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Sample records for anthracis dihydrofolate reductase

  1. Targeted Mutations of Bacillus anthracis Dihydrofolate Reductase Condense Complex Structure-Activity Relationships

    Energy Technology Data Exchange (ETDEWEB)

    J Beierlein; N Karri; A Anderson

    2011-12-31

    Several antifolates, including trimethoprim (TMP) and a series of propargyl-linked analogues, bind dihydrofolate reductase from Bacillus anthracis (BaDHFR) with lower affinity than is typical in other bacterial species. To guide lead optimization for BaDHFR, we explored a new approach to determine structure-activity relationships whereby the enzyme is altered and the analogues remain constant, essentially reversing the standard experimental design. Active site mutants of the enzyme, Ba(F96I)DHFR and Ba(Y102F)DHFR, were created and evaluated with enzyme inhibition assays and crystal structures. The affinities of the antifolates increase up to 60-fold with the Y102F mutant, suggesting that interactions with Tyr 102 are critical for affinity. Crystal structures of the enzymes bound to TMP and propargyl-linked inhibitors reveal the basis of TMP resistance and illuminate the influence of Tyr 102 on the lipophilic linker between the pyrimidine and aryl rings. Two new inhibitors test and validate these conclusions and show the value of the technique for providing new directions during lead optimization.

  2. Tales of Dihydrofolate Binding to R67 Dihydrofolate Reductase.

    Science.gov (United States)

    Duff, Michael R; Chopra, Shaileja; Strader, Michael Brad; Agarwal, Pratul K; Howell, Elizabeth E

    2016-01-12

    Homotetrameric R67 dihydrofolate reductase possesses 222 symmetry and a single active site pore. This situation results in a promiscuous binding site that accommodates either the substrate, dihydrofolate (DHF), or the cofactor, NADPH. NADPH interacts more directly with the protein as it is larger than the substrate. In contrast, the p-aminobenzoyl-glutamate tail of DHF, as monitored by nuclear magnetic resonance and crystallography, is disordered when bound. To explore whether smaller active site volumes (which should decrease the level of tail disorder by confinement effects) alter steady state rates, asymmetric mutations that decreased the half-pore volume by ∼35% were constructed. Only minor effects on k(cat) were observed. To continue exploring the role of tail disorder in catalysis, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide-mediated cross-linking between R67 DHFR and folate was performed. A two-folate, one-tetramer complex results in the loss of enzyme activity where two symmetry-related K32 residues in the protein are cross-linked to the carboxylates of two bound folates. The tethered folate could be reduced, although with a ≤30-fold decreased rate, suggesting decreased dynamics and/or suboptimal positioning of the cross-linked folate for catalysis. Computer simulations that restrain the dihydrofolate tail near K32 indicate that cross-linking still allows movement of the p-aminobenzoyl ring, which allows the reaction to occur. Finally, a bis-ethylene-diamine-α,γ-amide folate adduct was synthesized; both negatively charged carboxylates in the glutamate tail were replaced with positively charged amines. The K(i) for this adduct was ∼9-fold higher than for folate. These various results indicate a balance between folate tail disorder, which helps the enzyme bind substrate while dynamics facilitates catalysis. PMID:26637016

  3. Isolation and expression of the Pneumocystis carinii dihydrofolate reductase gene

    DEFF Research Database (Denmark)

    Edman, J C; Edman, U; Cao, Mi-Mi;

    1989-01-01

    Pneumocystis carinii dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) cDNA sequences have been isolated by their ability to confer trimethoprim resistance to Escherichia coli. Consistent with the recent conclusion that P. carinii is a member of the Fungi...

  4. Expression and site-directed mutagenesis of human dihydrofolate reductase

    International Nuclear Information System (INIS)

    A procaryotic high-level expression vector for human dihydrofolate reductase has been constructed and the protein characterized as a first step toward structure-function studies of this enzyme. A vector bearing the tac promoter, four synthetic oligodeoxynucleotides, and a restriction fragment from the dihydrofolate reductase cDNA were ligated in a manner which optimized the transcriptional and translational frequency of the enzyme mRNA. The reductase, comprising ca. 17% of the total soluble protein in the host bacteria, was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and characterized by amino acid composition, partial amino acid sequence, and steady-sate kinetic analysis. This expression vector has been used as a template for double-stranded plasmid DNA site-specific mutagenesis. Functional studies on a Cys-6 → Ser-6 mutant enzyme support the contention that Cys-6 is obligatory for organomercurial activation of human dihydrofolate reductase. The Ser-6 mutant enzyme was not activated to any extent following a 24-h incubation with p-(hydroxymercuri)benzoate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), whereas the k/sub cat/ for Cys-6 reductase increased 2-fold under identical conditions. The specific activities of the Cys-6 and Ser-6 enzymes were virtually identical as determined by methotrexate titration as were the K/sub m/ values for both dihydrofolate and NADPH. The Ser-6 mutant showed a decreased temperature stability and was more sensitive to inactivation by α-chymotrypsin when compared to the wild-type enzyme. These results suggest that the Ser-6 mutant reductase is conformationally altered relative to the Cys-6 native enzyme

  5. Binding of Natural and Synthetic Polyphenols to Human Dihydrofolate Reductase

    Directory of Open Access Journals (Sweden)

    José Neptuno Rodríguez-López

    2009-12-01

    Full Text Available Dihydrofolate reductase (DHFR is the subject of intensive investigation since it appears to be the primary target enzyme for antifolate drugs. Fluorescence quenching experiments show that the ester bond-containing tea polyphenols (--epigallocatechin gallate (EGCG and (--epicatechin gallate (ECG are potent inhibitors of DHFR with dissociation constants (KD of 0.9 and 1.8 μM, respectively, while polyphenols lacking the ester bound gallate moiety [e.g., (--epigallocatechin (EGC and (--epicatechin (EC] did not bind to this enzyme. To avoid stability and bioavailability problems associated with tea catechins we synthesized a methylated derivative of ECG (3-O-(3,4,5-trimethoxybenzoyl-(--epicatechin; TMECG, which effectively binds to DHFR (KD = 2.1 μM. In alkaline solution, TMECG generates a stable quinone methide product that strongly binds to the enzyme with a KD of 8.2 nM. Quercetin glucuronides also bind to DHFR but its effective binding was highly dependent of the sugar residue, with quercetin-3-xyloside being the stronger inhibitor of the enzyme with a KD of 0.6 μM. The finding that natural polyphenols are good inhibitors of human DHFR could explain the epidemiological data on their prophylactic effects for certain forms of cancer and open a possibility for the use of natural and synthetic polyphenols in cancer chemotherapy.

  6. Side chain conformational averaging in human dihydrofolate reductase.

    Science.gov (United States)

    Tuttle, Lisa M; Dyson, H Jane; Wright, Peter E

    2014-02-25

    The three-dimensional structures of the dihydrofolate reductase enzymes from Escherichia coli (ecDHFR or ecE) and Homo sapiens (hDHFR or hE) are very similar, despite a rather low level of sequence identity. Whereas the active site loops of ecDHFR undergo major conformational rearrangements during progression through the reaction cycle, hDHFR remains fixed in a closed loop conformation in all of its catalytic intermediates. To elucidate the structural and dynamic differences between the human and E. coli enzymes, we conducted a comprehensive analysis of side chain flexibility and dynamics in complexes of hDHFR that represent intermediates in the major catalytic cycle. Nuclear magnetic resonance relaxation dispersion experiments show that, in marked contrast to the functionally important motions that feature prominently in the catalytic intermediates of ecDHFR, millisecond time scale fluctuations cannot be detected for hDHFR side chains. Ligand flux in hDHFR is thought to be mediated by conformational changes between a hinge-open state when the substrate/product-binding pocket is vacant and a hinge-closed state when this pocket is occupied. Comparison of X-ray structures of hinge-open and hinge-closed states shows that helix αF changes position by sliding between the two states. Analysis of χ1 rotamer populations derived from measurements of (3)JCγCO and (3)JCγN couplings indicates that many of the side chains that contact helix αF exhibit rotamer averaging that may facilitate the conformational change. The χ1 rotamer adopted by the Phe31 side chain depends upon whether the active site contains the substrate or product. In the holoenzyme (the binary complex of hDHFR with reduced nicotinamide adenine dinucleotide phosphate), a combination of hinge opening and a change in the Phe31 χ1 rotamer opens the active site to facilitate entry of the substrate. Overall, the data suggest that, unlike ecDHFR, hDHFR requires minimal backbone conformational rearrangement as

  7. Direct demonstration of genetic alterations at the dihydrofolate reductase locus after gamma irradiation.

    OpenAIRE

    Graf, L. H.; Chasin, L A

    1982-01-01

    Gamma ray-induced mutants of Chinese hamster ovary cells lacking dihydrofolate reductase activity were screened for DNA sequence changes at the locus specifying this activity by using a cloned cDNA probe. Two of nine mutants screened displayed an altered restriction fragment pattern suggesting the occurrence of DNA deletions or rearrangements.

  8. Dihydrofolate reductase amplification and sensitization to methotrexate of methotrexate-resistant colon cancer cells

    DEFF Research Database (Denmark)

    Morales Torres, Christina; García, Maria J; Ribas, Maria;

    2009-01-01

    have analyzed the structure and dynamics of dihydrofolate reductase (DHFR) gene amplification in HT29 cells treated with methotrexate (MTX). Analysis of the DHFR gene amplification process shows that the amplicon exhibits a complex structure that is consistently reproduced in independent treatments...

  9. Identification and characterization of an inborn error of metabolism caused by dihydrofolate reductase deficiency

    NARCIS (Netherlands)

    Banka, S.; Blom, H.J.; Walter, J.; Aziz, M.; Urquhart, J.; Clouthier, C.M.; Rice, G.I.; Brouwer, A.P.M. de; Hilton, E.; Vassallo, G.; Will, A.; Smith, D.E.; Smulders, Y.M.; Wevers, R.A.; Steinfeld, R.; Heales, S.; Crow, Y.J.; Pelletier, J.N.; Jones, S.; Newman, W.G.

    2011-01-01

    Dihydrofolate reductase (DHFR) is a critical enzyme in folate metabolism and an important target of antineoplastic, antimicrobial, and antiinflammatory drugs. We describe three individuals from two families with a recessive inborn error of metabolism, characterized by megaloblastic anemia and/or pan

  10. Screening for inhibitors of dihydrofolate reductase using pulsed ultrafiltration mass spectrometry.

    Science.gov (United States)

    Nikolic, D; van Breemen, R B

    1998-04-01

    A method of screening combinatorial libraries for inhibitors of eukaryotic dihydrofolate reductase has been developed using pulsed ultra-filtration electrospray mass spectrometry, which is a continuous-flow affinity separation system for extracting and identifying high affinity ligands in combinatorial libraries. In this application, pulsed ultrafiltration conditions were optimized for the isolation and identification of inhibitors of dihydrofolate reductase from a 22 compound library containing six known inhibitors of the enzyme including trimethoprim, aminopterin, methotrexate, pyrimethamine, folic acid, and folinic acid, and 16 compounds without known affinity. In order to optimize the screening method, sources of non-specific binding were identified and minimized. A significant source of non-specific binding for this set of library compounds was hydrophobic interaction with the surfaces of the ultrafiltration chamber. After affinity separation of bound (high affinity) versus free (low affinity) library compounds during pulsed ultrafiltration, receptor-bound ligands were released and eluted using either organic solvent or acidified mobile phase. Although 80% methanol easily disrupted the receptor-ligand complexes, organic solvent had the undesirable effect of releasing non-specifically bound compounds from the chamber and thereby increasing the background noise. Interference from non-specific binding was minimized by releasing bound ligands using a low pH mobile phase eluent instead of organic solvent. Under the conditions used, pulsed ultrafiltration mass spectrometry selectively identified the two library compounds with the highest affinity for dihydrofolate reductase, methotrexate and aminopterin.

  11. Molecular cloning and sequence analysis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene.

    OpenAIRE

    Bzik, D J; Li, W B; Horii, T; Inselburg, J

    1987-01-01

    Genomic DNA clones that coded for the bifunctional dihydrofolate reductase (DHFR) and thymidylate synthase (TS) (DHFR-TS) activities from a pyrimethamine-sensitive strain of Plasmodium falciparum were isolated and sequenced. The deduced DHFR-TS protein contained 608 amino acids (71,682 Da). The coding region for DHFR-TS contained no intervening sequences and had a high A + T content (75%). The DHFR domain, in the amino-terminal portion of the protein, was joined by a 94-amino acid junction se...

