati, Timothy J.; Hazlett, Karsten R. O.

    2011-01-01

    Background The gram-negative bacterium Francisella tularensis survives in arthropods, fresh water amoeba, and mammals with both intracellular and extracellular phases and could reasonably be expected to express distinct phenotypes in these environments. The presence of a capsule on this bacterium has been controversial with some groups finding such a structure while other groups report that no capsule could be identified. Previously we reported in vitro culture conditions for this bacterium which, in contrast to typical methods, yielded a bacterial phenotype that mimics that of the bacterium's mammalian, extracellular phase. Methods/Findings SDS-PAGE and carbohydrate analysis of differentially-cultivated F. tularensis LVS revealed that bacteria displaying the host-adapted phenotype produce both longer polymers of LPS O-antigen (OAg) and additional HMW carbohydrates/glycoproteins that are reduced/absent in non-host-adapted bacteria. Analysis of wildtype and OAg-mutant bacteria indicated that the induced changes in surface carbohydrates involved both OAg and non-OAg species. To assess the impact of these HMW carbohydrates on the access of outer membrane constituents to antibody we used differentially-cultivated bacteria in vitro to immunoprecipitate antibodies directed against outer membrane moieties. We observed that the surface-carbohydrates induced during host–adaptation shield many outer membrane antigens from binding by antibody. Similar assays with normal mouse serum indicate that the induced HMW carbohydrates also impede complement deposition. Using an in vitro macrophage infection assay, we find that the bacterial HMW carbohydrate impedes TLR2-dependent, pro-inflammatory cytokine production by macrophages. Lastly we show that upon host-adaptation, the human-virulent strain, F. tularensis SchuS4 also induces capsule production with the effect of reducing macrophage-activation and accelerating tularemia pathogenesis in mice. Conclusion F. tularensis undergoes

  13. Hare-to-human transmission of Francisella tularensis subsp. holarctica, Germany.

    PubMed

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

    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.

  14. Efficient Inactivation of Burkholderia pseudomallei or Francisella tularensis in infected Cells for Safe Removal from Biosafety Level 3 Containment Laboratories

    PubMed Central

    Emery, Felicia D.; Stabenow, Jennifer M.; Miller, Mark A.

    2014-01-01

    Working with infectious agents that require BSL-3 level containment agents offers many challenges for researchers. BSL-3 containment laboratories are usually not equipped with expensive specialty equipment that is needed for studies such as flow cytometric analysis, microscopy, and proteomic analyses. Therefore, for most researchers that are working with BSL-3 level infectious agents, removal of samples from BSL-3 labs for these types of studies is necessary, and methods for complete and dependable inactivation of the samples are required. In this report we have done a thorough characterization of the effectiveness of paraformaldehyde fixation for inactivation of cell samples infected with the intracellular bacterial agents Burkholderia pseudomallei (Bp) and Francisella tularensis (Ft), both of which are Tier 1 select agent pathogens that require BSL-3 containment. We have demonstrated that cells infected with these pathogens are completely inactivated via 5-minute treatment with 4% paraformaldehyde. Moreover, a 15-minute treatment with 2% paraformaldehyde completely sterilized both Bp- and Ft-infected cells. These studies also revealed that Bp is significantly more sensitive to paraformaldehyde treatment than Ft. Our findings have clearly demonstrated that a 15-minute treatment of Bp- or Ft-infected cells with 4% paraformaldehyde solution will allow for safe removal of the cell samples from BSL-3 labs for downstream studies. PMID:24449562

  15. Metabolism-directed structure optimization of benzimidazole-based Francisella tularensis enoyl-reductase (FabI) inhibitors.

    PubMed

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

    2014-05-01

    1. 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. 2. 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. 3. Metabolic hotspots on the benzimidazole core structure as well as the benzyl ring were identified and verified by metabolite identification studies of four model compounds. Interestingly, the proposed structure-metabolism relationships did not apply to nine newly synthesized cyclopentane or oxacyclopentane derivatives of N-benzyl benzimidazole. 4. 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 non-linear regression models based on the selected molecular descriptors were developed and validated. 5. The structure-metabolism relationships for N-benzyl benzimidazole compounds should help optimization of N-benzyl benzimidazole compounds for better pharmacokinetic behavior.

  16. 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

  17. 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.

  18. O-Linked Glycosylation of the PilA Pilin Protein of Francisella tularensis: Identification of the Endogenous Protein-Targeting Oligosaccharyltransferase and Characterization of the Native Oligosaccharide▿†

    PubMed Central

    Egge-Jacobsen, Wolfgang; Salomonsson, Emelie Näslund; Aas, Finn Erik; Forslund, Anna-Lena; Winther-Larsen, Hanne C.; Maier, Josef; Macellaro, Anna; Kuoppa, Kerstin; Oyston, Petra C. F.; Titball, Richard W.; Thomas, Rebecca M.; Forsberg, Åke; Prior, Joann L.; Koomey, Michael

    2011-01-01

    Findings from a number of studies suggest that the PilA pilin proteins may play an important role in the pathogenesis of disease caused by species within the genus Francisella. As such, a thorough understanding of PilA structure and chemistry is warranted. Here, we definitively identified the PglA protein-targeting oligosaccharyltransferase by virtue of its necessity for PilA glycosylation in Francisella tularensis and its sufficiency for PilA glycosylation in Escherichia coli. In addition, we used mass spectrometry to examine PilA affinity purified from Francisella tularensis subsp. tularensis and F. tularensis subsp. holarctica and demonstrated that the protein undergoes multisite, O-linked glycosylation with a pentasaccharide of the structure HexNac-Hex-Hex-HexNac-HexNac. Further analyses revealed microheterogeneity related to forms of the pentasaccharide carrying unusual moieties linked to the distal sugar via a phosphate bridge. Type A and type B strains of Francisella subspecies thus express an O-linked protein glycosylation system utilizing core biosynthetic and assembly pathways conserved in other members of the proteobacteria. As PglA appears to be highly conserved in Francisella species, O-linked protein glycosylation may be a feature common to members of this genus. PMID:21804002

  19. Comparative evaluation of automated and manual commercial DNA extraction methods for detection of Francisella tularensis DNA from suspensions and spiked swabs by real-time polymerase chain reaction.

    PubMed

    Dauphin, Leslie A; Walker, Roblena E; Petersen, Jeannine M; Bowen, Michael D

    2011-07-01

    This study evaluated commercial automated and manual DNA extraction methods for the isolation of Francisella tularensis DNA suitable for real-time polymerase chain reaction (PCR) analysis from cell suspensions and spiked cotton, foam, and polyester swabs. Two automated methods, the MagNA Pure Compact and the QIAcube, were compared to 4 manual methods, the IT 1-2-3 DNA sample purification kit, the MasterPure Complete DNA and RNA purification kit, the QIAamp DNA blood mini kit, and the UltraClean Microbial DNA isolation kit. The methods were compared using 6 F. tularensis strains representing the 2 subspecies which cause the majority of reported cases of tularemia in humans. Cell viability testing of the DNA extracts showed that all 6 extraction methods efficiently inactivated F. tularensis at concentrations of ≤10⁶ CFU/mL. Real-time PCR analysis using a multitarget 5' nuclease assay for F. tularensis revealed that the PCR sensitivity was equivalent using DNA extracted by the 2 automated methods and the manual MasterPure and QIAamp methods. These 4 methods resulted in significantly better levels of detection from bacterial suspensions and performed equivalently for spiked swab samples than the remaining 2. This study identifies optimal DNA extraction methods for processing swab specimens for the subsequent detection of F. tularensis DNA using real-time PCR assays. Furthermore, the results provide diagnostic laboratories with the option to select from 2 automated DNA extraction methods as suitable alternatives to manual methods for the isolation of DNA from F. tularensis.

  20. An In Vitro Co-culture Mouse Model Demonstrates Efficient Vaccine-Mediated Control of Francisella tularensis SCHU S4 and Identifies Nitric Oxide as a Predictor of Efficacy

    PubMed Central

    Golovliov, Igor; Lindgren, Helena; Eneslätt, Kjell; Conlan, Wayne; Mosnier, Amandine; Henry, Thomas; Sjöstedt, Anders

    2016-01-01

    Francisella tularensis is a highly virulent intracellular bacterium and cell-mediated immunity is critical for protection, but mechanisms of protection against highly virulent variants, such as the prototypic strain F. tularensis strain SCHU S4, are poorly understood. To this end, we established a co-culture system, based on splenocytes from naïve, or immunized mice and in vitro infected bone marrow-derived macrophages that allowed assessment of mechanisms controlling infection with F. tularensis. We utilized the system to understand why the clpB gene deletion mutant, ΔclpB, of SCHU S4 shows superior efficacy as a vaccine in the mouse model as compared to the existing human vaccine, the live vaccine strain (LVS). Compared to naïve splenocytes, ΔclpB-, or LVS-immune splenocytes conferred very significant control of a SCHU S4 infection and the ΔclpB-immune splenocytes were superior to the LVS-immune splenocytes. Cultures with the ΔclpB-immune splenocytes also contained higher levels of IFN-γ, IL-17, and GM-CSF and nitric oxide, and T cells expressing combinations of IFN-γ, TNF-α, and IL-17, than did cultures with LVS-immune splenocytes. There was strong inverse correlation between bacterial replication and levels of nitrite, an end product of nitric oxide, and essentially no control was observed when BMDM from iNOS−/− mice were infected. Collectively, the co-culture model identified a critical role of nitric oxide for protection against a highly virulent strain of F. tularensis. PMID:27933275

  1. 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.

  2. Structural and biological evaluation of a novel series of benzimidazole inhibitors of Francisella tularensis enoyl-ACP reductase (FabI).

    PubMed

    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

    2015-03-15

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

  3. 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.

  4. Recombinant Attenuated Listeria monocytogenes Vaccine Expressing Francisella tularensis IglC Induces Protection in Mice Against Aerosolized Type A F. tularensis

    PubMed Central

    Jia, Qingmei; Lee, Bai-Yu; Clemens, Daniel L.; Bowen, Richard A.; Horwitz, Marcus A.

    2009-01-01

    Fransicella tularensis, the causative agent of tularemia, is in the top category (Category A) of potential agents of bioterrorism. To develop a safer vaccine against aerosolized F. tularensis, we have employed an attenuated Listeria monocytogenes, which shares with F. tularensis an intracellular and extraphagosomal lifestyle, as a delivery vehicle for F. tularensis antigens. We constructed recombinant L. monocytogenes (rLm) vaccines stably expressing 7 F. tularensis proteins including IglC (rLm/iglC), and tested their immunogenicity and protective efficacy against lethal F. tularensis challenge in mice. Mice immunized intradermally with rLm/iglC developed significant cellular immune responses to F. tularensis IglC as evidenced by lymphocyte proliferation and CD4+ and CD8+ T-cell intracellular expression of interferon gamma. Moreover, mice immunized with rLm/iglC were protected against lethal challenge with F. tularensis LVS administered by the intranasal route, a route chosen to mimic airborne infection, and, most importantly, against aerosol challenge with the highly virulent Type A F. tularensis SchuS4 strain. PMID:19126421

  5. Characterization of two unusual clinically significant Francisella strains.

    PubMed Central

    Clarridge, J E; Raich, T J; Sjösted, A; Sandström, G; Darouiche, R O; Shawar, R M; Georghiou, P R; Osting, C; Vo, L

    1996-01-01

    We have isolated two phenotypically distinct nonfastidious Francisella strains (Fx1 and Fx2) from the blood of compromised patients with pneumonia and compared them with eight other Francisella strains, including Francisella tularensis biovar tularensis, F. tularensis biovar novicida, and F. philomiragia. Our isolates grew well on sheep blood agar, chocolate agar, modified Thayer-Martin agar, and Trypticase soy agar. Fx1 and Fx2 were determined to be within the Francisella genus by cellular fatty acid analysis and by the utilization of glucose, production of H2S and catalase, and lack of motility, oxidase, nitrate reductase, and gelatinase. They were additionally shown to belong to the species F. tularensis by sequencing of two variable regions comprising approximately 500 nucleotides of the 16S rRNA gene. Also, RNA probe hybridization confirmed their belonging to the species F. tularensis. However, the new strains, which are not identical, are distinguished from other F. tularensis strains by growth characteristics, repetitive extragenic palindromic PCR fragment pattern, and some biochemical tests. Key biochemical differences included the findings that Fx1 was positive for beta-galactosidase and arabinose hydrolysis and that both strains were citrulline ureidase positive and glycerol negative. Commercial F. tularensis antiserum agglutinated stock F. tularensis strains but not Fx1, Fx2, F. tularensis biovar novicida, or F. philomiragia; serum from either patient failed to agglutinate or only weakly agglutinated commercial antigen but showed agglutination when tested against each patient's respective isolate. Fx1 and Fx2 produced beta-lactamase. Because of their good growth, negative serology, and biochemical profile, the organisms could be misidentified in the clinical laboratory if standard strategies or commercial identification systems are used. PMID:8818897

  6. 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

  7. Large Direct Repeats Flank Genomic Rearrangements between a New Clinical Isolate of Francisella tularensis subsp. tularensis A1 and Schu S4

    DTIC Science & Technology

    2010-02-03

    Silver Spring, Maryland, United States of America, 7 Oak Ridge National Laboratory, Oak Ridge, Tennessee , United States of America Abstract Francisella...Interpolated variable order motifs for identification of horizontally acquired DNA: revisiting the Salmonella pathoge- nicity islands. Bioinformatics

  8. 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

  9. 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.

  10. Structural and biological evaluation of a novel series of benzimidazole inhibitors of Francisella tularensis enoyl-ACP reductase (FabI)

    SciTech Connect

    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.

    2015-01-29

    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. In the bacterial FASII pathway we found it 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. Furthermore, these compounds display an improved low nanomolar enzymatic activity as well as promising low microgram/mL antibacterial activity against both F. tularensis and Staphylococcus aureus and its methicillin-resistant strain (MRSA). Finally, 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.

  11. Structural and biological evaluation of a novel series of benzimidazole inhibitors of Francisella tularensis enoyl-ACP reductase (FabI)

    DOE PAGES

    Mehboob, Shahila; Song, Jinhua; Hevener, Kirk E.; ...

    2015-01-29

    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. In the bacterial FASII pathway we found it 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. Furthermore, these compounds display an improved low nanomolar enzymatic activity as well as promisingmore » low microgram/mL antibacterial activity against both F. tularensis and Staphylococcus aureus and its methicillin-resistant strain (MRSA). Finally, 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.« less

  12. CpG Oligodeoxyribonucleotides Protect Mice from Burkholderia Pseudomallei but not Francisella Tularensis Schu S4 Aerosols

    DTIC Science & Technology

    2010-01-01

    David A Rozak1*, Herbert C Gelhaus1,3, Mark Smith2, Mojgan Zadeh1,4, Louis Huzella2, David Waag1, Jeffrey J Adamovicz1,5 Abstract Studies have shown...tularensis LPS, and contributes to F. novicida murine pathogenesis. Microbes and infection/ Institut Pasteur 2003, 5(5):397-403. 12. Forsman M, Sandstrom G

  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 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

  14. Components of the type six secretion system are substrates of Francisella tularensis Schu S4 DsbA-like FipB protein

    PubMed Central

    Qin, Aiping; Zhang, Yan; Clark, Melinda E.; Moore, Emily A.; Rabideau, Meaghan M.; Moreau, G. Brett; Mann, Barbara J.

    2016-01-01

    ABSTRACT FipB, an essential virulence factor in the highly virulent Schu S4 strain of F. tularensis subsp. tularensis, shares sequence similarity with Disulfide Bond formation (Dsb) proteins, which can have oxidoreductase, isomerase, or chaperone activity. To further explore FipB's role in virulence potential substrates were identified by co-purification and 2D gel electrophoresis, followed by protein sequencing using mass spectrometry. A total of 119 potential substrates were identified. Proteins with predicted enzymatic activity were prevalent, and there were 19 proteins that had been previously identified as impacting virulence. Among the potential substrates were IglC, IglB, and PdpB, three components of the Francisella Type Six Secretion System (T6SS), which is also essential for virulence. T6SS are widespread in Gram-negative pathogens, but have not been reported to be dependent on Dsb-like proteins for assembly or function. The presented results suggest that FipB affects IglB and IglC substrates differently. In a fipB mutant there were differences in free sulfhydryl accessibility of IglC, but not IglB, when compared to wild-type bacteria. However, for both proteins FipB appears to act as a chaperone that facilitates proper folding and conformation. Understanding the role FipB plays the assembly and structure in this T6SS may reveal critical aspects of assembly that are common and novel among this widely distributed class of secretion systems. PMID:27028889

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

    DOE PAGES

    McGillick, Brian E.; Kumaran, Desigan; Vieni, Casey; ...

    2016-01-28

    The bacterial system for fatty acid biosynthesis (FAS) contains several enzymes whose sequence and structure are highly conserved across a vast array of pathogens. Coupled with their low homology and difference in organization compared to the equivalent system in humans, this 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. In addition, we havemore » 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. Our 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.« less

  16. Differential Cultivation of Francisella tularensis Induces Changes in the Immune Response to and Protective Efficacy of Whole Cell-Based Inactivated Vaccines

    PubMed Central

    Kumar, Sudeep; Sunagar, Raju; Pham, Giang; Franz, Brian J.; Rosa, Sarah J.; Hazlett, Karsten R. O.; Gosselin, Edmund J.

    2017-01-01

    Francisella tularensis (Ft) is a category A biothreat agent for which there is no Food and Drug Administration-approved vaccine. Ft can survive in a variety of habitats with a remarkable ability to adapt to changing environmental conditions. Furthermore, Ft expresses distinct sets of antigens (Ags) when inside of macrophages (its in vivo host) as compared to those grown in vitro with Mueller Hinton Broth (MHB). However, in contrast to MHB-grown Ft, Ft grown in Brain-Heart Infusion (BHI) more closely mimics the antigenic profile of macrophage-grown Ft. Thus, we anticipated that when used as a vaccine, BHI-grown Ft would provide better protection compared to MHB-grown Ft, primarily due to its greater antigenic similarity to Ft circulating inside the host (macrophages) during natural infection. Our investigation, however, revealed that inactivated Ft (iFt) grown in MHB (iFt-MHB) exhibited superior protective activity when used as a vaccine, as compared to iFt grown in BHI (iFt-BHI). The superior protection afforded by iFt-MHB compared to that of iFt-BHI was associated with significantly lower bacterial burden and inflammation in the lungs and spleens of vaccinated mice. Moreover, iFt-MHB also induced increased levels of Ft-specific IgG. Further evaluation of early immunological cues also revealed that iFt-MHB exhibits increased engagement of Ag-presenting cells including increased iFt binding to dendritic cells, increased expression of costimulatory markers, and increased secretion of pro-inflammatory cytokines. Importantly, these studies directly demonstrate that Ft growth conditions strongly impact Ft vaccine efficacy and that the growth medium used to produce whole cell vaccines to Ft must be a key consideration in the development of a tularemia vaccine. PMID:28119692

  17. Differential Cultivation of Francisella tularensis Induces Changes in the Immune Response to and Protective Efficacy of Whole Cell-Based Inactivated Vaccines.

    PubMed

    Kumar, Sudeep; Sunagar, Raju; Pham, Giang; Franz, Brian J; Rosa, Sarah J; Hazlett, Karsten R O; Gosselin, Edmund J

    2016-01-01

    Francisella tularensis (Ft) is a category A biothreat agent for which there is no Food and Drug Administration-approved vaccine. Ft can survive in a variety of habitats with a remarkable ability to adapt to changing environmental conditions. Furthermore, Ft expresses distinct sets of antigens (Ags) when inside of macrophages (its in vivo host) as compared to those grown in vitro with Mueller Hinton Broth (MHB). However, in contrast to MHB-grown Ft, Ft grown in Brain-Heart Infusion (BHI) more closely mimics the antigenic profile of macrophage-grown Ft. Thus, we anticipated that when used as a vaccine, BHI-grown Ft would provide better protection compared to MHB-grown Ft, primarily due to its greater antigenic similarity to Ft circulating inside the host (macrophages) during natural infection. Our investigation, however, revealed that inactivated Ft (iFt) grown in MHB (iFt-MHB) exhibited superior protective activity when used as a vaccine, as compared to iFt grown in BHI (iFt-BHI). The superior protection afforded by iFt-MHB compared to that of iFt-BHI was associated with significantly lower bacterial burden and inflammation in the lungs and spleens of vaccinated mice. Moreover, iFt-MHB also induced increased levels of Ft-specific IgG. Further evaluation of early immunological cues also revealed that iFt-MHB exhibits increased engagement of Ag-presenting cells including increased iFt binding to dendritic cells, increased expression of costimulatory markers, and increased secretion of pro-inflammatory cytokines. Importantly, these studies directly demonstrate that Ft growth conditions strongly impact Ft vaccine efficacy and that the growth medium used to produce whole cell vaccines to Ft must be a key consideration in the development of a tularemia vaccine.

  18. A 14.7 kDa protein from Francisella tularensis subsp. novicida (named FTN_1133), involved in the response to oxidative stress induced by organic peroxides, is not endowed with thiol-dependent peroxidase activity.

    PubMed

    Meireles, Diogo de Abreu; Alegria, Thiago Geronimo Pires; Alves, Simone Vidigal; Arantes, Carla Rani Rocha; Netto, Luis Eduardo Soares

    2014-01-01

    Francisella genus comprises Gram-negative facultative intracellular bacteria that are among the most infectious human pathogens. A protein of 14.7 KDa named as FTN_1133 was previously described as a novel hydroperoxide resistance protein in F. tularensis subsp. novicida, implicated in organic peroxide detoxification and virulence. Here, we describe a structural and biochemical characterization of FTN_1133. Contrary to previous assumptions, multiple amino acid sequence alignment analyses revealed that FTN_1133 does not share significant similarity with proteins of the Ohr/OsmC family or any other Cys-based, thiol dependent peroxidase, including conserved motifs around reactive cysteine residues. Circular dichroism analyses were consistent with the in silico prediction of an all-α-helix secondary structure. The pKa of its single cysteine residue, determined by a monobromobimane alkylation method, was shown to be 8.0±0.1, value that is elevated when compared with other Cys-based peroxidases, such as peroxiredoxins and Ohr/OsmC proteins. Attempts to determine a thiol peroxidase activity for FTN_1133 failed, using both dithiols (DTT, thioredoxin and lipoamide) and monothiols (glutathione or 2-mercaptoethanol) as reducing agents. Heterologous expression of FTN_1133 gene in ahpC and oxyR mutants of E. coli showed no complementation. Furthermore, analysis of FTN_1133 protein by non-reducing SDS-PAGE showed that an inter-molecular disulfide bond (not detected in Ohr proteins) can be generated under hydroperoxide treatment, but the observed rates were not comparable to those observed for other thiol-dependent peroxidases. All the biochemical and structural data taken together indicated that FTN_1133 displayed distinct characteristics from other thiol dependent peroxidases and, therefore, suggested that FTN_1133 is not directly involved in hydroperoxide detoxification.

  19. Whole-Genome Relationships among Francisella Bacteria of Diverse Origins Define New Species and Provide Specific Regions for Detection.

    PubMed

    Challacombe, Jean F; Petersen, Jeannine M; Gallegos-Graves, La Verne; Hodge, David; Pillai, Segaran; Kuske, Cheryl R

    2017-02-01

    Francisella tularensis is a highly virulent zoonotic pathogen that causes tularemia and, because of weaponization efforts in past world wars, is considered a tier 1 biothreat agent. Detection and surveillance of F. tularensis may be confounded by the presence of uncharacterized, closely related organisms. Through DNA-based diagnostics and environmental surveys, novel clinical and environmental Francisella isolates have been obtained in recent years. Here we present 7 new Francisella genomes and a comparison of their characteristics to each other and to 24 publicly available genomes as well as a comparative analysis of 16S rRNA and sdhA genes from over 90 Francisella strains. Delineation of new species in bacteria is challenging, especially when isolates having very close genomic characteristics exhibit different physiological features-for example, when some are virulent pathogens in humans and animals while others are nonpathogenic or are opportunistic pathogens. Species resolution within Francisella varies with analyses of single genes, multiple gene or protein sets, or whole-genome comparisons of nucleic acid and amino acid sequences. Analyses focusing on single genes (16S rRNA, sdhA), multiple gene sets (virulence genes, lipopolysaccharide [LPS] biosynthesis genes, pathogenicity island), and whole-genome comparisons (nucleotide and protein) gave congruent results, but with different levels of discrimination confidence. We designate four new species within the genus; Francisella opportunistica sp. nov. (MA06-7296), Francisella salina sp. nov. (TX07-7308), Francisella uliginis sp. nov. (TX07-7310), and Francisella frigiditurris sp. nov. (CA97-1460). This study provides a robust comparative framework to discern species and virulence features of newly detected Francisella bacteria.

  20. Discordant Results Obtained with Francisella tularensis during In Vitro and In Vivo Immunological Studies Are Attributable to Compromised Bacterial Structural Integrity

    PubMed Central

    Singh, Anju; Rahman, Tabassum; Malik, Meenakshi; Hickey, Anthony J.; Leifer, Cynthia A.; Hazlett, Karsten R. O.; Sellati, Timothy J.

    2013-01-01

    Francisella tularensis (Ft) is a highly infectious intracellular pathogen and the causative agent of tularemia. Because Ft can be dispersed via small droplet-aerosols and has a very low infectious dose it is characterized as a category A Select Agent of biological warfare. Respiratory infection with the attenuated Live Vaccine Strain (LVS) and the highly virulent SchuS4 strain of Ft engenders intense peribronchiolar and perivascular inflammation, but fails to elicit select pro-inflammatory mediators (e.g., TNF, IL-1β, IL-6, IL-12, and IFN-γ) within the first ∼72 h. This in vivo finding is discordant with the principally TH1-oriented response to Ft frequently observed in cell-based studies wherein the aforementioned cytokines are produced. An often overlooked confounding factor in the interpretation of experimental results is the influence of environmental cues on the bacterium's capacity to elicit certain host responses. Herein, we reveal that adaptation of Ft to its mammalian host imparts an inability to elicit select pro-inflammatory mediators throughout the course of infection. Furthermore, in vitro findings that non-host adapted Ft elicits such a response from host cells reflect aberrant recognition of the DNA of structurally-compromised bacteria by AIM2-dependent and -independent host cell cytosolic DNA sensors. Growth of Ft in Muller-Hinton Broth or on Muller-Hinton-based chocolate agar plates or genetic mutation of Ft was found to compromise the structural integrity of the bacterium thus rendering it capable of aberrantly eliciting pro-inflammatory mediators (e.g., TNF, IL-1β, IL-6, IL-12, and IFN-γ). Our studies highlight the profound impact of different growth conditions on host cell response to infection and demonstrate that not all in vitro-derived findings may be relevant to tularemia pathogenesis in the mammalian host. Rational development of a vaccine and immunotherapeutics can only proceed from a foundation of knowledge based upon in vitro

  1. Whole-genome relationships among Francisella bacteria of diverse origins define new species and provide specific regions for detection

    DOE PAGES

    Challacombe, Jean Faust; Petersen, Jeannine M.; Gallegos-Graves, La Verne A.; ...

    2016-11-23

    Francisella tularensis is a highly virulent zoonotic pathogen that causes tularemia and, because of weaponization efforts in past world wars, is considered a tier 1 biothreat agent. Detection and surveillance of F. tularensis may be confounded by the presence of uncharacterized, closely related organisms. Through DNA-based diagnostics and environmental surveys, novel clinical and environmental Francisella isolates have been obtained in recent years. Here we present 7 new Francisella genomes and a comparison of their characteristics to each other and to 24 publicly available genomes as well as a comparative analysis of 16S rRNA and sdhA genes from over 90 Francisellamore » strains. Delineation of new species in bacteria is challenging, especially when isolates having very close genomic characteristics exhibit different physiological features—for example, when some are virulent pathogens in humans and animals while others are nonpathogenic or are opportunistic pathogens. Species resolution within Francisella varies with analyses of single genes, multiple gene or protein sets, or whole-genome comparisons of nucleic acid and amino acid sequences. Analyses focusing on single genes (16S rRNA, sdhA), multiple gene sets (virulence genes, lipopolysaccharide [LPS] biosynthesis genes, pathogenicity island), and whole-genome comparisons (nucleotide and protein) gave congruent results, but with different levels of discrimination confidence. We designate four new species within the genus; Francisella opportunistica sp. nov. (MA06-7296), Francisella salina sp. nov. (TX07-7308), Francisella uliginis sp. nov. (TX07-7310), and Francisella frigiditurris sp. nov. (CA97-1460). Lastly, this study provides a robust comparative framework to discern species and virulence features of newly detected Francisella bacteria.« less

  2. Whole-genome relationships among Francisella bacteria of diverse origins define new species and provide specific regions for detection

    SciTech Connect

    Challacombe, Jean Faust; Petersen, Jeannine M.; Gallegos-Graves, La Verne A.; Hodge, David; Pillai, Segaran; Kuske, Cheryl Rae; Drake, Harold L.

    2016-11-23

    Francisella tularensis is a highly virulent zoonotic pathogen that causes tularemia and, because of weaponization efforts in past world wars, is considered a tier 1 biothreat agent. Detection and surveillance of F. tularensis may be confounded by the presence of uncharacterized, closely related organisms. Through DNA-based diagnostics and environmental surveys, novel clinical and environmental Francisella isolates have been obtained in recent years. Here we present 7 new Francisella genomes and a comparison of their characteristics to each other and to 24 publicly available genomes as well as a comparative analysis of 16S rRNA and sdhA genes from over 90 Francisella strains. Delineation of new species in bacteria is challenging, especially when isolates having very close genomic characteristics exhibit different physiological features—for example, when some are virulent pathogens in humans and animals while others are nonpathogenic or are opportunistic pathogens. Species resolution within Francisella varies with analyses of single genes, multiple gene or protein sets, or whole-genome comparisons of nucleic acid and amino acid sequences. Analyses focusing on single genes (16S rRNA, sdhA), multiple gene sets (virulence genes, lipopolysaccharide [LPS] biosynthesis genes, pathogenicity island), and whole-genome comparisons (nucleotide and protein) gave congruent results, but with different levels of discrimination confidence. We designate four new species within the genus; Francisella opportunistica sp. nov. (MA06-7296), Francisella salina sp. nov. (TX07-7308), Francisella uliginis sp. nov. (TX07-7310), and Francisella frigiditurris sp. nov. (CA97-1460). Lastly, this study provides a robust comparative framework to discern species and virulence features of newly detected Francisella bacteria.

  3. An improved Francisella tularensis Live Vaccine Strain (LVS) is well tolerated and highly immunogenic when administered to rabbits in escalating doses using various immunization routes

    PubMed Central

    Pasetti, Marcela F.; Cuberos, Lilian; Horn, Thomas L.; Shearer, Jeffry D.; Matthews, Stephen J.; House, Robert V.; Sztein, Marcelo B.

    2008-01-01

    Tularemia is a severe disease for which there is no licensed vaccine. An attenuated F. tularensis live vaccine strain (LVS) was protective when administered to humans but safety concerns precluded its licensure and use in large scale immunization. An improved F. tularensis LVS preparation was produced under current Good Manufacturing Practice (cGMP) guidelines for evaluation in clinical trials. Preclinical safety, tolerability and immunogenicity were investigated in rabbits that received LVS in escalating doses (1x105 to 1x109 CFU) by the intradermal, subcutaneous or percutaneous (scarification) route. This improved LVS formulation was well tolerated at all doses; no death or adverse clinical signs were observed and necropsies showed no signs of pathology. No live organisms were detected in liver or spleen. Transient local reactogenicity was observed after scarification injection. Erythema and edema developed after intradermal injection in the highest dose cohorts. High levels of F. tularensis-specific IgM, IgG and IgA developed early after immunization, in a dose-dependent fashion. Scarification elicited higher levels of IgA. Antibodies elicited by LVS also recognized F. tularensis Schu-S4 antigens and there was a significant correlation between antibody titers measured against both LVS and Schu-S4. The ELISA titers also correlated closely with those measured by microagglutination. This is the first report describing comprehensive toxicological and immunological studies of F. tularensis LVS in rabbits. This animal model, which closely resembles human disease, proved adequate to assess safety and immunogenicity of F. tularensis vaccine candidates. This new LVS vaccine preparation is being evaluated in human clinical studies. PMID:18308432

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

    DOE PAGES

    Johnson, Shannon L.; Daligault, Hajnalka E.; Davenport, Karen W.; ...

    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.

  5. 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.