  12. The kinetic mechanism of wild-type and mutant mouse dihydrofolate reductases.

    Science.gov (United States)

    Thillet, J; Adams, J A; Benkovic, S J

    1990-05-29

    A kinetic mechanism is presented for mouse dihydrofolate reductase that predicts all the steady-state parameters and full time-course kinetics. This mechanism was derived from association and dissociation rate constants and pre-steady-state transients by using stopped-flow fluorescence and absorbance measurements. The major features of this kinetic mechanism are as follows: (1) the two native enzyme conformers, E1 and E2, bind ligands with varying affinities although only one conformer, E1, can support catalysis in the forward direction, (2) tetrahydrofolate dissociation is the rate-limiting step under steady-state turnover at low pH, and (3) the pH-independent rate of hydride transfer from NADPH to dihydrofolate is fast (khyd = 9000 s-1) and favorable (Keq = 100). The overall mechanism is similar in form to the Escherichia coli kinetic scheme (Fierke et al., 1987), although several differences are observed: (1) substrates and products predominantly bind the same form of the E. coli enzyme, and (2) the hydride transfer rate from NADPH to either folate or dihydrofolate is considerably faster for the mouse enzyme. The role of Glu-30 (Asp-27 in E. coli) in mouse DHFR has also been examined by using site-directed mutagenesis as a potential source of these differences. While aspartic acid is strictly conserved in all bacterial DHFRs, glutamic acid is conserved in all known eucaryotes. The two major effects of substituting Asp for Glu-30 in the mouse enzyme are (1) a decreased rate of folate reduction and (2) an increased rate of hydride transfer from NADPH to dihydrofolate.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Selective killing of methotrexate-resistant cells carrying amplified dihydrofolate reductase genes

    International Nuclear Information System (INIS)

    A method for the selective killing of methotrexate (MTX)-resistant cells has been developed. The selection is based on the incorporation of tritiated deoxyuridine into the DNA of MTX-resistant cells but not normal MTX-sensitive cells in the presence of the drug. A Chinese hamster ovary cell mutant that overproduces dihydrofolate reductase was used as an example of a MTX-resistant cell line. In this system, a 10,000-fold enrichment for wild-type MTX-sensitive cells could be achieved after 24 hr of exposure to the drug combination. This selection technique was applied to the isolation of MTX-sensitive segregants from hybrid cells formed between the MTX-resistant mutant and wild-type cells. The loss of MTX resistance and dihydrofolate reductase overproduction was always accompanied by the loss of a homogeneously staining region on chromosome 2 of the resistant parent that contains the amplified genes specifying this enzyme. While this region is always lost, other parts of chromosome 2 are almost always retained, suggesting that deletion rather than chromosome loss underlies marker segregation in this case. When the selection was applied to the resistant mutant itself, no MTX-sensitive revertants were obtained among 10(5) cells screened, attesting to the stability of gene amplification in this clone. It is suggested that this combination of drugs may be useful for the elimination of MTX-resistant tumor cells that develop after MTX chemotherapy

  14. An inverted repeat motif stabilizes binding of E2F and enhances transcription of the dihydrofolate reductase gene

    DEFF Research Database (Denmark)

    Wade, M; Blake, M C; Jambou, R C;

    1995-01-01

    An overlapping inverted repeat sequence that binds the eukaryotic transcription factor E2F is 100% conserved near the major transcription start sites in the promoters of three mammalian genes encoding dihydrofolate reductase, and is also found in the promoters of several other important cellular ...

  15. Prognostic significance of numeric aberrations of genes for thymidylate synthase, thymidine phosphorylase and dihydrofolate reductase in colorectal cancer

    DEFF Research Database (Denmark)

    Jensen, Søren Astrup; Vainer, B.; Witton, C.J.;

    2008-01-01

    BACKGROUND: Most human cancer cells have structural aberrations of chromosomal regions leading to loss or gain of gene specific alleles. This study aimed to assess the range of gene copies per nucleus of thymidylate synthase (TYMS), thymidine phosphorylase (TP) and dihydrofolate reductase (DHFR) ...

  16. Preliminary neutron diffraction studies of Escherichia coli dihydrofolate reductase bound to the anticancer drug methotrexate

    Energy Technology Data Exchange (ETDEWEB)

    Bennett, Brad C. [University of Tennessee, Knoxville (UTK); Meilleur, Flora [Institut Laue-Langevin (ILL); Myles, Dean A A [ORNL; Howell, Elizabeth E. [University of Tennessee, Knoxville (UTK); Dealwis, Chris G. [University of Tennessee, Knoxville (UTK)

    2005-01-01

    The contribution of H atoms in noncovalent interactions and enzymatic reactions underlies virtually all aspects of biology at the molecular level, yet their 'visualization' is quite difficult. To better understand the catalytic mechanism of Escherichia coli dihydrofolate reductase (ecDHFR), a neutron diffraction study is under way to directly determine the accurate positions of H atoms within its active site. Despite exhaustive investigation of the catalytic mechanism of DHFR, controversy persists over the exact pathway associated with proton donation in reduction of the substrate, dihydrofolate. As the initial step in a proof-of-principle experiment which will identify ligand and residue protonation states as well as precise solvent structures, a neutron diffraction data set has been collected on a 0.3 mm{sup 3} D{sub 2}O-soaked crystal of ecDHFR bound to the anticancer drug methotrexate (MTX) using the LADI instrument at ILL. The completeness in individual resolution shells dropped to below 50% between 3.11 and 3.48 {angstrom} and the I/{sigma}(I) in individual shells dropped to below 2 at around 2.46 {angstrom}. However, reflections with I/{sigma}(I) greater than 2 were observed beyond these limits (as far out as 2.2 {angstrom}). To our knowledge, these crystals possess one of the largest primitive unit cells (P6{sub 1}, a = b = 92, c = 73 {angstrom}) and one of the smallest crystal volumes so far tested successfully with neutrons.

  17. Molecular cloning and sequence analysis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene.

    Science.gov (United States)

    Bzik, D J; Li, W B; Horii, T; Inselburg, J

    1987-12-01

    Genomic DNA clones that coded for the bifunctional dihydrofolate reductase (DHFR) and thymidylate synthase (TS) (DHFR-TS) activities from a pyrimethamine-sensitive strain of Plasmodium falciparum were isolated and sequenced. The deduced DHFR-TS protein contained 608 amino acids (71,682 Da). The coding region for DHFR-TS contained no intervening sequences and had a high A + T content (75%). The DHFR domain, in the amino-terminal portion of the protein, was joined by a 94-amino acid junction sequence to the TS domain in the carboxyl-terminal portion of the protein. The TS domain was more conserved than the DHFR domain and both P. falciparum domains were more homologous to eukaryotic than to prokaryotic forms of the enzymes. Predicted secondary structures of the DHFR and TS domains were nearly identical to the structures identified in other DHFR and TS enzymes. PMID:2825189

  18. Site-specific demethylation and normal chromatin structure of the human dihydrofolate reductase gene promoter after transfection into CHO cells.

    OpenAIRE

    Shimada, T.; Inokuchi, K; Nienhuis, A W

    1987-01-01

    The effect of in vitro methylation on the function and chromatin structure of the human dihydrofolate reductase (DHFR) promoter linked to the DHFR coding sequences (minigene) was studied after DNA-mediated gene transfer into DHFR- CHO cells. Methylation of HhaI sites reduced the transforming frequency to about 10% of control, whereas methylation of HpaII sites had a less significant effect. The integrated genes were demethylated at specific sites in the promoter sequence, namely, HpaII sites ...

  19. Histidine Hydrogen-Deuterium Exchange Mass Spectrometry for Probing the Microenvironment of Histidine Residues in Dihydrofolate Reductase

    OpenAIRE

    Masaru Miyagi; Qun Wan; Md. Faiz Ahmad; Giridharan Gokulrangan; Tomechko, Sara E; Brad Bennett; Chris Dealwis

    2011-01-01

    BACKGROUND: Histidine Hydrogen-Deuterium Exchange Mass Spectrometry (His-HDX-MS) determines the HDX rates at the imidazole C(2)-hydrogen of histidine residues. This method provides not only the HDX rates but also the pK(a) values of histidine imidazole rings. His-HDX-MS was used to probe the microenvironment of histidine residues of E. coli dihydrofolate reductase (DHFR), an enzyme proposed to undergo multiple conformational changes during catalysis. METHODOLOGY/PRINCIPAL FINDINGS: Using His-...

  20. New small-molecule inhibitors of dihydrofolate reductase inhibit Streptococcus mutans.

    Science.gov (United States)

    Zhang, Qiong; Nguyen, Thao; McMichael, Megan; Velu, Sadanandan E; Zou, Jing; Zhou, Xuedong; Wu, Hui

    2015-08-01

    Streptococcus mutans is a major aetiological agent of dental caries. Formation of biofilms is a key virulence factor of S. mutans. Drugs that inhibit S. mutans biofilms may have therapeutic potential. Dihydrofolate reductase (DHFR) plays a critical role in regulating the metabolism of folate. DHFR inhibitors are thus potent drugs and have been explored as anticancer and antimicrobial agents. In this study, a library of analogues based on a DHFR inhibitor, trimetrexate (TMQ), an FDA-approved drug, was screened and three new analogues that selectively inhibited S. mutans were identified. The most potent inhibitor had a 50% inhibitory concentration (IC50) of 454.0±10.2nM for the biofilm and 8.7±1.9nM for DHFR of S. mutans. In contrast, the IC50 of this compound for human DHFR was ca. 1000nM, a >100-fold decrease in its potency, demonstrating the high selectivity of the analogue. An analogue that exhibited the least potency for the S. mutans biofilm also had the lowest activity towards inhibiting S. mutans DHFR, further indicating that inhibition of biofilms is related to reduced DHFR activity. These data, along with docking of the most potent analogue to the modelled DHFR structure, suggested that the TMQ analogues indeed selectively inhibited S. mutans through targeting DHFR. These potent and selective small molecules are thus promising lead compounds to develop new effective therapeutics to prevent and treat dental caries. PMID:26022931

  1. Pyrimethamine resistant Plasmodium falciparum: overproduction of dihydrofolate reductase by a gene duplication.

    Science.gov (United States)

    Inselburg, J; Bzik, D J; Horii, T

    1987-11-01

    The accumulation of [3H]pyrimethamine by pyrimethamine-resistant (Pyrr) mutants of the Plasmodium falciparum strain FCR3 was examined by measuring the accumulation of drug in infected red blood cells. [3H]Pyrimethamine was stage specifically accumulated in trophozoites and schizont infected red blood cells. The mutant parasites accumulated drug as efficiently as FCR3. Pyrimethamine was associated with a high molecular weight protein that eluted from a Sephadex G200 column exactly as [3H]fluorodeoxyuridinemonophosphate (FdUMP) labeled parasite dihydrofolate reductase-thymidylate synthetase (DHFR-TS) enzyme. These results suggested that the pyrimethamine resistance was not associated with decreased drug permeability of the membrane. DHFR-TS-[3H]FdUMP enzyme complex of all the Pyrr mutants and FCR3 had a monomer of 70 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One highly resistant mutant, FCR3-D7, exhibited a 5-10 fold higher uptake of pyrimethamine and a proportionately higher amount of DHFR-TS protein than FCR3 but only a normal level of DHFR activity. The genomic DNA of FCR3-D7 was shown to contain at least twice as much DHFR-TS specific DNA than either FCR3-D8, another Pyrr mutant, or FCR3. Preliminary results suggested some of the DHFR-TS genetic material in FCR3-D7 is associated with a gene duplication. PMID:3323903

  2. Dihydrofolate reductase is required for the development of heart and outflow tract in zebrafish

    Institute of Scientific and Technical Information of China (English)

    Shuna Sun; Yonghao Gui; Qiu Jiang; Houyan Song

    2011-01-01

    Folic acid is very important for embryonic development and folic acid inhibition can cause congenital heart defects in vertebrates.Dihydrofolate reductase (DHFR) is a key enzyme in folate-mediated metabolism.The dysfunction of DHFR disrupts the key biological processes which folic acid participates in.DHFR gene is conserved during vertebrate evolution.It is important to investigate the roles of DHFR in cardiac developments.In this study,we showed that DHFR knockdown resulted in the abnormal developments of zebrafish embryos in the early stages.Obvious malformations in heart and outflow tract (OFT) were also observed in DHFR knockdown embryos.DHFR overexpression rescued the abnormal phenotypes in the DHFR knockdown group.DHFR knockdown had negative impacts on the expressions of NKX2.5 (NK2 transcription factor-related 5),MEF2C (myocyte-specific enhancer factor 2C),TBX20 (T-box 20),and TBX1 (T-box 1) which are important transcriptional factors during cardiac development process,while DHFR overexpression had positive effects.DHFR was required for Hedgehog pathway.DHFR knockdown caused reduced cell proliferation and increased apoptosis,while its overexpression promoted cell proliferation and inhibited apoptosis.Taken together,our study suggested that DHFR plays crucial roles in the development of heart and OFT in zebrafish by regulating gene transcriptions and affecting cell proliferation and apoptosis.

  3. Chemical Ligation and Isotope Labeling to Locate Dynamic Effects during Catalysis by Dihydrofolate Reductase.

    Science.gov (United States)

    Luk, Louis Y P; Ruiz-Pernía, J Javier; Adesina, Aduragbemi S; Loveridge, E Joel; Tuñón, Iñaki; Moliner, Vincent; Allemann, Rudolf K

    2015-07-27

    Chemical ligation has been used to alter motions in specific regions of dihydrofolate reductase from E. coli and to investigate the effects of localized motional changes on enzyme catalysis. Two isotopic hybrids were prepared; one with the mobile N-terminal segment containing heavy isotopes ((2) H, (13) C, (15) N) and the remainder of the protein with natural isotopic abundance, and the other one with only the C-terminal segment isotopically labeled. Kinetic investigations indicated that isotopic substitution of the N-terminal segment affected only a physical step of catalysis, whereas the enzyme chemistry was affected by protein motions from the C-terminal segment. QM/MM studies support the idea that dynamic effects on catalysis mostly originate from the C-terminal segment. The use of isotope hybrids provides insights into the microscopic mechanism of dynamic coupling, which is difficult to obtain with other studies, and helps define the dynamic networks of intramolecular interactions central to enzyme catalysis.

  4. Sequential conformation change and activation of chicken liver dihydrofolate reductase in low concentration of guanidine hydrochloride

    Institute of Scientific and Technical Information of China (English)

    范映辛; 朱笠; 周筠梅; 邹承鲁

    1997-01-01

    The conformation changes of dihydrofolate reductase (DHFR) from chicken liver in guanidine hy-drochloride were monitored by protein intrinsic fluorescence, hydrophobic fluorescence probe TNS and limited proteol-ysis by proteinase K. The kinetics of the enzyme denaturation were also studied and compared with its activity changes. It was indicated by the enhanced fluorescence of 2-p-toluidinylnaphthalene (TNS) that a subtle conforma-tional change of the enzyme in dilute GuHCl parallels GuHCl-induced activation. At GuHCl concentration higher than 0.75 mol/L, the conformational change can be detected by increased susceptibility of the enzyme to proteinase K, but no significant gross conformational change of the enzyme molecule is observed by intrinsic fluorescence up to a GuHCl concentration of 1.2 mol/L. The results suggest that the denaturation of DHFR by GuHCl does not follow strictly the two-state model. The enzyme seems to open up sequentially with increasing concentrations of denaturants, mainly at th

  5. Fitness trade-offs in the evolution of dihydrofolate reductase and drug resistance in Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Marna S Costanzo

    Full Text Available BACKGROUND: Patterns of emerging drug resistance reflect the underlying adaptive landscapes for specific drugs. In Plasmodium falciparum, the parasite that causes the most serious form of malaria, antifolate drugs inhibit the function of essential enzymes in the folate pathway. However, a handful of mutations in the gene coding for one such enzyme, dihydrofolate reductase, confer drug resistance. Understanding how evolution proceeds from drug susceptibility to drug resistance is critical if new antifolate treatments are to have sustained usefulness. METHODOLOGY/PRINCIPAL FINDINGS: We use a transgenic yeast expression system to build on previous studies that described the adaptive landscape for the antifolate drug pyrimethamine, and we describe the most likely evolutionary trajectories for the evolution of drug resistance to the antifolate chlorcycloguanil. We find that the adaptive landscape for chlorcycloguanil is multi-peaked, not all highly resistant alleles are equally accessible by evolution, and there are both commonalities and differences in adaptive landscapes for chlorcycloguanil and pyrimethamine. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that cross-resistance between drugs targeting the same enzyme reflect the fitness landscapes associated with each particular drug and the position of the genotype on both landscapes. The possible public health implications of these findings are discussed.

  6. Thermal stabilization of dihydrofolate reductase using monte carlo unfolding simulations and its functional consequences.

    Directory of Open Access Journals (Sweden)

    Jian Tian

    2015-04-01

    Full Text Available Design of proteins with desired thermal properties is important for scientific and biotechnological applications. Here we developed a theoretical approach to predict the effect of mutations on protein stability from non-equilibrium unfolding simulations. We establish a relative measure based on apparent simulated melting temperatures that is independent of simulation length and, under certain assumptions, proportional to equilibrium stability, and we justify this theoretical development with extensive simulations and experimental data. Using our new method based on all-atom Monte-Carlo unfolding simulations, we carried out a saturating mutagenesis of Dihydrofolate Reductase (DHFR, a key target of antibiotics and chemotherapeutic drugs. The method predicted more than 500 stabilizing mutations, several of which were selected for detailed computational and experimental analysis. We find a highly significant correlation of r=0.65-0.68 between predicted and experimentally determined melting temperatures and unfolding denaturant concentrations for WT DHFR and 42 mutants. The correlation between energy of the native state and experimental denaturation temperature was much weaker, indicating the important role of entropy in protein stability. The most stabilizing point mutation was D27F, which is located in the active site of the protein, rendering it inactive. However for the rest of mutations outside of the active site we observed a weak yet statistically significant positive correlation between thermal stability and catalytic activity indicating the lack of a stability-activity tradeoff for DHFR. By combining stabilizing mutations predicted by our method, we created a highly stable catalytically active E. coli DHFR mutant with measured denaturation temperature 7.2°C higher than WT. Prediction results for DHFR and several other proteins indicate that computational approaches based on unfolding simulations are useful as a general technique to discover

  7. Dihydrofolate reductase mutations and chromosomal changes associated with pyrimethamine resistance of Plasmodium falciparum.

    Science.gov (United States)

    Tanaka, M; Gu, H M; Bzik, D J; Li, W B; Inselburg, J W

    1990-02-01

    The nucleotide sequence of the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene in pyrimethamine-resistant (PyrR) mutants of Plasmodium falciparum selected in vitro was examined to determine if specific mutations in DHFR were associated with drug resistance. We analysed the sequence of genomic DNA from strain FCR3, from eight previously isolated PyrR parasites derived from FCR3, and from strain Honduras-1. We found that: (1) five PyrR FCR3 mutants, FCR3-D4-D8, had an identical nucleotide change and a novel single amino acid change (Phe to Ser) at amino acid 223 of DHFR; (2) our originally reported nucleotide sequence of the DHFR-TS gene was of the PyrR strain Honduras-1, and was not of FCR3; (3) three PyrR mutants, FCR3-D1, D2, and D3, thought to have been derived from the FCR3 strain, were in fact isolates of Honduras-1. We also examined the chromosomal DNA of PyrR mutants by pulsed-field gradient gel (PFG) electrophoresis. The PyrR mutants FCR3-D1, D2, and D3 had several chromosome size polymorphisms compared to FCR3. In two of the PyrR FCR3 mutants, FCR3-D7 and D8, the chromosome 4-size DNA of FCR3 that the DHFR-TS probe normally hybridised to was not observed. Instead, in FCR3-D7, a chromosome larger than the chromosome 4-size DNA was observed to hybridise to the DHFR-TS probe. In FCR3-D8, two chromosomes that hybridised to the DHFR-TS probe were found. One of them was larger than FCR-3 chromosome 4-size DNA, and the other was smaller than FCR3 chromosome 1-size DNA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2406591

  8. Interaction Energy Analysis of Nonclassical Antifolates with Pneumocystis carinii Dihydrofolate Reductase

    Directory of Open Access Journals (Sweden)

    William M. Southerland

    2002-11-01

    Full Text Available Abstract: The x-ray structure of the Pneumocystis carinii dihydrofolate reductase (DHFR:trimethoprim:NADPH ternary complex obtained from the Protein Databank was used as a structural template to generate models for the following complexes: P. carinii DHFR:piritrexim:NADPH, P. carinii DHFR:epiroprim:NADPH, and P. carinii DHFR:trimetrexate:NADPH. Each of these complexes, including the original trimethoprim complex was then modeled in 60 angstrom cubes of explicit water and minimized to a rms gradient between 1.0 to 3.0 x 10-5 kcal/angstrom. Subsequently, each antifolate structure was subdivided into distinct substructural regions. The minimized complexes were used to calculate interaction energies for each intact antifolate and its corresponding substructural regions with the P. carinii DHFR binding site residues, the DHFR protein, the solvated complex ( which consists of P. carinii DHFR, NADPH, and solvent water, solvent water alone, and NADPH. Antifolate substructural regions which contained nitrogen and carbon atoms in an aromatic environment (i. e. the pteridyl, pyridopyrimidinyl, and diaminopyrimidinyl subregions contributed most to the stability of antifolate interactions, while interaction energies for the hydrocarbon aromatic rings, methoxy, and ethoxy groups were much less stable. Additionally, interaction energy analyses were calculated for carbon and nitrogen atoms of the pteridyl, pyridopyrimidinyl, and diaminopyrimidinyl subregions and for the carbon and oxygen atoms of methoxy and ethoxy subregions. The contributions of hydrogen atoms were included with those of the carbon, nitrogen and oxygen atoms to which they are attached. These analyses revealed that the carbon atoms of the pteridyl, pyridopyrimidinyl, and diaminopyrimidinyl subregions generally contributed most to the stability of those regions. Carbon atoms also contributed favorably to the stability of the methoxy group interactions. Those substructural regions which exhibit

  9. Molecular monitoring of the Leu-164 mutation of dihydrofolate reductase in a highly sulfadoxine/pyrimethamine-resistant area in Africa

    Directory of Open Access Journals (Sweden)

    Mutabingwa Theonest

    2003-12-01

    Full Text Available Abstract The selection of point mutation at codon 164 (from isoleucine to leucine of the dihydrofolate reductase (DHFR enzyme in Plasmodium falciparum is associated with high sulfadoxine /pyrimethamine (SP resistance. Using the yeast expression system that allows the detection of dhfr allele present at low level, the presence of this mutation had previously been reported between 1998–1999 in Muheza, Tanzania, an area of high SP resistance. Eighty five P. falciparum isolates, obtained from the same area between 2002 and 2003, were analysed for the presence of Leu-164 mutation, using standard protocol based on PCR-RFLP. None of the isolates had the Leu-164 mutation.

  10. Multiple origins of resistance-conferring mutations in Plasmodium vivax dihydrofolate reductase

    Directory of Open Access Journals (Sweden)

    O'Neil Michael T

    2008-04-01

    Full Text Available Abstract Background In order to maximize the useful therapeutic life of antimalarial drugs, it is crucial to understand the mechanisms by which parasites resistant to antimalarial drugs are selected and spread in natural populations. Recent work has demonstrated that pyrimethamine-resistance conferring mutations in Plasmodium falciparum dihydrofolate reductase (dhfr have arisen rarely de novo, but spread widely in Asia and Africa. The origin and spread of mutations in Plasmodium vivax dhfr were assessed by constructing haplotypes based on sequencing dhfr and its flanking regions. Methods The P. vivax dhfr coding region, 792 bp upstream and 683 bp downstream were amplified and sequenced from 137 contemporary patient isolates from Colombia, India, Indonesia, Papua New Guinea, Sri Lanka, Thailand, and Vanuatu. A repeat motif located 2.6 kb upstream of dhfr was also sequenced from 75 of 137 patient isolates, and mutational relationships among the haplotypes were visualized using the programme Network. Results Synonymous and non-synonymous single nucleotide polymorphisms (SNPs within the dhfr coding region were identified, as was the well-documented in-frame insertion/deletion (indel. SNPs were also identified upstream and downstream of dhfr, with an indel and a highly polymorphic repeat region identified upstream of dhfr. The regions flanking dhfr were highly variable. The double mutant (58R/117N dhfr allele has evolved from several origins, because the 58R is encoded by at least 3 different codons. The triple (58R/61M/117T and quadruple (57L/61M/117T/173F, 57I/58R/61M/117T and 57L/58R/61M/117T mutant alleles had at least three independent origins in Thailand, Indonesia, and Papua New Guinea/Vanuatu. Conclusion It was found that the P. vivax dhfr coding region and its flanking intergenic regions are highly polymorphic and that mutations in P. vivax dhfr that confer antifolate resistance have arisen several times in the Asian region. This contrasts

  11. X-ray structure of the ternary MTX·NADPH complex of the anthrax dihydrofolate reductase: A pharmacophore for dual-site inhibitor design

    Energy Technology Data Exchange (ETDEWEB)

    Bennett, Brad C.; Wan, Qun; Ahmad, Md Faiz; Langan, Paul; Dealwis, Chris G.; (Case Western); (LANL)

    2009-11-18

    For reasons of bioterrorism and drug resistance, it is imperative to identify and develop new molecular points of intervention against anthrax. Dihydrofolate reductase (DHFR) is a highly conserved enzyme and an established target in a number of species for a variety of chemotherapeutic programs. Recently, the crystal structure of B. anthracis DHFR (baDHFR) in complex with methotrexate (MTX) was determined and, based on the structure, proposals were made for drug design strategies directed against the substrate binding site. However, little is gleaned about the binding site for NADPH, the cofactor responsible for hydride transfer in the catalytic mechanism. In the present study, X-ray crystallography at 100 K was used to determine the structure of baDHFR in complex with MTX and NADPH. Although the NADPH binding mode is nearly identical to that seen in other DHFR ternary complex structures, the adenine moiety adopts an off-plane tilt of nearly 90 deg. and this orientation is stabilized by hydrogen bonds to functionally conserved Arg residues. A comparison of the binding site, focusing on this region, between baDHFR and the human enzyme is discussed, with an aim at designing species-selective therapeutics. Indeed, the ternary model, refined to 2.3{angstrom} resolution, provides an accurate template for testing the feasibility of identifying dual-site inhibitors, compounds that target both the substrate and cofactor binding site. With the ternary model in hand, using in silico methods, several compounds were identified which could potentially form key bonding contacts in the substrate and cofactor binding sites. Ultimately, two structurally distinct compounds were verified that inhibit baDHFR at low {mu}M concentrations. The apparent K{sub d} for one of these, (2-(3-(2-(hydroxyimino)-2-(pyridine-4-yl)-6,7-dimethylquinoxalin-2-yl)-1-(pyridine-4-yl)ethanone oxime), was measured by fluorescence spectroscopy to be 5.3 {mu}M.