  6. 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

    2016-08-03

    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.

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

    SciTech Connect

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

    2016-01-28

    The bacterial system for fatty acid biosynthesis (FAS) contains several enzymes whose sequence and structure are highly conserved across a vast array of pathogens. Coupled with their low homology and difference in organization compared to the equivalent system in humans, this 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. In addition, 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. Our 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.

  8. Identification of Ciprofloxacin Resistance by SimpleProbe (trademark), High Resolution Melt and Pyrosequencing (trademark) Nucleic Acid Analysis in Biothreat Agents: Bacillus anthracis, Yersinia pestis and Francisella tularensis

    DTIC Science & Technology

    2010-01-01

    strains critical for the implementation of suitable infection control measures. The fluorinated quinolone , ciprofloxacin, is an antibiotic effective for...encode the subunits of DNA gyrase (gyrA and gyrB) and topo IV (par C and parE) contain hotspots within an area known as the quinolone resistance...designated the fluorinated quinolone , ciprofloxacin, as one of the antibiotics of choice for treatingB. anthracis, Yersinia pestis, and Francisella

  9. Importance of Metabolic Adaptations in Francisella Pathogenesis

    PubMed Central

    Ziveri, Jason; Barel, Monique; Charbit, Alain

    2017-01-01

    Francisella tularensis is a highly infectious Gram-negative bacterium and the causative agent of the zoonotic disease tularemia. This bacterial pathogen can infect a broad variety of animal species and can be transmitted to humans in numerous ways with various clinical outcomes. Although, Francisella possesses the capacity to infect numerous mammalian cell types, the macrophage constitutes the main intracellular niche, used for in vivo bacterial dissemination. To survive and multiply within infected macrophages, Francisella must imperatively escape from the phagosomal compartment. In the cytosol, the bacterium needs to control the host innate immune response and adapt its metabolism to this nutrient-restricted niche. Our laboratory has shown that intracellular Francisella mainly relied on host amino acid as major gluconeogenic substrates and provided evidence that the host metabolism was also modified upon Francisella infection. We will review here our current understanding of how Francisella copes with the available nutrient sources provided by the host cell during the course of infection.

  10. 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

  11. 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.

  12. Immunity to Francisella

    PubMed Central

    Cowley, Siobhán C.; Elkins, Karen L.

    2011-01-01

    In recent years, studies on the intracellular pathogen Francisella tularensis have greatly intensified, generating a wealth of new information on the interaction of this organism with the immune system. Here we review the basic elements of the innate and adaptive immune responses that contribute to protective immunity against Francisella species, with special emphasis on new data that has emerged in the last 5 years. Most studies have utilized the mouse model of infection, although there has been an expansion of work on human cells and other new animal models. In mice, basic immune parameters that operate in defense against other intracellular pathogen infections, such as interferon gamma, TNF-α, and reactive nitrogen intermediates, are central for control of Francisella infection. However, new important immune mediators have been revealed, including IL-17A, Toll-like receptor 2, and the inflammasome. Further, a variety of cell types in addition to macrophages are now recognized to support Francisella growth, including epithelial cells and dendritic cells. CD4+ and CD8+ T cells are clearly important for control of primary infection and vaccine-induced protection, but new T cell subpopulations and the mechanisms employed by T cells are only beginning to be defined. A significant role for B cells and specific antibodies has been established, although their contribution varies greatly between bacterial strains of lower and higher virulence. Overall, recent data profile a pathogen that is adept at subverting host immune responses, but susceptible to many elements of the immune system's antimicrobial arsenal. PMID:21687418

  13. Reliable detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis by using multiplex qPCR including internal controls for nucleic acid extraction and amplification

    PubMed Central

    2010-01-01

    Background Several pathogens could seriously affect public health if not recognized timely. To reduce the impact of such highly pathogenic micro-organisms, rapid and accurate diagnostic tools are needed for their detection in various samples, including environmental samples. Results Multiplex real-time PCRs were designed for rapid and reliable detection of three major pathogens that have the potential to cause high morbidity and mortality in humans: B. anthracis, F. tularensis and Y. pestis. The developed assays detect three pathogen-specific targets, including at least one chromosomal target, and one target from B. thuringiensis which is used as an internal control for nucleic acid extraction from refractory spores as well as successful DNA amplification. Validation of the PCRs showed a high analytical sensitivity, specificity and coverage of diverse pathogen strains. Conclusions The multiplex qPCR assays that were developed allow the rapid detection of 3 pathogen-specific targets simultaneously, without compromising sensitivity. The application of B. thuringiensis spores as internal controls further reduces false negative results. This ensures highly reliable detection, while template consumption and laboratory effort are kept at a minimum PMID:21143837

  14. O-Antigen-Deficient Francisella tularensis Live Vaccine Strain Mutants Are Ingested via an Aberrant Form of Looping Phagocytosis and Show Altered Kinetics of Intracellular Trafficking in Human Macrophages

    PubMed Central

    Lee, Bai-Yu; Horwitz, Marcus A.

    2012-01-01

    We examined the uptake and intracellular trafficking of F. tularensis Live Vaccine Strain (LVS) and LVS with disruptions of wbtDEF and wbtI genes essential for synthesis of the O antigen of lipopolysaccharide. Unlike parental bacteria, O-antigen-deficient LVS is efficiently killed by serum with intact complement but not by serum lacking terminal complement components. Opsonization of O-antigen-deficient LVS in serum lacking terminal complement components allows efficient uptake of these live bacteria by macrophages. In the presence of complement, whereas parental F. tularensis LVS is internalized within spacious pseudopod loops, mutant LVS is internalized within tightly juxtaposed multiple onion-like layers of pseudopodia. Without complement, both parental and mutant LVSs are internalized within spacious pseudopod loops. Thus, molecules other than O antigen are important in triggering dramatic pseudopod extensions and uptake by spacious pseudopod loops. Following uptake, both parental and mutant LVSs enter compartments that show limited staining for the lysosomal membrane glycoprotein CD63 and little fusion with secondary lysosomes. Subsequently, both parental and mutant LVSs lose their CD63 staining. Whereas the majority of parental LVS escapes into the cytosol by 6 h after uptake, mutant LVS shows a marked lag but does escape by 1 day after uptake. Despite the altered kinetics of phagosome escape, both mutant and parental strains grow to high levels within human macrophages. Thus, the O antigen plays a role in the morphology of uptake in the presence of complement and the kinetics of intracellular growth but is not essential for escape, survival, altered membrane trafficking, or intramacrophage growth. PMID:22202123

  15. 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

  16. Medical Countermeasure Models. Volume 4. Francisella tularensis

    DTIC Science & Technology

    2013-04-12

    outcome in terms of morbidity, mortality and loss of work due to both the agent itself, and any adverse medical countermeasure side effects . The...MCM Model Inputs Input Category Explanation of Input Dose of Agent Inhaled dose Designates the number of inhaled organisms. Vaccination Inputs...The length of the incubation period is dose dependent; therefore, individuals that inhale large doses of agent have a shorter average incubation

  17. 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.

  18. 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.

  19. Development of functional and molecular correlates of vaccine-induced protection for a model intracellular pathogen, F. tularensis LVS.

    PubMed

    De Pascalis, Roberto; Chou, Alicia Y; Bosio, Catharine M; Huang, Chiung-Yu; Follmann, Dean A; Elkins, Karen L

    2012-01-01

    In contrast with common human infections for which vaccine efficacy can be evaluated directly in field studies, alternative strategies are needed to evaluate efficacy for slowly developing or sporadic diseases like tularemia. For diseases such as these caused by intracellular bacteria, serological measures of antibodies are generally not predictive. Here, we used vaccines varying in efficacy to explore development of clinically useful correlates of protection for intracellular bacteria, using Francisella tularensis as an experimental model. F. tularensis is an intracellular bacterium classified as Category A bioterrorism agent which causes tularemia. The primary vaccine candidate in the U.S., called Live Vaccine Strain (LVS), has been the subject of ongoing clinical studies; however, safety and efficacy are not well established, and LVS is not licensed by the U.S. FDA. Using a mouse model, we compared the in vivo efficacy of a panel of qualitatively different Francisella vaccine candidates, the in vitro functional activity of immune lymphocytes derived from vaccinated mice, and relative gene expression in immune lymphocytes. Integrated analyses showed that the hierarchy of protection in vivo engendered by qualitatively different vaccines was reflected by the degree of lymphocytes' in vitro activity in controlling the intramacrophage growth of Francisella. Thus, this assay may be a functional correlate. Further, the strength of protection was significantly related to the degree of up-regulation of expression of a panel of genes in cells recovered from the assay. These included IFN-γ, IL-6, IL-12Rβ2, T-bet, SOCS-1, and IL-18bp. Taken together, the results indicate that an in vitro assay that detects control of bacterial growth, and/or a selected panel of mediators, may ultimately be developed to predict the outcome of vaccine efficacy and to complement clinical trials. The overall approach may be applicable to intracellular pathogens in general.

  20. 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

  1. Enhanced survival but not amplification of Francisella spp. in the presence of free-living amoebae.

    PubMed

    Buse, Helen Y; Schaefer, Frank W; Rice, Eugene W

    2016-12-08

    Transmission of Francisella tularensis, the etiologic agent of tularemia, has been associated with various water sources. Survival of many waterborne pathogens within free-living amoeba (FLA) is well documented; however, the role of amoebae in the environmental persistence of F. tularensis is unclear. In this study, axenic FLA cultures of Acanthamoeba castellanii, Acanthamoeba polyphaga, and Vermamoeba vermiformis were each inoculated with virulent strains of F. tularensis (Types A and B), the attenuated live vaccine strain, and Francisella novicida. Experimental parameters included low and high multiplicity of infection and incubation temperatures of 25 and 30 °C for 0-10 days. Francisella spp. survival was enhanced by the presence of FLA; however, bacterial growth and protozoa infectivity were not observed. In contrast, co-infections of A. polyphaga and Legionella pneumophila, used as an amoeba pathogen control, resulted in bacterial proliferation, cytopathic effects, and amoebal lysis. Collectively, even though short-term incubation with FLA was beneficial, the long-term effects on Francisella survival are unknown, especially given the expenditure of available amoebal derived nutrients and the fastidious nature of Francisella spp. These factors have clear implications for the role of FLA in Francisella environmental persistence.

  2. Benzimidazole-Based Antibacterial Agents Against F. tularensis

    PubMed Central

    Kumar, Kunal; Awasthi, Divya; Lee, Seung-Yub; Cummings, Jason E.; Knudson, Susan E.; Slayden, Richard A.; Ojima, Iwao

    2013-01-01

    Francisella tularensis is a highly virulent pathogenic bacterium. In order to identify novel potential antibacterial agents against F. tularensis, libraries of trisubstituted benzimidazoles were screened against F. tularensis LVS strain. In a preliminary screening assay, remarkably, 23 of 2,5,6- and 2,5,7-trisubstituted benzimidazoles showed excellent activity exhibiting greater than 90 % growth inhibition at 1 µg/mL. Among those hits, 21 compounds showed MIC90 values in the range of 0.35–48.6 µg/mL after accurate MIC determination. In ex-vivo efficacy assays, four of these compounds exhibited 2–3 Log reduction in colony forming units (CFU) per mL at concentrations of 10 and 50 µg/mL. PMID:23623254

  3. 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

  4. 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...

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

    DOE PAGES

    Jaing, Crystal J.; McLoughlin, Kevin S.; Thissen, James B.; ...

    2016-09-26

    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, providemore » 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 then 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. For genes containing mutations encode DNA gyrase subunit A, a hypothetical protein, an asparagine synthase, a sugar transamine/perosamine synthetase and others. Finally, structural modeling performed on these proteins provides insights into the potential function of these proteins and how they might contribute to Cipro resistance mechanisms.« less

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

    SciTech Connect

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

    2016-09-26

    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 then 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. For genes containing mutations encode DNA gyrase subunit A, a hypothetical protein, an asparagine synthase, a sugar transamine/perosamine synthetase and others. Finally, structural modeling performed on these proteins provides insights into the potential function of these proteins and how they might contribute to Cipro resistance mechanisms.

  7. 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

  8. Review of Processing and Analytical Methods for Francisella ...

    EPA Pesticide Factsheets

    Journal Article The etiological agent of tularemia, Francisella tularensis, is a resilient organism within the environment and can be acquired many ways (infectious aerosols and dust, contaminated food and water, infected carcasses, and arthropod bites). However, isolating F. tularensis from environmental samples can be challenging due to its nutritionally fastidious and slow-growing nature. In order to determine the current state of the science regarding available processing and analytical methods for detection and recovery of F. tularensis from water and soil matrices, a review of the literature was conducted. During the review, analysis via culture, immunoassays, and genomic identification were the most commonly found methods for F. tularensis detection within environmental samples. Other methods included combined culture and genomic analysis for rapid quantification of viable microorganisms and use of one assay to identify multiple pathogens from a single sample. Gaps in the literature that were identified during this review suggest that further work to integrate culture and genomic identification would advance our ability to detect and to assess the viability of Francisella spp. The optimization of DNA extraction, whole genome amplification with inhibition-resistant polymerases, and multiagent microarray detection would also advance biothreat detection.

  9. Francisella species in ticks and animals, Iberian Peninsula.

    PubMed

    Lopes de Carvalho, I; Toledo, A; Carvalho, C L; Barandika, J F; Respicio-Kingry, L B; Garcia-Amil, C; García-Pérez, A L; Olmeda, A S; Zé-Zé, L; Petersen, J M; Anda, P; Núncio, M S; Escudero, R

    2016-02-01

    The presence of Francisella species in 2134 ticks, 93 lagomorphs and 280 small mammals from the Iberian Peninsula was studied. Overall, 19 ticks and 6 lagomorphs were positive for Francisella tularensis subsp. holarctica, suggesting, as described for other regions, that lagomorphs may have an important role in the maintenance of F. tularensis in nature. Of the 6 positive lagomorphs, 4 were identified as the European rabbit, Oryctogalus cuniculus. Additionally, 353 ticks and 3 small mammals were PCR positive for Francisella-like endosymbionts (FLEs) and one small mammal was also positive for Francisella hispaniensis-like DNA sequences. Among FLE positive specimens, a variety of sequence types were detected: ticks were associated with 5 lpnA sequence types, with only one type identified per tick, in contrast to 2 lpnA sequence types detected in a single wood mouse (Apodemus sylvaticus). To our knowledge, this is the first report of FLEs in free-living small mammals as well as the first detection of F. hispaniensis-like sequences in a natural setting.

  10. Characterization of Francisella sp., GM2212, the first Francisella isolate from marine fish, Atlantic cod (Gadus morhua).

    PubMed

    Ottem, Karl F; Nylund, Are; Karlsbakk, Egil; Friis-Møller, Alice; Krossøy, Bjørn

    2007-05-01

    A Francisella sp., isolate GM2212(T), previously isolated from diseased farmed Atlantic cod Gadus morhua in Norway is characterized. The complete 16S rDNA, 16S-23S intergenic spacer, 23S rDNA, 23S-5S intergenic spacer, 5S rDNA, FopA, lipoprotein TUL4 (LpnA), malate dehydrogenase and a hypothetical lipoprotein (LpnB) is sequenced and compared with Francisella tularensis and Francisella philomiragia. All these sequences support a close relationship between GM2212(T) and F. philomiragia. The bacterium grows at 10-25 degrees C with an optimum at about 20 degrees C, a temperature range clearly different from F. tularensis and F. philomiragia. GM2212(T) is catalase-positive, indole positive, oxidase-negative, do not produce H(2)S in Triple Sugar Iron agar, and does not hydrolyze gelatin, is resistant to erythromycin and susceptible to ceftazidime, the latter five characteristics separating it from F. philomiragia. Cysteine enhances growth. Acid is produced from D: -glucose, maltose, sucrose (weak) but not from lactose or glycerol. GM2212(T) grows on both MacConkey agar and in nutrient broth (6% NaCl). The bacterium is resistant to trimethoprim-sulfamethoxazole, penicillines, cefuroxime and erythromycin; but is susceptible to ceftazidime, tetracycline, gentamicin, ciprofloxacin. Based on the molecular and phenotypical characteristics, we suggest that this GM2212 isolate, may represent a new species of Francisella. Isolate GM2212(T) (=CNCM I-3481(T) = CNCM I-3511(T) = DSM 18777(T)).

  11. 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.

  12. 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

  13. Establishment of Three Francisella Infections in Zebrafish Embryos at Different Temperatures

    PubMed Central

    Brudal, Espen; Ulanova, Lilia S.; O. Lampe, Elisabeth; Rishovd, Anne-Lise; Winther-Larsen, Hanne C.

    2014-01-01

    Francisella spp. are facultative intracellular pathogens identified in increasingly diverse hosts, including mammals. F. noatunensis subsp. orientalis and F. noatunensis subsp. noatunensis infect fish inhabiting warm and cold waters, respectively, while F. tularensis subsp. novicida is highly infectious for mice and has been widely used as a model for the human pathogen F. tularensis. Here, we established zebrafish embryo infection models of fluorescently labeled F. noatunensis subsp. noatunensis, F. noatunensis subsp. orientalis, and F. tularensis subsp. novicida at 22, 28, and 32°C, respectively. All infections led to significant bacterial growth, as shown by reverse transcription-quantitative PCR (RT-qPCR), and to a robust proinflammatory immune response, dominated by increased transcription of tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β). F. noatunensis subsp. orientalis was the most virulent, F. noatunensis subsp. noatunensis caused chronic infection, and F. tularensis subsp. novicida showed moderate virulence and led to formation of relatively small granuloma-like structures. The use of transgenic zebrafish strains with enhanced green fluorescent protein (EGFP)-labeled immune cells revealed their detailed interactions with Francisella species. All three strains entered preferentially into macrophages, which eventually assembled into granuloma-like structures. Entry into neutrophils was also observed, though the efficiency of this event depended on the route of infection. The results demonstrate the usefulness of the zebrafish embryo model for studying infections caused by different Francisella species at a wide range of temperatures and highlight their interactions with immune cells. PMID:24614659

  14. 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

  15. Multiple Locus Variable Number Tandem Repeat Analysis of Francisella tularensis

    DTIC Science & Technology

    2009-10-01

    Original signed by Chad Stratilo Chad Stratilo Approved by Original signed by Dr. L. Nagata Dr. L. Nagata Head, Biotechnology Section Approved...for release by Original signed by Dr. P. D’Agostino Dr. P. D’Agostino Chairman, Document Review Panel © Her Majesty the Queen in Right of...information as to the origin of fourteen of these strains. With so few strains of known pedigree, further characterization of the genetic diversity

  16. Purification and Partial Characterization of Monoclonal Antibodies of Francisella tularensis

    DTIC Science & Technology

    1992-07-01

    incubated for 2 h at 37*C. Plates were washed with 0.05% Tween 20-PBS three times. Fifty pl of a 1:2500 dilution of goat anti-mouse IgG and IgM...Cherwonogrodzky, J.W., Dubray, G., Moreno, E. and Nmyer, H. 1990. Antigens of Brucella, p. 19-63 In K. Nielsen and J.R. Duncan (ed.) Animal Brucellosis , CRC

  17. Francisella tularensis Molecular Typing Using Differential Insertion Sequence Amplification

    DTIC Science & Technology

    2011-08-01

    were homogenized in cold phosphate-buffered saline (PBS) within containment by using a Mini Beadbeater-8 instrument (Bio Spec Products, Inc...from the University of Nehrao;ka Medical Center stock collection. ’ 1 Previously known at Bacillus sul>tilis var. niger . • Abbreviations: LVS. live

  18. A 52 Kilodalton Protein Vaccine Candidate for Francisella tularensis

    DTIC Science & Technology

    2004-12-01

    stratdgie pour la mise au point de vaccins sub- cellulaires s~curitaires et efficaces contre les agents biologiques dangereux tels que la tular6mie. La...production d’oxyde de diazote chez certaines lign6es cellulaires mammaliennes et qu’en grandes quantit6s, elle cause la mort de la cellule. La vaccination de...60%) unless antibiotic therapy is given [1]. The bacterium is readily grown on simple medium, is highly virulent when delivered as an aerosol or in

  19. Rapid Identification of Francisella tularensis by a Fluorogenic Enzyme Immunoassay

    DTIC Science & Technology

    1990-11-01

    Benjamin, D.C. and Wagner, R.R., "Monoclonal antibodies to the glycoprotein of vesicular stomatitis virus: comparative neutralizing activity", J. Virol... feline leukemia virus and its neutralization", J. Immunol. Methods, 114 (1988) pp. 253-260. UNCLASSIFIED UNCLASSIFIED 22 29. Palfree, R.G.E. and

  20. Rapid Generation of Specific Protective Immunity to Francisella tularensis

    DTIC Science & Technology

    1992-11-01

    tu/arensis. To characterize the time course of development of diluent PBS was used for priming 3 days previously. The protective immunity, we...were inoculated with 104 LVS or the LVS, or the control diluent , PBS, i.d., i.v., or s.c. as indicated. Three days control diluent . PBS, i.d. at the...or the control diluent , PBS (tinprimed). Three davy later. they were challenged with i .... INS l.’ i.p. ranging from 10W to 101; actual (1(0) dose ot

  1. NaxD is a deacetylase required for lipid A modification and Francisella pathogenesis

    PubMed Central

    Llewellyn, Anna C.; Zhao, Jinshi; Song, Feng; Parvathareddy, Jyothi; Xu, Qian; Napier, Brooke A.; Laroui, Hamed; Merlin, Didier; Bina, James E.; Cotter, Peggy A.; Miller, Mark A.; Raetz, Christian R. H.; Weiss, David S.

    2013-01-01

    Summary Modification of specific Gram-negative bacterial cell envelope components, such as capsule, O-antigen and lipid A, are often essential for the successful establishment of infection. Francisella species express lipid A molecules with unique characteristics involved in circumventing host defences, which significantly contribute to their virulence. In this study, we show that NaxD, a member of the highly conserved YdjC superfamily, is a deacetylase required for an important modification of the outer membrane component lipid A in Francisella. Mass spectrometry analysis revealed that NaxD is essential for the modification of a lipid A phosphate with galactosamine in Francisella novicida, a model organism for the study of highly virulent Francisella tularensis. Significantly, enzymatic assays confirmed that this protein is necessary for deacetylation of its substrate. In addition, NaxD was involved in resistance to the antimicrobial peptide polymyxin B and critical for replication in macrophages and in vivo virulence. Importantly, this protein is also required for lipid A modification in F. tularensis as well as Bordetella bronchiseptica. Since NaxD homologues are conserved among many Gram-negative pathogens, this work has broad implications for our understanding of host subversion mechanisms of other virulent bacteria. PMID:22966934

  2. Guanylate-binding proteins promote activation of the AIM2 inflammasome during infection with Francisella novicida.

    PubMed

    Meunier, Etienne; Wallet, Pierre; Dreier, Roland F; Costanzo, Stéphanie; Anton, Leonie; Rühl, Sebastian; Dussurgey, Sébastien; Dick, Mathias S; Kistner, Anne; Rigard, Mélanie; Degrandi, Daniel; Pfeffer, Klaus; Yamamoto, Masahiro; Henry, Thomas; Broz, Petr

    2015-05-01

    The AIM2 inflammasome detects double-stranded DNA in the cytosol and induces caspase-1-dependent pyroptosis as well as release of the inflammatory cytokines interleukin 1β (IL-1β) and IL-18. AIM2 is critical for host defense against DNA viruses and bacteria that replicate in the cytosol, such as Francisella tularensis subspecies novicida (F. novicida). The activation of AIM2 by F. novicida requires bacteriolysis, yet whether this process is accidental or is a host-driven immunological mechanism has remained unclear. By screening nearly 500 interferon-stimulated genes (ISGs) through the use of small interfering RNA (siRNA), we identified guanylate-binding proteins GBP2 and GBP5 as key activators of AIM2 during infection with F. novicida. We confirmed their prominent role in vitro and in a mouse model of tularemia. Mechanistically, these two GBPs targeted cytosolic F. novicida and promoted bacteriolysis. Thus, in addition to their role in host defense against vacuolar pathogens, GBPs also facilitate the presentation of ligands by directly attacking cytosolic bacteria.

  3. 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.

  4. Possible Links Between Stress Defense and the Tricarboxylic Acid (TCA) Cycle in Francisella Pathogenesis*

    PubMed Central

    Dieppedale, Jennifer; Gesbert, Gael; Ramond, Elodie; Chhuon, Cerina; Dubail, Iharilalao; Dupuis, Marion; Guerrera, Ida Chiara; Charbit, Alain

    2013-01-01

    Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularemia. In vivo, this facultative intracellular bacterium survives and replicates mainly in the cytoplasm of infected cells. We have recently identified a genetic locus, designated moxR that is important for stress resistance and intramacrophage survival of F. tularensis. In the present work, we used tandem affinity purification coupled to mass spectrometry to identify in vivo interacting partners of three proteins encoded by this locus: the MoxR-like ATPase (FTL_0200), and two proteins containing motifs predicted to be involved in protein–protein interactions, bearing von Willebrand A (FTL_0201) and tetratricopeptide (FTL_0205) motifs. The three proteins were designated here for simplification, MoxR, VWA1, and TPR1, respectively. MoxR interacted with 31 proteins, including various enzymes. VWA1 interacted with fewer proteins, but these included the E2 component of 2-oxoglutarate dehydrogenase and TPR1. The protein TPR1 interacted with one hundred proteins, including the E1 and E2 subunits of both oxoglutarate and pyruvate dehydrogenase enzyme complexes, and their common E3 subunit. Remarkably, chromosomal deletion of either moxR or tpr1 impaired pyruvate dehydrogenase and oxoglutarate dehydrogenase activities, supporting the hypothesis of a functional role for the interaction of MoxR and TPR1 with these complexes. Altogether, this work highlights possible links between stress resistance and metabolism in F. tularensis virulence. PMID:23669032

  5. 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

  6. Secreted Effectors Encoded within and outside of the Francisella Pathogenicity Island Promote Intramacrophage Growth.

    PubMed

    Eshraghi, Aria; Kim, Jungyun; Walls, Alexandra C; Ledvina, Hannah E; Miller, Cheryl N; Ramsey, Kathryn M; Whitney, John C; Radey, Matthew C; Peterson, S Brook; Ruhland, Brittany R; Tran, Bao Q; Goo, Young Ah; Goodlett, David R; Dove, Simon L; Celli, Jean; Veesler, David; Mougous, Joseph D

    2016-11-09

    The intracellular bacterial pathogen Francisella tularensis causes tularemia, a zoonosis that can be fatal. The type VI secretion system (T6SS) encoded by the Francisella pathogenicity island (FPI) is critical for the virulence of this organism. Existing studies suggest that the complete repertoire of T6SS effectors delivered to host cells is encoded by the FPI. Using a proteome-wide approach, we discovered that the FPI-encoded T6SS exports at least three effectors encoded outside of the island. These proteins share features with virulence determinants of other pathogens, and we provide evidence that they can contribute to intramacrophage growth. The remaining proteins that we identified are encoded within the FPI. Two of these FPI-encoded proteins constitute effectors, whereas the others form a unique complex required for core function of the T6SS apparatus. The discovery of secreted effectors mediating interactions between Francisella and its host significantly advances our understanding of the pathogenesis of this organism.

  7. Essential genes from Arctic bacteria used to construct stable, temperature-sensitive bacterial vaccines.

    PubMed

    Duplantis, Barry N; Osusky, Milan; Schmerk, Crystal L; Ross, Darrell R; Bosio, Catharine M; Nano, Francis E

    2010-07-27

    All bacteria share a set of evolutionarily conserved essential genes that encode products that are required for viability. The great diversity of environments that bacteria inhabit, including environments at extreme temperatures, place adaptive pressure on essential genes. We sought to use this evolutionary diversity of essential genes to engineer bacterial pathogens to be stably temperature-sensitive, and thus useful as live vaccines. We isolated essential genes from bacteria found in the Arctic and substituted them for their counterparts into pathogens of mammals. We found that substitution of nine different essential genes from psychrophilic (cold-loving) bacteria into mammalian pathogenic bacteria resulted in strains that died below their normal-temperature growth limits. Substitution of three different psychrophilic gene orthologs of ligA, which encode NAD-dependent DNA ligase, resulted in bacterial strains that died at 33, 35, and 37 degrees C. One ligA gene was shown to render Francisella tularensis, Salmonella enterica, and Mycobacterium smegmatis temperature-sensitive, demonstrating that this gene functions in both Gram-negative and Gram-positive lineage bacteria. Three temperature-sensitive F. tularensis strains were shown to induce protective immunity after vaccination at a cool body site. About half of the genes that could be tested were unable to mutate to temperature-resistant forms at detectable levels. These results show that psychrophilic essential genes can be used to create a unique class of bacterial temperature-sensitive vaccines for important human pathogens, such as S. enterica and Mycobacterium tuberculosis.

  8. 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.

  9. 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.

  10. 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

  11. Francisella philomiragia Infection and Lethality in Mammalian Tissue Culture Cell Models, Galleria mellonella, and BALB/c Mice.

    PubMed

    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 × 10(3), and BALB/c mice by intranasal infection, with a LD50 of 3.45 × 10(3). 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

  12. Francisella novicida pathogenicity island encoded proteins were secreted during infection of macrophage-like cells.

    PubMed

    Hare, Rebekah F; Hueffer, Karsten

    2014-01-01

    Intracellular pathogens and other organisms have evolved mechanisms to exploit host cells for their life cycles. Virulence genes of some intracellular bacteria responsible for these mechanisms are located in pathogenicity islands, such as secretion systems that secrete effector proteins. The Francisella pathogenicity island is required for phagosomal escape, intracellular replication, evasion of host immune responses, virulence, and encodes a type 6 secretion system. We hypothesize that some Francisella novicida pathogenicity island proteins are secreted during infection of host cells. To test this hypothesis, expression plasmids for all Francisella novicida FPI-encoded proteins with C-terminal and N-terminal epitope FLAG tags were developed. These plasmids expressed their respective epitope FLAG-tagged proteins at their predicted molecular weights. J774 murine macrophage-like cells were infected with Francisella novicida containing these plasmids. The FPI proteins expressed from these plasmids successfully restored the intramacrophage growth phenotype in mutants of the respective genes that were deficient for intramacrophage growth. Using these expression plasmids, the localization of the Francisella pathogenicity island proteins were examined via immuno-fluorescence microscopy within infected macrophage-like cells. Several Francisella pathogenicity island encoded proteins (IglABCDEFGHIJ, PdpACE, DotU and VgrG) were detected extracellularly and they were co-localized with the bacteria, while PdpBD and Anmk were not detected and thus remained inside bacteria. Proteins that were co-localized with bacteria had different patterns of localization. The localization of IglC was dependent on the type 6 secretion system. This suggests that some Francisella pathogenicity island proteins were secreted while others remain within the bacterium during infection of host cells as structural components of the secretion system and were necessary for secretion.

  13. Importance of Host Cell Arginine Uptake in Francisella Phagosomal Escape and Ribosomal Protein Amounts*

    PubMed Central

    Ramond, Elodie; Gesbert, Gael; Guerrera, Ida Chiara; Chhuon, Cerina; Dupuis, Marion; Rigard, Mélanie; Henry, Thomas; Barel, Monique; Charbit, Alain

    2015-01-01

    Upon entry into mammalian host cells, the pathogenic bacterium Francisella must import host cell arginine to multiply actively in the host cytoplasm. We identified and functionally characterized an arginine transporter (hereafter designated ArgP) whose inactivation considerably delayed bacterial phagosomal escape and intracellular multiplication. Intramacrophagic growth of the ΔargP mutant was fully restored upon supplementation of the growth medium with excess arginine, in both F. tularensis subsp. novicida and F. tularensis subsp. holarctica LVS, demonstrating the importance of arginine acquisition in these two subspecies. High-resolution mass spectrometry revealed that arginine limitation reduced the amount of most of the ribosomal proteins in the ΔargP mutant. In response to stresses such as nutritional limitation, repression of ribosomal protein synthesis has been observed in all kingdoms of life. Arginine availability may thus contribute to the sensing of the intracellular stage of the pathogen and to trigger phagosomal egress. All MS data have been deposited in the ProteomeXchange database with identifier PXD001584 (http://proteomecentral.proteomexchange.org/dataset/PXD001584). PMID:25616868

  14. 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.