  12. A 19-base pair deletion polymorphism in dihydrofolate reductase is associated with increased unmetabolized folic acid in plasma and decreased red blood cell folate

    Science.gov (United States)

    Dihydrofolate reductase (DHFR) catalyzes the reduction of folic acid to tetrahydrofolate (THF). A 19-bp noncoding deletion allele maps to intron 1, beginning 60 bases from the splice donor site, and has been implicated in neural tube defects and cancer, presumably by influencing folate metabolism. T...

  13. The bifunctional dihydrofolate reductase thymidylate synthase of Tetrahymena thermophila provides a tool for molecular and biotechnology applications

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    Tiedtke Arno

    2006-03-01

    Full Text Available Abstract Background Dihydrofolate reductase (DHFR and thymidylate synthase (TS are crucial enzymes in DNA synthesis. In alveolata both enzymes are expressed as one bifunctional enzyme. Results Loss of this essential enzyme activities after successful allelic assortment of knock out alleles yields an auxotrophic marker in ciliates. Here the cloning, characterisation and functional analysis of Tetrahymena thermophila's DHFR-TS is presented. A first aspect of the presented work relates to destruction of DHFR-TS enzyme function in an alveolate thereby causing an auxotrophy for thymidine. A second aspect is to knock in an expression cassette encoding for a foreign gene with subsequent expression of the target protein. Conclusion This system avoids the use of antibiotics or other drugs and therefore is of high interest for biotechnological applications.

  14. Dihydrofolate reductase and dihydropteroate synthase genotypes associated with in vitro resistance of Plasmodium falciparum to pyrimethamine, trimethoprim, sulfadoxine, and sulfamethoxazole

    DEFF Research Database (Denmark)

    Khalil, Insaf; Rønn, Anita M; Alifrangis, Michael;

    2003-01-01

    A total of 70 Plasmodium falciparum isolates were tested in vitro against pyrimethamine (PYR), trimethoprim (TRM), sulfadoxine (SDX), and sulfamethoxazole (SMX), and their dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genotypes were determined. dhfr genotypes correlated.......46 +/- 0.86 (PYR) and 2.90 +/- 0.59 microg/100 microl (TRM). For both drugs, the differences in the mean IC50 between wild and mutant parasites were statistically significant (P resistance...... with PYR and TRM drug responses (r = 0.93 and 0.85). Isolates with wild-type alleles showed mean half inhibitory concentrations (IC50 +/- SD) of 0.10 +/- 0.10 and 0.15 +/- 0.06 microg/100 microl for PYR and TRM. Parasites with mutations at codons 108 and 51 alone or combined with codon 59 have IC50 of 11...

  15. High prevalence of mutations in the dihydrofolate reductase gene of Plasmodium falciparum in isolates from Tanzania without evidence of an association to clinical sulfadoxine/pyrimethamine resistance

    DEFF Research Database (Denmark)

    Jelinek, T; Rønn, A M; Curtis, J;

    1997-01-01

    Recently the efficacy of sulfadoxine/pyrimethamine (S/P) in treatment of uncomplicated falciparum malaria in Tanzania has been seriously compromised by the development of resistance. The occurrence of active site mutations in the Plasmodium falciparum gene sequence coding for dihydrofolate...... resistance to the S/P combination. It has been proposed earlier that sulfadoxine could itself act on DHFR, because of a false dihydrofolate produced by drug metabolism through DHPS and dihydrofolate synthase. The results of this treatment study suggest that such a possibility is unlikely....... reductase (DHFR) is known to confer resistance to pyrimethamine. This study investigates the occurrence of these mutations in infected blood samples taken from Tanzanian children before treatment with S/P and their relationship to parasite breakthrough by day 7. The results confirm the occurrence of one...

  16. Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography [Neutron and ultrahigh resolution X-ray crystallography reveals water as the proton donor in the catalytic mechanism of dihydrofolate reductase

    International Nuclear Information System (INIS)

    Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate (THF). An important step in the mechanism involves proton donation to the N5 atom of DHF. The inability to determine the protonation states of active site residues and substrate has led to the lack of consensus on a catalytic mechanism. To resolve this ambiguity, we conducted neutron and ultrahigh resolution X-ray crystallographic studies of the pseudo-Michaelis ternary complex of DHFR with folate and NADP+ from E. coli. The neutron data were collected to 2.0 Å resolution using a 3.6 mm3 crystal with the quasi-Laue technique, and the structure reveals that the N3 atom of folate is protonated while Asp27 is negatively charged. Previous mechanisms have proposed a keto-to-enol tautomerization of the substrate to facilitate protonation of the N5 atom. The structure supports the existence of the keto tautomer due to protonation of the N3 atom, suggesting tautomerization is unnecessary for catalysis. In the 1.05 Å resolution X-ray structure of the ternary complex, conformational disorder of the Met20 side chain is coupled to electron density for a partially occupied water within hydrogen-bonding distance of the N5 atom of folate; this suggests direct protonation of substrate by solvent. We propose a catalytic mechanism for DHFR that involves stabilization of the keto tautomer of the substrate, elevation of the pKa of the N5 atom of DHF by Asp27, and protonation of N5 by water whose access to the active site is gated by fluctuation of the Met20 side chain even though the Met-20 loop is closed

  17. Biosynthetic incorporation of telluromethionine into dihydrofolate reductase and crystallographic analysis of the distribution of tellurium atoms in the protein molecule

    Energy Technology Data Exchange (ETDEWEB)

    Kunkle, M.G.; Lewinski, K.; Boles, J.O.; Dunlap, R.B.; Odom, J.D.; Lebioda, L. [Univ. of South Carolina, Columbia, SC (United States)

    1994-12-01

    Recent successes in crystallographic studies of proteins with methionine (Met) residues replaced with SeMet, pioneered by Hendrickson and coworkers, inspired us to replace Met with TeMet in Escherichia coli dihydrofolate reductase (DHFR). E. coli DHFR, which catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate, consists of 159 residues, 5 of which are Met. TeMet was incorporated into DHFR using the Met auxotroph, E. coli DL41, carrying the expression vector pWT8 with an IPTG inducible promoter and ampicillin resistance gene. The enzyme was purified by successive chromatography on Q-Sepharose and PHenyl Sepharose resins, yielding milligram quantities of homogeneous enzyme with a specific activity of 40 units/mg. TeMet DHFR exhibits kinetic properties similar to those of wt DHFR. Amino acid analysis indicated 3 authentic Met residues in TeMet DHFR, whereas atomic absorption spectroscopy detected 2 Te per protein molecule. Amino acid sequence analysis results suggested that only authentic Met was present in the first three Met positions (1,16,and 20). Crystals of Te-DHFR were grown in the presence of methotrexate from PEG 4000 and were isomorphous with wt-DHFR crystals grown from ethanol. Difference Fourier maps and restrained least-squares refinement show very little, if any, Te in the first three Met positions: Met{sup 1}, Met{sup 16}, and Met{sup 20}, whereas the occupancy of Te in positions 42 and 92 is 0.64. Apparently, the process of folding, subsequent purification, and crystallization select DHFR molecules with Te in Met{sup 42} and Met{sup 92}. Replacing Met with TeMet provides an internal probe that should facilitate structural and mechanistic studies of proteins.

  18. Structural comparison of chromosomal and exogenous dihydrofolate reductase from Staphylococcus aureus in complex with the potent inhibitor trimethoprim

    Energy Technology Data Exchange (ETDEWEB)

    Heaslet, Holly; Harris, Melissa; Fahnoe, Kelly; Sarver, Ronald; Putz, Henry; Chang, Jeanne; Subramanyam, Chakrapani; Barreiro, Gabriela; Miller, J. Richard; Pfizer

    2010-09-02

    Dihydrofolate reductase (DHFR) is the enzyme responsible for the NADPH-dependent reduction of 5,6-dihydrofolate to 5,6,7,8-tetrahydrofolate, an essential cofactor in the synthesis of purines, thymidylate, methionine, and other key metabolites. Because of its importance in multiple cellular functions, DHFR has been the subject of much research targeting the enzyme with anticancer, antibacterial, and antimicrobial agents. Clinically used compounds targeting DHFR include methotrexate for the treatment of cancer and diaminopyrimidines (DAPs) such as trimethoprim (TMP) for the treatment of bacterial infections. DAP inhibitors of DHFR have been used clinically for >30 years and resistance to these agents has become widespread. Methicillin-resistant Staphylococcus aureus (MRSA), the causative agent of many serious nosocomial and community acquired infections, and other gram-positive organisms can show resistance to DAPs through mutation of the chromosomal gene or acquisition of an alternative DHFR termed 'S1 DHFR.' To develop new therapies for health threats such as MRSA, it is important to understand the molecular basis of DAP resistance. Here, we report the crystal structure of the wild-type chromosomal DHFR from S. aureus in complex with NADPH and TMP. We have also solved the structure of the exogenous, TMP resistant S1 DHFR, apo and in complex with TMP. The structural and thermodynamic data point to important molecular differences between the two enzymes that lead to dramatically reduced affinity of DAPs to S1 DHFR. These differences in enzyme binding affinity translate into reduced antibacterial activity against strains of S. aureus that express S1 DHFR.

  19. Effects of point mutations in Plasmodium falciparum dihydrofolate reductase and dihydropterate synthase genes on clinical outcomes and in vitro susceptibility to sulfadoxine and pyrimethamine.

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    David J Bacon

    Full Text Available BACKGROUND: Sulfadoxine-pyrimethamine was a common first line drug therapy to treat uncomplicated falciparum malaria, but increasing therapeutic failures associated with the development of significant levels of resistance worldwide has prompted change to alternative treatment regimes in many national malaria control programs. METHODOLOGY AND FINDING: We conducted an in vivo therapeutic efficacy trial of sulfadoxine-pyrimethamine at two locations in the Peruvian Amazon enrolling 99 patients of which, 86 patients completed the protocol specified 28 day follow up. Our objective was to correlate the presence of polymorphisms in P. falciparum dihydrofolate reductase and dihydropteroate synthase to in vitro parasite susceptibility to sulfadoxine and pyrimethamine and to in vivo treatment outcomes. Inhibitory concentration 50 values of isolates increased with numbers of mutations (single [108N], sextuplet [BR/51I/108N/164L and 437G/581G] and septuplet (BR/51I/108N/164L and 437G/540E/581G with geometric means of 76 nM (35-166 nM, 582 nM (49-6890- nM and 4909 (3575-6741 nM nM for sulfadoxine and 33 nM (22-51 nM, 81 nM (19-345 nM, and 215 nM (176-262 nM for pyrimethamine. A single mutation present in the isolate obtained at the time of enrollment from either dihydrofolate reductase (164L or dihydropteroate synthase (540E predicted treatment failure as well as any other single gene alone or in combination. Patients with the dihydrofolate reductase 164L mutation were 3.6 times as likely to be treatment failures [failures 85.4% (164L vs 23.7% (I164; relative risk = 3.61; 95% CI: 2.14 - 6.64] while patients with the dihydropteroate synthase 540E were 2.6 times as likely to fail treatment (96.7% (540E vs 37.5% (K540; relative risk = 2.58; 95% CI: 1.88 - 3.73. Patients with both dihydrofolate reductase 164L and dihydropteroate synthase 540E mutations were 4.1 times as likely to be treatment failures [96.7% vs 23.7%; RR = 4.08; 95% CI: 2.45 - 7.46] compared to

  20. Assessment of Folic Acid Supplementation in Pregnant Women by Estimation of Serum Levels of Tetrahydrofolic Acid, Dihydrofolate Reductase, and Homocysteine.

    Science.gov (United States)

    Naithani, Manisha; Saxena, Vartika; Mirza, Anissa Atif; Kumari, Ranjeeta; Sharma, Kapil; Bharadwaj, Jyoti

    2016-01-01

    Background. Status of folic acid use in pregnant women of the hilly regions in North India was little known. This study was carried out to assess the folic acid use and estimate folate metabolites in pregnant women of this region. Materials and Methods. This cross-sectional study is comprised of 76 pregnant women, whose folic acid supplementation was assessed by a questionnaire and serum levels of homocysteine, tetrahydrofolic acid (THFA), and dihydrofolate reductase (DHFR) were estimated using Enzyme Linked Immunoassays. Results. The study data revealed awareness of folic acid use during pregnancy was present in 46.1% and 23.7% were taking folic acid supplements. The study depicted that there was no statistically significant difference between serum levels of THFA and DHFR in pregnant women with and without folic acid supplements (p = 0.790). Hyperhomocysteinemia was present in 15.78% of the participants. Conclusion. Less awareness about folic acid supplementation and low use of folic acid by pregnant women were observed in this region. Sufficient dietary ingestion may suffice for the escalated requirements in pregnancy, but since this cannot be ensured, hence folic acid supplementation should be made as an integral part of education and reproductive health programs for its better metabolic use, growth, and development of fetus.