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

    PubMed Central

    Mailman, Timothy L; Schmidt, Matthias H

    2005-01-01

    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. PMID:18159552

  16. Discrimination of Pathogenic vs. Nonpathogenic Francisella tularensis and Burkholderia pseudomallei Using Proteomics Mass Spectrometry

    DTIC Science & Technology

    2011-03-01

    GroEL AhpC/TSA family protein hypothetical protein FTL0617 heat shock protein DnaK succinyl-CoA synthetase subunit beta hypothetical protein...lipoprotein chaperonin GroEL co-chaperonin GroES DNA-directed RNA polymerase subunit beta intracellular growth locus, subunit C 3.2 Differentiation...thailandensis E264 Unique Proteins Whole Cell Lysates OMPs putative lipoprotein glucan 1,4-a-glucosidase glycosy hydrolase family protein putative

  17. Protein, Lipid, Chemical and Structural Signatures of Differentially-Cultivated Francisella tularensis and Acinetobactor baumannii

    DTIC Science & Technology

    2014-03-05

    Resistant Strains of Acinetobacter baumannii , Antimicrobial Agents and Chemotherapy, (07 2013): 0. doi: 10.1128/AAC.00865-13 Bibiana V Iglesias...Q. Shanks, Y. Doi. Activities of Vancomycin-Containing Regimens against Colistin-Resistant Acinetobacter baumannii Clinical Strains, Antimicrobial... Acinetobacter baumannii , Antimicrobial Agents and Chemotherapy (04 2013) TOTAL: 1 Received Paper TOTAL: PERCENT_SUPPORTEDNAME FTE Equivalent: Total

  18. Transcriptome Analysis of Human Immune Responses Following Live Vaccine Strain (LVS) Francisella Tularensis Vaccination

    DTIC Science & Technology

    2007-03-08

    CSF2RB Colony-stimulating factor 2 receptor, beta, low-affinity (granulocyte-macrophage) – IL3/ IL5 receptor low-affinity Antimicrobial humoral...STAT6, IFN gamma, IL5 , IL7, IL13, IL19 3597 SCAP2 src family-associated phosphoprotein 2 Protein complex assembly, signal transduction, associated with

  19. Multiple mechanisms of NADPH oxidase inhibition by type A and type B Francisella tularensis

    PubMed Central

    McCaffrey, Ramona L.; Schwartz, Justin T.; Lindemann, Stephen R.; Moreland, Jessica G.; Buchan, Blake W.; Jones, Bradley D.; Allen, Lee-Ann H.

    2010-01-01

    Ft is a facultative intracellular pathogen that infects many cell types, including neutrophils. In previous work, we demonstrated that the type B Ft strain LVS disrupts NADPH oxidase activity throughout human neutrophils, but how this is achieved is incompletely defined. Here, we used several type A and type B strains to demonstrate that Ft-mediated NADPH oxidase inhibition is more complex than appreciated previously. We confirm that phagosomes containing Ft opsonized with AS exclude flavocytochrome b558 and extend previous results to show that soluble phox proteins were also affected, as indicated by diminished phosphorylation of p47phox and other PKC substrates. However, a different mechanism accounts for the ability of Ft to inhibit neutrophil activation by formyl peptides, Staphylococcus aureus, OpZ, and phorbol esters. In this case, enzyme targeting and assembly were normal, and impaired superoxide production was characterized by sustained membrane accumulation of dysfunctional NADPH oxidase complexes. A similar post-assembly inhibition mechanism also diminished the ability of anti-Ft IS to confer neutrophil activation and bacterial killing, consistent with the limited role for antibodies in host defense during tularemia. Studies of mutants that we generated in the type A Ft strain Schu S4 demonstrate that the regulatory factor fevR is essential for NADPH oxidase inhibition, whereas iglI and iglJ, candidate secretion system effectors, and the acid phosphatase acpA are not. As Ft uses multiple mechanisms to block neutrophil NADPH oxidase activity, our data strongly suggest that this is a central aspect of virulence. PMID:20610796

  20. Rapid Countermeasure Discovery against Francisella tularensis Based on a Metabolic Network Reconstruction

    DTIC Science & Technology

    2013-05-21

    Streptococcus pneumonia chorismate synthase inhibitor [40]. Although the computed PK parameters were in the good to moderate ranges and the compound was non...DMEM; Gibco Invitrogen) containing 10% fetal calf serum (FBS), penicillin (100 U/ml), and streptomycin (100 mg/ml) were added, and cells were incubated

  1. Transcriptional Profiling of Francisella tularensis Infected Peripheral Blood Monomuclear Cells: A Predictive Tool for Tularemia

    DTIC Science & Technology

    2008-01-01

    Infections with intracellular pathogens, such as Toxoplasma gondii (Masek et al., 2006), Salmonella (Gewirtz et al., 2000), and mycobacteria (Yadav et al...recognition of Toxoplasma gondii. J Cell Sci 119: 4565–4573. Munn DH, Shafizadeh E, Attwood JT, Bondarev I, Pashine A & Mellor AL (1999) Inhibition of T cell

  2. Dominance of Human Innate Immune Responses in Primary Francisella tularensis Live Vaccine Strain Vaccination

    DTIC Science & Technology

    2006-03-31

    Diseases, Bacteriology Division, 425 Porter St, Frederick , MD 21702-5011. Dr Brittingham is the recipient of the National Research Council Fellowship...tularemia vaccine strain) infection by the sera of human recipients of the live tula- remia vaccine. Am J Med Sci 1994;308:83-7. 10. Herzberg VL

  3. 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.

  4. 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.

  5. The transcription factor IRF1 and guanylate-binding proteins target activation of the AIM2 inflammasome by Francisella infection.

    PubMed

    Man, Si Ming; Karki, Rajendra; Malireddi, R K Subbarao; Neale, Geoffrey; Vogel, Peter; Yamamoto, Masahiro; Lamkanfi, Mohamed; Kanneganti, Thirumala-Devi

    2015-05-01

    Inflammasomes are critical for mounting host defense against pathogens. The molecular mechanisms that control activation of the AIM2 inflammasome in response to different cytosolic pathogens remain unclear. Here we found that the transcription factor IRF1 was required for activation of the AIM2 inflammasome during infection with the Francisella tularensis subspecies novicida (F. novicida), whereas engagement of the AIM2 inflammasome by mouse cytomegalovirus (MCMV) or transfected double-stranded DNA did not require IRF1. Infection of F. novicida detected by the DNA sensor cGAS and its adaptor STING induced type I interferon-dependent expression of IRF1, which drove the expression of guanylate-binding proteins (GBPs); this led to intracellular killing of bacteria and DNA release. Our results reveal a specific requirement for IRF1 and GBPs in the liberation of DNA for sensing by AIM2 depending on the pathogen encountered by the cell.

  6. 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

    2010-09-02

    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.

  7. 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.

  8. VIRULENCE AND CITRULLINE UREIDASE ACTIVITY OF PASTEURELLA TULARENSIS12

    PubMed Central

    Marchette, Nyven J.; Nicholes, Paul S.

    1961-01-01

    Marchette, Nyven J. (University of Utah, Salt Lake City), and Paul S. Nicholes. Virulence and citrulline ureidase activity of Pasteurella tularensis. J. Bacteriol. 82:26–32. 1961.—The presence of a citrulline ureidase system in Pasteurella tularensis strains of high virulence, and its absence in avirulent strains and strains of low virulence was confirmed. The presence of this system, however, was shown to be not directly related to virulence. The only wild strain of P. tularensis tested that lacked a citrulline ureidase system was isolated from a rodent. All the strains, isolated from rabbits, rabbit ticks, a human being, and a horse, that were tested possessed this system. The existence of two North American varieties of P. tularensis was postulated on the basis of virulence and citrulline ureidase activity. PMID:13766500

  9. Virulence and citrulline ureidase activity of Pasteurella tularensis.

    PubMed

    MARCHETTE, N J; NICHOLES, P S

    1961-07-01

    Marchette, Nyven J. (University of Utah, Salt Lake City), and Paul S. Nicholes. Virulence and citrulline ureidase activity of Pasteurella tularensis. J. Bacteriol. 82:26-32. 1961.-The presence of a citrulline ureidase system in Pasteurella tularensis strains of high virulence, and its absence in avirulent strains and strains of low virulence was confirmed. The presence of this system, however, was shown to be not directly related to virulence. The only wild strain of P. tularensis tested that lacked a citrulline ureidase system was isolated from a rodent. All the strains, isolated from rabbits, rabbit ticks, a human being, and a horse, that were tested possessed this system. The existence of two North American varieties of P. tularensis was postulated on the basis of virulence and citrulline ureidase activity.

  10. Francisella IglG protein and the DUF4280 proteins: PAAR-like proteins in non-canonical Type VI secretion systems?

    PubMed Central

    Lays, Claire; Tannier, Eric; Henry, Thomas

    2016-01-01

    Type VI secretion systems (T6SS) are bacterial molecular machines translocating effector proteins into target cells. T6SS are widely present in Gram-negative bacteria where they predominantly act to kill neighboring bacteria. This secretion system is reminiscent of the tail of contractile bacteriophages and consists of a contractile sheath anchored in the bacterial envelope and an inner tube made of stacks of the Hcp protein. The Hcp tube is capped with a VgrG trimer and a spike protein termed PAAR, which acts as the membrane-puncturing device. Francisella tularensis, the agent of tularemia, is an intracellular bacterium replicating within the host cytosol. Upon entry into the host cell, F. tularensis rapidly lyses the host vacuolar membrane to reach the host cytosol. This escape is dependent on the Francisella Pathogenicity Island (FPI), which is encoding an atypical T6SS. Among the 17 proteins encoded by the FPI, most of them required for virulence, eight have some homology to canonical T6SS proteins. We recently identified the function of one protein of unknown function encoded within the FPI, IglG. By three-dimensional modelling and following validation by different techniques, we found that IglG adopts a fold resembling the one of PAAR proteins. Importantly, IglG features a domain of unknown function DUF4280, present in numerous bacterial species. We thus propose to rename this domain of unknown function, PAAR-like domain, and discuss here the characteristics of this domain and its distribution in both Gram-negative and Gram-positive bacteria. PMID:28357328

  11. 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.

  12. Live Vaccine Strain Francisella tularensis is Detectable at the Inoculation Site but Not in Blood after Vaccination Against Tularemia

    DTIC Science & Technology

    2006-08-10

    The culture and PCR results of all blood samples were negative. Results of real - time PCR from the inoculation site samples were positive for 41 (100...LVS vaccination, with real - time PCR being more sensitive than culture. Our data suggest that bacteremia does not occur after LVS vaccination in normal, healthy human volunteers.

  13. 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...

  14. Anti-Francisella tularensis DNA Aptamers Detect Tularemia Antigen from Different Subspecies by Aptamer-Linked Immobilized Sorbent Assay

    DTIC Science & Technology

    2006-01-01

    Biobehavior, Bioassessment & Biosurveillance Branch 711 Human Performance Wing 2486 Gillingham Dr Human Effectiveness Directorate Brooks City-Base, TX... Biosurveillance Branch Brooks City-Base, TX 78235 10. SPONSOR/MONITOR’S ACRONYM(S) 711 HPW/RHPF; 711 HPW/RHPFB 11. SPONSOR/MONITOR’S REPORT... portal of infection. The clinical appearance varies from skin lesions to multi-organ involvement. Furthermore, the severity depends on the dose and the

  15. 3-Substituted Indole Inhibitors Against Francisella tularensis FabI Identified by Structure-Based Virtual Screening

    DTIC Science & Technology

    2013-07-01

    Seto, W. H.; Ng, T. K.; Yam, W. C.; Ng, W. W. Increasing resistance of Streptococcus pneumoniae to fluoroquinolones: results of a Hong Kong...Brenwald, N. P.; Gill, M. J.; Walker, R. A.; Livermore, D. M.; George, R. C. Emergence of a fluoroquinolone-resistant strain of Streptococcus pneumoniae ...Payne, D. J.; Rock, C. O.; Wallis, N. G. Characterization of Streptococcus pneumoniae enoyl-(acyl-carrier protein) reductase (FabK). Biochem. J. 2003

  16. 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

    2015-12-28

    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.

  17. 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

  18. A statistical framework for improving genomic annotations of prokaryotic essential genes.

    PubMed

    Deng, Jingyuan; Su, Shengchang; Lin, Xiaodong; Hassett, Daniel J; Lu, Long Jason

    2013-01-01

    Large-scale systematic analysis of gene essentiality is an important step closer toward unraveling the complex relationship between genotypes and phenotypes. Such analysis cannot be accomplished without unbiased and accurate annotations of essential genes. In current genomic databases, most of the essential gene annotations are derived from whole-genome transposon mutagenesis (TM), the most frequently used experimental approach for determining essential genes in microorganisms under defined conditions. However, there are substantial systematic biases associated with TM experiments. In this study, we developed a novel Poisson model-based statistical framework to simulate the TM insertion process and subsequently correct the experimental biases. We first quantitatively assessed the effects of major factors that potentially influence the accuracy of TM and subsequently incorporated relevant factors into the framework. Through iteratively optimizing parameters, we inferred the actual insertion events occurred and described each gene's essentiality on probability measure. Evaluated by the definite mapping of essential gene profile in Escherichia coli, our model significantly improved the accuracy of original TM datasets, resulting in more accurate annotations of essential genes. Our method also showed encouraging results in improving subsaturation level TM datasets. To test our model's broad applicability to other bacteria, we applied it to Pseudomonas aeruginosa PAO1 and Francisella tularensis novicida TM datasets. We validated our predictions by literature as well as allelic exchange experiments in PAO1. Our model was correct on six of the seven tested genes. Remarkably, among all three cases that our predictions contradicted the TM assignments, experimental validations supported our predictions. In summary, our method will be a promising tool in improving genomic annotations of essential genes and enabling large-scale explorations of gene essentiality. Our

  19. Biofilm formation of Francisella noatunensis subsp. orientalis

    USGS Publications Warehouse

    Soto, Esteban; Halliday-Wimmonds, Iona; Francis , Stewart; 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.

  20. Host Genes and Resistance/Sensitivity to Military Priority Pathogens

    DTIC Science & Technology

    2013-06-01

    Vargas , A. Velds, T. R. Brummelkamp, A. M. Dirac, T. K. Sixma, and R. Bernards. 2005. A genomic and functional inventory of deubiquitinating enzymes. Cell...JW, Kayagaki N, Broz P, Henry T, Newton K, O’Rourke K, Chan S, Dong J, Qu Y, Roose-Girma M, et al: Absent in melanoma 2 is required for innate immune...increased proinflammatory responses from host cells. Infect Immun 2010, 78(3):1022-1031. 62. Weiss DS, Henry T, Monack DM: Francisella tularensis

  1. Multiplexed identification of blood-borne bacterial pathogens by use of a novel 16S rRNA gene PCR-ligase detection reaction-capillary electrophoresis assay.

    PubMed

    Pingle, Maneesh R; Granger, Kathleen; Feinberg, Philip; Shatsky, Rebecca; Sterling, Bram; Rundell, Mark; Spitzer, Eric; Larone, Davise; Golightly, Linnie; Barany, Francis

    2007-06-01

    We have developed a novel high-throughput PCR-ligase detection reaction-capillary electrophoresis (PCR-LDR-CE) assay for the multiplexed identification of 20 blood-borne pathogens (Staphylococcus epidermidis, Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Acinetobacter baumannii, Neisseria meningitidis, Bacteroides fragilis, Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Brucella abortus), the last four of which are biothreat agents. The method relies on the amplification of two regions within the bacterial 16S rRNA gene, using universal PCR primers and querying the identity of specific single-nucleotide polymorphisms within the amplified regions in a subsequent LDR. The ligation products vary in color and size and are separated by CE. Each organism generates a specific pattern of ligation products, which can be used to distinguish the pathogens using an automated software program we developed for that purpose. The assay has been verified on 315 clinical isolates and demonstrated a detection sensitivity of 98%. Additionally, 484 seeded blood cultures were tested, with a detection sensitivity of 97.7%. The ability to identify geographically variant strains of the organisms was determined by testing 132 isolates obtained from across the United States. In summary, the PCR-LDR-CE assay can successfully identify, in a multiplexed fashion, a panel of 20 blood-borne pathogens with high sensitivity and specificity.

  2. Identification of the clpB and bipA genes and an evaluation of their expression as related to intracellular survival for the bacterial pathogen Piscirickettsia salmonis.

    PubMed

    Isla, A; Haussmann, D; Vera, T; Kausel, G; Figueroa, J

    2014-10-10

    Piscirickettsia salmonis is the pathogen responsible for salmonid rickettsial septicemia (SRS), a disease that affects a wide variety of marine cultivated fish species and causes economic losses for the aquaculture industry worldwide. Many in vitro studies have reported on the capacity of this microorganism to replicate in the interior of cytoplasmic vesicles from varied fish cell lines. However, the mechanisms used by this bacteria to survive, replicate, and propagate in cell lines, especially in macrophages and monocytes, are unknown. A number of studies have described the diverse proteins in pathogens such as Legionella pneumophila, Coxiella burnetii, and Francisella tularensis which allow these to evade the cellular immune response and replicate in the interior of macrophages in different hosts. Some of these proteins are the virulence factor BipA/TypA and the heat shock protein ClpB, both of which have been widely characterized. The results of the current study present the complete coding sequence of the genes clpB and bipA from the P. salmonis genome. Moreover, the experimental results suggest that during the infectious process of the SHK-1 cellular line in P. salmonis, the pathogen significantly increases the expression of proteins ClpB and BipA. This would permit the pathogen to adapt to the hostile conditions produced by the macrophage and thus evade mechanisms of cellular degradation while facilitating replication in the interior of this salmon cell line.

  3. Cathepsin B modulates lysosomal biogenesis and host defense against Francisella novicida infection

    PubMed Central

    Malireddi, R.K. Subbarao; Karki, Rajendra; Lupfer, Christopher; Gurung, Prajwal; Lamkanfi, Mohamed

    2016-01-01

    Lysosomal cathepsins regulate an exquisite range of biological functions, and their deregulation is associated with inflammatory, metabolic, and degenerative diseases in humans. In this study, we identified a key cell-intrinsic role for cathepsin B as a negative feedback regulator of lysosomal biogenesis and autophagy. Mice and macrophages lacking cathepsin B activity had increased resistance to the cytosolic bacterial pathogen Francisella novicida. Genetic deletion or pharmacological inhibition of cathepsin B down-regulated mechanistic target of rapamycin activity and prevented cleavage of the lysosomal calcium channel TRPML1. These events drove transcription of lysosomal and autophagy genes via transcription factor EB, which increased lysosomal biogenesis and activation of autophagy initiation kinase ULK1 for clearance of the bacteria. Our results identified a fundamental biological function of cathepsin B in providing a checkpoint for homeostatic maintenance of lysosome populations and basic recycling functions in the cell. PMID:27551156

  4. Discovery of a novel and potent class of F. tularensis enoyl-reductase (FabI) inhibitors by molecular shape and electrostatic matching.

    PubMed

    Hevener, Kirk E; Mehboob, Shahila; Su, Pin-Chih; Truong, Kent; Boci, Teuta; Deng, Jiangping; Ghassemi, Mahmood; Cook, James L; Johnson, Michael E

    2012-01-12

    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 submicromolar 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.

  5. Link between intraphagosomal biotin and rapid phagosomal escape in Francisella

    PubMed Central

    Napier, Brooke A.; Meyer, Lena; Bina, James E.; Miller, Mark A.; Sjöstedt, Anders; Weiss, David S.

    2012-01-01

    Cytosolic bacterial pathogens require extensive metabolic adaptations within the host to replicate intracellularly and cause disease. In phagocytic cells such as macrophages, these pathogens must respond rapidly to nutrient limitation within the harsh environment of the phagosome. Many cytosolic pathogens escape the phagosome quickly (15–60 min) and thereby subvert this host defense, reaching the cytosol where they can replicate. Although a great deal of research has focused on strategies used by bacteria to resist antimicrobial phagosomal defenses and transiently pass through this compartment, the metabolic requirements of bacteria in the phagosome are largely uncharacterized. We previously identified a Francisella protein, FTN_0818, as being essential for intracellular replication and involved in virulence in vivo. We now show that FTN_0818 is involved in biotin biosynthesis and required for rapid escape from the Francisella-containing phagosome (FCP). Addition of biotin complemented the phagosomal escape defect of the FTN_0818 mutant, demonstrating that biotin is critical for promoting rapid escape during the short time that the bacteria are in the phagosome. Biotin also rescued the attenuation of the FTN_0818 mutant during infection in vitro and in vivo, highlighting the importance of this process. The key role of biotin in phagosomal escape implies biotin may be a limiting factor during infection. We demonstrate that a bacterial metabolite is required for phagosomal escape of an intracellular pathogen, providing insight into the link between bacterial metabolism and virulence, likely serving as a paradigm for other cytosolic pathogens. PMID:23071317

  6. 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.

  7. 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.

  8. Efficacy of Resistance to Francisella Imparted by ITY/NRAMP/SLC11A1 Depends on Route of Infection.

    PubMed

    Powell, Daniel A; Frelinger, Jeffrey A

    2017-01-01

    Natural resistance-associated macrophage protein (NRAMP) encoded by the Slc11a1 gene is a membrane-associated transporter of divalent metal ions. Murine Slc11a1 has two known alleles, a functional Slc11a1(Gly169), which is found in DBA2/J, NOD/LtJ, and 129p3/J and related mouse strains, and a non-functional Slc11a1(Asp169), that is found in C56Bl/6J (B6) and BALB/cJ mice. B6 mice congenic for Slc11a1(Gly169) (B6-Slc11a1(G169) ) are markedly resistant to the intracellular pathogens Salmonella, Leishmania, and Mycobacterium tuberculosis. We examined the host cell response and replication of Francisella in B6-Slc11a1(G169) mice. Bone marrow-derived macrophages from either B6-Slc11a1(G169) or B6 mice were both effectively invaded by Francisella live vaccine strain (LVS). However, at 16 hours post-infection (hpi), the number of LVS bacteria recovered from B6 macrophages had increased roughly 100-fold, while in B6-Slc11a1(G169) mice the number decreased 10-fold. When the mice were challenged intranasally (i.n.) B6 mice lost significant amounts (~15%) of weight, where as B6-Slc11a1(G169) mice lost no weight. Three days after infection in B6-Slc11a1(G169) mice, we failed to recover viable Francisella from the lungs, livers, or spleens. By contrast, B6 mice had bacterial burdens approaching 1 × 10(6) CFU/organ in all three organs. To further examine the degree of resistance imparted by Slc11a1(Gly169) expression, we challenged mice deficient in TLR2, TLR4, and TLR9, but expressing the functional Slc11a1 (B6-Slc11a1(G169)Tlr2/4/9(-/-) ). Surprisingly, B6-Slc11a1(G169)Tlr2/4/9(-/-) mice had no notable weight loss. Eighty percent of B6-Slc11a1(G169)Tlr2/4/9(-)(/)(-) mice yielded no detectable Francisella in any organ tested. Additionally, Slc11a1(G169) produced little detectable cytokine either in the lung or serum compared to B6 mice. Mice expressing Slc11a1(Gly169) survived even high doses (~80 LD50) of LVS inoculation. These data taken together serve to highlight

  9. Efficacy of Resistance to Francisella Imparted by ITY/NRAMP/SLC11A1 Depends on Route of Infection

    PubMed Central

    Powell, Daniel A.; Frelinger, Jeffrey A.

    2017-01-01

    Natural resistance-associated macrophage protein (NRAMP) encoded by the Slc11a1 gene is a membrane-associated transporter of divalent metal ions. Murine Slc11a1 has two known alleles, a functional Slc11a1Gly169, which is found in DBA2/J, NOD/LtJ, and 129p3/J and related mouse strains, and a non-functional Slc11a1Asp169, that is found in C56Bl/6J (B6) and BALB/cJ mice. B6 mice congenic for Slc11a1Gly169 (B6-Slc11a1G169) are markedly resistant to the intracellular pathogens Salmonella, Leishmania, and Mycobacterium tuberculosis. We examined the host cell response and replication of Francisella in B6-Slc11a1G169 mice. Bone marrow-derived macrophages from either B6-Slc11a1G169 or B6 mice were both effectively invaded by Francisella live vaccine strain (LVS). However, at 16 hours post-infection (hpi), the number of LVS bacteria recovered from B6 macrophages had increased roughly 100-fold, while in B6-Slc11a1G169 mice the number decreased 10-fold. When the mice were challenged intranasally (i.n.) B6 mice lost significant amounts (~15%) of weight, where as B6-Slc11a1G169 mice lost no weight. Three days after infection in B6-Slc11a1G169 mice, we failed to recover viable Francisella from the lungs, livers, or spleens. By contrast, B6 mice had bacterial burdens approaching 1 × 106 CFU/organ in all three organs. To further examine the degree of resistance imparted by Slc11a1Gly169 expression, we challenged mice deficient in TLR2, TLR4, and TLR9, but expressing the functional Slc11a1 (B6-Slc11a1G169Tlr2/4/9−/−). Surprisingly, B6-Slc11a1G169Tlr2/4/9−/− mice had no notable weight loss. Eighty percent of B6-Slc11a1G169Tlr2/4/9−/− mice yielded no detectable Francisella in any organ tested. Additionally, Slc11a1G169 produced little detectable cytokine either in the lung or serum compared to B6 mice. Mice expressing Slc11a1Gly169 survived even high doses (~80 LD50) of LVS inoculation. These data taken together serve to highlight that functional Slc11a1Gly169 can

  10. Effects of Francisella tularensis, Salmonella typhimurium and Streptococcus pneumoniae Infections on Oxidative, Glycolytic and Lysosomal Enzyme Activity in Red and White Skeletal Muscle in the Rat,

    DTIC Science & Technology

    1981-03-30

    Miller (18) reported an inhibition of succinic dehydrogenase but not of cytochrome c oxidase activity in rabbit skeletal muscle by meningococci and...determination of the activities of cytochrome c oxidase (CYTOX; E.G. 1.9.3.1) (23), citrate synthase (CS; E.C. 4.1.3.7) (22), glyceraldehyde-3

  11. Whole-Genome Single Nucleotide Polymorphism Based Phylogeny of Francisella tularensis and Its Application to the Development of a Strain Typing Assay

    DTIC Science & Technology

    2009-10-07

    gen- erated using the MrBayes program, version 3.1.2 [15-17]. The program was run for 200,000 generations, using a haploid model. The root of the...provide real biological insights into the adaptation and evolution of a species. Such phylogenetic-based comparative analysis can cap- ture genomic...States. Emerg Infect Dis 2005, 11(12):1835-1841. 23. Svensson K, Larsson P, Johansson D, Bystrom M, Forsman M, Johans- son A: Evolution of subspecies of

  12. Guanylate-binding proteins promote AIM2 inflammasome activation during Francisella novicida infection by inducing cytosolic bacteriolysis and DNA release

    PubMed Central

    Dreier, Roland F.; Costanzo, Stéphanie; Anton, Leonie; Rühl, Sebastian; Dussurgey, Sébastien; Dick, Mathias S.; Kistner, Anne; Rigard, Mélanie; Degrandi, Daniel; Pfeffer, Klaus; Yamamoto, Masahiro; Henry, Thomas; Broz, Petr

    2015-01-01

    The AIM2 inflammasome detects double-stranded DNA in the cytosol and induces caspase-1-dependent pyroptosis as well as release of the inflammatory cytokines IL-1β and IL-18. AIM2 is critical for host defense against DNA viruses and bacteria that replicate in the cytosol, such as Francisella novicida. AIM2 activation by F. novicida requires bacteriolysis, yet whether this process is accidental or a host-driven immune mechanism remained unclear. Using siRNA screening for nearly 500 interferon-stimulated genes, we identified guanylate-binding proteins GBP2 and GBP5 as key AIM2 activators during F. novicida infection. Their prominent role was validated in vitro and in a mouse model of tularemia. Mechanistically, these two GBPs target cytosolic F. novicida and promote bacteriolysis. Thus, besides their role in host defense against vacuolar pathogens, GBPs also facilitate the presentation of ligands by directly attacking cytosolic bacteria. PMID:25774716

  13. Detection of Francisella piscicida in Atlantic cod (Gadus morhua L) by the loop-mediated isothermal amplification (LAMP) reaction.

    PubMed

    Caipang, Christopher M A; Kulkarni, Amod; Brinchmann, Monica F; Korsnes, Kjetil; Kiron, Viswanath

    2010-06-01

    The loop-mediated isothermal amplification (LAMP) reaction was evaluated for its speed and sensitivity in detecting the presence of Francisella piscicida, the causative agent of francisellosis in Atlantic cod (Gadus morhua). Four primer sets, consisting of two outer and two inner, were designed from the groEL gene of the pathogen. The LAMP reaction was optimised at 63 degrees C for 1h using bacterial genomic DNA as the template and the products were visualised under ultra-violet light and analysed by agarose gel electrophoresis. A ladder-like pattern of bands, specific for F. piscicida, was amplified from positive samples. The method was highly specific for the detection of F. piscicida and was 100 times more sensitive than conventional PCR. In addition, the LAMP assay was able to detect the pathogen in kidney and splenic samples of naturally-infected Atlantic cod.

  14. 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.

  15. 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

  16. Structure and Engineering of Francisella novicida Cas9

    PubMed Central

    Hirano, Hisato; Gootenberg, Jonathan S.; Horii, Takuro; Abudayyeh, Omar O.; Kimura, Mika; Hsu, Patrick D.; Nakane, Takanori; Ishitani, Ryuichiro; Hatada, Izuho; Zhang, Feng; Nishimasu, Hiroshi; Nureki, Osamu

    2016-01-01

    Summary The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets complementary to the guide RNA, and has been applied to programmable genome editing. Cas9-mediated cleavage requires a protospacer adjacent motif (PAM) juxtaposed with the DNA target sequence, thus constricting the range of targetable sites. Here, we report the 1.7 Å resolution crystal structures of Cas9 from Francisella novicida (FnCas9), one of the largest Cas9 orthologs, in complex with a guide RNA and its PAM-containing DNA targets. A structural comparison of FnCas9 with other Cas9 orthologs revealed striking conserved and divergent features among distantly related CRISPR-Cas9 systems. We found that FnCas9 recognizes the 5′-NGG-3′ PAM, and used the structural information to create a variant that can recognize the more relaxed 5′-YG-3′ PAM. Furthermore, we demonstrated that pre-assembled FnCas9 ribonucleoprotein complexes can be microinjected into mouse zygotes to edit endogenous sites with the 5′-YG-3′ PAMs, thus expanding the target space of the CRISPR-Cas9 toolbox. PMID:26875867

  17. Francisella noatunensis subsp. orientalis pathogenesis analyzed by experimental immersion challenge in Nile tilapia, Oreochromis niloticus (L.).

    PubMed

    Soto, Esteban; Kidd, Scott; Mendez, Susan; Marancik, David; Revan, Floyd; Hiltchie, David; Camus, Alvin

    2013-05-31

    Francisella noatunensis subsp. orientalis (Fno) (syn. F. asiatica) is an emergent warmwater fish pathogen and the causative agent of francisellosis in tilapia (Oreochromis sp). To study the pathogenesis of this bacterium, tilapia fingerlings were experimentally infected by immersion challenge with wild type (WT) Fno and the distribution of bacteria to multiple organs, as well as associated lesion development, investigated after 3, 24, 48, 96, and 192h by real-time PCR and histopathological examination. Surface mucus collected 3h post-infection contained the highest number of Fno genome equivalents (GE). After 96h, marked increases of WT Fno GE were detected in spleen, anterior kidney, posterior kidney, gill, heart, liver, brain, gonad, and the gastrointestinal tract. Increases in bacterial GE also corresponded to the appearance, size and number of granulomas typical of francisellosis, predominantly in the spleen and anterior and posterior kidney segments. A simultaneous comparison was also made in tilapia challenged with an attenuated Fno strain containing a mutation in the intracellular growth locus C (iglC) gene, essential to intracellular survival. Compared to the WT, the mutant iglC strain was present in most tissues in similar numbers prior to 48h post-challenge. While the mutant did not replicate significantly or produce lesions in any tissue, it persisted for up to 192h. These findings provide insight into the pathophysiology of francisellosis in tilapia, which may also prove useful as a model for the study of mammalian tularemia, and advance our understanding of the utility of the ΔiglC mutant as a potential vaccine candidate.

  18. Finished genome assembly of warm spring isolate Francisella novicida DPG 3A-IS

    DOE PAGES

    Johnson, Shannon L.; Minogue, Timothy D.; Daligault, Hajnalka E.; ...

    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.

  19. 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.

  20. 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.

  1. 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

  2. Light Addressable Potentiometric Immunoassays for Identification of Biological Agents: NATO SIBCA Exercise I

    DTIC Science & Technology

    2001-11-01

    developed: Bacillus anthracis, Brucella melitensis , Francisella tularensis, Burkholderia mallei, Yersinia pestis, Venezuelan Equine Encephalitis virus...tularensis, Brucella metitensis. Burkholderia mallei, Yellow Fever virus, Vaccinia virus, or Coxiella burnetii. The participating laboratory for Canada was

  3. 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...

  4. Mortality and Morbidity Avoidance/Reduction of Respiratory Sickness Immediately Following Exposure to Bioweaponized Microbial Pathogens

    DTIC Science & Technology

    2007-11-02

    vegetative cells of Bacillus anthracis, Clostridium spp., fungal lung pathogens in general and cells of Yersinia pestis, Francisella tularensis, Brucella suis, Salmonella typhi and related bacterial species.

  5. 42 CFR 73.9 - Responsible Official.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ..., or e-mail: Bacillus anthracis, Botulinum neurotoxins, Botulinum neurotoxin producing species of Clostridium, Burkholderia mallei, Burkholderia pseudomallei Francisella tularensis, Ebola viruses, ,...

  6. 42 CFR 73.9 - Responsible Official.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ..., or e-mail: Bacillus anthracis, Botulinum neurotoxins, Botulinum neurotoxin producing species of Clostridium, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Ebola viruses,...

  7. 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

  8. 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.

  9. Efficient targeted mutagenesis of rice and tobacco genomes using Cpf1 from Francisella novicida

    PubMed Central

    Endo, Akira; Masafumi, Mikami; Kaya, Hidetaka; Toki, Seiichi

    2016-01-01

    CRISPR/Cas9 systems are nowadays applied extensively to effect genome editing in various organisms including plants. CRISPR from Prevotella and Francisella 1 (Cpf1) is a newly characterized RNA-guided endonuclease that has two distinct features as compared to Cas9. First, Cpf1 utilizes a thymidine-rich protospacer adjacent motif (PAM) while Cas9 prefers a guanidine-rich PAM. Cpf1 could be used as a sequence-specific nuclease to target AT-rich regions of a genome that Cas9 had difficulty accessing. Second, Cpf1 generates DNA ends with a 5′ overhang, whereas Cas9 creates blunt DNA ends after cleavage. “Sticky” DNA ends should increase the efficiency of insertion of a desired DNA fragment into the Cpf1-cleaved site using complementary DNA ends. Therefore, Cpf1 could be a potent tool for precise genome engineering. To evaluate whether Cpf1 can be applied to plant genome editing, we selected Cpf1 from Francisella novicida (FnCpf1), which recognizes a shorter PAM (TTN) within known Cpf1 proteins, and applied it to targeted mutagenesis in tobacco and rice. Our results show that targeted mutagenesis had occurred in transgenic plants expressing FnCpf1 with crRNA. Deletions of the targeted region were the most frequently observed mutations. Our results demonstrate that FnCpf1 can be applied successfully to genome engineering in plants. PMID:27905529

  10. Antibiotic uptake by cultured Atlantic cod leucocytes and effect on intracellular Francisella noatunensis subsp. noatunensis replication.

    PubMed

    Kaldestad, Marte; Haugland, Gyri T; Rønneseth, Anita; Wergeland, Heidrun I; Samuelsen, Ole Bent

    2014-02-04

    The granuloma disease caused by Francisella noatunensis subsp. noatunensis in farmed Atlantic cod has not been successfully treated by use of antibacterials, even when antibacterial resistance testing indicates a sufficient effect. The reason for this treatment failure may be the intracellular existence of the bacteria within immune cells, mainly macrophages. To investigate the effect of antibacterials on intracellular Francisella replication, we established a protocol for the detection of drugs within Atlantic cod immune cells using high-performance liquid chromatography (HPLC). When the uptake and intracellular concentrations of oxolinic acid and flumequine were analysed in isolated adherent head kidney leucocytes (HKLs) by HPLC, we found that uptake was rapid and the intracellular concentrations reflected the extracellular exposure concentrations. To investigate the effect of the antibacterial compounds on intracellular bacterial replication, adherent HKLs experimentally infected with the bacteria were analysed using flow cytometry and intracellular labelling of bacteria by specific antibodies. We found that flumequine did not inhibit intracellular bacterial replication. Unexpectedly, the results indicated that the intracellularly effiacy of the drug was reduced. The HPLC method used proved to be highly applicable for accurate determination of intracellular drug concentrations. When combined with sensitive and specific flow cytometry analyses for identification and measurement of intracellular bacterial replication, we suggest that this approach can be very valuable for the design of antibacterial treatments of intracellular pathogens.

  11. Francisella philomiragia biofilm formation and interaction with the aquatic protist Acanthamoeba castellanii.

    PubMed

    Verhoeven, Anne B; Durham-Colleran, Meghan W; Pierson, Tony; Boswell, William T; Van Hoek, Monique L

    2010-10-01

    The bacterium Francisella philomiragia has been isolated from environmental samples originating from around the globe. F. philomiragia-related strains cause francisellosis of both farmed and wild fish. In addition, occasional human infections caused by F. philomiragia are found in victims of near-drowning and patients with chronic granulomatous disease. We have shown that F. philomiragia forms in vitro biofilms with increased formation at 25 °C over 37 °C conditions. We found that F. philomiragia can form a biofilm in a co-culture with live Acanthamoeba castellanii, an aquatic amoeba. Interestingly, amoeba-conditioned supernatant has an inhibitory effect on production of biofilm by F. philomiragia, whereas Francisella-conditioned supernatant has no effect on growth of amoebae. We have shown that F. philomiragia can infect A. castellanii after only 5 days of co-incubation and that it infects A. castellanii more quickly than the related species F. novicida does. Our studies point to a potentially overlooked interaction between F. philomiragia and Acanthamoeba. This relationship in the marine lifecycle of F. philomiragia may support the persistence of the bacterium in waterways and its ability to infect fish. An understanding of the persistence of this organism in aquatic systems through biofilm formation and its interaction with Acanthamoeba will be important in developing prevention strategies for this pathogen.

  12. 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

  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.

  14. A High-Throughput Pipeline for Designing Microarray-Based Pathogen Diagnostic Assays

    DTIC Science & Technology

    2008-04-10

    tularensis (1892819) Francisella philomiragia (2049711) 22 5 440% Burkholderia mallei (5835527) Burkholderia thailandensis (6723972) 21 7 300% Brucella ...836 Francisella tularensis 1469 2028 Burkholderia mallei 572 1146 Brucella melitensis 1352 7659Page 8 of 13 (page number not for citation purposes

  15. 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.

  16. Genus Francisella.

    DTIC Science & Technology

    1981-05-15

    ferrous sulfate per liter of medium. Incorporation of antibiotics (penicillin, polvmyxin B and cycloheximide) is required when clinical specimens...inoculated with infected tissue will initially support growth Of F. tullarensis. The addition of colistin , mystatin, lincomycin and trimethroprim to...100,000 units/ml; polymyxin B sulfate , 100,000 units/ml and cycloheximide, 0.1 mg/ml) is recommended when specimens are expected to contain abundant

  17. 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.                   

  18. The Atypical Occurrence of Two Biotin Protein Ligases in Francisella novicida Is Due to Distinct Roles in Virulence and Biotin Metabolism

    PubMed Central

    Feng, Youjun; Chin, Chui-Yoke; Chakravartty, Vandana; Gao, Rongsui; Crispell, Emily K.

    2015-01-01

    ABSTRACT The physiological function of biotin requires biotin protein ligase activity in order to attach the coenzyme to its cognate proteins, which are enzymes involved in central metabolism. The model intracellular pathogen Francisella novicida is unusual in that it encodes two putative biotin protein ligases rather than the usual single enzyme. F. novicida BirA has a ligase domain as well as an N-terminal DNA-binding regulatory domain, similar to the prototypical BirA protein in E. coli. However, the second ligase, which we name BplA, lacks the N-terminal DNA binding motif. It has been unclear why a bacterium would encode these two disparate biotin protein ligases, since F. novicida contains only a single biotinylated protein. In vivo complementation and enzyme assays demonstrated that BirA and BplA are both functional biotin protein ligases, but BplA is a much more efficient enzyme. BirA, but not BplA, regulated transcription of the biotin synthetic operon. Expression of bplA (but not birA) increased significantly during F. novicida infection of macrophages. BplA (but not BirA) was required for bacterial replication within macrophages as well as in mice. These data demonstrate that F. novicida has evolved two distinct enzymes with specific roles; BplA possesses the major ligase activity, whereas BirA acts to regulate and thereby likely prevent wasteful synthesis of biotin. During infection BplA seems primarily employed to maximize the efficiency of biotin utilization without limiting the expression of biotin biosynthetic genes, representing a novel adaptation strategy that may also be used by other intracellular pathogens. PMID:26060274

  19. Draft Genome Sequence of Francisella noatunensis subsp. orientalis STIR-GUS-F2f7, a Highly Virulent Strain Recovered from Diseased Red Nile Tilapia Farmed in Europe

    PubMed Central

    Larsson, Pär; Wehner, Stefanie; Bekaert, Michaël; Öhrman, Caroline; Metselaar, Matthijs; Thompson, Kimberly Dawn; Richards, Randolph Harvey; Penman, David James; Adams, Alexandra

    2017-01-01

    ABSTRACT A highly virulent strain of Francisella noatunensis subsp. orientalis, STIR-GUS-F2f7, was isolated from moribund red Nile tilapia (Oreochromis niloticus) farmed in Europe. In this communication, the complete genome sequencing of this bacterium is reported. PMID:28302784

  20. Attenuated Francisella asiatica iglC mutant induces protective immunity to francisellosis in tilapia.

    PubMed

    Soto, Esteban; Wiles, Judy; Elzer, Philip; Macaluso, Kevin; Hawke, John P

    2011-01-10

    Francisella asiatica is a Gram-negative, facultative intracellular bacteria that causes fish francisellosis. Fish francisellosis is a severe sub-acute to chronic granulomatous disease with high mortalities and high infectivity rates in cultured and wild fish. To date, there is no approved vaccine for this widespread emergent disease. The goal of this study was to characterize the efficacy of a defined F. asiatica mutant (ΔiglC) as a live attenuated vaccine against subsequent immersion challenge with the wild-type (WT) organism. In previous work, the ΔiglC was found to be attenuated upon intraperitoneal injection and immersion challenges. In vitro, the ΔiglC exhibited reduced growth in tilapia head-kidney derived macrophages, and was significantly attenuated (p<0.001) as demonstrated by cytopathogenic and apoptosis assays. In this study, the ΔiglC was tested to determine its ability to protect tilapia against challenge with high doses (lethal dose 80) of WT bacteria. Naïve tilapia vaccinated by immersion with a suspension of the ΔiglC and subsequently challenged with WT F. asiatica were protected (90% mean percent survival) from the lethal challenges. F. asiatica-specific antibodies produced in response to immunization with the ΔiglC were subsequently found to protect naïve tilapia against high-dose F. asiatica challenge in passive immunization experiments. Significant protection (p<0.001) was obtained when fish were passively immunized and challenged with 10(4) and 10(5)CFU/fish of WT F. asiatica; but not when challenged with 10(6)CFU/fish. This is the first report of a defined live attenuated strain providing protection against F. asiatica in fish.

  1. Prevalence of Francisella noatunensis subsp. orientalis in cultured tilapia on the island of Oahu, Hawaii.

    PubMed

    Soto, Esteban; McGovern-Hopkins, Kathleen; Klinger-Bowen, Ruth; Fox, Bradley K; Brock, James; Antonio, Nathene; Waal, Zelda van der; Rushton, Stephen; Mill, Aileen; Tamaru, Clyde S

    2013-06-01

    Francisellosis is an emergent disease in cultured and wild aquatic animals. The causative agent, Francisella noatunensis subsp. orientalis (Fno), is a gram-negative bacterium recognized as one of the most virulent pathogens of warmwater fish. The main objective of this project was to investigate the prevalence of Fno in cultured tilapia (specifically, Mozambique Tilapia Oreochromis mossambicus, Koilapia [also known as Wami Tilapia] O. hornorum, Blue Tilapia O. aureus, and Nile Tilapia O. niloticus hybrids) on the island of Oahu, Hawaii, using conventional and real-time PCR assays followed by statistical modeling to compare the different diagnostic methods and identify potential risk factors. During 2010 and 2012, 827 fish were collected from different geographical locations throughout the island of Oahu. Upon collection of fish, the water temperature in the rearing system and the length of individual fish were measured. Extraction of DNA from different tissues collected aseptically during necropsy served as a template for molecular diagnosis. High correlation between both molecular methods was observed. Moreover, the bacterium was isolated from infected tilapia on selective media and confirmed to be Fno utilizing a species-specific Taqman-based real-time PCR assay. Although a direct comparison of the prevalence of Fno between the different geographical areas was not possible, the results indicate a high prevalence of Fno DNA in cultured tilapia throughout the farm sites located on Oahu. Of the different tilapia species and hybrids currently cultured in Hawaii, Mozambique Tilapia were more susceptible to infection than Koilapia. Water temperature in the rearing systems and fish size also had a strong effect on the predicted level of infection, with fish held at lower temperatures and smaller fish being more susceptible to piscine francisellosis.

  2. Effects of temperature and salt concentration on Francisella noatunensis subsp. orientalis infections in Nile tilapia Oreochromis niloticus.

    PubMed

    Soto, Esteban; Abrams, Stephanie B; Revan, Floyd

    2012-11-19

    Little is known about the environmental conditions that allow Francisella noatunensis subsp. orientalis, a worldwide emergent bacterial fish pathogen, to colonize and infect wild and cultured fish. We evaluated the effect of temperature and salinity on the infectivity of F. noatunensis subsp. orientalis in Nile tilapia Oreochromis niloticus (L). Immersion challenges of tilapia with F. noatunensis subsp. orientalis at water temperatures of 25 and 30°C in both sea and fresh water were conducted for 14 d. Morbidity and mortality were recorded daily, and at the completion of the study, a quantitative assessment of the splenic bacterial burden was performed in surviving fish. Fish maintained at 25°C developed francisellosis and had considerably higher mortality and splenic bacterial concentrations compared to control fish and fish maintained at 30°C. Moreover, increasing the water temperature from 25 to 30°C prevented the development of clinical signs and mortality in Francisella-challenged fish. In conclusion, temperature significantly influenced the development of francisellosis in tilapia, whereas salinity had no effect. Our findings may be useful in the establishment of improved prophylactic practices and in the management of outbreaks of francisellosis in the aquaculture industry.

  3. Revisiting the Gram-Negative Lipoprotein Paradigm

    PubMed Central

    LoVullo, Eric D.; Wright, Lori F.; Isabella, Vincent; Huntley, Jason F.

    2015-01-01

    ABSTRACT The processing of lipoproteins (Lpps) in Gram-negative bacteria is generally considered an essential pathway. Mature lipoproteins in these bacteria are triacylated, with the final fatty acid addition performed by Lnt, an apolipoprotein N-acyltransferase. The mature lipoproteins are then sorted by the Lol system, with most Lpps inserted into the outer membrane (OM). We demonstrate here that the lnt gene is not essential to the Gram-negative pathogen Francisella tularensis subsp. tularensis strain Schu or to the live vaccine strain LVS. An LVS Δlnt mutant has a small-colony phenotype on sucrose medium and increased susceptibility to globomycin and rifampin. We provide data indicating that the OM lipoprotein Tul4A (LpnA) is diacylated but that it, and its paralog Tul4B (LpnB), still sort to the OM in the Δlnt mutant. We present a model in which the Lol sorting pathway of Francisella has a modified ABC transporter system that is capable of recognizing and sorting both triacylated and diacylated lipoproteins, and we show that this modified system is present in many other Gram-negative bacteria. We examined this model using Neisseria gonorrhoeae, which has the same Lol architecture as that of Francisella, and found that the lnt gene is not essential in this organism. This work suggests that Gram-negative bacteria fall into two groups, one in which full lipoprotein processing is essential and one in which the final acylation step is not essential, potentially due to the ability of the Lol sorting pathway in these bacteria to sort immature apolipoproteins to the OM. IMPORTANCE This paper describes the novel finding that the final stage in lipoprotein processing (normally considered an essential process) is not required by Francisella tularensis or Neisseria gonorrhoeae. The paper provides a potential reason for this and shows that it may be widespread in other Gram-negative bacteria. PMID:25755189

  4. In vitro and in vivo efficacy of florfenicol for treatment of Francisella asiatica infection in tilapia.

    PubMed

    Soto, Esteban; Endris, Richard G; Hawke, John P

    2010-11-01

    Francisella asiatica is a recently described, Gram-negative, facultative intracellular fish pathogen, known to be the causative agent of francisellosis in warm-water fish. Francisellosis outbreaks have increased in frequency among commercial aquaculture operations and have caused severe economic losses in every case reported. The lack of effective treatments for piscine francisellosis led us to investigate the potential efficacy of florfenicol for inhibition of F. asiatica in vitro and as an oral therapeutic agent in vivo. The MIC of florfenicol for F. asiatica, as determined by the broth dilution method, was 2 μg/ml, which indicates its potential efficacy as a therapeutic agent for treatment of francisellosis. The intracellular susceptibility of the bacterium to florfenicol in tilapia head kidney-derived macrophages (THKDM) was also investigated. Addition of florfenicol to the medium at 10 μg/ml was sufficient to significantly reduce bacterial loads in the THKDM in vitro. Cytotoxicity assays done in infected THKDM also demonstrated drug efficacy in vivo, as determined by lactate dehydrogenase (LDH) release. Levels of LDH released from infected THKDM were significantly lower in macrophages treated with florfenicol (P < 0.001) than in untreated cells. In medicated-feed trials, fish were fed 15 mg of florfenicol/kg of fish body weight for 10 days, and the feeding was initiated at either 1, 3, or 6 days postchallenge. Immersion challenges resulted in 30% mean percent survival in nontreated fish, and fish receiving medicated feed administered at 1 and 3 days postinfection showed higher mean percent survival (100% and 86.7%, respectively). A significant decrease (P < 0.001) in bacterial numbers (number of CFU/g of spleen tissue) was observed in treated groups compared to nontreated infected fish at both 1 and 3 days postchallenge. There were no differences in bacterial burden in the spleens between fish treated 6 days postchallenge and untreated controls. In

  5. Characterization and Vaccine Potential of Membrane Vesicles Produced by Francisella noatunensis sup. orientalis in an Adult Zebrafish Model.

    PubMed

    Lagos, Leidy; Tandberg, Julia I; Repnik, Urska; Boysen, Preben; Ropstad, Erik; Varkey, Deepa; Paulsen, Ian T; Winther-Larsen, Hanne C

    2017-03-22

    Vaccine development against extracellular bacteria has been important for the sustainability of the aquaculture industry. In contrast, infections with intracellular pathogens remain largely an unresolved problem. Francisella noatunensis subspecies orientalis (Fno) are Gram-negative, facultative intracellular bacteria that cause the disease francisellosis in fish. Francisellosis is commonly characterized as a chronic granulomatous disease with high morbidity and can result in high mortality depending on the host. In this study, we explored the potential of bacterial membrane vesicles (MVs) as a vaccine agent against Fno Bacterial MVs are spherical structures naturally released from the membrane of bacteria and are often enriched with selected bacterial components such as toxins and signaling molecules. In the present work, MVs were isolated from broth-cultured Fno and proteomic analysis by mass spectrometry revealed that MVs contained a variety of immunogenic factors, including the intracellular growth proteins IglC and IglB, known to be part of a Francisella Pathogenicity Island (FPI), as well as outer membrane protein OmpA, chaperonin GroEL and chaperone ClpB. By using flow cytometry and electron microscopy, we observed that Fno mainly infect myelomonocytic cells, both in vivo and in vitro. Immunization with MVs isolated from Fno protect zebrafish from subsequent challenge with a lethal dose of Fno To determine if MVs induce a typical acute inflammatory response, mRNA expression level were assessed by quantitative real-time PCR. The expression of tnfa, il1b, ifng, as well as mhcii, mpeg1.1 and ighm was upregulated, thus confirming the immunogenic properties of Fno derived MVs.

  6. 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.

  7. 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

  8. 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...

  9. 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...

  10. 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).

  11. Outbreaks and genetic diversity of Francisella noatunensis subsp orientalis isolated from farm-raised Nile tilapia (Oreochromis niloticus) in Brazil.

    PubMed

    Leal, C A G; Tavares, G C; Figueiredo, H C P

    2014-07-25

    Francisella noatunensis subsp orientalis (FNO) is an emerging pathogen of warm water tilapia in a number of different countries. The disease caused by this bacterium in fish is characterized by a systemic granulomatous infection that causes high mortality rates during outbreaks. FNO has been previously described in Asia, Europe, and Central and North America. Its occurrence in South America has never been described. Since 2012, outbreaks of a granulomatous disease have been recorded in cage farms of Nile tilapia (Oreochromis niloticus L.) in Brazil. The current study aimed to identify the etiologic agent of recent francisellosis outbreaks at Brazilian tilapia farms, and to characterize the genetic diversity of the pathogen from farms with distinct geographic origins and without epidemiological connections. Bacteriological analysis of 44 diseased Nile tilapia collected from five cage farms in Brazil was performed during 2012 and 2013. The farms were in different locations and had no recent history of animal or biological material transport between each other. Sixty-two FNO isolates were identified on the basis of FNO-specific qPCR. The main predisposing factors for the occurrence of outbreaks on Brazilian farms were lower water temperature (<22°C) and life stage of fish, affecting mainly fry, fingerlings and young adults (live weight <100 g). The genetic diversity of the Brazilian FNO isolates was evaluated using repetitive extragenic palindromic-PCR. The isolates from different origins were shown to be clonally related. This is the first report of the occurrence and genetic diversity of FNO in South America.

  12. Genes

    MedlinePlus

    ... Search Search MedlinePlus GO GO About MedlinePlus Site Map FAQs Customer Support Health Topics Drugs & Supplements Videos & Tools Español You Are Here: Home → Medical Encyclopedia → Genes URL of this page: //medlineplus.gov/ency/article/ ...

  13. Outer membrane vesicles displaying engineered glycotopes elicit protective antibodies

    PubMed Central

    Valentine, Jenny L.; Huang, Chung-Jr; Endicott, Christine E.; Moeller, Tyler D.; Rasmussen, Jed A.; Fletcher, Joshua R.; Boll, Joseph M.; Rosenthal, Joseph A.; Dobruchowska, Justyna; Wang, Zhirui; Heiss, Christian; Azadi, Parastoo; Putnam, David; Trent, M. Stephen; Jones, Bradley D.; DeLisa, Matthew P.

    2016-01-01

    The O-antigen polysaccharide (O-PS) component of lipopolysaccharides on the surface of gram-negative bacteria is both a virulence factor and a B-cell antigen. Antibodies elicited by O-PS often confer protection against infection; therefore, O-PS glycoconjugate vaccines have proven useful against a number of different pathogenic bacteria. However, conventional methods for natural extraction or chemical synthesis of O-PS are technically demanding, inefficient, and expensive. Here, we describe an alternative methodology for producing glycoconjugate vaccines whereby recombinant O-PS biosynthesis is coordinated with vesiculation in laboratory strains of Escherichia coli to yield glycosylated outer membrane vesicles (glycOMVs) decorated with pathogen-mimetic glycotopes. Using this approach, glycOMVs corresponding to eight different pathogenic bacteria were generated. For example, expression of a 17-kb O-PS gene cluster from the highly virulent Francisella tularensis subsp. tularensis (type A) strain Schu S4 in hypervesiculating E. coli cells yielded glycOMVs that displayed F. tularensis O-PS. Immunization of BALB/c mice with glycOMVs elicited significant titers of O-PS–specific serum IgG antibodies as well as vaginal and bronchoalveolar IgA antibodies. Importantly, glycOMVs significantly prolonged survival upon subsequent challenge with F. tularensis Schu S4 and provided complete protection against challenge with two different F. tularensis subsp. holarctica (type B) live vaccine strains, thereby demonstrating the vaccine potential of glycOMVs. Given the ease with which recombinant glycotopes can be expressed on OMVs, the strategy described here could be readily adapted for developing vaccines against many other bacterial pathogens. PMID:27274048

  14. Francisella–Arthropod Vector Interaction and its Role in Patho-Adaptation to Infect Mammals

    PubMed Central

    Akimana, Christine; Kwaik, Yousef Abu

    2011-01-01

    Francisella tularensis is a Gram-negative, intracellular, zoonotic bacterium, and is the causative agent of tularemia with a broad host range. Arthropods such as ticks, mosquitoes, and flies maintain F. tularensis in nature by transmitting the bacteria among small mammals. While the tick is largely believed to be a biological vector of F. tularensis, transmission by mosquitoes and flies is largely believed to be mechanical on the mouthpart through interrupted feedings. However, the mechanism of infection of the vectors by F. tularensis is not well understood. Since F. tularensis has not been localized in the salivary gland of the primary human biting ticks, it is thought that bacterial transmission by ticks is through mechanical inoculation of tick feces containing F. tularensis into the skin wound. Drosophila melanogaster is an established good arthropod model for arthropod vectors of tularemia, where F. tularensis infects hemocytes, and is found in hemolymph, as seen in ticks. In addition, phagosome biogenesis and robust intracellular proliferation of F. tularensis in arthropod-derived cells are similar to that in mammalian macrophages. Furthermore, bacterial factors required for infectivity of mammals are often required for infectivity of the fly by F. tularensis. Several host factors that contribute to F. tularensis intracellular pathogenesis in D. melanogaster have been identified, and F. tularensis targets some of the evolutionarily conserved eukaryotic processes to enable intracellular survival and proliferation in evolutionarily distant hosts. PMID:21687425

  15. 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.

  16. Agricultural Bioterrorism What Challenges and Actions Remain

    DTIC Science & Technology

    2006-03-10

    Midwest annually produces more than 80 million cattle, hogs, sheep, goats and bison and is more economically exposed to the threat of agroterrorism...pestis (plaque), Francisella tularensis (tularemia), Coxiella burnetii (Q fever), Venezuelan equine encephalitis virus, Brucella suis ( brucellosis ), and

  17. Operational Art and the Incident Command System: Public Health’s Bridge in Bioterrorism Preparedness and Response

    DTIC Science & Technology

    2007-11-02

    from botulinum toxins), Francisella tularensis (Tularemia), and Filoviruses and Arenaviruses like Ebola virus and Lassa virus (Viral Hemorrhagic fevers...an extremely fragile virus , but anthrax spores can “shelter in place” indefinitely). Virulence (lethality) refers to the agent’s ability to produce

  18. Feasibility of Screening for Antibiotic Resistance-Part II

    DTIC Science & Technology

    2005-08-01

    however, that biological (warfare) agents with multiple resistance characteristics can be encountered. TNO report IDV2 2005-A050 12 /38 2 Materials and...Gram-negative bacteria: TNO report [ DV2 2005-A050 25/38 Brucella melitensis, Brucella suis, Citrobacterfreundii, Coxiella burnetii, Enterobacter ... aerogenes , Erwinia carotovora, Escherichia coli K12, Francisella tularensis, Klebsiella pneumoniae, Neisseria gonorrhoeae, Providencia stuartii

  19. Unmanned Aerial Vehicles and Weapons of Mass Destruction: A Lethal Combination?

    DTIC Science & Technology

    1997-08-01

    90 Tularemia Francisella 1-10 days 5-20 Tularensis Cholera Vibrio Cholerae 2-5 days 25-50 Venezuelan VEE Virus 2-5 days ə Equine Encephalitis Q Fever...Storm. 1995. 69 pages. FELMAN, Marc D., Lt Col, USAF (T-2). The Military/Media Clash and the New Principle of War: Media Spin. 1993. 42 pages. FISCHER

  20. 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

  1. Report of the Working Group on Strengthening the Biosecurity of the United States

    DTIC Science & Technology

    2009-10-01

    Coccidioides posadasii/Coccidioides immitis Coxiella burnetii Francisella tularensis Rickettsia prowazekii Rickettsia rickettsii Yersinia...Biosafety Level, Agent type ( bacteria , virus, etc.) 4. Storage location (building, room number, freezer number) 5. Storage conditions (refrigerator...16, 2008. APPENDIX 1-C 83 HHS SELECT AGENTS AND TOXINS Bacteria Viruses Botulinum neurotoxin producing species of Clostridium

  2. Possible Impacts of Major Counter Terrorism Security Actions on Research, Development, and Higher Education

    DTIC Science & Technology

    2002-04-08

    botulinum, Francisella tularensis, Yersinia pestis; Rickettsiae : Coxiella burnetii, Rickettsia prowazekii, Rickettsia rickettsii ; Fungi: Coccidioides...virus (smallpox virus), Venezuelan equine encephalitis virus, Viruses causing hantavirus pulmonary syndrome, Yellow fever virus; Bacteria : Bacillus...select” agents (viruses, bacteria , fungi, and toxins, including genetically modified or genetic material from those listed agents),61 as well as

  3. Biological Warfare Agents, Toxins, Vectors and Pests as Biological Terrorism Agents

    DTIC Science & Technology

    2003-07-01

    virus Omsk fever virus Human pathogens ( bacteria , rickettsiae , protozoa and fungi) as biological terrorism agents: Bacteria / Rickettsia 1...Bacillus anthracis 2. Yersinia pestis 3. Francisella tularensis 4. Rickettsia prowazekii 5. Rickettsia rickettsii 6. Bulkholderia (Pseudomonas) mallei...assessment according to criteria for selecting pathogens as biological terrorism agents. Table 1b. Human pathogens ( bacteria , rickettsiae , protozoa

  4. Targeted next generation sequencing for the detection of ciprofloxacin resistance markers using molecular inversion probes

    DTIC Science & Technology

    2016-07-06

    detection of genetic variants known to confer ciprofloxacin resistance in Bacillus anthracis, Yersinia pestis, and Francisella tularensis. Sequencing...results demonstrate MIPs capture and amplify targeted regions of interest at significant levels of coverage. Depending on the genetic variant...multiple downstream molecular assays for the detection of targeted genetic regions. TR-16-130 DISTRIBUTION STATEMENT A: Approved for public release

  5. Genes and Gene Therapy

    MedlinePlus

    ... correctly, a child can have a genetic disorder. Gene therapy is an experimental technique that uses genes to ... or prevent disease. The most common form of gene therapy involves inserting a normal gene to replace an ...

  6. Comparison of Radii Sets, Entropy, QM Methods, and Sampling on MM-PBSA, MM-GBSA, and QM/MM-GBSA Ligand Binding Energies of F. tularensis Enoyl-ACP Reductase (FabI)

    PubMed Central

    Su, Pin-Chih; Tsai, Cheng-Chieh; Mehboob, Shahila; Hevener, Kirk E.; Johnson, Michael E.

    2015-01-01

    To validate a method for predicting the binding affinities of FabI inhibitors, three implicit solvent methods, MM-PBSA, MM-GBSA and QM/MM GBSA were carefully compared using sixteen benzimidazole inhibitors in complex with F. tularensis FabI. The data suggests that the prediction results are sensitive to radii sets, GB methods, QM Hamiltonians, sampling protocols, and simulation length, if only one simulation trajectory is used for each ligand. In this case, QM/MM-GBSA using 6 ns MD simulation trajectories together with GBneck2, PM3, and the mbondi2 radii set, generate the closest agreement with experimental values (r2= 0.88). However, if the three implicit solvent methods are averaged from six 1 ns MD simulations for each ligand (called “multiple independent sampling”), the prediction results are relatively insensitive to all the tested parameters. Moreover, MM/GBSA together with GBHCT and mbondi, using 600 frames extracted evenly from six 0.25 ns MD simulations, can also provide accurate prediction to experimental values (r2 = 0.84). Therefore, the multiple independent sampling method can be more efficient than a single, long simulation method. Since future scaffold expansions may significantly change the benzimidazole's physiochemical properties (charges, etc.) and possibly binding modes, which may affect the sensitivities of various parameters, the relatively insensitive “multiple independent sampling method” may avoid the need of an entirely new validation study. Moreover, due to large fluctuating entropy values, (QM/)MM-P(G)BSA were limited to inhibitors’ relative affinity prediction, but not the absolute affinity. The developed protocol will support an ongoing benzimidazole lead optimization program. PMID:26216222

  7. Investigating an airborne tularemia outbreak, Germany.

    PubMed

    Hauri, Anja M; Hofstetter, Iris; Seibold, Erik; Kaysser, Philip; Eckert, Juergen; Neubauer, Heinrich; Splettstoesser, Wolf D

    2010-02-01

    In November 2005, an outbreak of tularemia occurred among 39 participants in a hare hunt in Hesse, Germany. Previously reported tularemia outbreaks in Germany dated back to the 1950s. We conducted a retrospective cohort study among participants and investigated the environment to identify risk factors for infection. Ten participants had serologic evidence of acute Francisella tularensis infection; 1 other participant died before laboratory confirmation was obtained. Presence within 5 meters of the place where disemboweled hares were rinsed with a water hose was the risk factor most strongly associated with infection (risk ratio 22.1; 95% confidence interval 13.2-154.3). Swabs taken at the game chamber and water samples were PCR negative for F. tularensis. Eleven of 14 hare parts showed low-level concentrations of F. tularensis, compatible with cross-contamination. More than half of case-patients may have acquired infection through inhalation of aerosolized droplets containing F. tularensis generated during rinsing of infected hares.