  1. Assessment of Folic Acid Supplementation in Pregnant Women by Estimation of Serum Levels of Tetrahydrofolic Acid, Dihydrofolate Reductase, and Homocysteine

    Directory of Open Access Journals (Sweden)

    Manisha Naithani

    2016-01-01

    Full Text Available Background. Status of folic acid use in pregnant women of the hilly regions in North India was little known. This study was carried out to assess the folic acid use and estimate folate metabolites in pregnant women of this region. Materials and Methods. This cross-sectional study is comprised of 76 pregnant women, whose folic acid supplementation was assessed by a questionnaire and serum levels of homocysteine, tetrahydrofolic acid (THFA, and dihydrofolate reductase (DHFR were estimated using Enzyme Linked Immunoassays. Results. The study data revealed awareness of folic acid use during pregnancy was present in 46.1% and 23.7% were taking folic acid supplements. The study depicted that there was no statistically significant difference between serum levels of THFA and DHFR in pregnant women with and without folic acid supplements (p=0.790. Hyperhomocysteinemia was present in 15.78% of the participants. Conclusion. Less awareness about folic acid supplementation and low use of folic acid by pregnant women were observed in this region. Sufficient dietary ingestion may suffice for the escalated requirements in pregnancy, but since this cannot be ensured, hence folic acid supplementation should be made as an integral part of education and reproductive health programs for its better metabolic use, growth, and development of fetus.

  2. Comparative study on dihydrofolate reductases from Shewanella species living in deep-sea and ambient atmospheric-pressure environments.

    Science.gov (United States)

    Murakami, Chiho; Ohmae, Eiji; Tate, Shin-ichi; Gekko, Kunihiko; Nakasone, Kaoru; Kato, Chiaki

    2011-03-01

    To examine whether dihydrofolate reductase (DHFR) from deep-sea bacteria has undergone molecular evolution to adapt to high-pressure environments, we cloned eight DHFRs from Shewanella species living in deep-sea and ambient atmospheric-pressure environments, and subsequently purified six proteins to compare their structures, stabilities, and functions. The DHFRs showed 74-90% identity in primary structure to DHFR from S. violacea, but only 55% identity to DHFR from Escherichia coli (ecDHFR). Far-ultraviolet circular dichroism and fluorescence spectra suggested that the secondary and tertiary structures of these DHFRs were similar. In addition, no significant differences were found in structural stability as monitored by urea-induced unfolding and the kinetic parameters, K(m) and k(cat); although the DHFRs from Shewanella species were less stable and more active (2- to 4-fold increases in k(cat)/K(m)) than ecDHFR. Interestingly, the pressure effects on enzyme activity revealed that DHFRs from ambient-atmospheric species are not necessarily incompatible with high pressure, and DHFRs from deep-sea species are not necessarily tolerant of high pressure. These results suggest that the DHFR molecule itself has not evolved to adapt to high-pressure environments, but rather, those Shewanella species with enzymes capable of retaining functional activity under high pressure migrated into the deep-sea.

  3. Towards the Understanding of Resistance Mechanisms in Clinically Isolated Trimethoprim-resistant, Methicillin-resistant Staphylococcus aureus Dihydrofolate Reductase

    Energy Technology Data Exchange (ETDEWEB)

    Frey, K.; Lombardo, M; Wright, D; Anderson, A

    2010-01-01

    Resistance to therapeutics such as trimethoprim-sulfamethoxazole has become an increasing problem in strains of methicillin-resistant Staphylococcus aureus (MRSA). Clinically isolated trimethoprim-resistant strains reveal a double mutation, H30N/F98Y, in dihydrofolate reductase (DHFR). In order to develop novel and effective therapeutics against these resistant strains, we evaluated a series of propargyl-linked antifolate lead compounds for inhibition of the mutant enzyme. For the propargyl-linked antifolates, the F98Y mutation generates minimal (between 1.2- and 6-fold) losses of affinity and the H30N mutation generates greater losses (between 2.4- and 48-fold). Conversely, trimethoprim affinity is largely diminished by the F98Y mutation (36-fold) and is not affected by the H30N mutation. In order to elucidate a mechanism of resistance, we determined a crystal structure of a complex of this double mutant with a lead propargyl-linked antifolate. This structure suggests a resistance mechanism consistent both for the propargyl-linked class of antifolates and for trimethoprim that is based on the loss of a conserved water-mediated hydrogen bond.

  4. Effect of dihydrofolate reductase gene knock-down on the expression of heart and neural crest derivatives expressed transcript 2 in zebrafish cardiac development

    Institute of Scientific and Technical Information of China (English)

    SUN Shu-na; GUI Yong-hao; WANG Yue-xiang; QIAN Lin-xi; JIANG Qiu; LIU Dong; SONG Hou-yan

    2007-01-01

    Background Folic acid is very important for embryonic development and dihydrofolate reductase is one of the key enzymes in the process of folic acid performing its biological function. Therefore, the dysfunction of dihydrofolate reductase can inhibit the function of folic acid and finally cause the developmental malformations. In this study, we observed the abnormal cardiac phenotypes in dihydrofolate reductase (DHFR) gene knock-down zebrafish embryos,investigated the effect of DHFR on the expression of heart and neural crest derivatives expressed transcript 2 (HAND2)and explored the possible mechanism of DHFR knock-down inducing zebrafish cardiac malformations.Methods Morpholino oligonucleotides were microinjected into fertilized eggs to knock down the functions of DHFR or HAND2. Full length of HAND2 mRNA which was transcribed in vitro was microinjected into fertilized eggs to overexpress HAND2. The cardiac morphologies, the heart rates and the ventricular shortening fraction were observed and recorded under the microscope at 48 hours post fertilization. Whole-mount in situ hybridization and real-time PCR were performed to detect HAND2 expression.Results DHFR or HAND2 knock-down caused the cardiac malformation in zebrafish. The expression of HAND2 was obviously reduced in DHFR knock-down embryos (P<0.05). Microinjecting HAND2 mRNA into fertilized eggs can induce HAND2 overexpression. HAND2 overexpression rescued the cardiac malformation phenotypes of DHFR knock-down embryos.Conclusions DHFR plays a crucial role in cardiac development. The down-regulation of HAND2 caused by DHFR knock-down is the possible mechanism of DHFR knock-down inducing the cardiac malformation.

  5. The effects of differential polyadenylation on expression of the dihydrofolate reductase-encoding gene in Chinese hamster lung cells.

    Science.gov (United States)

    Yang, H; Hussain, A; Melera, P W

    1995-10-01

    Three differently sized mRNAs are expressed from each of two DHFR (encoding dihydrofolate reductase) alleles present in the Chinese hamster lung (CHL) cell line, DC-3F. The relative abundancy of the transcripts produced from each allele differs dramatically as a result of differential utilization of the multiple poly(A) sites present in the DHFR DHFR gene and a genetic polymorphism located within the third poly(A) signal of one allele. We sought to determine whether such differences in polyadenylation affect the steady-state levels of DHFR and mRNAs expressed from either allele and, in a more general sense, to ask whether differences in 3' end RNA processing in a gene containing multiple poly(A) sites affects the final level of gene expression. An SV40 promoter-based transient expression system producing chimeric cat::DHFR transcripts was developed to regenerate the in vivo mRNA polyadenylation patterns associated with each of the two DHFR alleles. The results demonstrate that the total amount of polyadenylated RNA expressed from each of these constructs in vitro is the same regardless of the differential utilization of the poly(A) signals that occurs between them. Moreover, measurement of the individual turnover rates of the DHFR mRNAs expressed in vivo from each allele, as determined by pulse-chase labeling and actinomycin D inhibition studies, revealed no significant allele-specific differences in transcript half-lives. Finally, measuring the steady-state levels of DHFR poly(A)+ mRNA in parental DC-3F cells demonstrated that both alleles are expressed to the same extent during normal growth. Thus, even though dramatic allele-specific differences in 3' end processing of DHFR transcripts occur in vivo, such differences do not appear to influence the steady-state levels of DHFR gene expression. PMID:7590264

  6. Mutant dihydrofolate reductase-thymidylate synthase genes in pyrimethamine-resistant Plasmodium falciparum with polymorphic chromosome duplications.

    Science.gov (United States)

    Tanaka, M; Gu, H M; Bzik, D J; Li, W B; Inselburg, J

    1990-08-01

    We have identified dihydrofolate reductase (DHFR) gene point mutations and chromosomal changes in pyrimethamine-resistant mutants selected in vitro of Plasmodium falciparum strain FCR3. A pyrimethamine-resistant derivative of the pyrimethamine-sensitive strain FCR3, FCR3-D8, that had been grown in the absence of pyrimethamine for an extended time, was grown in two concentrations of pyrimethamine, and surviving drug-resistant parasites were subcloned. One selected mutant, FCR3-D81, that grew at 1 X 10(-6) M pyrimethamine, contained a single point mutation in the DHFR domain which caused an amino acid change (Phe to Ser) at amino acid 223, whereas another mutant, FCR3-D85, that grew at 5 X 10(-6) M pyrimethamine had that same mutation and an additional point mutation that changed amino acid 54 (Asp to Asn). The selection of FCR3-D85, whose nucleotide sequence was identical to that previously reported for FCR3-D8, confirmed that the original FCR3-D8 parasite population had changed during extended growth in vitro in the absence of drug pressure. FCR3-D81 and FCR3-D85 cells contained different pairs of polymorphic chromosomes that hybridized to a DHFR-TS probe as well as to three other chromosome 4 specific DNAs, indicating that at least part of chromosome 4 had been duplicated and that these parasites were aneuploid with 15 rather than 14 chromosomes. The mutant DHFR-TS genes were diploid. We consider the roles of the polymorphic chromosome duplications and DHFR point mutation(s) as causes of pyrimethamine resistance. PMID:2233901

  7. Trypanosoma brucei DHFR-TS Revisited: Characterisation of a Bifunctional and Highly Unstable Recombinant Dihydrofolate Reductase-Thymidylate Synthase.

    Science.gov (United States)

    Gibson, Marc W; Dewar, Simon; Ong, Han B; Sienkiewicz, Natasha; Fairlamb, Alan H

    2016-05-01

    Bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a chemically and genetically validated target in African trypanosomes, causative agents of sleeping sickness in humans and nagana in cattle. Here we report the kinetic properties and sensitivity of recombinant enzyme to a range of lipophilic and classical antifolate drugs. The purified recombinant enzyme, expressed as a fusion protein with elongation factor Ts (Tsf) in ThyA- Escherichia coli, retains DHFR activity, but lacks any TS activity. TS activity was found to be extremely unstable (half-life of 28 s) following desalting of clarified bacterial lysates to remove small molecules. Stability could be improved 700-fold by inclusion of dUMP, but not by other pyrimidine or purine (deoxy)-nucleosides or nucleotides. Inclusion of dUMP during purification proved insufficient to prevent inactivation during the purification procedure. Methotrexate and trimetrexate were the most potent inhibitors of DHFR (Ki 0.1 and 0.6 nM, respectively) and FdUMP and nolatrexed of TS (Ki 14 and 39 nM, respectively). All inhibitors showed a marked drop-off in potency of 100- to 1,000-fold against trypanosomes grown in low folate medium lacking thymidine. The most potent inhibitors possessed a terminal glutamate moiety suggesting that transport or subsequent retention by polyglutamylation was important for biological activity. Supplementation of culture medium with folate markedly antagonised the potency of these folate-like inhibitors, as did thymidine in the case of the TS inhibitors raltitrexed and pemetrexed. PMID:27175479

  8. High pressure NMR reveals active-site hinge motion of folate-bound Escherichia coli dihydrofolate reductase.

    Science.gov (United States)

    Kitahara, R; Sareth, S; Yamada, H; Ohmae, E; Gekko, K; Akasaka, K

    2000-10-24

    A high-pressure (15)N/(1)H two-dimensional NMR study has been carried out on folate-bound dihydrofolate reductase (DHFR) from Escherichia coli in the pressure range between 30 and 2000 bar. Several cross-peaks in the (15)N/(1)H HSQC spectrum are split into two with increasing pressure, showing the presence of a second conformer in equilibrium with the first. Thermodynamic analysis of the pressure and temperature dependencies indicates that the second conformer is characterized by a smaller partial molar volume (DeltaV = -25 mL/mol at 15 degrees C) and smaller enthalpy and entropy values, suggesting that the second conformer is more open and hydrated than the first. The splittings of the cross-peaks (by approximately 1 ppm on (15)N axis at 2000 bar) arise from the hinges of the M20 loop, the C-helix, and the F-helix, all of which constitute the major binding site for the cofactor NADPH, suggesting that major differences in conformation occur in the orientations of the NADPH binding units. The Gibbs free energy of the second, open conformer is 5.2 kJ/mol above that of the first at 1 bar, giving an equilibrium population of about 10%. The second, open conformer is considered to be crucial for NADPH binding, and the NMR line width indicates that the upper limit for the rate of opening is 20 s(-)(1) at 2000 bar. These experiments show that high pressure NMR is a generally useful tool for detecting and analyzing "open" structures of a protein that may be directly involved in function.

  9. Island-wide diversity in single nucleotide polymorphisms of the Plasmodium vivax dihydrofolate reductase and dihydropteroate synthetase genes in Sri Lanka

    DEFF Research Database (Denmark)

    Schousboe, Mette L; Rajakaruna, Rupika S; Salanti, Ali;

    2007-01-01

    BACKGROUND: Single nucleotide polymorphisms (SNPs) in the Plasmodium vivax dihydrofolate reductase (Pfdhfr) and dihydropteroate synthetase (Pvdhps) genes cause parasite resistance to the antifolate drug combination, sulphadoxine/pyrimethamine (SP). Monitoring these SNPs provide insights...... into the level of drug pressure caused by SP use and presumably other antifolate drugs. In Sri Lanka, chloroquine (CQ) with primaquine (PQ) and SP with PQ is used as first and second line treatment, respectively, against uncomplicated Plasmodium falciparum and/or P. vivax infections. CQ/PQ is still efficacious...... and diversity of Pvdhfr mutations was unexpected indicating the emergence of drug resistant parasites despite a low level of SP drug pressure....

  10. Photoaffinity analogues of methotrexate as folate antagonist binding probes. 1. Photoaffinity labeling of murine L1210 dihydrofolate reductase and amino acid sequence of the binding region

    International Nuclear Information System (INIS)

    N/sup α/-(4-Amino-4-deoxy-10-methylpteroyl)-N/sup epsilon/-(4-azido-5-[125I]iodosalicylyl)-L-lysine, a photoaffinity analogue of methotrexate, is only 2-fold less potent than methotrexate in the inhibition of murine L1210 dihydrofolate reductase. Irradiation of the enzyme in the presence of an equimolar concentration of the 125I-labeled analogue ultimately leads to an 8% incorporation of the photoprobe. A 100-fold molar excess of methotrexate essentially blocks this incorporation. Cyanogen bromide digestion of the labeled enzyme, followed by high-pressure liquid chromatography purification of the generated peptides, indicates that greater than 85% of the total radioactivity is incorporated into a single cyanogen bromide peptide. Sequence analysis revealed this peptide to be residues 53-111, with a majority of the radioactivity centered around residues 63-65 (Lys-Asn-Arg). These data demonstrate that the photoaffinity analogue specifically binds to dihydrofolate reductase and covalently modifies the enzyme following irradiation and is therefore a photolabeling agent useful for probing the inhibitor binding domain of the enzyme

  11. Stable transformation of Toxoplasma gondii based on a pyrimethamine resistant trifunctional dihydrofolate reductase-cytosine deaminase-thymidylate synthase gene that confers sensitivity to 5-fluorocytosine.

    Science.gov (United States)

    Fox, B A; Belperron, A A; Bzik, D J

    1999-01-01

    To improve genetic models available for the analysis of apicomplexan protozoan parasites, bacterial sequences encoding the 427 amino acid cytosine deaminase (CD) gene were fused, in-frame, to an engineered linker domain of the high level pyrimethamine resistant form of the parasite bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene. Toxoplasma gondii was transformed with the plasmid containing the fused pyrimethamine resistant dihydrofolate reductase-cytosine deaminase-thymidylate synthase (DHFRm2m3-CD-TS) gene and parasites were selected in a high level of pyrimethamine. Transfected parasites that acquired resistance to pyrimethamine were cloned and evaluated for expression of the CD genetic marker. CD transgenic parasites acquired a high sensitivity to 5-fluorocytosine due to the intraparasitic conversion of this non-toxic prodrug to the cytotoxic compound 5-fluorouracil. Exogenously supplied cytosine or uracil rescued the growth of CD transgenic T. gondii parasites that were cultured in the presence of cytotoxic concentrations of 5-fluorouracil or 5-fluorocytosine. Bacterial CD fused to the pyrimethamine resistant DHFR-TS marker provides a novel genetic tool for new positive and negative genetic selection strategies in several protozoan parasites. An advantage of the CD genetic marker is that it is derived from a bacterial gene and can therefore be used in nearly any parasite genetic background for negative selection. This novel system should facilitate new approaches for the development of improved model genetic systems for the biological investigation of apicomplexan parasites. PMID:10029312

  12. Evaluation of dihydrofolate reductase and dihydropteroate synthetase genotypes that confer resistance to sulphadoxine-pyrimethamine in Plasmodium falciparum in Haiti

    Directory of Open Access Journals (Sweden)

    Carter Tamar E

    2012-08-01

    Full Text Available Abstract Background Malaria caused by Plasmodium falciparum infects roughly 30,000 individuals in Haiti each year. Haiti has used chloroquine (CQ as a first-line treatment for malaria for many years and as a result there are concerns that malaria parasites may develop resistance to CQ over time. Therefore it is important to prepare for alternative malaria treatment options should CQ resistance develop. In many other malaria-endemic regions, antifolates, particularly pyrimethamine (PYR and sulphadoxine (SDX treatment combination (SP, have been used as an alternative when CQ resistance has developed. This study evaluated mutations in the dihydrofolate reductase (dhfr and dihydropteroate synthetase (dhps genes that confer PYR and SDX resistance, respectively, in P. falciparum to provide baseline data in Haiti. This study is the first comprehensive study to examine PYR and SDX resistance genotypes in P. falciparum in Haiti. Methods DNA was extracted from dried blood spots and genotyped for PYR and SDX resistance mutations in P. falciparum using PCR and DNA sequencing methods. Sixty-one samples were genotyped for PYR resistance in codons 51, 59, 108 and 164 of the dhfr gene and 58 samples were genotyped for SDX resistance codons 436, 437, 540 of the dhps gene in P. falciparum. Results Thirty-three percent (20/61 of the samples carried a mutation at codon 108 (S108N of the dhfr gene. No mutations in dhfr at codons 51, 59, 164 were observed in any of the samples. In addition, no mutations were observed in dhps at the three codons (436, 437, 540 examined. No significant difference was observed between samples collected in urban vs rural sites (Welch’s T-test p-value = 0.53 and permutations p-value = 0.59. Conclusion This study has shown the presence of the S108N mutation in P. falciparum that confers low-level PYR resistance in Haiti. However, the absence of SDX resistance mutations suggests that SP resistance may not be present in Haiti. These

  13. Detection of long-lived bound water molecules in complexes of human dihydrofolate reductase with methotrexate and NADPH.

    Science.gov (United States)

    Meiering, E M; Wagner, G

    1995-03-24

    The locations of long-lived bound water molecules in the binary complex of human dihydrofolate reductase (hDHFR) with methotrexate (MTX) and the ternary complex of hDHFR with MTX and NADPH have been investigated using 15N-resolved, three-dimensional ROESY-HMQC and NOESY-HSQC spectra acquired at 25 degrees C and 8 degrees C. NOEs with NH groups of the protein are detected for five bound water molecules in the binary complex and six bound water molecules in the ternary complex. Inspection of crystal structures of hDHFR reveals that the bound water molecules perform structural and functional roles in the complexes. Two water molecules located outside the active site, WatA and WatB, have similar NOEs in the binary and ternary complexes. These water molecules from multiple hydrogen bonds bridging loops and/or secondary structural elements in crystal structures of hDHFR and so stabilize the tertiary fold of the enzyme. Two water molecules in the active site, WatC and WatD, also have similar NOEs in both complexes. In crystal structures of hDHFR, WatC is involved in MTX binding by forming hydrogen bonds to the ligand and protein, while WatD stabilizes WatC by hydrogen bonding to it and the protein. A third active-site water molecule, WatE, has a markedly stronger NOE in the ternary complex than in the binary complex. Differences in the binding of WatE in the binary and ternary complexes are important for understanding the mechanism of DHFR, since this water molecule is believed to be involved in substrate protonation. Although the increased NOE intensity for WatE could be caused by a change in the position of water molecule, it may also be caused by an increase in its lifetime, since structural fluctuations in the active site are decreased upon cofactor binding. NOEs for one other water molecule, WatF, may be observed in the ternary complex but not the binary complex. WatF forms hydrogen bonds bridging the cofactor and the protein in crystal structures of hDHFR.

  14. Structures of dihydrofolate reductase-thymidylate synthase of Trypanosoma cruzi in the folate-free state and in complex with two antifolate drugs, trimetrexate and methotrexate

    Energy Technology Data Exchange (ETDEWEB)

    Senkovich, Olga; Schormann, Norbert; Chattopadhyay, Debasish; (UAB)

    2010-11-22

    The flagellate protozoan parasite Trypanosoma cruzi is the pathogenic agent of Chagas disease (also called American trypanosomiasis), which causes approximately 50 000 deaths annually. The disease is endemic in South and Central America. The parasite is usually transmitted by a blood-feeding insect vector, but can also be transmitted via blood transfusion. In the chronic form, Chagas disease causes severe damage to the heart and other organs. There is no satisfactory treatment for chronic Chagas disease and no vaccine is available. There is an urgent need for the development of chemotherapeutic agents for the treatment of T. cruzi infection and therefore for the identification of potential drug targets. The dihydrofolate reductase activity of T. cruzi, which is expressed as part of a bifunctional enzyme, dihydrofolate reductase-thymidylate synthase (DHFR-TS), is a potential target for drug development. In order to gain a detailed understanding of the structure-function relationship of T. cruzi DHFR, the three-dimensional structure of this protein in complex with various ligands is being studied. Here, the crystal structures of T. cruzi DHFR-TS with three different compositions of the DHFR domain are reported: the folate-free state, the complex with the lipophilic antifolate trimetrexate (TMQ) and the complex with the classical antifolate methotrexate (MTX). These structures reveal that the enzyme is a homodimer with substantial interactions between the two TS domains of neighboring subunits. In contrast to the enzymes from Cryptosporidium hominis and Plasmodium falciparum, the DHFR and TS active sites of T. cruzi lie on the same side of the monomer. As in other parasitic DHFR-TS proteins, the N-terminal extension of the T. cruzi enzyme is involved in extensive interactions between the two domains. The DHFR active site of the T. cruzi enzyme shows subtle differences compared with its human counterpart. These differences may be exploited for the development of

  15. Molecular dynamics simulation of dihydrofolate reductase in salt solution%二氢叶酸还原酶在盐溶液中的分子动力学模拟

    Institute of Scientific and Technical Information of China (English)

    王四华; 付晓平; 王文研; 张光亚

    2012-01-01

    In oder to find how halophilic enzyme maintain stability and activity in high salt concentration.dihydrofolate reductase from the Haloferax volcanii and Escherichia coli has been chosen as an model respectively, and suffer molecular dynamics simulation in five different concentration of salt solution. Molecular dynamics trajectories of the dihydrofolate reductases in the five different salt solutions were obtained after 9 ns of simulation, by a detailed analysis of the trajectories, we obtain their dynamical characteristics in different salt solution. The results showed that the salt bridge and protein-solvent H-bond of dihydrofolate reductase from the Haloferax volcanii are significantly more than that of dihydrofolate reductase from Escherichia coli, while the solvent accessible surface area of dihydrofolate reductase from the Escherichia coli is significantly large than that of dihydrofolate reductase from the Haloferax volcanii. The RMSD and each amino acids RMSF value were also addressed.%为了研究嗜盐酶如何在高盐环境下维持稳定性与活性,本文以沃尔卡尼极嗜盐菌及大肠杆菌的二氢叶酸还原酶(DHFR)为模型,将二者分别置于5种不同盐浓度的水溶液中进行分子动力学模拟.经9 ns动力学模拟,得到了二者在不同浓度盐溶液中的运动轨迹,通过对运动轨迹的分析,获取了二者在不同盐浓度下的动力学特性.结果发现嗜盐古生菌的二氢叶酸还原酶自身所形成盐桥及与溶剂所形成的氢键均比大肠杆菌的二氢叶酸还原酶多,而溶剂可及性表面则要小,二者差异均达极显著水平.同时还分析了这两种分子及其氨基酸残基的柔性等.

  16. In Silico Screening, Synthesis and In Vitro Evaluation of Some Quinazolinone and Pyridine Derivatives as Dihydrofolate Reductase Inhibitors for Anticancer Activity

    Directory of Open Access Journals (Sweden)

    A. G. Nerkar

    2009-01-01

    Full Text Available Dihydrofolate reductase (DHFR is the important target for anticancer drugs belonging to the class of antimetabolites as the enzyme plays important role in the de novo purine synthesis. We here report the in silico screening to obtain best fit molecules as DHFR inhibitors, synthesis of some ʻbest fitʼ quinazolinone from 2-phenyl-3-(substituted-benzilidine-amino quinazolinones (Quinazolinone Shiff's bases QSB1-5 and pyridine-4-carbohydrazide Shiff's bases (ISB1-5 derivatives and their in vitro anticancer assay. Synthesis of the molecules was performed using microwave assisted synthesis. The structures of these molecules were elucidated by IR and 1H-NMR. These compounds were then subjected for in vitro anticancer evaluation against five human cancer cell-lines for anticancer cyto-toxicity assay. Methotrexate (MTX was used as standard for this evaluation to give a comparable inhibition of the cell proliferation by DHFR inhibition. Placlitaxel, adriamycin and 5-fluoro-uracil were also used as standard to give a comparable activity of these compounds with other mechanism of anticancer activity. ISB3 (4-(N, N-dimethyl-amino-phenyl Schiff''s base derivative of pyridine carbohydrazide showed equipotent activity with the standards used in in vitro anticancer assay as per the NCI (National Cancer Institute guidelines.