  8. Cas9-dependent endogenous gene regulation is required for bacterial virulence.

    PubMed

    Sampson, Timothy R; Weiss, David S

    2013-12-01

    CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems are known to mediate bacterial defence against foreign nucleic acids. We recently demonstrated a non-canonical role for a CRISPR-Cas system in controlling endogenous gene expression, which had not previously been appreciated. In the present article, we describe the studies that led to this discovery, beginning with an unbiased genome-wide screen to identify virulence genes in the intracellular pathogen Francisella novicida. A gene annotated as encoding a hypothetical protein, but which we now know encodes the Cas protein Cas9, was identified as one of the most critical to the ability of F. novicida to replicate and survive during murine infection. Subsequent studies revealed a role for this protein in evasion of the host innate immune response. Specifically, Cas9 represses the expression of a BLP (bacterial lipoprotein) that could otherwise be recognized by TLR2 (Toll-like receptor 2), a host protein involved in initiating an antibacterial pro-inflammatory response. By repressing BLP levels, Cas9 mediates evasion of TLR2, promoting bacterial virulence. Finally, we described the molecular mechanism by which Cas9 functions in complex with two small RNAs to target the mRNA encoding the BLP for degradation. This work greatly broadened the paradigm for CRISPR-Cas function, highlighting a role in gene regulation that could be conserved in numerous bacteria, and elucidating its integral contribution to bacterial pathogenesis.

  9. Prevalence and distribution of soil-borne zoonotic pathogens in Lahore district of Pakistan

    PubMed Central

    Shabbir, Muhammad Z.; Jamil, Tariq; Ali, Asad A.; Ahmad, Arfan; Naeem, Muhammad; Chaudhary, Muhammad H.; Bilal, Muhammad; Ali, Muhammad A.; Muhammad, Khushi; Yaqub, Tahir; Bano, Asghari; Mirza, Ali I.; Shabbir, Muhammad A. B.; McVey, Walter R.; Patel, Ketan; Francesconi, Stephen; Jayarao, Bhushan M.; Rabbani, Masood

    2015-01-01

    A multidisciplinary, collaborative project was conducted to determine the prevalence and distribution of soil-borne zoonotic pathogens in Lahore district of Pakistan and ascertain its Public Health Significance. Using a grid-based sampling strategy, soil samples (n = 145) were collected from villages (n = 29, 5 samples/village) and examined for Bacillus anthracis, Burkholderia mallei/pseudomallei, Coxiella burnetii, Francisella tularensis, and Yersinia pestis using real time PCR assays. Chemical analysis of soil samples was also performed on these samples. The relationship between soil composition and absence or presence of the pathogen, and seven risk factors was evaluated. DNA of B. anthracis (CapB), B. mallei/pseudomallei (chromosomal gene), C. burnetii (IS1111, transposase gene), and F. tularensis (lipoprotein/outer membrane protein) was detected in 9.6, 1.4, 4.8, and 13.1% of soil samples, respectively. None of the samples were positive for protective antigen plasmid (PA) of B. anthracis and Y. pestis (plasminogen activating factor, pPla gene). The prevalence of B. anthracis (CapB) was found to be associated with organic matter, magnesium (Mg), copper (Cu), chromium (Cr), manganese (Mn), cobalt (Co), cadmium (Cd), sodium (Na), ferrous (Fe), calcium (Ca), and potassium (K). Phosphorous (P) was found to be associated with prevalence of F. tularensis while it were Mg, Co, Na, Fe, Ca, and K for C. burnetii. The odds of detecting DNA of F. tularensis were 2.7, 4.1, and 2.7 higher when soil sample sites were >1 km from animal markets, >500 m from vehicular traffic roads and animal density of < 1000 animals, respectively. While the odds of detecting DNA of C. burnetii was 32, 11.8, and 5.9 higher when soil sample sites were >500 m from vehicular traffic roads, presence of ground cover and animal density of < 1000 animals, respectively. In conclusion, the distribution pattern of the soil-borne pathogens in and around the areas of Lahore district puts both human and

  10. Seroepidemiologic study of three zoonoses (leptospirosis, Q fever, and tularemia) among trappers in Québec, Canada.

    PubMed Central

    Lévesque, B; De Serres, G; Higgins, R; D'Halewyn, M A; Artsob, H; Grondin, J; Major, M; Garvie, M; Duval, B

    1995-01-01

    This study was undertaken to evaluate the prevalence of antibodies against Francisella tularensis, Coxiella burnetii, and certain serovars of Leptospira interrogans among trappers in Québec, Canada. Muskrat trapping was identified as a risk factor for F. tularensis infection, whereas having a cat at home apparently protected trappers against infection by L. interrogans. High percentages of control sera were positive for antibodies against C. burnetii (15%) and L. interrogans (5%), most frequently serovar bratislava. This is the first report of human infection by serovar bratislava in North America. PMID:7583933

  11. Toward an Understanding of the Perpetuation of the Agent of Tularemia

    PubMed Central

    Telford, Sam R.; Goethert, Heidi K.

    2011-01-01

    The epidemiology of tularemia has influenced, perhaps incorrectly skewed, our views on the ecology of the agent of tularemia. In particular, the central role of lagomorphs needs to be reexamined. Diverse observations, some incidental, and some that are more generally reproducible, have not been synthesized so that the critical elements of the perpetuation of Francisella tularensis can be identified. Developing a quantitative model of the basic reproduction number of F. tularensis may require separate treatments for Type A and Type B given the fundamental differences in their ecology. PMID:21687803

  12. A bioinformatic approach to understanding antibiotic resistance in intracellular bacteria through whole genome analysis.

    PubMed

    Biswas, Silpak; Raoult, Didier; Rolain, Jean-Marc

    2008-09-01

    Intracellular bacteria survive within eukaryotic host cells and are difficult to kill with certain antibiotics. As a result, antibiotic resistance in intracellular bacteria is becoming commonplace in healthcare institutions. Owing to the lack of methods available for transforming these bacteria, we evaluated the mechanisms of resistance using molecular methods and in silico genome analysis. The objective of this review was to understand the molecular mechanisms of antibiotic resistance through in silico comparisons of the genomes of obligate and facultative intracellular bacteria. The available data on in vitro mutants reported for intracellular bacteria were also reviewed. These genomic data were analysed to find natural mutations in known target genes involved in antibiotic resistance and to look for the presence or absence of different resistance determinants. Our analysis revealed the presence of tetracycline resistance protein (Tet) in Bartonella quintana, Francisella tularensis and Brucella ovis; moreover, most of the Francisella strains possessed the blaA gene, AmpG protein and metallo-beta-lactamase family protein. The presence or absence of folP (dihydropteroate synthase) and folA (dihydrofolate reductase) genes in the genome could explain natural resistance to co-trimoxazole. Finally, multiple genes encoding different efflux pumps were studied. This in silico approach was an effective method for understanding the mechanisms of antibiotic resistance in intracellular bacteria. The whole genome sequence analysis will help to predict several important phenotypic characteristics, in particular resistance to different antibiotics. In the future, stable mutants should be obtained through transformation methods in order to demonstrate experimentally the determinants of resistance in intracellular bacteria.

  13. CGUG: in silico proteome and genome parsing tool for the determination of "core" and unique genes in the analysis of genomes up to ca. 1.9 Mb

    PubMed Central

    Mahadevan, Padmanabhan; King, John F; Seto, Donald

    2009-01-01

    Background Viruses and small-genome bacteria (~2 megabases and smaller) comprise a considerable population in the biosphere and are of interest to many researchers. These genomes are now sequenced at an unprecedented rate and require complementary computational tools to analyze. "CoreGenesUniqueGenes" (CGUG) is an in silico genome data mining tool that determines a "core" set of genes from two to five organisms with genomes in this size range. Core and unique genes may reflect similar niches and needs, and may be used in classifying organisms. Findings CGUG is available at as a web-based on-the-fly tool that performs iterative BLASTP analyses using a reference genome and up to four query genomes to provide a table of genes common to these genomes. The result is an in silico display of genomes and their proteomes, allowing for further analysis. CGUG can be used for "genome annotation by homology", as demonstrated with Chlamydophila and Francisella genomes. Conclusion CGUG is used to reanalyze the ICTV-based classifications of bacteriophages, to reconfirm long-standing relationships and to explore new classifications. These genomes have been problematic in the past, due largely to horizontal gene transfers. CGUG is validated as a tool for reannotating small genome bacteria using more up-to-date annotations by similarity or homology. These serve as an entry point for wet-bench experiments to confirm the functions of these "hypothetical" and "unknown" proteins. PMID:19706165

  14. Studying Genes

    MedlinePlus

    ... Area What are genes? Genes are sections of DNA that contain instructions for making the molecules—many ... material in an organism. This includes genes and DNA elements that control the activity of genes. Does ...

  15. Tularaemia in southwest Germany: Three cases of tick-borne transmission.

    PubMed

    Boone, I; Hassler, D; Nguyen, T; Splettstoesser, W D; Wagner-Wiening, C; Pfaff, G

    2015-07-01

    Tularaemia, caused by Francisella tularensis, is an endemic zoonosis frequently occurring in southwest Germany. Since 2005 there is an increase in the number of reported cases of tularaemia in Germany. We report on two cases of ulceroglandular tularaemia and one case of glandular tularaemia that occurred in the summer of 2012 and 2013 in two counties in the Federal State of Baden-Wuerttemberg. Bacteria were transmitted through tick bites, which to date has only rarely been reported in Germany. Inadequate treatment of the patients and an aggravation of clinical symptoms were caused by a delay between disease onset and the detection of the pathogen. Although contact to or consumption of infected hares are the most often reported transmission routes of tularaemia in Germany, tick-bites should also be taken into account. Health professionals should include Francisella tularensis in the differential diagnosis of patients with fever and/or ulcerative lymphadenopathy following a tick bite.

  16. 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.

  17. Unique Structural Modifications Are Present in the Lipopolysaccharide from Colistin-Resistant Strains of Acinetobacter baumannii

    DTIC Science & Technology

    2013-10-01

    Glucosamine hydrochlo- ride, N-acetylglucosamine, 2-amino-2-methyl-1, and 3-propanediol were from Sigma. 2-Galactoseamine hydrochloride was from...ethanolamine, aminoarabinose, and glucosamine to lipid A modulates CAMP resistance (Fig. 1) (7–9). A previous report shows that a colistin-resistant...ethanolamine, aminoarabinose, and glucosamine , alters resistance to CAMPs. (A) Salmonella Typhimurium; (B) Francisella tularensis. Pelletier et al. 4832

  18. Aptamer Selection Express: A Novel Method for Rapid Single-Step Selection and Sensing of Aptamers

    DTIC Science & Technology

    2008-12-01

    This process has been used to select aptamers against different types of targets ( Bacillus anthracis spores, Bacillus thuringiensis spores, MS-2...aptamers of Bacillus anthracis (Ba), Shiga toxin, botulinum neurotoxin (BoNT), and Francisella tularensis bacteria (all selected by SELEX) have been...preparation procedure for DNA capture element sensing system. quantum dots were used here for the DNA capture element sys- tems of Bacillus anthracis

  19. 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

  20. Entry of Yersinia Pestis into the Viable but Nonculturable State in a Low-Temperature Tap Water Microcosm

    DTIC Science & Technology

    2011-03-16

    to enter the VBNC state [17,18,19,20]. In addition, many other bacterial pathogens, including Francisella tularensis, Vibrio cholerae , and Escherichia... Vibrio cholerae , and Escherichia coli enter the VBNC state [12,21]. In addition, the two closest pathogenic relatives of Y. pestis, namely Y...2001) Viability of the Nonculturable Vibrio cholerae O1 and O139. Systematic and Applied Microbiology 24: 331–341. 38. Jang KI, Kim MG, Ha SD, Kim KS

  1. Demonstration of a Robot-Based Raman Spectroscopic Detector for the Identification of CBE Threat Agents

    DTIC Science & Technology

    2006-11-01

    Brucella abortus Bacillus anthracis (LD Viable) Raman...Yersinia pestis Burkholderia mallei Brucella abortus Bacillus anthracis (LD Viable) Raman Shift (cm-1 ) O f f s e t I n t e n s i t y © ChemImage...Hierarchical Cluster Analysis 0 0.5 1 1.5 2 2.5 3 Burkholderia mallei Ricin Brucella abortus Francisella tularensis Yersina pestis Bacillus anthracis

  2. Enzyme-Linked Immunosorbant Assays for Identification of Biological Agents in Sample Unknowns: NATO SIBCA. Exercise 5

    DTIC Science & Technology

    2004-12-01

    des organismes irradi6 par des rayons gamma Bacillus anthracis, Yersinia pestis. Brucella melitensis , Francisella tularensis, Vibrio cholerae... Brucella melitensis , VEE virus, Burkholderia mallei, Vaccinia virus, and Yellow fever virus were used to screen SIBCA samples for homologous agents...cholerae, Brucella melitensis , le virus EEV, Burkholderia mnallei, le virus de la vaccine et le virus de la fi~vre jaune ont k6 utilis~es pour analyser les

  3. A More Flexible Lipoprotein Sorting Pathway

    PubMed Central

    Chahales, Peter

    2015-01-01

    Lipoprotein biogenesis in Gram-negative bacteria occurs by a conserved pathway, each step of which is considered essential. In contrast to this model, LoVullo and colleagues demonstrate that the N-acyl transferase Lnt is not required in Francisella tularensis or Neisseria gonorrhoeae. This suggests the existence of a more flexible lipoprotein pathway, likely due to a modified Lol transporter complex, and raises the possibility that pathogens may regulate lipoprotein processing to modulate interactions with the host. PMID:25755190

  4. Oligonucleotide Fingerprint Identification for Microarray-Based Pathogen Diagnostic Assays

    DTIC Science & Technology

    2006-10-01

    Yersinia species), or a set of organisms that may or may not have any phylogenetic relationship (e.g. to detect a viral or a bacterial family) and (2) the... phylogenetic tree or published data. The target and neighbor will contain common DNA sequences, which, obviously, cannot be used as DNA fingerprints. DNA... Yersinia pestis and 250 fingerprints for Francisella tularensis are presented. Availability: The implemented algorithm is available upon request

  5. Vaccines, Pharmaceutical Products, and Bioterrorism: Challenges for the U.S. Food and Drug Administration

    DTIC Science & Technology

    1999-08-01

    plague (Yersinia pestis), tularemia (Francisella tularensis), brucellosis ( Brucella abortus, B. melitensis , B. suis, B. canis), Q fever (Coxiella...Special Issue 20011029 090 Vaccines, Pharmaceutical Products, and Bioterrorism: Challenges for the U.S. Food and Drug Administration Kathryn C...Zoon U.S. Food and Drug Administration, Rockville, Maryland, USA In regards to bioterrorism, the goal of the U.S. Food and Drug Administration (FDA

  6. Tularaemia in a brown hare (Lepus europaeus) in 2013: first case in the Netherlands in 60 years.

    PubMed

    Rijks, J M; Kik, M; Koene, M G; Engelsma, M Y; van Tulden, P; Montizaan, M G; Oomen, T; Spierenburg, M A; Ijzer, J; van der Giessen, J W; Gröne, A; Roest, H J

    2013-12-05

    Tularaemia has not been reported in Dutch wildlife since 1953. To enhance detection, as of July 2011, brown hares (Lepus europaeus) submitted for postmortem examination in the context of non-targeted wildlife disease surveillance, were routinely tested for tularaemia by polymerase chain reaction (PCR). Francisella tularensis subspecies holarctica infection was confirmed in a hare submitted in May 2013. The case occurred in Limburg, near the site of the 1953 case. Further surveillance should clarify the significance of this finding.

  7. A sensitive liquid chromatography/mass spectrometry-based assay for quantitation of amino-containing moieties in lipid A

    PubMed Central

    Kalhorn, Thomas F.; Kiavand, Anahita; Cohen, Ilana E.; Nelson, Amanda K.; Ernst, Robert K.

    2009-01-01

    A novel sensitive liquid chromatography/mass spectrometry-based assay was developed for the quantitation of aminosugars, including 2-amino-2-deoxyglucose (glucosamine, GlcN), 2-amino-2-deoxygalactose (galactosamine, GalN), and 4-amino-4-deoxyarabinose (aminoarabinose, AraN), and for ethanolamine (EtN), present in lipid A. This assay enables the identification and quantitation of all amino-containing moieties present in lipopolysaccharide or lipid A from a single sample. The method was applied to the analysis of lipid A (endotoxin) isolated from a variety of biosynthetic and regulatory mutants of Salmonella enterica serovar Typhimurium and Francisella tularensis subspecies novicida. Lipid A is treated with trifluoroacetic acid to liberate and deacetylate individual aminosugars and mass tagged with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, which reacts with primary and secondary amines. The derivatives are separated using reversed-phase chromatography and analyzed using a single quadrupole mass spectrometer to detect quantities as small as 20 fmol. GalN was detected only in Francisella and AraN only in Salmonella, while GlcN was detected in lipid A samples from both species of bacteria. Additionally, we found an approximately 10-fold increase in the level of AraN in lipid A isolated from Salmonella grown in magnesium-limited versus magnesium-replete conditions. Salmonella with defined mutations in lipid A synthesis and regulatory genes were used to further validate the assay. Salmonella with null mutations in the phoP, pmrE, and prmF genes were unable to add AraN to their lipid A, while Salmonella with constitutively active phoP and pmrA exhibited AraN modification of lipid A even in the normally repressive magnesium-replete growth condition. The described assay produces excellent repeatability and reproducibility for the detection of amino-containing moieties in lipid A from a variety of bacterial sources. PMID:19130491

  8. Tularaemia seroprevalence of captured and wild animals in Germany: the fox (Vulpes vulpes) as a biological indicator.

    PubMed

    Kuehn, A; Schulze, C; Kutzer, P; Probst, C; Hlinak, A; Ochs, A; Grunow, R

    2013-04-01

    A total of 2475 animals from Germany, both captive and wild, were tested for antibodies against Francisella tularensis to obtain more knowledge about the presence of this pathogen in Germany. An indirect and a competitive ELISA served as screening methods, positive and inconclusive samples were confirmed by Western blot. Of the zoo animals sampled between 1992 and 2007 (n = 1122), three (0·3%) were seropositive. The seroconversion of a hippopotamus in Berlin Zoo was documented. From 1353 serum samples of wild foxes (Vulpes vulpes), raccoon dogs (Nyctereutes procyonoides) and wild boars (Sus scrofa), collected between 2005 and 2009 in the federal state of Brandenburg (surrounding Berlin), a total of 101 (7·5%) tested positive for antibodies to F. tularensis lipopolysaccharide. Our results indicate a higher seroprevalence of F. tularensis in wildlife in eastern Germany than commonly assumed. Furthermore, we found foxes and raccoon dogs to be biological indicators for tularaemia.

  9. Landscape Epidemiology of Tularemia Outbreaks in Sweden

    PubMed Central

    Svensson, Kerstin; Bäck, Erik; Eliasson, Henrik; Berglund, Lennart; Granberg, Malin; Karlsson, Linda; Larsson, Pär; Forsman, Mats

    2009-01-01

    Summer outbreaks of tularemia that occurred from 1995 through 2005 in 2 locations in Sweden affected 441 persons. We performed an epidemiologic investigation of these outbreaks using a novel strategy, involving high-resolution genotyping of Francisella tularensis isolates obtained from 136 patients (using 18 genetic markers developed from 6 F. tularensis genome sequences) and interviews with the patients. Strong spatial associations were found between F. tularensis subpopulations and the places of disease transmission; infection by some subpopulations occurred within areas as small as 2 km2, indicating unidentified environmental point sources of tularemia. In both locations, disease clusters were associated with recreational areas beside water, and genetic subpopulations were present throughout the tularemia season and persisted over years. High-resolution genotyping in combination with patients’ statements about geographic places of disease transmission provided valuable indications of likely sources of infection and the causal genotypes during these tularemia outbreaks. PMID:19961673

  10. Septic tularemia in 2 cottontop tamarins(Sanguinus oedipus).

    PubMed

    Guthrie, Amanda L; Gailbreath, Katherine L; Cienava, Elizabeth A; Bradway, Daniel S; Munoz Gutierrez, Juan F

    2012-06-01

    Two captive cottontop tamarins (Sanguinus oedipus) died within 5 d of each other from systemic infection by Francisella tularensis (tularemia). One tamarin experienced mild clinical signs, including malaise, anorexia, and a mucoid nasal discharge for 4 d before death, whereas the other experienced a more rapid progression of disease that lasted less than 24 h. Differential diagnoses included gram-negative septicemia by an organism such as Escherichia coli, Salmonella, or Yersinia; protozoal infection such as Toxoplasma gondii or an acute viral infection such as lymphocytic choriomeningitis. F. tularensis infection was identified by F. tularensis-specific PCR in both primates. Possible sources of infection include aerosol, biting arthropod vectors, and transmission via a rodent reservoir. This case report highlights the importance of tularemia as a differential diagnosis in acute febrile illness in captive nonhuman primates.

  11. [Epidemiological evaluation of a rapidly-prevented tularemia outbreak in Canakkale province, Turkey].

    PubMed

    Tatman Otkun, Müşerref; Akçalı, Alper; Karadenizli, Aynur; Ozbey, Nilgün; Gazel, Deniz; Sener, Alper; Güçlü, Oğuz; Tanrıöver, Arzu; Otkun, Metin

    2011-01-01

    Tularemia is a disease caused by Francisella tularensis and widely seen at northern hemisphere of the world. In Turkey, oropharyngeal infections caused by a less virulent serotype F.tularensis subsp. Holarctica are more prevalent. The aim of this study was to present the results of an epidemiological research performed after the detection of tularemia cases from Biga county of Canakkale province, Turkey, in December 2009. Following the report of two tularemia suspected cases from two villages (Baliklicesme and Sinekci) of Biga, an epidemiological investigation was undertaken to inspect the situation in this area. Water samples, clinical samples as throat swabs, wound swabs and serum samples were collected. Samples were cultured on heart agar supplemented with sheep blood, cysteine and antibiotics. Cultures were incubated at 37°C in 5% CO(2) and followed for 10 days. Suspected colonies were identified by slide agglutination test using F.tularensis antisera. F.tularensis antibodies were investigated by standard tube agglutination method. Positive results obtained with agglutination test were also checked for a probable crossreaction with Brucella antibodies by Rose-Bengal test. Water and wound samples were investigated using real-time polymerase chain reaction (RT Taqman PCR; Quantica, Techne Inc, UK) with probe and primers specific for ISFtu2 gene. All of the cultures yielded negative results, however eight of 16 water samples, one lymph node aspirate and one throat sample were found positive in F.tularensis TaqMan RT-PCR test. In tube agglutination test positive antibody titers between 1:20-1:1280 were detected in 36 of 115 serum samples. Two cases with antibody titers of 1:1280 and accompanying acute clinical findings, were diagnosed as tularemia and treated accordingly. Lymphatic drainage fluid samples obtained from one of these patients yielded positive result in PCR, however clinical sample could not be obtained from the other patient. The only epidemiological

  12. Advances with using CRISPR/Cas-mediated gene editing to treat infections with hepatitis B virus and hepatitis C virus.

    PubMed

    Moyo, Buhle; Bloom, Kristie; Scott, Tristan; Ely, Abdullah; Arbuthnot, Patrick

    2017-01-10

    Chronic infections with hepatitis B and hepatitis C viruses (HBV and HCV) account for the majority of cases of cirrhosis and hepatocellular carcinoma. Current therapies for the infections have limitations and improved efficacy is necessary to prevent complications in carriers of the viruses. In the case of HBV persistence, the replication intermediate comprising covalently closed circular DNA (cccDNA) is particularly problematic. Licensed therapies have little effect on cccDNA and HBV replication relapses following treatment withdrawal. Disabling cccDNA is thus key to curing HBV infections and application of gene editing technology, such as harnessing the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system, has curative potential. Several studies have reported good efficacy when employing CRISPR/Cas technologies to disable HBV replication in cultured cells and in hydrodynamically injected mice. Recent advances with HCV drug development have revolutionized treatment of the infection. Nevertheless, individuals may be refractory to treatment. Targeting RNA from HCV with CRISPR/Cas isolated from Francisella novicida may have therapeutic utility. Although preclinical work shows that CRISPR/Cas technology has potential to overcome infection with HBV and HCV, significant challenges need to be met. Ensuring specificity for viral targets and efficient delivery of the gene editing sequences to virus-infected cells are particularly important. The field is at an interesting stage and the future of curative antiviral drug regimens, particularly for treatment of chronic HBV infection, may well entail use of combinations that include derivatives of CRISPR/Cas.

  13. Gene Therapy

    MedlinePlus

    ... cells in an effort to treat or stop disease. Genes contain your DNA — the code that controls much of your body's form and function, from making you grow taller to regulating your body systems. Genes that don't work properly can cause disease. Gene therapy replaces a faulty gene or adds ...

  14. Bis-indolic compounds as potential new therapeutic alternatives for tularaemia

    PubMed Central

    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

  15. Gene Positioning

    PubMed Central

    Ferrai, Carmelo; de Castro, Inês Jesus; Lavitas, Liron; Chotalia, Mita; Pombo, Ana

    2010-01-01

    Eukaryotic gene expression is an intricate multistep process, regulated within the cell nucleus through the activation or repression of RNA synthesis, processing, cytoplasmic export, and translation into protein. The major regulators of gene expression are chromatin remodeling and transcription machineries that are locally recruited to genes. However, enzymatic activities that act on genes are not ubiquitously distributed throughout the nucleoplasm, but limited to specific and spatially defined foci that promote preferred higher-order chromatin arrangements. The positioning of genes within the nuclear landscape relative to specific functional landmarks plays an important role in gene regulation and disease. PMID:20484389

  16. Gene doping.

    PubMed

    Azzazy, Hassan M E

    2010-01-01

    Gene doping abuses the legitimate approach of gene therapy. While gene therapy aims to correct genetic disorders by introducing a foreign gene to replace an existing faulty one or by manipulating existing gene(s) to achieve a therapeutic benefit, gene doping employs the same concepts to bestow performance advantages on athletes over their competitors. Recent developments in genetic engineering have contributed significantly to the progress of gene therapy research and currently numerous clinical trials are underway. Some athletes and their staff are probably watching this progress closely. Any gene that plays a role in muscle development, oxygen delivery to tissues, neuromuscular coordination, or even pain control is considered a candidate for gene dopers. Unfortunately, detecting gene doping is technically very difficult because the transgenic proteins expressed by the introduced genes are similar to their endogenous counterparts. Researchers today are racing the clock because assuring the continued integrity of sports competition depends on their ability to develop effective detection strategies in preparation for the 2012 Olympics, which may mark the appearance of genetically modified athletes.

  17. Gene therapy.

    PubMed

    Williamson, B

    1982-07-29

    Gene therapy is not yet possible, but may become feasible soon, particularly for well understood gene defects. Although treatment of a patient raises no ethical problems once it can be done well, changing the genes of an early embryo is more difficult, controversial and unlikely to be required clinically.

  18. Targeted next-generation sequencing for the detection of ciprofloxacin resistance markers using molecular inversion probes

    PubMed Central

    Stefan, Christopher P.; Koehler, Jeffrey W.; Minogue, Timothy D.

    2016-01-01

    Antibiotic resistance (AR) is an epidemic of increasing magnitude requiring rapid identification and profiling for appropriate and timely therapeutic measures and containment strategies. In this context, ciprofloxacin is part of the first-line of countermeasures against numerous high consequence bacteria. Significant resistance can occur via single nucleotide polymorphisms (SNP) and deletions within ciprofloxacin targeted genes. Ideally, use of ciprofloxacin would be prefaced with AR determination to avoid overuse or misuse of the antibiotic. Here, we describe the development and evaluation of a panel of 44 single-stranded molecular inversion probes (MIPs) coupled to next-generation sequencing (NGS) for the detection of genetic variants known to confer ciprofloxacin resistance in Bacillus anthracis, Yersinia pestis, and Francisella tularensis. Sequencing results demonstrate MIPs capture and amplify targeted regions of interest at significant levels of coverage. Depending on the genetic variant, limits of detection (LOD) for high-throughput pooled sequencing ranged from approximately 300–1800 input genome copies. LODs increased 10-fold in the presence of contaminating human genome DNA. In addition, we show that MIPs can be used as an enrichment step with high resolution melt (HRM) real-time PCR which is a sensitive assay with a rapid time-to-answer. Overall, this technology is a multiplexable upfront enrichment applicable with multiple downstream molecular assays for the detection of targeted genetic regions. PMID:27174456

  19. Gene dispensability.

    PubMed

    Korona, Ryszard

    2011-08-01

    Genome-wide mutagenesis studies indicate that up to about 90% of genes in bacteria and 80% in eukaryotes can be inactivated individually leaving an organism viable, often seemingly unaffected. Several strategies are used to learn what these apparently dispensable genes contribute to fitness. Assays of growth under hundreds of physical and chemical stresses are among the most effective experimental approaches. Comparative studies of genomic DNA sequences continue to be valuable in discriminating between the core bacterial genome and the more variable niche-specific genes. The concept of the core genome appears currently unfeasible for eukaryotes but progress has been made in understanding why they contain numerous gene duplicates.

  20. Trichoderma genes

    DOEpatents

    Foreman, Pamela [Los Altos, CA; Goedegebuur, Frits [Vlaardingen, NL; Van Solingen, Pieter [Naaldwijk, NL; Ward, Michael [San Francisco, CA

    2012-06-19

    Described herein are novel gene sequences isolated from Trichoderma reesei. Two genes encoding proteins comprising a cellulose binding domain, one encoding an arabionfuranosidase and one encoding an acetylxylanesterase are described. The sequences, CIP1 and CIP2, contain a cellulose binding domain. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.

  1. Gene therapy.

    PubMed

    Drugan, A; Miller, O J; Evans, M I

    1987-01-01

    Severe genetic disorders are potentially correctable by the addition of a normal gene into tissues. Although the technical problems involving integration, stable expression, and insertional damage to the treated cell are not yet fully solved, enough scientific progress has already been made to consider somatic cell gene therapy acceptable from both the ethical and scientific viewpoints. The resolutions to problems evolving from somatic cell gene therapy will help to overcome the technical difficulties encountered presently with germ line gene manipulation. This procedure would then become morally permissible as it will cause, in time, a reduction in the pool of abnormal genes in the population. Enhancement genetic engineering is technically feasible but morally unacceptable. Eugenic genetic engineering is not technically possible or ethically permissible in the foreseeable future.

  2. [Gene and gene sequence patenting].

    PubMed

    Bergel, S D

    1998-01-01

    According to the author, the patenting of elements isolated or copied from the human body boils down to the issue of genes and gene sequences. He describes the current situation from the comparative law standpoint (U.S. and Spanish law mainly) and then esamines the biotechnology industry's position.

  3. F. novicida-Infected A. castellanii Does Not Enhance Bacterial Virulence in Mice.

    PubMed

    Ozanic, Mateja; Gobin, Ivana; Brezovec, Martin; Marecic, Valentina; Trobonjaca, Zlatko; Abu Kwaik, Yousef; Santic, Marina

    2016-01-01

    Francisella tularensis is a facultative intracellular bacterium that causes tularemia in humans and animals. Epidemiology of tularemia worldwide is often associated with water-borne transmission, which includes mosquitoes and amoebae as the potential host reservoirs of the bacteria in water environment. In vitro studies showed intracellular replication of F. tularensis within Acanthamoeba castellanii and Hartmanella vermiformis cells. While infection of amoeba by Legionella pneumophila has been shown to enhance infectivity of L. pneumophila the role of F. tularensis-infected protozoa in the pathogenesis of tularemia is not known. We used 6 h coculture of A. castellanii and F. novicida for investigation of the effect of inhaled amoeba on the pathogenesis of tularemia on in vivo model. Balb/c mice were infected intratracheally with F. novicida or with F. novicida-infected A. castellanii. Surprisingly, infection with F. novicida-infected A. castellanii did not lead to bronchopneumonia in Balb/c mice, and Francisella did not disseminate into the liver and spleen. Upon inhalation, F. novicida infects a variety of host cells, though neutrophils are the predominant cells early during infection in the lung infiltrates of pulmonary tularemia. The numbers of neutrophils in the lungs of Balb/c mice were significantly lower in the infection of mice with F. novicida-infected A. castellanii in comparison to group of mice infected only with F. novicida. These results demonstrate that following inoculation of mice with F. novicida-infected A. castellanii, mice did not develop tularemia.