  17. Towards understanding the origins of the different specificities of binding the reduced (NADPH) and oxidised (NADP +) forms of nicotinamide adenine dinucleotide phosphate coenzyme to dihydrofolate reductase

    Science.gov (United States)

    Polshakov, Vladimir I.; Biekofsky, Rodolfo R.; Birdsall, Berry; Feeney, James

    2002-01-01

    Lactobacillus casei dihydrofolate reductase (DHFR) binds more than a thousand times tighter to NADPH than to NADP +. The origins of the difference in binding affinity to DHFR between NADPH and NADP + are investigated in the present study using experimental NMR data and hybrid density functional, B3LYP, calculations. Certain protein residues (Ala 6, Gln 7, Ile 13 and Gly 14) that are directly involved in hydrogen bonding with the nicotinamide carboxamide group show consistent differences in 1H and 15N chemical shift between NADPH and NADP + in a variety of ternary complexes. B3LYP calculations in model systems of protein-coenzyme interactions show differences in the H-bond geometry and differences in charge distribution between the oxidised and reduced forms of the nicotinamide ring. GIAO isotropic nuclear shieldings calculated for nuclei in these systems reproduce the experimentally observed trends in magnitudes and signs of the chemical shifts. The experimentally observed reduction in binding of NADP + compared with NADPH results partly from NADP + having to change its nicotinamide amide group from a cis- to a trans-conformation on binding and partly from the oxidised nicotinamide ring of NADP + being unable to take up its optimal hydrogen bonding geometry in its interactions with protein residues.

  18. Declining trend of Plasmodium falciparum dihydrofolate reductase (dhfr and dihydropteroate synthase (dhps mutant alleles after the withdrawal of Sulfadoxine-Pyrimethamine in North Western Ethiopia.

    Directory of Open Access Journals (Sweden)

    Sofonias K Tessema

    Full Text Available Antimalarial drug resistance is one of the major challenges in global efforts of malaria control and elimination. In 1998, chloroquine was abandoned and replaced with sulfadoxine/pyrimethamine, which in turn was replaced with artemether/lumefantrine for the treatment of uncomplicated falciparum malaria in 2004. Sulfadoxine/pyrimethamine resistance is associated with mutations in dihydrofolate reductase (Pfdhfr and dihydropteroate synthase (Pfdhps genes. The prevalence of mutation in Pfdhfr and Pfdhps genes were evaluated and compared for a total of 159 isolates collected in two different time points, 2005 and 2007/08, from Pawe hospital, in North Western Ethiopia. The frequency of triple Pfdhfr mutation decreased significantly from 50.8% (32/63 to 15.9% (10/63 (P<0.001, while Pfdhps double mutation remained high and changed only marginally from 69.2% (45/65 to 55.4% (40/65 (P = 0.08. The combined Pfdhfr/Pfdhps quintuple mutation, which is strongly associated with sulfadoxine/pyrimethamine resistance, was significantly decreased from 40.7% (24/59 to 13.6% (8/59 (P<0.0001. On the whole, significant decline in mutant alleles and re-emergence of wild type alleles were observed. The change in the frequency is explained by the reduction of residual drug-resistant parasites caused by the strong drug pressure imposed when sulfadoxine/pyrimethamine was the first-line drug, followed by lower fitness of these resistant parasites in the absence of drug pressure. Despite the decrease in the frequency of mutant alleles, higher percentages of mutation remain prevalent in the study area in 2007/08 in both Pfdhfr and Pfdhps genes. Therefore, further multi-centered studies in different parts of the country will be required to assess the re-emergence of sulfadoxine/pyrimethamine sensitive parasites and to monitor and prevent the establishment of multi drug resistant parasites in this region.

  19. Declining trend of Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) mutant alleles after the withdrawal of Sulfadoxine-Pyrimethamine in North Western Ethiopia.

    Science.gov (United States)

    Tessema, Sofonias K; Kassa, Moges; Kebede, Amha; Mohammed, Hussein; Leta, Gemechu Tadesse; Woyessa, Adugna; Guma, Geremew Tasew; Petros, Beyene

    2015-01-01

    Antimalarial drug resistance is one of the major challenges in global efforts of malaria control and elimination. In 1998, chloroquine was abandoned and replaced with sulfadoxine/pyrimethamine, which in turn was replaced with artemether/lumefantrine for the treatment of uncomplicated falciparum malaria in 2004. Sulfadoxine/pyrimethamine resistance is associated with mutations in dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes. The prevalence of mutation in Pfdhfr and Pfdhps genes were evaluated and compared for a total of 159 isolates collected in two different time points, 2005 and 2007/08, from Pawe hospital, in North Western Ethiopia. The frequency of triple Pfdhfr mutation decreased significantly from 50.8% (32/63) to 15.9% (10/63) (P<0.001), while Pfdhps double mutation remained high and changed only marginally from 69.2% (45/65) to 55.4% (40/65) (P = 0.08). The combined Pfdhfr/Pfdhps quintuple mutation, which is strongly associated with sulfadoxine/pyrimethamine resistance, was significantly decreased from 40.7% (24/59) to 13.6% (8/59) (P<0.0001). On the whole, significant decline in mutant alleles and re-emergence of wild type alleles were observed. The change in the frequency is explained by the reduction of residual drug-resistant parasites caused by the strong drug pressure imposed when sulfadoxine/pyrimethamine was the first-line drug, followed by lower fitness of these resistant parasites in the absence of drug pressure. Despite the decrease in the frequency of mutant alleles, higher percentages of mutation remain prevalent in the study area in 2007/08 in both Pfdhfr and Pfdhps genes. Therefore, further multi-centered studies in different parts of the country will be required to assess the re-emergence of sulfadoxine/pyrimethamine sensitive parasites and to monitor and prevent the establishment of multi drug resistant parasites in this region. PMID:26431464

  20. Structural comparison of complexes of methotrexate analogues with Lactobacillus casei dihydrofolate reductase by two-dimensional 1H NMR at 500 MHz

    International Nuclear Information System (INIS)

    The authors have used two-dimensional (2D) NMR methods to examine complexes of Lactobacillus casei dihydrofolate reductase and methotrexate (MTX) analogues having structural modifications of the benzoyl ring and also the glutamic acid moiety. Assignments of the 1H signals in the spectra of the various complexes were made by comparison of their 2D spectra with those complexes containing methotrexate where we have previously assigned resonances from 32 of the 162 amino acid residues. In the complexes formed with the dihalomethotrexate analogues, the glutamic acid and pteridine ring moieties were shown to bind to the enzyme in a manner similar to that found in the methotrexate-enzyme complex. Perturbations in 1H chemical shifts of protons in Phe-49, Leu-54, and Leu-27 and the methotrexate H7 and NMe protons were observed in the different complexes and were accounted for by changes in orientation of the benzoyl ring in the various complexes. Binding of oxidized or reduced coenzyme to the binary complexes did not result in different shifts for Leu-27, Leu-54, or Leu-19 protons, and thus, the orientation of the benzoyl ring of the methotrexate analogues is not perturbed greatly by the presence of either oxidized or reduced coenzyme. In the complex with the γ-monoamide analog, the 1H signals of assigned residues in the protein had almost identical shifts with the corresponding protons in the methotrexate-enzyme complex for all residues except His-28 and, to a lesser extent, Leu-27. This indicates that while the His-28 interaction with the MTX γ-CO2- is no longer present in this complex with the γ-amide, there has not been a major change in the overall structure of the two complexes. This behavior contrasts to that of the α-amide complex where 1H signals from protons in several amino acid residues are different compared with their values in the complex formed with methotrexate

  1. Site-specific bioconjugation of a murine dihydrofolate reductase enzyme by copper(I-catalyzed azide-alkyne cycloaddition with retained activity.

    Directory of Open Access Journals (Sweden)

    Sung In Lim

    Full Text Available Cu(I-catalyzed azide-alkyne cycloaddition (CuAAC is an efficient reaction linking an azido and an alkynyl group in the presence of copper catalyst. Incorporation of a non-natural amino acid (NAA containing either an azido or an alkynyl group into a protein allows site-specific bioconjugation in mild conditions via CuAAC. Despite its great potential, bioconjugation of an enzyme has been hampered by several issues including low yield, poor solubility of a ligand, and protein structural/functional perturbation by CuAAC components. In the present study, we incorporated an alkyne-bearing NAA into an enzyme, murine dihydrofolate reductase (mDHFR, in high cell density cultivation of Escherichia coli, and performed CuAAC conjugation with fluorescent azide dyes to evaluate enzyme compatibility of various CuAAC conditions comprising combination of commercially available Cu(I-chelating ligands and reductants. The condensed culture improves the protein yield 19-fold based on the same amount of non-natural amino acid, and the enzyme incubation under the optimized reaction condition did not lead to any activity loss but allowed a fast and high-yield bioconjugation. Using the established conditions, a biotin-azide spacer was efficiently conjugated to mDHFR with retained activity leading to the site-specific immobilization of the biotin-conjugated mDHFR on a streptavidin-coated plate. These results demonstrate that the combination of reactive non-natural amino acid incorporation and the optimized CuAAC can be used to bioconjugate enzymes with retained enzymatic activity.

  2. Crystal Structures of Wild-type and Mutant Methicillin-resistant Staphylococcus aureus Dihydrofolate Reductase Reveal an Alternative Conformation of NADPH that may be Linked to Trimethoprim Resistance

    Energy Technology Data Exchange (ETDEWEB)

    Frey, K.; Liu, J; Lombardo, M; Bolstad, D; Wright, D; Anderson, A

    2009-01-01

    Both hospital- and community-acquired Staphylococcus aureus infections have become major health concerns in terms of morbidity, suffering and cost. Trimethoprim-sulfamethoxazole (TMP-SMZ) is an alternative treatment for methicillin-resistant S. aureus (MRSA) infections. However, TMP-resistant strains have arisen with point mutations in dihydrofolate reductase (DHFR), the target for TMP. A single point mutation, F98Y, has been shown biochemically to confer the majority of this resistance to TMP. Using a structure-based approach, we have designed a series of novel propargyl-linked DHFR inhibitors that are active against several trimethoprim-resistant enzymes. We screened this series against wild-type and mutant (F98Y) S. aureus DHFR and found that several are active against both enzymes and specifically that the meta-biphenyl class of these inhibitors is the most potent. In order to understand the structural basis of this potency, we determined eight high-resolution crystal structures: four each of the wild-type and mutant DHFR enzymes bound to various propargyl-linked DHFR inhibitors. In addition to explaining the structure-activity relationships, several of the structures reveal a novel conformation for the cofactor, NADPH. In this new conformation that is predominantly associated with the mutant enzyme, the nicotinamide ring is displaced from its conserved location and three water molecules complete a network of hydrogen bonds between the nicotinamide ring and the protein. In this new position, NADPH has reduced interactions with the inhibitor. An equilibrium between the two conformations of NADPH, implied by their occupancies in the eight crystal structures, is influenced both by the ligand and the F98Y mutation. The mutation induced equilibrium between two NADPH-binding conformations may contribute to decrease TMP binding and thus may be responsible for TMP resistance.

  3. Structure-based approach to pharmacophore identification, in silico screening, and three-dimensional quantitative structure-activity relationship studies for inhibitors of Trypanosoma cruzi dihydrofolate reductase function

    Energy Technology Data Exchange (ETDEWEB)

    Schormann, N.; Senkovich, O.; Walker, K.; Wright, D.L.; Anderson, A.C.; Rosowsky, A.; Ananthan, S.; Shinkre, B.; Velu, S.; Chattopadhyay, D. (UAB); (Connecticut); (Southern Research); (DFCI)

    2009-07-10

    We have employed a structure-based three-dimensional quantitative structure-activity relationship (3D-QSAR) approach to predict the biochemical activity for inhibitors of T. cruzi dihydrofolate reductase-thymidylate synthase (DHFR-TS). Crystal structures of complexes of the enzyme with eight different inhibitors of the DHFR activity together with the structure in the substrate-free state (DHFR domain) were used to validate and refine docking poses of ligands that constitute likely active conformations. Structural information from these complexes formed the basis for the structure-based alignment used as input for the QSAR study. Contrary to indirect ligand-based approaches the strategy described here employs a direct receptor-based approach. The goal is to generate a library of selective lead inhibitors for further development as antiparasitic agents. 3D-QSAR models were obtained for T. cruzi DHFR-TS (30 inhibitors in learning set) and human DHFR (36 inhibitors in learning set) that show a very good agreement between experimental and predicted enzyme inhibition data. For crossvalidation of the QSAR model(s), we have used the 10% leave-one-out method. The derived 3D-QSAR models were tested against a few selected compounds (a small test set of six inhibitors for each enzyme) with known activity, which were not part of the learning set, and the quality of prediction of the initial 3D-QSAR models demonstrated that such studies are feasible. Further refinement of the models through integration of additional activity data and optimization of reliable docking poses is expected to lead to an improved predictive ability.