  4. F. novicida-Infected A. castellanii Does Not Enhance Bacterial Virulence in Mice

    PubMed Central

    Ozanic, Mateja; Gobin, Ivana; Brezovec, Martin; Marecic, Valentina; Trobonjaca, Zlatko; Abu Kwaik, Yousef; Santic, Marina

    2016-01-01

    Francisella tularensis is a facultative intracellular bacterium that causes tularemia in humans and animals. Epidemiology of tularemia worldwide is often associated with water-borne transmission, which includes mosquitoes and amoebae as the potential host reservoirs of the bacteria in water environment. In vitro studies showed intracellular replication of F. tularensis within Acanthamoeba castellanii and Hartmanella vermiformis cells. While infection of amoeba by Legionella pneumophila has been shown to enhance infectivity of L. pneumophila the role of F. tularensis-infected protozoa in the pathogenesis of tularemia is not known. We used 6 h coculture of A. castellanii and F. novicida for investigation of the effect of inhaled amoeba on the pathogenesis of tularemia on in vivo model. Balb/c mice were infected intratracheally with F. novicida or with F. novicida-infected A. castellanii. Surprisingly, infection with F. novicida-infected A. castellanii did not lead to bronchopneumonia in Balb/c mice, and Francisella did not disseminate into the liver and spleen. Upon inhalation, F. novicida infects a variety of host cells, though neutrophils are the predominant cells early during infection in the lung infiltrates of pulmonary tularemia. The numbers of neutrophils in the lungs of Balb/c mice were significantly lower in the infection of mice with F. novicida-infected A. castellanii in comparison to group of mice infected only with F. novicida. These results demonstrate that following inoculation of mice with F. novicida-infected A. castellanii, mice did not develop tularemia. PMID:27242974

  5. 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

  6. Genes V.

    SciTech Connect

    Lewin, B.

    1994-12-31

    This fifth edition book encompasses a wide range of topics covering 1,272 pages. The book is arranged into nine parts with a total of 36 chapters. These nine parts include Introduction; DNA as a Store of Information; Translation; Constructing Cells; Control of Prokaryotypic Gene Expression; Perpetuation of DNA; Organization of the Eukaryotypic Genome; Eukaryotypic Transcription and RNA Processing; The Dynamic Genome; and Genes in Development.

  7. Attention Genes

    ERIC Educational Resources Information Center

    Posner, Michael I.; Rothbart, Mary K.; Sheese, Brad E.

    2007-01-01

    A major problem for developmental science is understanding how the cognitive and emotional networks important in carrying out mental processes can be related to individual differences. The last five years have seen major advances in establishing links between alleles of specific genes and the neural networks underlying aspects of attention. These…

  8. Designer Genes.

    ERIC Educational Resources Information Center

    Miller, Judith; Miller, Mark

    1983-01-01

    Genetic technologies may soon help fill some of the most important needs of humanity from food to energy to health care. The research of major designer genes companies and reasons why the initial mad rush for biotechnology has slowed are reviewed. (SR)

  9. Genome sequence of the endosymbiont Rickettsia peacockii and comparison with virulent Rickettsia rickettsii: identification of virulence factors.

    PubMed

    Felsheim, Roderick F; Kurtti, Timothy J; Munderloh, Ulrike G

    2009-12-21

    Rickettsia peacockii, also known as the East Side Agent, is a non-pathogenic obligate intracellular bacterium found as an endosymbiont in Dermacentor andersoni ticks in the western USA and Canada. Its presence in ticks is correlated with reduced prevalence of Rickettsia rickettsii, the agent of Rocky Mountain Spotted Fever. It has been proposed that a virulent SFG rickettsia underwent changes to become the East Side Agent. We determined the genome sequence of R. peacockii and provide a comparison to a closely related virulent R. rickettsii. The presence of 42 chromosomal copies of the ISRpe1 transposon in the genome of R. peacockii is associated with a lack of synteny with the genome of R. rickettsii and numerous deletions via recombination between transposon copies. The plasmid contains a number of genes from distantly related organisms, such as part of the glycosylation island of Pseudomonas aeruginosa. Genes deleted or mutated in R. peacockii which may relate to loss of virulence include those coding for an ankyrin repeat containing protein, DsbA, RickA, protease II, OmpA, ScaI, and a putative phosphoethanolamine transferase. The gene coding for the ankyrin repeat containing protein is especially implicated as it is mutated in R. rickettsii strain Iowa, which has attenuated virulence. Presence of numerous copies of the ISRpe1 transposon, likely acquired by lateral transfer from a Cardinium species, are associated with extensive genomic reorganization and deletions. The deletion and mutation of genes possibly involved in loss of virulence have been identified by this genomic comparison. It also illustrates that the introduction of a transposon into the genome can have varied effects; either correlating with an increase in pathogenicity as in Francisella tularensis or a loss of pathogenicity as in R. peacockii and the recombination enabled by multiple transposon copies can cause significant deletions in some genomes while not in others.

  10. Tick-borne zoonotic bacteria in ticks collected from central Spain.

    PubMed

    Toledo, Alvaro; Olmeda, A Sonia; Escudero, Raquel; Jado, Isabel; Valcárcel, Félix; Casado-Nistal, Miguel A; Rodríguez-Vargas, Manuela; Gil, Horacio; Anda, Pedro

    2009-07-01

    The prevalence of tick-borne and related bacteria infecting adult ticks in central Spain was assessed by molecular methods. Six areas were sampled monthly during a 2-year longitudinal study. A total of 1,038 questing and 442 feeding ticks, belonging to eight different species, were tested. The most abundant species were Hyalomma lusitanicum (54% of captures), followed by Dermacentor marginatus (23%) and Rhipicephalus sanguineus (10%). Four human pathogens, including seven Rickettsia species, Anaplasma phagocytophilum, Borrelia burgdorferi, and Francisella tularensis, were detected at percentages of 19.0, 2.2, 1.7, and 0.5, respectively, whereas Bartonella spp. was never detected. In terms of infection and tick abundance, H. lusitanicum seems to be the most significant tick species in the area, carrying three of the five agents tested, and the anthropophilic tick, D. marginatum, infected with Rickettsia spp. and F. tularensis, is the most relevant in terms of public health. The significance of these data is discussed.

  11. 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.

  12. Tularemia type A in captive Bornean orangutans (Pongo pygmaeus pygmaeus).

    PubMed

    Ketz-Riley, Cornelia J; Kennedy, George A; Carpenter, James W; Zeidner, Nordin S; Petersen, Jeannine M

    2009-06-01

    In 2003, tularemia was suspected to be the cause of severe illness in two orangutans (Pongo pygmaeus pygmaeus) and the cause of death in a third orangutan at an urban zoo. The two sick orangutans were treated two times under chemical immobilization with i.v. doxycycline, fluids, and antipyretic drugs, followed by a sustained course of oral doxycycline. The rest of the orangutan group was treated prophylactically with oral doxycycline. Postmortem diagnosis was obtained via immunohistochemistry and bacterial culture that revealed Francisella tularensis type A. Tularemia was also confirmed in the two surviving orangutans via paired serology testing. In addition, F. tularensis was identified in two wild rabbit carcasses submitted during a die-off, several weeks prior to the tularemia outbreak in the apes, indicating that rabbits were possibly a reservoir for tularemia within the zoo premises.

  13. 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.

  14. 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.

  15. Epidemiologic and Molecular Analysis of Human Tularemia, United States, 1964–2004

    PubMed Central

    Staples, J. Erin; Kubota, Kristy A.; Chalcraft, Linda G.; Mead, Paul S.

    2006-01-01

    Tularemia in the United States is caused by 2 subspecies of Francisella tularensis, subspecies tularensis (type A) and subspecies holarctica (type B). We compared clinical and demographic features of human tularemia cases from 1964 to 2004 from 39 states in which an isolate was recovered and subtyped. Our data indicate that type A and type B infections differ with respect to affected populations, anatomic site of isolation, and geographic distribution. Molecular subtyping with pulsed-field gel electrophoresis further defined 2 subpopulations of type A (type A-east and type A-west) that differ with respect to geographic distribution, disease outcome, and transmission. Our data suggest that type A-west infections are less severe than either type B or type A-east infections. Through a combined epidemiologic and molecular approach to human cases of tularemia, we provide new insights into the disease for future investigation. PMID:16836829

  16. Endothelial Genes

    DTIC Science & Technology

    2005-06-01

    Suppression subtractive hybridization re- Cancer: principles and practice of oncology. Philadelphia: Lippincott- vealed an RNA sequence (GenBank accession...Lau YC, Campbell AP, et al. Suppression subtractive hybridization : A method for generating differentially regulated or tissue-tissues, EG-1 appears to...this gene, we investigated its interaction with Src and members of the called suppression subtractive hybridization (12). In human mitogen-activated

  17. Laboratory Analysis of Tularemia in Wild-Trapped, Commercially Traded Prairie Dogs, Texas, 2002

    PubMed Central

    Schriefer, Martin E.; Carter, Leon G.; Zhou, Yan; Sealy, Tara; Bawiec, Darcy; Yockey, Brook; Urich, Sandra; Zeidner, Nordin S.; Avashia, Swati; Kool, Jacob L.; Buck, Jan; Lindley, Connie; Celeda, Leos; Monteneiri, John A.; Gage, Kenneth L.; Chu, May C.

    2004-01-01

    Oropharyngeal tularemia was identified as the cause of a die-off in captured wild prairie dogs at a commercial exotic animal facility in Texas. From this point source, Francisella tularensis–infected prairie dogs were traced to animals distributed to the Czech Republic and to a Texas pet shop. F. tularensis culture isolates were recovered tissue specimens from 63 prairie dogs, including one each from the secondary distribution sites. Molecular and biochemical subtyping indicated that all isolates were F. tularensis subsp. holarctica (Type B). Microagglutination assays detected antibodies against F. tularensis, with titers as great as 1:4,096 in some live animals. All seropositive animals remained culture positive, suggesting that prairie dogs may act as chronic carriers of F. tularensis. These findings demonstrate the need for additional studies of tularemia in prairie dogs, given the seriousness of the resulting disease, the fact that prairie dogs are sold commercially as pets, and the risk for pet-to-human transmission. PMID:15109407

  18. Seroprevalence of tularemia in wild bears and hares in Japan.

    PubMed

    Hotta, A; Tanabayashi, K; Yamamoto, Y; Fujita, O; Uda, A; Mizoguchi, T; Yamada, A

    2012-03-01

    Tularemia is a zoonotic disease caused by Francisella tularensis. The distribution of the pathogen in Japan has not been studied well. In this study, seroprevalence of tularemia among wild black bears and hares in Japan was determined. Blood samples collected from 431 Japanese black bears (Ursus thibetanus japonicus) and 293 Japanese hares (Lepus brachurus) between 1998 and 2009 were examined for antibodies against F. tularensis by micro-agglutination test (MA) or enzyme-linked immunosorbent assay. By subsequent confirmatory tests using western blot (WB) and indirect immunofluorescence assay (IFA), eight sera from Japanese black bears were definitely shown to be seropositive. All of these eight bears were residents of the northeastern part of main-island of Japan, where human tularemia had been reported. On the other hand, no seropositive Japanese hares were found. These results suggest that Japanese black bears can serve as sentinel for tularemia surveillance and may help understand the distribution of F. tularensis throughout the country. This is the first report on detection of antibody to F. tularensis in black bears of Japan.

  19. Transmission of tularemia from a water source by transstadial maintenance in a mosquito vector

    NASA Astrophysics Data System (ADS)

    Bäckman, Stina; Näslund, Jonas; Forsman, Mats; Thelaus, Johanna

    2015-01-01

    Mosquitoes are thought to function as mechanical vectors of Francisella tularensis subspecies holarctica (F. t. holarctica) causing tularemia in humans. We investigated the clinical relevance of transstadially maintained F. t. holarctica in mosquitoes. Aedes egypti larvae exposed to a fully virulent F. t. holarctica strain for 24 hours, were allowed to develop into adults when they were individually homogenized. Approximately 24% of the homogenates tested positive for F. t. DNA in PCR. Mice injected with the mosquito homogenates acquired tularemia within 5 days. This novel finding demonstrates the possibility of transmission of bacteria by adult mosquitoes having acquired the pathogen from their aquatic larval habitats.

  20. Tularemia in a kidney transplant recipient: an unsuspected case and literature review.

    PubMed

    Khoury, Jad A; Bohl, Daniel L; Hersh, Michael J; Argoudelis, Alexis C; Brennan, Daniel C

    2005-05-01

    Tularemia is a zoonotic infection that has rarely been reported in transplant recipients. The authors present a case of unsuspected tularemia in a kidney transplant patient that was diagnosed by isolation of Francisella tularensis in the blood. The patient was treated successfully with antibiotics. During diagnostic workup, a laboratory technician was exposed to tularemia by inhalation of the culture plate and received postexposure prophylaxis. This report emphasizes the importance of exposure history in the investigation of fever in an immunocompromised host and the special precautions needed when a virulent infectious organism is suspected.

  1. 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.

  2. Tularemia presenting as pulmonary nodules in an immunocompromised patient

    PubMed Central

    Fallahzadeh, Mohammad Kazem; Berhe, Mezgebe

    2017-01-01

    Tularemia is a zoonotic disease caused by Francisella tularensis that can be transmitted to humans when they handle rabbits, receive tick bites, consume contaminated water, or inhale aerosolized particles. We present the case of a 51-year-old white man with rheumatoid arthritis who was taking immunosuppressive medications and presented with tularemia. Our patient acquired the typhoidal form of tularemia, which is a severe systemic illness that manifests with fevers, headaches, myalgias, vomiting, diarrhea, and neurological symptoms, due to his immunocompromised state. The diagnosis was made through biopsy of a pulmonary nodule found incidentally on computed tomography scan.

  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. Two-Color Multiplex Assay for the Identification of Orthopox Viruses with Real-Time LUX-PCR

    DTIC Science & Technology

    2005-07-14

    perfringens 13124 1000 0/2 0/2 Francisella tularensis NA 1000 0/2 0/2 Haemophlius influenzae 10211 1000 0/2 0/2 Listeia monocytogenes 15313 1000 0/2 0/2...Loveless BM, Norwood D, Zwiers SH, Mucker E, Hartmann C, et al. Smallpox and pan-orthopox virus detection by real-time 3 0-minor groove binder TaqMan ...Whitehouse CA, Hartmann C, Mucker E, et al. Monkeypox virus detection in rodents using real-time 3 0-minor groove binder TaqMan assays on the Roche

  5. [Culture properties of the causative agent of tularemia isolated in natural foci in Stavropol Territory, the Kalmyk ASSR and the Armenian SSR].

    PubMed

    Basilova, G I; Nekrasov, A A; Pilipenko, V G

    1983-01-01

    The strains isolated in natural foci of the Stavropol Territory and the Armenian SSR have been found to belong to the holarctic race of Francisella tularensis, biovar II. In natural foci of the Kalmyk ASSR the strains belonging to biovars I and II have been isolated. The study of the tularecinogenicity of the cultures has revealed the existence of strains which are not sensitive to their own tularecins. The phenomenon of tularecinogenicity in F. novocida has been established. Avirulent strain 319/38 belonging to the non-arctic race is recommended as an indicator strain for the determination of tularecinogenicity.

  6. Quaternary Structure of Fur Proteins, a New Subfamily of Tetrameric Proteins.

    PubMed

    Pérard, Julien; Covès, Jacques; Castellan, Mathieu; Solard, Charles; Savard, Myriam; Miras, Roger; Galop, Sandra; Signor, Luca; Crouzy, Serge; Michaud-Soret, Isabelle; de Rosny, Eve

    2016-03-15

    The ferric uptake regulator (Fur) belongs to the family of the DNA-binding metal-responsive transcriptional regulators. Fur is a global regulator found in all proteobacteria. It controls the transcription of a wide variety of genes involved in iron metabolism but also in oxidative stress or virulence factor synthesis. When bound to ferrous iron, Fur can bind to specific DNA sequences, called Fur boxes. This binding triggers the repression or the activation of gene expression, depending on the regulated genes. As a general view, Fur proteins are considered to be dimeric proteins both in solution and when bound to DNA. In this study, we have purified Fur from four pathogenic strains (Pseudomonas aeruginosa, Francisella tularensis, Yersinia pestis, and Legionella pneumophila) and compared them to Fur from Escherichia coli (EcFur), the best characterized of this family. By using a series of "in solution" techniques, including multiangle laser light scattering and small-angle X-ray scattering, as well as cross-linking experiments, we have shown that the Fur proteins can be classified into two groups, according to their quaternary structure. The group of dimers is represented by EcFur and YpFur and the group of very stable tetramers by PaFur, FtFur, and LpFur. Using PaFur as a case study, we also showed that the dissociation of the tetramers into dimers is necessary for binding of Fur to DNA, and that this dissociation requires the combined effect of metal ion binding and DNA proximity.

  7. Compare Gene Profiles

    SciTech Connect

    2014-05-31

    Compare Gene Profiles (CGP) performs pairwise gene content comparisons among a relatively large set of related bacterial genomes. CGP performs pairwise BLAST among gene calls from a set of input genome and associated annotation files, and combines the results to generate lists of common genes, unique genes, homologs, and genes from each genome that differ substantially in length from corresponding genes in the other genomes. CGP is implemented in Python and runs in a Linux environment in serial or parallel mode.

  8. Gene and enhancer traps for gene discovery.

    PubMed

    Rojas-Pierce, Marcela; Springer, Patricia S

    2003-01-01

    Gene traps and enhancer traps provide a valuable tool for gene discovery. With this system, genes can be identified based solely on the expression pattern of an inserted reporter gene. The use of a reporter gene, such as beta-glucuoronidase (GUS), provides a very sensitive assay for the identification of tissue- and cell-type specific expression patterns. In this chapter, protocols for examining and documenting GUS reporter gene activity in individual lines are described. Methods for the amplification of sequences flanking transposant insertions and subsequent molecular and genetic characterization of individual insertions are provided.

  9. Gene doping: gene delivery for olympic victory.

    PubMed

    Gould, David

    2013-08-01

    With one recently recommended gene therapy in Europe and a number of other gene therapy treatments now proving effective in clinical trials it is feasible that the same technologies will soon be adopted in the world of sport by unscrupulous athletes and their trainers in so called 'gene doping'. In this article an overview of the successful gene therapy clinical trials is provided and the potential targets for gene doping are highlighted. Depending on whether a doping gene product is secreted from the engineered cells or is retained locally to, or inside engineered cells will, to some extent, determine the likelihood of detection. It is clear that effective gene delivery technologies now exist and it is important that detection and prevention plans are in place.

  10. Expression cloning and biochemical characterization of a Rhizobium leguminosarum lipid A 1-phosphatase.

    PubMed

    Karbarz, Mark J; Kalb, Suzanne R; Cotter, Robert J; Raetz, Christian R H

    2003-10-10

    Lipid A of Rhizobium leguminosarum, a nitrogen-fixing plant endosymbiont, displays several significant structural differences when compared with Escherichia coli. An especially striking feature of R. leguminosarum lipid A is that it lacks both the 1- and 4'-phosphate groups. Distinct lipid A phosphatases that attack either the 1 or the 4' positions have previously been identified in extracts of R. leguminosarum and Rhizobium etli but not Sinorhizobium meliloti or E. coli. Here we describe the identification of a hybrid cosmid (pMJK-1) containing a 25-kb R. leguminosarum 3841 DNA insert that directs the overexpression of the lipid A 1-phosphatase. Transfer of pMJK-1 into S. meliloti 1021 results in heterologous expression of 1-phosphatase activity, which is normally absent in extracts of strain 1021, and confers resistance to polymyxin. Sequencing of a 7-kb DNA fragment derived from the insert of pMJK-1 revealed the presence of a lipid phosphatase ortholog (designated LpxE). Expression of lpxE in E. coli behind the T7lac promoter results in the appearance of robust 1-phosphatase activity, which is normally absent in E. coli membranes. Matrix-assisted laser-desorption/time of flight and radiochemical analysis of the product generated in vitro from the model substrate lipid IVA confirms the selective removal of the 1-phosphate group. These findings show that lpxE is the structural gene for the 1-phosphatase. The availability of lpxE may facilitate the re-engineering of lipid A structures in diverse Gram-negative bacteria and allow assessment of the role of the 1-phosphatase in R. leguminosarum symbiosis with plants. Possible orthologs of LpxE are present in some intracellular human pathogens, including Francisella tularensis, Brucella melitensis, and Legionella pneumophila.

  11. The genome of the amoeba symbiont "Candidatus Amoebophilus asiaticus" reveals common mechanisms for host cell interaction among amoeba-associated bacteria.

    PubMed

    Schmitz-Esser, Stephan; Tischler, Patrick; Arnold, Roland; Montanaro, Jacqueline; Wagner, Michael; Rattei, Thomas; Horn, Matthias

    2010-02-01

    Protozoa play host for many intracellular bacteria and are important for the adaptation of pathogenic bacteria to eukaryotic cells. We analyzed the genome sequence of "Candidatus Amoebophilus asiaticus," an obligate intracellular amoeba symbiont belonging to the Bacteroidetes. The genome has a size of 1.89 Mbp, encodes 1,557 proteins, and shows massive proliferation of IS elements (24% of all genes), although the genome seems to be evolutionarily relatively stable. The genome does not encode pathways for de novo biosynthesis of cofactors, nucleotides, and almost all amino acids. "Ca. Amoebophilus asiaticus" encodes a variety of proteins with predicted importance for host cell interaction; in particular, an arsenal of proteins with eukaryotic domains, including ankyrin-, TPR/SEL1-, and leucine-rich repeats, which is hitherto unmatched among prokaryotes, is remarkable. Unexpectedly, 26 proteins that can interfere with the host ubiquitin system were identified in the genome. These proteins include F- and U-box domain proteins and two ubiquitin-specific proteases of the CA clan C19 family, representing the first prokaryotic members of this protein family. Consequently, interference with the host ubiquitin system is an important host cell interaction mechanism of "Ca. Amoebophilus asiaticus". More generally, we show that the eukaryotic domains identified in "Ca. Amoebophilus asiaticus" are also significantly enriched in the genomes of other amoeba-associated bacteria (including chlamydiae, Legionella pneumophila, Rickettsia bellii, Francisella tularensis, and Mycobacterium avium). This indicates that phylogenetically and ecologically diverse bacteria which thrive inside amoebae exploit common mechanisms for interaction with their hosts, and it provides further evidence for the role of amoebae as training grounds for bacterial pathogens of humans.

  12. Autism and Genes

    ERIC Educational Resources Information Center

    National Institutes of Health, 2005

    2005-01-01

    This document defines and discusses autism and how genes play a role in the condition. Answers to the following questions are covered: (1) What are genes? (2) What is autism? (3) What causes autism? (4) Why study genes to learn about autism? (5) How do researchers look for the genes involved in autism? (screen the whole genome; conduct cytogenetic…

  13. Compare Gene Calls

    SciTech Connect

    Ecale Zhou, Carol L.

    2016-07-05

    Compare Gene Calls (CGC) is a Python code used for combining and comparing gene calls from any number of gene callers. A gene caller is a computer program that predicts the extends of open reading frames within genomes of biological organisms.

  14. 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.

  15. [Incidence of zoonoses in petting zoos and evaluation of hygiene measures to prevent the transmission to humans].

    PubMed

    Bütikofer, B; Bissig-Choisat, B; Regula, G; Corboz, L; Wittwer, M; Danuser, J

    2005-12-01

    In summer 2003, a study was performed in thirty Swiss petting zoos with the objective to determine the prevalence of zoonotic agents, and to describe hygiene measures implemented to reduce the risk of human infection. Fecal samples from different animal species were collected from the floor of pens to determine the prevalence of Salmonella spp., Campylobacter spp., verocytotoxin producing E. coli/ VTEC and Francisella tularensis. A questionnaire on hygiene measures, number of animals per species, housing system, care procedures and feeding was administered to every petting zoo to estimate exposure of visitors to zoonotic microorganisms. In total, 423 fecal samples were examined. Of these samples, 41 were positive for Campylobacter spp., which were mainly isolates from pigs and poultry (35% positive samples from each species). In pigs, 50% of the positive samples (6 samples) were typed as C. jejuni. The others were typed as C. coli (3) and C lan' (3), respectively. Five poultry isolates were typed as C. jejuni, and two as C. coli. Two samples were positive for Salmonella spp. Salmonella typhimurium was isolated from a goat, the other isolate could not be identified by serotyping. Neither Francisella tularensis nor verocytotoxin producing E. coli/ VTEC were found. The low prevalence of zoonotic microorganisms in Swiss petting zoos could be attributed to the cleanness of enclosures and animals, low stocking rates and good animal care. However, there is room for improvement concerning visitors' information on hygiene and hand washing. Furthermore, a strict separation between picnic - areas and animals should be enforced.

  16. Epilepsy-associated genes.

    PubMed

    Wang, Jie; Lin, Zhi-Jian; Liu, Liu; Xu, Hai-Qing; Shi, Yi-Wu; Yi, Yong-Hong; He, Na; Liao, Wei-Ping

    2017-01-01

    Development in genetic technology has led to the identification of an increasing number of genes associated with epilepsy. These discoveries will both provide the basis for including genetic tests in clinical practice and improve diagnosis and treatment of epilepsy. By searching through several databases (OMIM, HGMD, and EpilepsyGene) and recent publications on PubMed, we found 977 genes that are associated with epilepsy. We classified these genes into 4 categories according to the manifestation of epilepsy in phenotypes. We found 84 genes that are considered as epilepsy genes: genes that cause epilepsies or syndromes with epilepsy as the core symptom. 73 genes were listed as neurodevelopment-associated genes: genes associated with both brain-development malformations and epilepsy. Several genes (536) were epilepsy-related: genes associated with both physical or other systemic abnormalities and epilepsy or seizures. We found 284 additional genes putatively associated with epilepsy; this requires further verification. These integrated data will provide new insights useful for both including genetic tests in the clinical practice and evaluating the results of genetic tests. We also summarized the epilepsy-associated genes according to their function, with the goal to better characterize the association between genes and epilepsies and to further understand the mechanisms underlying epilepsy.

  17. Gene regulation in cancer gene therapy strategies.

    PubMed

    Scanlon, Ian; Lehouritis, Panos; Niculescu-Duvaz, Ion; Marais, Richard; Springer, Caroline J

    2003-10-01

    Regulation of expression in gene therapy is considered to be a very desirable goal, preventing toxic effects and improving biological efficacy. A variety of systems have been reported in an ever widening range of applications, this paper describes these systems with specific reference to cancer gene therapy.

  18. Human Gene Therapy: Genes without Frontiers?

    ERIC Educational Resources Information Center

    Simon, Eric J.

    2002-01-01

    Describes the latest advancements and setbacks in human gene therapy to provide reference material for biology teachers to use in their science classes. Focuses on basic concepts such as recombinant DNA technology, and provides examples of human gene therapy such as severe combined immunodeficiency syndrome, familial hypercholesterolemia, and…

  19. Human gene therapy.

    PubMed

    Sandhu, J S; Keating, A; Hozumi, N

    1997-01-01

    Human gene therapy and its application for the treatment of human genetic disorders, such as cystic fibrosis, cancer, and other diseases, are discussed. Gene therapy is a technique in which a functioning gene is inserted into a human cell to correct a genetic error or to introduce a new function to the cell. Many methods, including retroviral vectors and non-viral vectors, have been developed for both ex vivo and in vivo gene transfer into cells. Vectors need to be developed that efficiently transfer genes to target cells, and promoter systems are required that regulate gene expression according to physiologic needs of the host cell. There are several safety and ethical issues related to manipulating the human genome that need to be resolved. Current gene therapy efforts focus on gene insertion into somatic cells only. Gene therapy has potential for the effective treatment of genetic disorders, and gene transfer techniques are being used for basic research, for example, in cancer, to examine the underlying mechanism of disease. There are still many technical obstacles to be overcome before human gene therapy can become a routine procedure. The current human genome project provides the sequences of a vast number of human genes, leading to the identification, characterization, and understanding of genes that are responsible for many human diseases.

  20. Prevalence of tick-borne pathogens in Ixodes ricinus and Dermacentor reticulatus ticks from different geographical locations in Belarus.

    PubMed

    Reye, Anna L; Stegniy, Valentina; Mishaeva, Nina P; Velhin, Sviataslau; Hübschen, Judith M; Ignatyev, George; Muller, Claude P

    2013-01-01

    Worldwide, ticks are important vectors of human and animal pathogens. Besides Lyme Borreliosis, a variety of other bacterial and protozoal tick-borne infections are of medical interest in Europe. In this study, 553 questing and feeding Ixodes ricinus (n = 327) and Dermacentor reticulatus ticks (n = 226) were analysed by PCR for Borrelia, Rickettsia, Anaplasma, Coxiella, Francisella and Babesia species. Overall, the pathogen prevalence in ticks was 30.6% for I. ricinus and 45.6% for D. reticulatus. The majority of infections were caused by members of the spotted-fever group rickettsiae (24.4%), 9.4% of ticks were positive for Borrelia burgdorferi sensu lato, with Borrelia afzelii being the most frequently detected species (40.4%). Pathogens with low prevalence rates in ticks were Anaplasma phagocytophilum (2.2%), Coxiella burnetii (0.9%), Francisella tularensis subspecies (0.7%), Bartonella henselae (0.7%), Babesia microti (0.5%) and Babesia venatorum (0.4%). On a regional level, hotspots of pathogens were identified for A. phagocytophilum (12.5-17.2%), F. tularensis ssp. (5.5%) and C. burnetii (9.1%), suggesting established zoonotic cycles of these pathogens at least at these sites. Our survey revealed a high burden of tick-borne pathogens in questing and feeding I. ricinus and D. reticulatus ticks collected in different regions in Belarus, indicating a potential risk for humans and animals. Identified hotspots of infected ticks should be included in future surveillance studies, especially when F. tularensis ssp. and C. burnetii are involved.

  1. Gene therapy for blindness.

    PubMed

    Sahel, José-Alain; Roska, Botond

    2013-07-08

    Sight-restoring therapy for the visually impaired and blind is a major unmet medical need. Ocular gene therapy is a rational choice for restoring vision or preventing the loss of vision because most blinding diseases originate in cellular components of the eye, a compartment that is optimally suited for the delivery of genes, and many of these diseases have a genetic origin or genetic component. In recent years we have witnessed major advances in the field of ocular gene therapy, and proof-of-concept studies are under way to evaluate the safety and efficacy of human gene therapies. Here we discuss the concepts and recent advances in gene therapy in the retina. Our review discusses traditional approaches such as gene replacement and neuroprotection and also new avenues such as optogenetic therapies. We conjecture that advances in gene therapy in the retina will pave the way for gene therapies in other parts of the brain.

  2. Scientists Spot 'Teetotaler' Gene

    MedlinePlus

    ... gov/news/fullstory_162265.html Scientists Spot 'Teetotaler' Gene Discovery might one day lead to drugs to ... HealthDay News) -- Scientists say they've identified a gene variant that dampens the desire to drink alcohol. ...

  3. Genes and Hearing Loss

    MedlinePlus

    ... gametes (reproductive cells). One gamete will carry the mutant form of the gene of interest, and the ... by having parents who are heterozygous carriers for mutant forms of the gene in question but are ...

  4. Myocardial gene therapy

    NASA Astrophysics Data System (ADS)

    Isner, Jeffrey M.

    2002-01-01

    Gene therapy is proving likely to be a viable alternative to conventional therapies in coronary artery disease and heart failure. Phase 1 clinical trials indicate high levels of safety and clinical benefits with gene therapy using angiogenic growth factors in myocardial ischaemia. Although gene therapy for heart failure is still at the pre-clinical stage, experimental data indicate that therapeutic angiogenesis using short-term gene expression may elicit functional improvement in affected individuals.

  5. Tick species, tick-borne pathogens and symbionts in an insular environment off the coast of Western France.

    PubMed

    Michelet, Lorraine; Joncour, Guy; Devillers, Elodie; Torina, Alessandra; Vayssier-Taussat, Muriel; Bonnet, Sarah I; Moutailler, Sara

    2016-10-01

    Insular environments provide ideal natural conditions to study disease ecology, especially emerging diseases, due to clear differentiation between local and long-distance transmission. Such environments are of particular interest regarding tick-borne pathogens (TBP), since animal exchange with the mainland (along with any ticks they carry) is limited, and because such locations could lie on migratory routes for birds carrying ticks. Therefore both tick species and TBP may display different prevalence than those observed on the continent. As such, an epidemiological survey was performed on Belle-Ile-en-Mer, an island off the coast of Western France, in order to estimate the prevalence of tick species and the microorganisms they carried. Three tick species, Dermacentor marginatus, D. reticulatus, and Haemaphysalis punctata were collected at five different sites in 2010 and 2011. All ticks were tested for pathogen's and symbiont's DNA by (i) PCR for Anaplasma spp., Borrelia spp., Rickettsia spp.; (ii) real-time PCR for Francisella tularensis, Francisella-like endosymbionts (FLE) and Coxiella spp. and (iii) PCR-RLB for Babesia-Theileria spp. Pathogen DNA detected in D. marginatus including Borrelia spp. (18%), Rickettsia spp. (13%) which was identified as R. slovaca, Babesia spp. (8%), and Theileria spp. (1%). Pathogens detected in D. reticulatus including Rickettsia spp. (31%) identified as R. raoulti, Francisella-like endosymbiont (86%), and Babesia spp (21%). Pathogens detected in H. punctata including Rickettsia spp. (1%) identified as R. aeschlimannii, FLE (0.4%), Babesia spp. (18%), and Theileria spp. (7%). Anaplasma spp., F. tularensis, or Coxiella spp. were not detected in any of the collected ticks. This study represents the first epidemiological survey of the insular Belle-Ile-en-Mer environment. It demonstrated the presence of expected pathogens, consistent with reports from island veterinarians or physicians, as well as unexpected pathogens, raising

  6. Reading and Generalist Genes

    ERIC Educational Resources Information Center

    Haworth, Claire M. A.; Meaburn, Emma L.; Harlaar, Nicole; Plomin, Robert

    2007-01-01

    Twin-study research suggests that many (but not all) of the same genes contribute to genetic influence on diverse learning abilities and disabilities, a hypothesis called "generalist genes". This generalist genes hypothesis was tested using a set of 10 DNA markers (single nucleotide polymorphisms [SNPs]) found to be associated with early reading…

  7. The potential of liposome-encapsulated ciprofloxacin as a tularemia therapy.

    PubMed

    Hamblin, Karleigh A; Wong, Jonathan P; Blanchard, James D; Atkins, Helen S

    2014-01-01

    Liposome-encapsulation has been suggested as method to improve the efficacy of ciprofloxacin against the intracellular pathogen, Francisella tularensis. Early work with a prototype formulation, evaluated for use against the F. tularensis live vaccine strain, showed that a single dose of liposomal ciprofloxacin given by the intranasal or inhalational route could provide protection in a mouse model of pneumonic tularemia. Liposomal ciprofloxacin offered better protection than ciprofloxacin given by the same routes. Liposomal ciprofloxacin has been further developed by Aradigm Corporation for Pseudomonas aeruginosa infections in patients with cystic fibrosis and non-cystic fibrosis bronchiectasis. This advanced development formulation is safe, effective and well tolerated in human clinical trials. Further evaluation of the advanced liposomal ciprofloxacin formulation against the highly virulent F. tularensis Schu S4 strain has shown that aerosolized CFI (Ciprofloxacin encapsulated in liposomes for inhalation) provides significantly better protection than oral ciprofloxacin. Thus, liposomal ciprofloxacin is a promising treatment for tularemia and further research with the aim of enabling licensure under the animal rule is warranted.

  8. Dynamics of a Tularemia Outbreak in a Closely Monitored Free-Roaming Population of Wild House Mice

    PubMed Central

    Dobay, Akos; Pilo, Paola; Lindholm, Anna K.; Origgi, Francesco; Bagheri, Homayoun C.; König, Barbara

    2015-01-01

    Infectious disease outbreaks can be devastating because of their sudden occurrence, as well as the complexity of monitoring and controlling them. Outbreaks in wildlife are even more challenging to observe and describe, especially when small animals or secretive species are involved. Modeling such infectious disease events is relevant to investigating their dynamics and is critical for decision makers to accomplish outbreak management. Tularemia, caused by the bacterium Francisella tularensis, is a potentially lethal zoonosis. Of the few animal outbreaks that have been reported in the literature, only those affecting zoo animals have been closely monitored. Here, we report the first estimation of the basic reproduction number R0 of an outbreak in wildlife caused by F. tularensis using quantitative modeling based on a susceptible-infected-recovered framework. We applied that model to data collected during an extensive investigation of an outbreak of tularemia caused by F. tularensis subsp. holarctica (also designated as type B) in a closely monitored, free-roaming house mouse (Mus musculus domesticus) population in Switzerland. Based on our model and assumptions, the best estimated basic reproduction number R0 of the current outbreak is 1.33. Our results suggest that tularemia can cause severe outbreaks in small rodents. We also concluded that the outbreak self-exhausted in approximately three months without administrating antibiotics. PMID:26536232

  9. 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.

  10. Possible interaction between a rodenticide treatment and a pathogen in common vole (Microtus arvalis) during a population peak.

    PubMed

    Vidal, D; Alzaga, V; Luque-Larena, J J; Mateo, R; Arroyo, L; Viñuela, J

    2009-12-20

    A common vole (Microtus arvalis) population peak in Northern Spain in 2007 was treated with large scale application of chlorophacinone, an anticoagulant rodenticide of the indandione family. Voles found dead and trapped alive were collected in treated and untreated areas. Residues of chlorophacinone were analyzed in liver of voles by HPLC-UV. Also, the presence of the pathogen Francisella tularensis was analyzed by PCR in samples of vole spleen. Chlorophacinone (82-3800 ng/g; wet weight liver) was only detected in voles found dead in treated areas (55.5%). The prevalence of F. tularensis in voles found dead in treated areas was also particularly high (66.7%). Moreover, chlorophacinone levels were lower in voles that were PCR-positive for F. tularensis (geometric mean [95% CI], 418 [143-1219] ng/g) than in those that were PCR-negative (1084 [581-2121] ng/g). Interactions between pathogens and rodenticides might be considered to reduce the doses used in baits or to avoid the use of the more toxic 2nd generation anticoagulant rodenticides.

  11. Seroprevalence of seven pathogens transmitted by the Ixodes ricinus tick in forestry workers in France.

    PubMed

    Rigaud, E; Jaulhac, B; Garcia-Bonnet, N; Hunfeld, K-P; Féménia, F; Huet, D; Goulvestre, C; Vaillant, V; Deffontaines, G; Abadia-Benoist, G

    2016-08-01

    In order to assess the level of occupational exposure to the main pathogens transmitted by the Ixodes ricinus tick, a seroprevalence study was performed on serum samples collected in 2003 from 2975 forestry workers of northeastern France. The global seroprevalence estimated for the seven pathogens studied was 14.1% (419/2975) for Borrelia burgdorferi sl, 5.7% (164/2908) for Francisella tularensis, 2.3% (68/2941) for tick-borne encephalitis virus, 1.7% (50/2908) for Anaplasma phagocytophilum and 1.7% (48/2908) for Bartonella henselae. The seroprevalences of Babesia divergens and Babesia microti studied in a subgroup of participants seropositive for at least one of these latter pathogens were 0.1% (1/810) and 2.5% (20/810), respectively. Borrelia burgdorferi sl seroprevalence was significantly higher in Alsace and Lorraine and F. tularensis seroprevalence was significantly higher in Champagne-Ardenne and Franche-Comté. The results of this survey also suggest low rates of transmission of Bartonella henselae and F. tularensis by ticks and a different west/east distribution of Babesia species in France. The frequency and potential severity of these diseases justify continued promotion of methods of prevention of I. ricinus bites.

  12. Gene hunting in autoinflammation

    PubMed Central

    2013-01-01

    Steady progress in our understanding of the genetic basis of autoinflammatory diseases has been made over the past 16 years. Since the discovery of the familial Mediterranean fever gene MEFV (also known as marenostrin) in 1997, 18 other genes responsible for monogenic autoinflammatory diseases have been identified to date. The discovery of these genes was made through the utilisation of many genetic mapping techniques, including next generation sequencing platforms. This review article clearly describes the gene hunting approaches, methods of data analysis and the technological platforms used, which has relevance to all those working within the field of gene discovery for Mendelian disorders. PMID:24070009

  13. Gene therapy review.

    PubMed

    Moss, Joseph Anthony

    2014-01-01

    The use of genes to treat disease, more commonly known as gene therapy, is a valid and promising tool to manage and treat diseases that conventional drug therapies cannot cure. Gene therapy holds the potential to control a wide range of diseases, including cystic fibrosis, heart disease, diabetes, cancer, and blood diseases. This review assesses the current status of gene therapy, highlighting therapeutic methodologies and applications, terminology, and imaging strategies. This article presents an overview of roadblocks associated with each therapeutic methodology, along with some of the scientific, social, and ethical issues associated with gene therapy.

  14. Regulated Gene Therapy.

    PubMed

    Breger, Ludivine; Wettergren, Erika Elgstrand; Quintino, Luis; Lundberg, Cecilia

    2016-01-01

    Gene therapy represents a promising approach for the treatment of monogenic and multifactorial neurological disorders. It can be used to replace a missing gene and mutated gene or downregulate a causal gene. Despite the versatility of gene therapy, one of the main limitations lies in the irreversibility of the process: once delivered to target cells, the gene of interest is constitutively expressed and cannot be removed. Therefore, efficient, safe and long-term gene modification requires a system allowing fine control of transgene expression.Different systems have been developed over the past decades to regulate transgene expression after in vivo delivery, either at transcriptional or post-translational levels. The purpose of this chapter is to give an overview on current regulatory system used in the context of gene therapy for neurological disorders. Systems using external regulation of transgenes using antibiotics are commonly used to control either gene expression using tetracycline-controlled transcription or protein levels using destabilizing domain technology. Alternatively, specific promoters of genes that are regulated by disease mechanisms, increasing expression as the disease progresses or decreasing expression as disease regresses, are also examined. Overall, this chapter discusses advantages and drawbacks of current molecular methods for regulated gene therapy in the central nervous system.

  15. Gene therapy in periodontics.

    PubMed

    Chatterjee, Anirban; Singh, Nidhi; Saluja, Mini

    2013-03-01

    GENES are made of DNA - the code of life. They are made up of two types of base pair from different number of hydrogen bonds AT, GC which can be turned into instruction. Everyone inherits genes from their parents and passes them on in turn to their children. Every person's genes are different, and the changes in sequence determine the inherited differences between each of us. Some changes, usually in a single gene, may cause serious diseases. Gene therapy is 'the use of genes as medicine'. It involves the transfer of a therapeutic or working gene copy into specific cells of an individual in order to repair a faulty gene copy. Thus it may be used to replace a faulty gene, or to introduce a new gene whose function is to cure or to favorably modify the clinical course of a condition. It has a promising era in the field of periodontics. Gene therapy has been used as a mode of tissue engineering in periodontics. The tissue engineering approach reconstructs the natural target tissue by combining four elements namely: Scaffold, signaling molecules, cells and blood supply and thus can help in the reconstruction of damaged periodontium including cementum, gingival, periodontal ligament and bone.

  16. Genes, dreams, and cancer.

    PubMed

    Sikora, K

    1994-05-07

    There have been tremendous advances in our understanding of cancer from the application of molecular biology over the past decade. The disease is caused by a series of defects in the genes that accelerate growth--oncogenes--and those that slow down cellular turnover--tumour suppressor genes. The proteins they encode provide a promising hunting ground in which to design and test new anticancer drugs. Several treatment strategies are now under clinical trial entailing direct gene transfer. These include the use of gene marking to detect minimal residual disease, the production of novel cancer vaccines by the insertion of genes which uncloak cancer cells so making them visible to the host's immune system, the isolation and coupling of cancer specific molecular switches upstream of drug activating genes, and the correction of aberrant oncogenes or tumour suppressor genes. The issues in these approaches are likely to have a profound impact on the management of cancer patients as we enter the next century.

  17. Conventional murine gene targeting.

    PubMed

    Zimmermann, Albert G; Sun, Yue

    2013-01-01

    Murine gene knockout models engineered over the last two decades have continued to demonstrate their potential as invaluable tools in understanding the role of gene function in the context of normal human development and disease. The more recent elucidation of the human and mouse genomes through sequencing has opened up the capability to elucidate the function of every human gene. State-of-the-art mouse model generation allows, through a multitude of experimental steps requiring careful standardization, gene function to be reliably and predictably ablated in a live model system. The application of these standardized methodologies to directly target gene function through murine gene knockout has to date provided comprehensive and verifiable genetic models that have contributed tremendously to our understanding of the cellular and molecular pathways underlying normal and disease states in humans. The ensuing chapter provides an overview of the latest steps and procedures required to ablate gene function in a murine model.

  18. Primetime for Learning Genes.

    PubMed

    Keifer, Joyce

    2017-02-11

    Learning genes in mature neurons are uniquely suited to respond rapidly to specific environmental stimuli. Expression of individual learning genes, therefore, requires regulatory mechanisms that have the flexibility to respond with transcriptional activation or repression to select appropriate physiological and behavioral responses. Among the mechanisms that equip genes to respond adaptively are bivalent domains. These are specific histone modifications localized to gene promoters that are characteristic of both gene activation and repression, and have been studied primarily for developmental genes in embryonic stem cells. In this review, studies of the epigenetic regulation of learning genes in neurons, particularly the brain-derived neurotrophic factor gene (BDNF), by methylation/demethylation and chromatin modifications in the context of learning and memory will be highlighted. Because of the unique function of learning genes in the mature brain, it is proposed that bivalent domains are a characteristic feature of the chromatin landscape surrounding their promoters. This allows them to be "poised" for rapid response to activate or repress gene expression depending on environmental stimuli.

  19. Human HOX gene disorders.

    PubMed

    Quinonez, Shane C; Innis, Jeffrey W

    2014-01-01

    The Hox genes are an evolutionarily conserved family of genes, which encode a class of important transcription factors that function in numerous developmental processes. Following their initial discovery, a substantial amount of information has been gained regarding the roles Hox genes play in various physiologic and pathologic processes. These processes range from a central role in anterior-posterior patterning of the developing embryo to roles in oncogenesis that are yet to be fully elucidated. In vertebrates there are a total of 39 Hox genes divided into 4 separate clusters. Of these, mutations in 10 Hox genes have been found to cause human disorders with significant variation in their inheritance patterns, penetrance, expressivity and mechanism of pathogenesis. This review aims to describe the various phenotypes caused by germline mutation in these 10 Hox genes that cause a human phenotype, with specific emphasis paid to the genotypic and phenotypic differences between allelic disorders. As clinical whole exome and genome sequencing is increasingly utilized in the future, we predict that additional Hox gene mutations will likely be identified to cause distinct human phenotypes. As the known human phenotypes closely resemble gene-specific murine models, we also review the homozygous loss-of-function mouse phenotypes for the 29 Hox genes without a known human disease. This review will aid clinicians in identifying and caring for patients affected with a known Hox gene disorder and help recognize the potential for novel mutations in patients with phenotypes informed by mouse knockout studies.

  20. Primetime for Learning Genes

    PubMed Central

    Keifer, Joyce

    2017-01-01

    Learning genes in mature neurons are uniquely suited to respond rapidly to specific environmental stimuli. Expression of individual learning genes, therefore, requires regulatory mechanisms that have the flexibility to respond with transcriptional activation or repression to select appropriate physiological and behavioral responses. Among the mechanisms that equip genes to respond adaptively are bivalent domains. These are specific histone modifications localized to gene promoters that are characteristic of both gene activation and repression, and have been studied primarily for developmental genes in embryonic stem cells. In this review, studies of the epigenetic regulation of learning genes in neurons, particularly the brain-derived neurotrophic factor gene (BDNF), by methylation/demethylation and chromatin modifications in the context of learning and memory will be highlighted. Because of the unique function of learning genes in the mature brain, it is proposed that bivalent domains are a characteristic feature of the chromatin landscape surrounding their promoters. This allows them to be “poised” for rapid response to activate or repress gene expression depending on environmental stimuli. PMID:28208656

  1. Do Housekeeping Genes Exist?

    PubMed Central

    Sun, Bingyun

    2015-01-01

    The searching of human housekeeping (HK) genes has been a long quest since the emergence of transcriptomics, and is instrumental for us to understand the structure of genome and the fundamentals of biological processes. The resolved genes are frequently used in evolution studies and as normalization standards in quantitative gene-expression analysis. Within the past 20 years, more than a dozen HK-gene studies have been conducted, yet none of them sampled human tissues completely. We believe an integration of these results will help remove false positive genes owing to the inadequate sampling. Surprisingly, we only find one common gene across 15 examined HK-gene datasets comprising 187 different tissue and cell types. Our subsequent analyses suggest that it might not be appropriate to rigidly define HK genes as expressed in all tissue types that have diverse developmental, physiological, and pathological states. It might be beneficial to use more robustly identified HK functions for filtering criteria, in which the representing genes can be a subset of genome. These genes are not necessarily the same, and perhaps need not to be the same, everywhere in our body. PMID:25970694

  2. Parkinson's disease: gene therapies.

    PubMed

    Coune, Philippe G; Schneider, Bernard L; Aebischer, Patrick

    2012-04-01

    With the recent development of effective gene delivery systems, gene therapy for the central nervous system is finding novel applications. Here, we review existing viral vectors and discuss gene therapy strategies that have been proposed for Parkinson's disease. To date, most of the clinical trials were based on viral vectors to deliver therapeutic transgenes to neurons within the basal ganglia. Initial trials used genes to relieve the major motor symptoms caused by nigrostriatal degeneration. Although these new genetic approaches still need to prove more effective than existing symptomatic treatments, there is a need for disease-modifying strategies. The investigation of the genetic factors implicated in Parkinson's disease is providing precious insights in disease pathology that, combined with innovative gene delivery systems, will hopefully offer novel opportunities for gene therapy interventions to slow down, or even halt disease progression.

  3. Green genes gleaned.

    PubMed

    Beale, Samuel I

    2005-07-01

    A recent paper by Ayumi Tanaka and colleagues identifying an Arabidopsis thaliana gene for 3,8-divinyl(proto)chlorophyllide 8-vinyl reductase brings a satisfying conclusion to the hunt for genes encoding enzymes for the steps in the chlorophyll biosynthetic pathway. Now, at least in angiosperm plants represented by Arabidopsis, genes for all 15 steps in the pathway from glutamyl-tRNA to chlorophylls a and b have been identified.

  4. Gene-Category Analysis.

    PubMed

    Bauer, Sebastian

    2017-01-01

    Gene-category analysis is one important knowledge integration approach in biomedical sciences that combines knowledge bases such as Gene Ontology with lists of genes or their products, which are often the result of high-throughput experiments, gained from either wet-lab or synthetic experiments. In this chapter, we will motivate this class of analyses and describe an often used variant that is based on Fisher's exact test. We show that this approach has some problems in the context of Gene Ontology of which users should be aware. We then describe some more recent algorithms that try to address some of the shortcomings of the standard approach.

  5. Antiangiogenic Eye Gene Therapy.

    PubMed

    Corydon, Thomas J

    2015-08-01

    The idea of treating disease in humans with genetic material was conceived over two decades ago and with that a promising journey involving development and efficacy studies in cells and animals of a large number of novel therapeutic reagents unfolded. In the footsteps of this process, successful gene therapy treatment of genetic conditions in humans has shown clear signs of efficacy. Notably, significant advancements using gene supplementation and silencing strategies have been made in the field of ocular gene therapy, thereby pinpointing ocular gene therapy as one of the compelling "actors" bringing gene therapy to the clinic. Most of all, this success has been facilitated because of (1) the fact that the eye is an effortlessly accessible, exceedingly compartmentalized, and immune-privileged organ offering a unique advantage as a gene therapy target, and (2) significant progress toward efficient, sustained transduction of cells within the retina having been achieved using nonintegrating vectors based on recombinant adeno-associated virus and nonintegrating lentivirus vectors. The results from in vivo experiments and trials suggest that treatment of inherited retinal dystrophies, ocular angiogenesis, and inflammation with gene therapy can be both safe and effective. Here, the progress of ocular gene therapy is examined with special emphasis on the potential use of RNAi- and protein-based antiangiogenic gene therapy to treat exudative age-related macular degeneration.

  6. History of gene therapy.

    PubMed

    Wirth, Thomas; Parker, Nigel; Ylä-Herttuala, Seppo

    2013-08-10

    Two decades after the initial gene therapy trials and more than 1700 approved clinical trials worldwide we not only have gained much new information and knowledge regarding gene therapy in general, but also learned to understand the concern that has persisted in society. Despite the setbacks gene therapy has faced, success stories have increasingly emerged. Examples for these are the positive recommendation for a gene therapy product (Glybera) by the EMA for approval in the European Union and the positive trials for the treatment of ADA deficiency, SCID-X1 and adrenoleukodystrophy. Nevertheless, our knowledge continues to grow and during the course of time more safety data has become available that helps us to develop better gene therapy approaches. Also, with the increased understanding of molecular medicine, we have been able to develop more specific and efficient gene transfer vectors which are now producing clinical results. In this review, we will take a historical view and highlight some of the milestones that had an important impact on the development of gene therapy. We will also discuss briefly the safety and ethical aspects of gene therapy and address some concerns that have been connected with gene therapy as an important therapeutic modality.

  7. Towards Consensus Gene Ages

    PubMed Central

    Liebeskind, Benjamin J.; McWhite, Claire D.; Marcotte, Edward M.

    2016-01-01

    Correctly estimating the age of a gene or gene family is important for a variety of fields, including molecular evolution, comparative genomics, and phylogenetics, and increasingly for systems biology and disease genetics. However, most studies use only a point estimate of a gene’s age, neglecting the substantial uncertainty involved in this estimation. Here, we characterize this uncertainty by investigating the effect of algorithm choice on gene-age inference and calculate consensus gene ages with attendant error distributions for a variety of model eukaryotes. We use 13 orthology inference algorithms to create gene-age datasets and then characterize the error around each age-call on a per-gene and per-algorithm basis. Systematic error was found to be a large factor in estimating gene age, suggesting that simple consensus algorithms are not enough to give a reliable point estimate. We also found that different sources of error can affect downstream analyses, such as gene ontology enrichment. Our consensus gene-age datasets, with associated error terms, are made fully available at so that researchers can propagate this uncertainty through their analyses (geneages.org). PMID:27259914

  8. Cell and gene therapy.

    PubMed

    Rao, Rajesh C; Zacks, David N

    2014-01-01

    Replacement or repair of a dysfunctional gene combined with promoting cell survival is a two-pronged approach that addresses an unmet need in the therapy of retinal degenerative diseases. In this chapter, we discuss various strategies toward achieving both goals: transplantation of wild-type cells to replace degenerating cells and to rescue gene function, sequential gene and cell therapy, and in vivo reprogramming of rods to cones. These approaches highlight cutting-edge advances in cell and gene therapy, and cellular lineage conversion in order to devise new therapies for various retinal degenerative diseases.

  9. Smart Genes, Stupid Science.

    ERIC Educational Resources Information Center

    Randerson, Sherman; Mahadeva, Madhu N.

    1983-01-01

    Because many people still believe that specific, identifiable genes dictate the level of human intelligence and that the number/quality of these genes can be evaluated, presents evidence from human genetics (related to nervous system development) to counter this view. Also disputes erroneous assumptions made in "heritability studies" of human…

  10. Genes, genome and Gestalt.

    PubMed

    Grisolia, Cesar Koppe

    2005-03-31

    According to Gestalt thinking, biological systems cannot be viewed as the sum of their elements, but as processes of the whole. To understand organisms we must start from the whole, observing how the various parts are related. In genetics, we must observe the genome over and above the sum of its genes. Either loss or addition of one gene in a genome can change the function of the organism. Genomes are organized in networks of genes, which need to be well integrated. In the case of genetically modified organisms (GMOs), for example, soybeans, rats, Anopheles mosquitoes, and pigs, the insertion of an exogenous gene into a receptive organism generally causes disturbance in the networks, resulting in the breakdown of gene interactions. In these cases, genetic modification increased the genetic load of the GMO and consequently decreased its adaptability (fitness). Therefore, it is hard to claim that the production of such organisms with an increased genetic load does not have ethical implications.

  11. [Gene therapy and ethics].

    PubMed

    Müller, H; Rehmann-Sutter, C

    1995-01-10

    Gene therapy represents a new strategy to treat human disorders. It was originally conceived as a cure for severe monogenetic disorders. Since its conception, the spectrum of possible application for gene therapy has been to include the treatment of acquired diseases, such as various forms of cancer and some viral infections, most notably human immune deficiency virus (HIV) and hepatitis B virus. Since somatic gene therapy does not cause substantially new ethical problems, it has gained broad approval. This is by no means the case with germ-line gene therapy. Practically all bodies who were evaluating the related ethical aspects wanted to ban its medical application on grounds of fundamental and pragmatic considerations. In this review, practical and ethical views concerning gene therapy are summarized which were presented at the "Junitagung 1994" of the Swiss Society for Biomedical Ethics in Basle.

  12. Introduction and persistence of tularemia in Bulgaria

    PubMed Central

    Myrtennäs, Kerstin; Marinov, Krustyu; Johansson, Anders; Niemcewicz, Marcin; Karlsson, Edvin; Byström, Mona; Forsman, Mats

    2016-01-01

    Introduction Outbreaks of the zoonotic disease tularemia occurred in north-east Bulgaria in the 1960s. Then came 30 years of epidemiological silence until new outbreaks occurred in west Bulgaria in the 1990s. To investigate how bacterial strains of Francisella tularensis causing tularemia in wildlife and humans in the 1960s and the 1990s were related, we explored their genetic diversity. Material and methods Ten F. tularensis genomes from the 1960s (n=3) and the 1990s (n=7) were sequenced, assigned to canonical single-nucleotide polymorphism (canSNP) clades, and compared to reference genomes. We developed four new canSNP polymerase chain reaction (PCR) assays based on the genome sequence information. Results and discussion The genetic analysis showed that the outbreaks in the 1960s as well as in the 1990s involved multiple clones and new genetic diversity. The smallest genetic difference found between any of the Bulgarian strains was five SNPs between the strains L2 and 81 isolated 43 years apart, indicating that F. tularensis may persist locally over long time periods without causing outbreaks. The existence of genetically highly similar strain-pairs isolated the same year in the same area from different hosts supports a hypothesis of local expansion of clones during outbreaks. Close relationship (two SNPs) was found between one strain isolated 1961 in northeast Bulgaria and one strain isolated 5 years before in USSR. Historical data coinciding with the actual time point describe the introduction of water rats from USSR into the Bulgarian outbreak area, which may explain the close genetic relationship and the origin of the outbreak. Conclusion Genome analysis of strains from two outbreaks in the 1960s and the 1990s provided valuable information on the genetic diversity and persistence of F. tularensis in Bulgaria. PMID:27790972

  13. Phenotypic, Morphological, and Functional Heterogeneity of Splenic Immature Myeloid Cells in the Host Response to Tularemia

    PubMed Central

    Rasmussen, John W.; Tam, Jason W.; Okan, Nihal A.; Mena, Patricio; Furie, Martha B.; Thanassi, David G.; Benach, Jorge L.

    2012-01-01

    Recent studies have linked accumulation of the Gr-1+ CD11b+ cell phenotype with functional immunosuppression in diverse pathological conditions, including bacterial and parasitic infections and cancer. Gr-1+ CD11b+ cells were the largest population of cells present in the spleens of mice infected with sublethal doses of the Francisella tularensis live vaccine strain (LVS). In contrast, the number of T cells present in the spleens of these mice did not increase during early infection. There was a significant delay in the kinetics of accumulation of Gr-1+ CD11b+ cells in the spleens of B-cell-deficient mice, indicating that B cells play a role in recruitment and maintenance of this population in the spleens of mice infected with F. tularensis. The splenic Gr-1+ CD11b+ cells in tularemia were a heterogeneous population that could be further subdivided into monocytic (mononuclear) and granulocytic (polymorphonuclear) cells using the Ly6C and Ly6G markers and differentiated into antigen-presenting cells following ex vivo culture. Monocytic, CD11b+ Ly6Chi Ly6G− cells but not granulocytic, CD11b+ Ly6Cint Ly6G+ cells purified from the spleens of mice infected with F. tularensis suppressed polyclonal T-cell proliferation via a nitric oxide-dependent pathway. Although the monocytic, CD11b+ Ly6Chi Ly6G− cells were able to suppress the proliferation of T cells, the large presence of Gr-1+ CD11b+ cells in mice that survived F. tularensis infection also suggests a potential role for these cells in the protective host response to tularemia. PMID:22526678

  14. 4. AERIAL VIEW OF GENE WASH RESERVOIR AND GENE CAMP ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    4. AERIAL VIEW OF GENE WASH RESERVOIR AND GENE CAMP LOOKING SOUTHWEST. DAM AND SPILLWAY VISIBLE IN BOTTOM OF PHOTO. - Gene Wash Reservoir & Dam, 2 miles west of Parker Dam, Parker Dam, San Bernardino County, CA

  15. Fecundity genes in sheep.

    PubMed

    Davis, G H

    2004-07-01

    Since 1980 there has been increasing interest in the identification and utilisation of major genes for prolificacy in sheep. Mutations that increase ovulation rate have been discovered in the BMPR-1B, BMP15 and GDF9 genes, and others are known to exist from the expressed inheritance patterns although the mutations have not yet been located. In the case of BMP15, four different mutations have been discovered but each produces the same phenotype. The modes of inheritance of the different prolificacy genes include autosomal dominant genes with additive effects on ovulation rate (BMPR-1B; Lacaune), autosomal over-dominant genes with infertility in homozygous females (GDF9), X-linked over-dominant genes with infertility in homozygous females (BMP15), and X-linked maternally imprinted genes (FecX2). The size of the effect of one copy of a mutation on ovulation rate ranges from an extra 0.4 ovulations per oestrus for the FecX2 mutation to an extra 1.5 ovulations per oestrus for the BMPR-1B mutation. DNA tests enable some of these mutations to be used in genetic improvement programmes based on marker assisted selection.

  16. Gene therapy for hemophilia.

    PubMed

    Chuah, M K; Evens, H; VandenDriessche, T

    2013-06-01

    Hemophilia A and B are X-linked monogenic disorders resulting from deficiencies of factor VIII and FIX, respectively. Purified clotting factor concentrates are currently intravenously administered to treat hemophilia, but this treatment is non-curative. Therefore, gene-based therapies for hemophilia have been developed to achieve sustained high levels of clotting factor expression to correct the clinical phenotype. Over the past two decades, different types of viral and non-viral gene delivery systems have been explored for hemophilia gene therapy research with a variety of target cells, particularly hepatocytes, hematopoietic stem cells, skeletal muscle cells, and endothelial cells. Lentiviral and adeno-associated virus (AAV)-based vectors are among the most promising vectors for hemophilia gene therapy. In preclinical hemophilia A and B animal models, the bleeding phenotype was corrected with these vectors. Some of these promising preclinical results prompted clinical translation to patients suffering from a severe hemophilic phenotype. These patients receiving gene therapy with AAV vectors showed long-term expression of therapeutic FIX levels, which is a major step forwards in this field. Nevertheless, the levels were insufficient to prevent trauma or injury-induced bleeding episodes. Another challenge that remains is the possible immune destruction of gene-modified cells by effector T cells, which are directed against the AAV vector antigens. It is therefore important to continuously improve the current gene therapy approaches to ultimately establish a real cure for hemophilia.

  17. Differentially Coexpressed Disease Gene Identification Based on Gene Coexpression Network.

    PubMed

    Jiang, Xue; Zhang, Han; Quan, Xiongwen

    2016-01-01

    Screening disease-related genes by analyzing gene expression data has become a popular theme. Traditional disease-related gene selection methods always focus on identifying differentially expressed gene between case samples and a control group. These traditional methods may not fully consider the changes of interactions between genes at different cell states and the dynamic processes of gene expression levels during the disease progression. However, in order to understand the mechanism of disease, it is important to explore the dynamic changes of interactions between genes in biological networks at different cell states. In this study, we designed a novel framework to identify disease-related genes and developed a differentially coexpressed disease-related gene identification method based on gene coexpression network (DCGN) to screen differentially coexpressed genes. We firstly constructed phase-specific gene coexpression network using time-series gene expression data and defined the conception of differential coexpression of genes in coexpression network. Then, we designed two metrics to measure the value of gene differential coexpression according to the change of local topological structures between different phase-specific networks. Finally, we conducted meta-analysis of gene differential coexpression based on the rank-product method. Experimental results demonstrated the feasibility and effectiveness of DCGN and the superior performance of DCGN over other popular disease-related gene selection methods through real-world gene expression data sets.

  18. Differentially Coexpressed Disease Gene Identification Based on Gene Coexpression Network

    PubMed Central

    Quan, Xiongwen

    2016-01-01

    Screening disease-related genes by analyzing gene expression data has become a popular theme. Traditional disease-related gene selection methods always focus on identifying differentially expressed gene between case samples and a control group. These traditional methods may not fully consider the changes of interactions between genes at different cell states and the dynamic processes of gene expression levels during the disease progression. However, in order to understand the mechanism of disease, it is important to explore the dynamic changes of interactions between genes in biological networks at different cell states. In this study, we designed a novel framework to identify disease-related genes and developed a differentially coexpressed disease-related gene identification method based on gene coexpression network (DCGN) to screen differentially coexpressed genes. We firstly constructed phase-specific gene coexpression network using time-series gene expression data and defined the conception of differential coexpression of genes in coexpression network. Then, we designed two metrics to measure the value of gene differential coexpression according to the change of local topological structures between different phase-specific networks. Finally, we conducted meta-analysis of gene differential coexpression based on the rank-product method. Experimental results demonstrated the feasibility and effectiveness of DCGN and the superior performance of DCGN over other popular disease-related gene selection methods through real-world gene expression data sets. PMID:28042568

  19. Genes and social behavior.

    PubMed

    Robinson, Gene E; Fernald, Russell D; Clayton, David F

    2008-11-07

    What genes and regulatory sequences contribute to the organization and functioning of neural circuits and molecular pathways in the brain that support social behavior? How does social experience interact with information in the genome to modulate brain activity? Here, we address these questions by highlighting progress that has been made in identifying and understanding two key "vectors of influence" that link genes, the brain, and social behavior: (i) Social information alters gene expression in the brain to influence behavior, and (ii) genetic variation influences brain function and social behavior. We also discuss how evolutionary changes in genomic elements influence social behavior and outline prospects for a systems biology of social behavior.

  20. "Bad genes" & criminal responsibility.

    PubMed

    González-Tapia, María Isabel; Obsuth, Ingrid

    2015-01-01

    The genetics of the accused is trying to break into the courts. To date several candidate genes have been put forward and their links to antisocial behavior have been examined and documented with some consistency. In this paper, we focus on the so called "warrior gene", or the low-activity allele of the MAOA gene, which has been most consistently related to human behavior and specifically to violence and antisocial behavior. In preparing this paper we had two objectives. First, to summarize and analyze the current scientific evidence, in order to gain an in depth understanding of the state of the issue and determine whether a dominant line of generally accepted scientific knowledge in this field can be asserted. Second, to derive conclusions and put forward recommendations related to the use of genetic information, specifically the presence of the low-activity genotype of the MAOA gene, in modulation of criminal responsibility in European and US courts.

  1. Genes underlying altruism.

    PubMed

    Thompson, Graham J; Hurd, Peter L; Crespi, Bernard J

    2013-01-01

    William D. Hamilton postulated the existence of 'genes underlying altruism', under the rubric of inclusive fitness theory, a half-century ago. Such genes are now poised for discovery. In this article, we develop a set of intuitive criteria for the recognition and analysis of genes for altruism and describe the first candidate genes affecting altruism from social insects and humans. We also provide evidence from a human population for genetically based trade-offs, underlain by oxytocin-system polymorphisms, between alleles for altruism and alleles for non-social cognition. Such trade-offs between self-oriented and altruistic behaviour may influence the evolution of phenotypic diversity across all social animals.

  2. Clock genes and sleep.

    PubMed

    Landgraf, Dominic; Shostak, Anton; Oster, Henrik

    2012-01-01

    In most species--from cyanobacteria to humans--endogenous clocks have evolved that drive 24-h rhythms of behavior and physiology. In mammals, these circadian rhythms are regulated by a hierarchical network of cellular oscillators controlled by a set of clock genes organized in a system of interlocked transcriptional feedback loops. One of the most prominent outputs of the circadian system is the synchronization of the sleep-wake cycle with external (day-) time. Clock genes also have a strong impact on many other biological functions, such as memory formation, energy metabolism, and immunity. Remarkably, large overlaps exist between clock gene and sleep (loss) mediated effects on these processes. This review summarizes sleep clock gene interactions for these three phenomena, highlighting potential mediators linking sleep and/or clock function to physiological output in an attempt to better understand the complexity of diurnal adaptation and its consequences for health and disease.

  3. GeneLab

    NASA Technical Reports Server (NTRS)

    Berrios, Daniel C.; Thompson, Terri G.

    2015-01-01

    NASA GeneLab is expected to capture and distribute omics data and experimental and process conditions most relevant to research community in their statistical and theoretical analysis of NASAs omics data.

  4. Evolutionary Fingerprinting of Genes

    PubMed Central

    Kosakovsky Pond, Sergei L.; Scheffler, Konrad; Gravenor, Michael B.; Poon, Art F.Y.; Frost, Simon D.W.

    2010-01-01

    Over time, natural selection molds every gene into a unique mosaic of sites evolving rapidly or resisting change—an “evolutionary fingerprint” of the gene. Aspects of this evolutionary fingerprint, such as the site-specific ratio of nonsynonymous to synonymous substitution rates (dN/dS), are commonly used to identify genetic features of potential biological interest; however, no framework exists for comparing evolutionary fingerprints between genes. We hypothesize that protein-coding genes with similar protein structure and/or function tend to have similar evolutionary fingerprints and that comparing evolutionary fingerprints can be useful for discovering similarities between genes in a way that is analogous to, but independent of, discovery of similarity via sequence-based comparison tools such as Blast. To test this hypothesis, we develop a novel model of coding sequence evolution that uses a general bivariate discrete parameterization of the evolutionary rates. We show that this approach provides a better fit to the data using a smaller number of parameters than existing models. Next, we use the model to represent evolutionary fingerprints as probability distributions and present a methodology for comparing these distributions in a way that is robust against variations in data set size and divergence. Finally, using sequences of three rapidly evolving RNA viruses (HIV-1, hepatitis C virus, and influenza A virus), we demonstrate that genes within the same functional group tend to have similar evolutionary fingerprints. Our framework provides a sound statistical foundation for efficient inference and comparison of evolutionary rate patterns in arbitrary collections of gene alignments, clustering homologous and nonhomologous genes, and investigation of biological and functional correlates of evolutionary rates. PMID:19864470

  5. Cystic fibrosis modifier genes.

    PubMed Central

    Davies, Jane; Alton, Eric; Griesenbach, Uta

    2005-01-01

    Since the recognition that CFTR genotype was not a good predictor of pulmonary disease severity in CF, several candidate modifier genes have been identified. It is unlikely that a single modifier gene will be found, but more probable that several haplotypes in combination may contribute, which in itself presents a major methodological challenge. The aims of such studies are to increase our understanding of disease pathogenesis, to aid prognosis and ultimately to lead to the development of novel treatments. PMID:16025767

  6. Evidence for homosexuality gene

    SciTech Connect

    Pool, R.

    1993-07-16

    A genetic analysis of 40 pairs of homosexual brothers has uncovered a region on the X chromosome that appears to contain a gene or genes for homosexuality. When analyzing the pedigrees of homosexual males, the researcheres found evidence that the trait has a higher likelihood of being passed through maternal genes. This led them to search the X chromosome for genes predisposing to homosexuality. The researchers examined the X chromosomes of pairs of homosexual brothers for regions of DNA that most or all had in common. Of the 40 sets of brothers, 33 shared a set of five markers in the q28 region of the long arm of the X chromosome. The linkage has a LOD score of 4.0, which translates into a 99.5% certainty that there is a gene or genes in this area that predispose males to homosexuality. The chief researcher warns, however, that this one site cannot explain all instances of homosexuality, since there were some cases where the trait seemed to be passed paternally. And even among those brothers where there was no evidence that the trait was passed paternally, seven sets of brothers did not share the Xq28 markers. It seems likely that homosexuality arises from a variety of causes.

  7. GeneClinics

    PubMed Central

    Tarczy-Hornoch, Peter; Shannon, Paul; Baskin, Patty; Espeseth, Miriam; Pagon, Roberta A.

    2000-01-01

    GeneClinics is an online genetic information resource consisting of descriptions of specific inherited disorders (“disease profiles”) as well as information on the role of genetic testing in the diagnosis, management, and genetic counseling of patients with these inherited conditions. GeneClinics is intended to promote the use of genetic services in medical care and personal decision making by providing health care practitioners and patients with information on genetic testing for specific inherited disorders. GeneClinics is implemented as an object-oriented database containing a combination of data and semistructured text that is rendered as HTML for publishing a given “disease profile” on the Web. Content is acquired from authors via templates, converted to an XML document reflecting the underlying database schema (with tagging of embedded data), and then loaded into the database and subjected to peer review. The initial implementation of a production system and the first phase of population of the GeneClinics database content are complete. Further expansion of the content to cover more disease, significant scaling up of rate of content creation, and evaluation redesign are under way. The ultimate goal is to have an entry in GeneClinics for each entry in the GeneTests directory of medical genetics laboratories—that is, for each disease for which clinical genetic testing is available. PMID:10833163

  8. Gene therapy for newborns.

    PubMed

    Kohn, D B; Parkman, R

    1997-07-01

    Application of gene therapy to treat genetic and infectious diseases may have several advantages if performed in newborns. Because of the minimal adverse effect of the underlying disease on cells of the newborn, the relatively small size of infants, and the large amount of future growth, gene therapy may be more successful in newborns than in older children or adults. The presence of umbilical cord blood from newborns provides a unique and susceptible target for the genetic modification of hematopoietic stem cells. In our first trial of gene therapy in newborns, we inserted a normal adenosine deaminase gene into umbilical cord blood cells of three neonates with a congenital immune deficiency. The trial demonstrated the successful transduction and engraftment of stem cells, which continue to contribute to leukocyte production more than 3 years later. A similar approach may be taken to insert genes that inhibit replication of HIV-1 into umbilical cord blood cells of HIV-1-infected neonates. Many other metabolic and infectious disorders could be treated by gene therapy during the neonatal period if prenatal diagnoses are made and the appropriate technical and regulatory requirements have been met.

  9. Gene indexing: characterization and analysis of NLM's GeneRIFs.

    PubMed

    Mitchell, Joyce A; Aronson, Alan R; Mork, James G; Folk, Lillian C; Humphrey, Susanne M; Ward, Janice M

    2003-01-01

    We present an initial analysis of the National Library of Medicine's (NLM) Gene Indexing initiative. Gene Indexing occurs at the time of indexing for all 4600 journals and over 500,000 articles added to PubMed/MEDLINE each year. Gene Indexing links articles about the basic biology of a gene or protein within eight model organisms to a specific record in the NLM's LocusLink database of gene products. The result is an entry called a Gene Reference Into Function (GeneRIF) within the LocusLink database. We analyzed the numbers of GeneRIFs produced in the first year of GeneRIF production. 27,645 GeneRIFs were produced, pertaining to 9126 loci over eight model organisms. 60% of these were associated with human genes and 27% with mouse genes. About 80% discuss genes with an established MeSH Heading or other MeSH term. We developed a prototype functional alerting system for researchers based on the GeneRIFs, and a strategy to find all of the literature related to genes. We conclude that the Gene Indexing initiative adds considerable value to the life sciences research community.

  10. Harnessing gene expression networks to prioritize candidate epileptic encephalopathy genes.

    PubMed

    Oliver, Karen L; Lukic, Vesna; Thorne, Natalie P; Berkovic, Samuel F; Scheffer, Ingrid E; Bahlo, Melanie

    2014-01-01

    We apply a novel gene expression network analysis to a cohort of 182 recently reported candidate Epileptic Encephalopathy genes to identify those most likely to be true Epileptic Encephalopathy genes. These candidate genes were identified as having single variants of likely pathogenic significance discovered in a large-scale massively parallel sequencing study. Candidate Epileptic Encephalopathy genes were prioritized according to their co-expression with 29 known Epileptic Encephalopathy genes. We utilized developing brain and adult brain gene expression data from the Allen Human Brain Atlas (AHBA) and compared this to data from Celsius: a large, heterogeneous gene expression data warehouse. We show replicable prioritization results using these three independent gene expression resources, two of which are brain-specific, with small sample size, and the third derived from a heterogeneous collection of tissues with large sample size. Of the nineteen genes that we predicted with the highest likelihood to be true Epileptic Encephalopathy genes, two (GNAO1 and GRIN2B) have recently been independently reported and confirmed. We compare our results to those produced by an established in silico prioritization approach called Endeavour, and finally present gene expression networks for the known and candidate Epileptic Encephalopathy genes. This highlights sub-networks of gene expression, particularly in the network derived from the adult AHBA gene expression dataset. These networks give clues to the likely biological interactions between Epileptic Encephalopathy genes, potentially highlighting underlying mechanisms and avenues for therapeutic targets.

  11. 5. OVERHEAD VIEW OF GENE CAMP LOOKING SOUTH. GENE PUMP ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    5. OVERHEAD VIEW OF GENE CAMP LOOKING SOUTH. GENE PUMP PLANT IS AT CENTER WITH ADMINISTRATIVE COMPLEX IN FOREGROUND AND RESIDENTIAL AREA BEYOND PLANT. - Gene Pump Plant, South of Gene Wash Reservoir, 2 miles west of Whitsett Pump Plant, Parker Dam, San Bernardino County, CA

  12. Classification of genes based on gene expression analysis

    SciTech Connect

    Angelova, M. Myers, C. Faith, J.

    2008-05-15

    Systems biology and bioinformatics are now major fields for productive research. DNA microarrays and other array technologies and genome sequencing have advanced to the point that it is now possible to monitor gene expression on a genomic scale. Gene expression analysis is discussed and some important clustering techniques are considered. The patterns identified in the data suggest similarities in the gene behavior, which provides useful information for the gene functionalities. We discuss measures for investigating the homogeneity of gene expression data in order to optimize the clustering process. We contribute to the knowledge of functional roles and regulation of E. coli genes by proposing a classification of these genes based on consistently correlated genes in expression data and similarities of gene expression patterns. A new visualization tool for targeted projection pursuit and dimensionality reduction of gene expression data is demonstrated.

  13. GeneCards Version 3: the human gene integrator.

    PubMed

    Safran, Marilyn; Dalah, Irina; Alexander, Justin; Rosen, Naomi; Iny Stein, Tsippi; Shmoish, Michael; Nativ, Noam; Bahir, Iris; Doniger, Tirza; Krug, Hagit; Sirota-Madi, Alexandra; Olender, Tsviya; Golan, Yaron; Stelzer, Gil; Harel, Arye; Lancet, Doron

    2010-08-05

    GeneCards (www.genecards.org) is a comprehensive, authoritative compendium of annotative information about human genes, widely used for nearly 15 years. Its gene-centric content is automatically mined and integrated from over 80 digital sources, resulting in a web-based deep-linked card for each of >73,000 human gene entries, encompassing the following categories: protein coding, pseudogene, RNA gene, genetic locus, cluster and uncategorized. We now introduce GeneCards Version 3, featuring a speedy and sophisticated search engine and a revamped, technologically enabling infrastructure, catering to the expanding needs of biomedical researchers. A key focus is on gene-set analyses, which leverage GeneCards' unique wealth of combinatorial annotations. These include the GeneALaCart batch query facility, which tabulates user-selected annotations for multiple genes and GeneDecks, which identifies similar genes with shared annotations, and finds set-shared annotations by descriptor enrichment analysis. Such set-centric features address a host of applications, including microarray data analysis, cross-database annotation mapping and gene-disorder associations for drug targeting. We highlight the new Version 3 database architecture, its multi-faceted search engine, and its semi-automated quality assurance system. Data enhancements include an expanded visualization of gene expression patterns in normal and cancer tissues, an integrated alternative splicing pattern display, and augmented multi-source SNPs and pathways sections. GeneCards now provides direct links to gene-related research reagents such as antibodies, recombinant proteins, DNA clones and inhibitory RNAs and features gene-related drugs and compounds lists. We also portray the GeneCards Inferred Functionality Score annotation landscape tool for scoring a gene's functional information status. Finally, we delineate examples of applications and collaborations that have benefited from the GeneCards suite. Database

  14. Neighboring Genes Show Correlated Evolution in Gene Expression.

    PubMed

    Ghanbarian, Avazeh T; Hurst, Laurence D

    2015-07-01

    When considering the evolution of a gene's expression profile, we commonly assume that this is unaffected by its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between neighboring genes in gene expression profiles in extant taxa. Indeed, in all eukaryotic genomes genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their expression or is gene expression evolution autonomous? To address this here we consider evolution of human gene expression since the human-chimp common ancestor, allowing for both variation in estimation of current expression level and error in Bayesian estimation of the ancestral state. We find that in all tissues and both sexes, the change in gene expression of a focal gene on average predicts the change in gene expression of neighbors. The effect is highly pronounced in the immediate vicinity (<100 kb) but extends much further. Sex-specific expression change is also genomically clustered. As genes increasing their expression in humans tend to avoid nuclear lamina domains and be enriched for the gene activator 5-hydroxymethylcytosine, we conclude that, most probably owing to chromatin level control of gene expression, a change in gene expression of one gene likely affects the expression evolution of neighbors, what we term expression piggybacking, an analog of hitchhiking.

  15. Hox genes and evolution.

    PubMed

    Hrycaj, Steven M; Wellik, Deneen M

    2016-01-01

    Hox proteins are a deeply conserved group of transcription factors originally defined for their critical roles in governing segmental identity along the antero-posterior (AP) axis in Drosophila. Over the last 30 years, numerous data generated in evolutionarily diverse taxa have clearly shown that changes in the expression patterns of these genes are closely associated with the regionalization of the AP axis, suggesting that Hox genes have played a critical role in the evolution of novel body plans within Bilateria. Despite this deep functional conservation and the importance of these genes in AP patterning, key questions remain regarding many aspects of Hox biology. In this commentary, we highlight recent reports that have provided novel insight into the origins of the mammalian Hox cluster, the role of Hox genes in the generation of a limbless body plan, and a novel putative mechanism in which Hox genes may encode specificity along the AP axis. Although the data discussed here offer a fresh perspective, it is clear that there is still much to learn about Hox biology and the roles it has played in the evolution of the Bilaterian body plan.

  16. Selenoprotein Gene Nomenclature.

    PubMed

    Gladyshev, Vadim N; Arnér, Elias S; Berry, Marla J; Brigelius-Flohé, Regina; Bruford, Elspeth A; Burk, Raymond F; Carlson, Bradley A; Castellano, Sergi; Chavatte, Laurent; Conrad, Marcus; Copeland, Paul R; Diamond, Alan M; Driscoll, Donna M; Ferreiro, Ana; Flohé, Leopold; Green, Fiona R; Guigó, Roderic; Handy, Diane E; Hatfield, Dolph L; Hesketh, John; Hoffmann, Peter R; Holmgren, Arne; Hondal, Robert J; Howard, Michael T; Huang, Kaixun; Kim, Hwa-Young; Kim, Ick Young; Köhrle, Josef; Krol, Alain; Kryukov, Gregory V; Lee, Byeong Jae; Lee, Byung Cheon; Lei, Xin Gen; Liu, Qiong; Lescure, Alain; Lobanov, Alexei V; Loscalzo, Joseph; Maiorino, Matilde; Mariotti, Marco; Sandeep Prabhu, K; Rayman, Margaret P; Rozovsky, Sharon; Salinas, Gustavo; Schmidt, Edward E; Schomburg, Lutz; Schweizer, Ulrich; Simonović, Miljan; Sunde, Roger A; Tsuji, Petra A; Tweedie, Susan; Ursini, Fulvio; Whanger, Philip D; Zhang, Yan

    2016-11-11

    The human genome contains 25 genes coding for selenocysteine-containing proteins (selenoproteins). These proteins are involved in a variety of functions, most notably redox homeostasis. Selenoprotein enzymes with known functions are designated according to these functions: TXNRD1, TXNRD2, and TXNRD3 (thioredoxin reductases), GPX1, GPX2, GPX3, GPX4, and GPX6 (glutathione peroxidases), DIO1, DIO2, and DIO3 (iodothyronine deiodinases), MSRB1 (methionine sulfoxide reductase B1), and SEPHS2 (selenophosphate synthetase 2). Selenoproteins without known functions have traditionally been denoted by SEL or SEP symbols. However, these symbols are sometimes ambiguous and conflict with the approved nomenclature for several other genes. Therefore, there is a need to implement a rational and coherent nomenclature system for selenoprotein-encoding genes. Our solution is to use the root symbol SELENO followed by a letter. This nomenclature applies to SELENOF (selenoprotein F, the 15-kDa selenoprotein, SEP15), SELENOH (selenoprotein H, SELH, C11orf31), SELENOI (selenoprotein I, SELI, EPT1), SELENOK (selenoprotein K, SELK), SELENOM (selenoprotein M, SELM), SELENON (selenoprotein N, SEPN1, SELN), SELENOO (selenoprotein O, SELO), SELENOP (selenoprotein P, SeP, SEPP1, SELP), SELENOS (selenoprotein S, SELS, SEPS1, VIMP), SELENOT (selenoprotein T, SELT), SELENOV (selenoprotein V, SELV), and SELENOW (selenoprotein W, SELW, SEPW1). This system, approved by the HUGO Gene Nomenclature Committee, also resolves conflicting, missing, and ambiguous designations for selenoprotein genes and is applicable to selenoproteins across vertebrates.

  17. Engineered Gene Circuits

    NASA Astrophysics Data System (ADS)

    Hasty, Jeff

    2003-03-01

    Uncovering the structure and function of gene regulatory networks has become one of the central challenges of the post-genomic era. Theoretical models of protein-DNA feedback loops and gene regulatory networks have long been proposed, and recently, certain qualitative features of such models have been experimentally corroborated. This talk will focus on model and experimental results that demonstrate how a naturally occurring gene network can be used as a ``parts list'' for synthetic network design. The model formulation leads to computational and analytical approaches relevant to nonlinear dynamics and statistical physics, and the utility of such a formulation will be demonstrated through the consideration of specific design criteria for several novel genetic devices. Fluctuations originating from small molecule-number effects will be discussed in the context of model predictions, and the experimental validation of these stochastic effects underscores the importance of internal noise in gene expression. Potential biotech applications will be highlighted within the framework of cellular control schemes. Specifically, the coupling of an oscillating cellular process to a synthetic oscillator will be considered, and the resulting model behavior will be analyzed in the context of synchronization. The underlying methodology highlights the utility of engineering-based methods in the design of synthetic gene regulatory networks.

  18. Hox genes and evolution

    PubMed Central

    Hrycaj, Steven M.; Wellik, Deneen M.

    2016-01-01

    Hox proteins are a deeply conserved group of transcription factors originally defined for their critical roles in governing segmental identity along the antero-posterior (AP) axis in Drosophila. Over the last 30 years, numerous data generated in evolutionarily diverse taxa have clearly shown that changes in the expression patterns of these genes are closely associated with the regionalization of the AP axis, suggesting that Hox genes have played a critical role in the evolution of novel body plans within Bilateria. Despite this deep functional conservation and the importance of these genes in AP patterning, key questions remain regarding many aspects of Hox biology. In this commentary, we highlight recent reports that have provided novel insight into the origins of the mammalian Hox cluster, the role of Hox genes in the generation of a limbless body plan, and a novel putative mechanism in which Hox genes may encode specificity along the AP axis. Although the data discussed here offer a fresh perspective, it is clear that there is still much to learn about Hox biology and the roles it has played in the evolution of the Bilaterian body plan. PMID:27239281

  19. Gene therapy prospects--intranasal delivery of therapeutic genes.

    PubMed

    Podolska, Karolina; Stachurska, Anna; Hajdukiewicz, Karolina; Małecki, Maciej

    2012-01-01

    Gene therapy is recognized to be a novel method for the treatment of various disorders. Gene therapy strategies involve gene manipulation on broad biological processes responsible for the spreading of diseases. Cancer, monogenic diseases, vascular and infectious diseases are the main targets of gene therapy. In order to obtain valuable experimental and clinical results, sufficient gene transfer methods are required. Therapeutic genes can be administered into target tissues via gene carriers commonly defined as vectors. The retroviral, adenoviral and adeno-associated virus based vectors are most frequently used in the clinic. So far, gene preparations may be administered directly into target organs or by intravenous, intramuscular, intratumor or intranasal injections. It is common knowledge that the number of gene therapy clinical trials has rapidly increased. However, some limitations such as transfection efficiency and stable and long-term gene expression are still not resolved. Consequently, great effort is focused on the evaluation of new strategies of gene delivery. There are many expectations associated with intranasal delivery of gene preparations for the treatment of diseases. Intranasal delivery of therapeutic genes is regarded as one of the most promising forms of pulmonary gene therapy research. Gene therapy based on inhalation of gene preparations offers an alternative way for the treatment of patients suffering from such lung diseases as cystic fibrosis, alpha-1-antitrypsin defect, or cancer. Experimental and first clinical trials based on plasmid vectors or recombinant viruses have revealed that gene preparations can effectively deliver therapeutic or marker genes to the cells of the respiratory tract. The noninvasive intranasal delivery of gene preparations or conventional drugs seems to be very encouraging, although basic scientific research still has to continue.

  20. FunGene: the functional gene pipeline and repository

    PubMed Central

    Fish, Jordan A.; Chai, Benli; Wang, Qiong; Sun, Yanni; Brown, C. Titus; Tiedje, James M.; Cole, James R.

    2013-01-01

    Ribosomal RNA genes have become the standard molecular markers for microbial community analysis for good reasons, including universal occurrence in cellular organisms, availability of large databases, and ease of rRNA gene region amplification and analysis. As markers, however, rRNA genes have some significant limitations. The rRNA genes are often present in multiple copies, unlike most protein-coding genes. The slow rate of change in rRNA genes means that multiple species sometimes share identical 16S rRNA gene sequences, while many more species share identical sequences in the short 16S rRNA regions commonly analyzed. In addition, the genes involved in many important processes are not distributed in a phylogenetically coherent manner, potentially due to gene loss or horizontal gene transfer. While rRNA genes remain the most commonly used markers, key genes in ecologically important pathways, e.g., those involved in carbon and nitrogen cycling, can provide important insights into community composition and function not obtainable through rRNA analysis. However, working with ecofunctional gene data requires some tools beyond those required for rRNA analysis. To address this, our Functional Gene Pipeline and Repository (FunGene; http://fungene.cme.msu.edu/) offers databases of many common ecofunctional genes and proteins, as well as integrated tools that allow researchers to browse these collections and choose subsets for further analysis, build phylogenetic trees, test primers and probes for coverage, and download aligned sequences. Additional FunGene tools are specialized to process coding gene amplicon data. For example, FrameBot produces frameshift-corrected protein and DNA sequences from raw reads while finding the most closely related protein reference sequence. These tools can help provide better insight into microbial communities by directly studying key genes involved in important ecological processes. PMID:24101916

  1. Gene Therapy for Skin Diseases

    PubMed Central

    Gorell, Emily; Nguyen, Ngon; Lane, Alfred; Siprashvili, Zurab

    2014-01-01

    The skin possesses qualities that make it desirable for gene therapy, and studies have focused on gene therapy for multiple cutaneous diseases. Gene therapy uses a vector to introduce genetic material into cells to alter gene expression, negating a pathological process. This can be accomplished with a variety of viral vectors or nonviral administrations. Although results are promising, there are several potential pitfalls that must be addressed to improve the safety profile to make gene therapy widely available clinically. PMID:24692191

  2. Characterizing gene family evolution

    PubMed Central

    Liberles, David A.

    2008-01-01

    Gene families are widely used in comparative genomics, molecular evolution, and in systematics. However, they are constructed in different manners, their data analyzed and interpreted differently, with different underlying assumptions, leading to sometimes divergent conclusions. In systematics, concepts like monophyly and the dichotomy between homoplasy and homology have been central to the analysis of phylogenies. We critique the traditional use of such concepts as applied to gene families and give examples of incorrect inferences they may lead to. Operational definitions that have emerged within functional genomics are contrasted with the common formal definitions derived from systematics. Lastly, we question the utility of layers of homology and the meaning of homology at the character state level in the context of sequence evolution. From this, we move forward to present an idealized strategy for characterizing gene family evolution for both systematic and functional purposes, including recent methodological improvements. PMID:19461954

  3. Alphaviruses in Gene Therapy

    PubMed Central

    Lundstrom, Kenneth

    2015-01-01

    Alphavirus vectors present an attractive approach for gene therapy applications due to the rapid and simple recombinant virus particle production and their broad range of mammalian host cell transduction. Mainly three types of alphavirus vectors, namely naked RNA, recombinant particles and DNA/RNA layered vectors, have been subjected to preclinical studies with the goal of achieving prophylactic or therapeutic efficacy, particularly in oncology. In this context, immunization with alphavirus vectors has provided protection against challenges with tumor cells. Moreover, alphavirus intratumoral and systemic delivery has demonstrated substantial tumor regression and significant prolonged survival rates in various animal tumor models. Recent discoveries of the strong association of RNA interference and disease have accelerated gene therapy based approaches, where alphavirus-based gene delivery can play an important role. PMID:25961488

  4. Virus induced gene silencing of Arabidopsis gene homologues in wheat identify genes conferring improved drought tolerance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In a non-model staple crop like wheat, functional validation of potential drought stress responsive genes identified in Arabidopsis could provide gene targets for wheat breeding. Virus induced gene silencing (VIGS) of genes of interest can overcome the inherent problems of polyploidy and limited tra...

  5. GeneTIER: prioritization of candidate disease genes using tissue-specific gene expression profiles

    PubMed Central

    Antanaviciute, Agne; Daly, Catherine; Crinnion, Laura A.; Markham, Alexander F.; Watson, Christopher M.; Bonthron, David T.; Carr, Ian M.

    2015-01-01

    Motivation: In attempts to determine the genetic causes of human disease, researchers are often faced with a large number of candidate genes. Linkage studies can point to a genomic region containing hundreds of genes, while the high-throughput sequencing approach will often identify a great number of non-synonymous genetic variants. Since systematic experimental verification of each such candidate gene is not feasible, a method is needed to decide which genes are worth investigating further. Computational gene prioritization presents itself as a solution to this problem, systematically analyzing and sorting each gene from the most to least likely to be the disease-causing gene, in a fraction of the time it would take a researcher to perform such queries manually. Results: Here, we present Gene TIssue Expression Ranker (GeneTIER), a new web-based application for candidate gene prioritization. GeneTIER replaces knowledge-based inference traditionally used in candidate disease gene prioritization applications with experimental data from tissue-specific gene expression datasets and thus largely overcomes the bias toward the better characterized genes/diseases that commonly afflict other methods. We show that our approach is capable of accurate candidate gene prioritization and illustrate its strengths and weaknesses using case study examples. Availability and Implementation: Freely available on the web at http://dna.leeds.ac.uk/GeneTIER/. Contact: umaan@leeds.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25861967

  6. Genes and functions controlled by floral organ identity genes.

    PubMed

    Sablowski, Robert

    2010-02-01

    Floral organ identity genes specify the identity of floral organs in a manner analogous to the specification of body segments by Hox genes in animals. Different combinations of organ identity genes co-ordinate the expression of genes required for the development of each type of floral organ, from organ initiation until final differentiation. Here, I review what is known about the genes and functions subordinate to the organ identity genes. The sets of target genes change as organ development progresses and ultimately organ identity genes modify the expression of thousands of genes with a multitude of predicted functions, particularly in reproductive organs. However, genes involved in transcriptional control and hormone functions feature prominently among the early and direct targets. Functional analysis showed that control of organ-specific tissues and structures can be delegated to specialised intermediate regulators, but organ identity genes also fine-tune genes with general roles in shoot organ development, consistent with the notion that organ identity genes modify a core leaf-like developmental program. Future challenges include obtaining data with cellular resolution, predictive modelling of the regulatory network, and quantitative analysis of how organ identity genes and their targets control cell behaviour and ultimately organ shape.

  7. Gene Therapy and Children (For Parents)

    MedlinePlus

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