  4. Disagreement in genotyping results of drug resistance alleles of the Plasmodium falciparum dihydrofolate reductase (Pfdhfr) gene by allele-specific PCR (ASPCR) assays and Sanger sequencing.

    Science.gov (United States)

    Sharma, Divya; Lather, Manila; Dykes, Cherry L; Dang, Amita S; Adak, Tridibes; Singh, Om P

    2016-01-01

    The rapid spread of antimalarial drug resistance in Plasmodium falciparum over the past few decades has necessitated intensive monitoring of such resistance for an effective malaria control strategy. P. falciparum dihydropteroate synthase (Pfdhps) and P. falciparum dihydrofolate reductase (Pfdhfr) genes act as molecular markers for resistance against the antimalarial drugs sulphadoxine and pyrimethamine, respectively. Resistance to pyrimethamine which is used as a partner drug in artemisinin combination therapy (ACT) is associated with several mutations in the Pfdhfr gene, namely A16V, N51I, C59R, S108N/T and I164L. Therefore, routine monitoring of Pfdhfr-drug-resistant alleles in a population may help in effective drug resistance management. Allele-specific PCR (ASPCR) is one of the commonly used methods for molecular genotyping of these alleles. In this study, we genotyped 55 samples of P. falciparum for allele discrimination at four codons of Pfdhfr (N51, C59, S108 and I164) by ASPCR using published methods and by Sanger's DNA sequencing method. We found that the ASPCR identified a significantly higher number of mutant alleles as compared to the DNA sequencing method. Such discrepancies arise due to the non-specificity of some of the allele-specific primer sets and due to the lack of sensitivity of Sanger's DNA sequencing method to detect minor alleles present in multiple clone infections. This study reveals the need of a highly specific and sensitive method for genotyping and detecting minor drug-resistant alleles present in multiple clonal infections.

  5. Island-wide diversity in single nucleotide polymorphisms of the Plasmodium vivax dihydrofolate reductase and dihydropteroate synthetase genes in Sri Lanka

    Directory of Open Access Journals (Sweden)

    Konradsen Flemming

    2007-03-01

    Full Text Available Abstract Background Single nucleotide polymorphisms (SNPs in the Plasmodium vivax dihydrofolate reductase (Pfdhfr and dihydropteroate synthetase (Pvdhps genes cause parasite resistance to the antifolate drug combination, sulphadoxine/pyrimethamine (SP. Monitoring these SNPs provide insights into the level of drug pressure caused by SP use and presumably other antifolate drugs. In Sri Lanka, chloroquine (CQ with primaquine (PQ and SP with PQ is used as first and second line treatment, respectively, against uncomplicated Plasmodium falciparum and/or P. vivax infections. CQ/PQ is still efficacious against P. vivax infections, thus SP is rarely used and it is assumed that the prevalence of SNPs related to P. vivax SP resistance is low. However, this has not been assessed in Sri Lanka as in most other parts of Asia. This study describes the prevalence and distribution of SNPs related to P. vivax SP resistance across Sri Lanka. Subjects and methods P. vivax-positive samples were collected from subjects presenting at government health facilities across nine of the major malaria endemic districts on the island. The samples were analysed for SNPs/haplotypes at codon 57, 58, 61 and 117 of the Pvdhfr gene and 383, 553 and 585 of the Pvdhps gene by applying PCR followed by a hybridization step using sequence specific oligonucleotide probes (SSOPs in an ELISA format. Results In the study period, the government of Sri Lanka recorded 2,149 P. vivax cases from the nine districts out of which, 454 (21.1% blood samples were obtained. Pvdhfr haplotypes could be constructed for 373 of these. The FSTS wild-haplotype was represented in 257 samples (68.9%, the double mutant LRTS haplotype was the most frequently observed mutant (24.4% while the triple mutation (LRTN was only identified once. Except for two samples of the single mutated Pvdhps GAV haplotype, the remaining samples were wildtype. Geographical differences were apparent, notably a significantly higher

  6. Change in mutation patterns of Plasmodium vivax dihydrofolate reductase (Pvdhfr and dihydropteroate synthase (Pvdhps in P. vivax isolates from malaria endemic areas of Thailand

    Directory of Open Access Journals (Sweden)

    Jiraporn Kuesap

    2011-08-01

    Full Text Available Malaria is the most important public health problem in several countries. In Thailand, co-infections of Plasmodium vivax and Plasmodium falciparum are common. We examined the prevalence and patterns of mutations in P. vivax dihydrofolate reductase (Pvdhfr and P. vivax dihydropteroate synthase (Pvdhps in 103 blood samples collected from patients with P. vivax infection who had attended the malaria clinic in Mae Sot, Tak Province during 2009 and 2010. Using nested polymerase chain reaction-restriction fragment length polymorfism, we examined single nucleotide polymorphisms-haplotypes at amino acid positions 13, 33, 57, 58, 61, 117 and 173 of Pvdhfr and 383 and 553 of Pvdhps. All parasite isolates carried mutant Pvdhfr alleles, of which the most common alleles were triple mutants (99%. Eight different types of Pvdhfr and combination alleles were found, as follows: 57I/58R/117T, 57I/58R/117T, 57I/58R/117T/N, 57L/58R/117T, 57L/58R/117T, 58R/61M/117N, 58R/61M/117N and 13L/57L/58R/117T. The most common Pvdhfr alleles were 57I/58R/117T (77.7%, 57I/58R/117T/N (1%, 57L/58R/117T (5.8% and 58R/61M/117N (14.5%. The most common Pvdhfr alleles were 57I/58R/117T (77.7%, 57I/58R/117T/N (1%, 57L/58R/117T (5.8% and 58R/61M/117N (14.5%. Additionally, we recovered one isolate of a carrying a quadruple mutant allele, 13L/57L/58R/117T. The most prevalent Pvdhps allele was a single mutation in amino acid 383 (82.5%, followed by the wild-type A383/A553 (17.5% allele. Results suggest that all P. vivax isolates in Thailand carry some combination of mutations in Pvdhfr and Pvdhps. Our findings demonstrate that development of new antifolate drugs effective against sulfadoxine-pyrimethamine-resistant P. vivax is required.

  7. Anti-folate drug resistance in Africa: meta-analysis of reported dihydrofolate reductase (dhfr and dihydropteroate synthase (dhps mutant genotype frequencies in African Plasmodium falciparum parasite populations

    Directory of Open Access Journals (Sweden)

    Sridaran Sankar

    2010-08-01

    Full Text Available Abstract Background Mutations in the dihydrofolate reductase (dhfr and dihydropteroate synthase (dhps genes of Plasmodium falciparum are associated with resistance to anti-folate drugs, most notably sulphadoxine-pyrimethamine (SP. Molecular studies document the prevalence of these mutations in parasite populations across the African continent. However, there is no systematic review examining the collective epidemiological significance of these studies. This meta-analysis attempts to: 1 summarize genotype frequency data that are critical for molecular surveillance of anti-folate resistance and 2 identify the specific challenges facing the development of future molecular databases. Methods This review consists of 220 studies published prior to 2009 that report the frequency of select dhfr and dhps mutations in 31 African countries. Maps were created to summarize the location and prevalence of the highly resistant dhfr triple mutant (N51I, C59R, S108N genotype and dhps double mutant (A437G and K540E genotype in Africa. A hierarchical mixed effects logistic regression was used to examine the influence of various factors on reported mutant genotype frequency. These factors include: year and location of study, age and clinical status of sampled population, and reporting conventions for mixed genotype data. Results A database consisting of dhfr and dhps mutant genotype frequencies from all African studies that met selection criteria was created for this analysis. The map illustrates particularly high prevalence of both the dhfr triple and dhps double mutant genotypes along the Kenya-Tanzania border and Malawi. The regression model shows a statistically significant increase in the prevalence of both the dhfr triple and dhps double mutant genotypes in Africa. Conclusion Increasing prevalence of the dhfr triple mutant and dhps double mutant genotypes in Africa are consistent with the loss of efficacy of SP for treatment of clinical malaria in most parts

  8. Pyridine Nucleotide Complexes with Bacillus anthracis Coenzyme A-Disulfide Reductase: A Structural Analysis of Dual NAD(P)H Specificity

    Energy Technology Data Exchange (ETDEWEB)

    Wallen,J.; Paige, C.; Mallett, T.; Karplus, P.; Claiborne, A.

    2008-01-01

    We have recently reported that CoASH is the major low-molecular weight thiol in Bacillus anthracis, and we have now characterized the kinetic and redox properties of the B. anthracis coenzyme A-disulfide reductase (CoADR, BACoADR) and determined the crystal structure at 2.30 Angstroms resolution. While the Staphylococcus aureus and Borrelia burgdorferi CoADRs exhibit strong preferences for NADPH and NADH, respectively, B. anthracis CoADR can use either pyridine nucleotide equally well. Sequence elements within the respective NAD(P)H-binding motifs correctly reflect the preferences for S. aureus and Bo. burgdorferi CoADRs, but leave questions as to how BACoADR can interact with both pyridine nucleotides. The structures of the NADH and NADPH complexes at ca. 2.3 Angstroms resolution reveal that a loop consisting of residues Glu180-Thr187 becomes ordered and changes conformation on NAD(P)H binding. NADH and NADPH interact with nearly identical conformations of this loop; the latter interaction, however, involves a novel binding mode in which the 2'-phosphate of NADPH points out toward solvent. In addition, the NAD(P)H-reduced BACoADR structures provide the first view of the reduced form (Cys42-SH/CoASH) of the Cys42-SSCoA redox center. The Cys42-SH side chain adopts a new conformation in which the conserved Tyr367'-OH and Tyr425'-OH interact with the nascent thiol(ate) on the flavin si-face. Kinetic data with Y367F, Y425F, and Y367, 425F BACoADR mutants indicate that Tyr425' is the primary proton donor in catalysis, with Tyr367' functioning as a cryptic alternate donor in the absence of Tyr425'.

  9. Prediction of Plasmodium falciparum resistance to sulfadoxine/pyrimethamine in vivo by mutations in the dihydrofolate reductase and dihydropteroate synthetase genes: a comparative study between sites of differing endemicity

    DEFF Research Database (Denmark)

    Alifrangis, Michael; Enosse, Sonja; Khalil, Insaf F;

    2003-01-01

    Plasmodium falciparum resistance to sulfadoxine/pyrimethamine (S/P) is due to mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhfr) genes. Large-scale screening of the prevalence of these mutations could facilitate the surveillance of the level of S/P resistance...... marker seems debatable. The differences may reflect variation in the duration and magnitude of S/P usage (or other antifolate drugs) between the sites. Thus, triple dhfr mutations may prove suitable only as a general guideline for detecting emerging S/P resistance in areas where S/P has been introduced...... in vivo. The prevalence of mutations in dhfr and dhps in relation to S/P efficacy was studied in four sites of differing endemicity in Sudan, Mozambique, and Tanzania. The sites were organized in order of increasing resistance and a significant increase in the prevalence of triple mutations in codons c51...

  10. Dihydrofolate Reductase Deficiency Due to a Homozygous DHFR Mutation Causes Megaloblastic Anemia and Cerebral Folate Deficiency Leading to Severe Neurologic Disease

    OpenAIRE

    Cario, Holger; Smith, Desirée E. C.; Blom, Henk; Blau, Nenad; Bode, Harald; Holzmann, Karlheinz; Pannicke, Ulrich; Hopfner, Karl-Peter; Rump, Eva-Maria; Ayric, Zuleya; Kohne, Elisabeth; Debatin, Klaus-Michael; Smulders, Yvo; Schwarz, Klaus

    2011-01-01

    The importance of intracellular folate metabolism is illustrated by the severity of symptoms and complications caused by inborn disorders of folate metabolism or by folate deficiency. We examined three children of healthy, distantly related parents presenting with megaloblastic anemia and cerebral folate deficiency causing neurologic disease with atypical childhood absence epilepsy. Genome-wide homozygosity mapping revealed a candidate region on chromosome 5 including the dihydrofolate reduct...

  11. Role of Mutations in Dihydrofolate Reductase DfrA (Rv2763c) and Thymidylate Synthase ThyA (Rv2764c) in Mycobacterium tuberculosis Drug Resistance

    KAUST Repository

    Koser, C. U.

    2010-09-17

    We would like to comment on a number of recent reports in this journal (6, 8, 12, 18) concerning Mycobacterium tuberculosis dihydrofolate reductase (DHFR), encoded by dfrA (Rv2763c). Around 36% of phenotypically para-aminosalicylic acid (PAS)-resistant M. tuberculosis strains harbor mutations in thyA (Rv2764c), which encodes a thymidylate synthase (20). In their effort to elucidate the remaining unknown resistance mechanism(s), Mathys et al. extended their sequence analysis to a number of additional genes, including dfrA (12). It was unclear whether the three dfrA mutations they identified in the PAS-resistant strains P-693 and P-3158 could contribute to PAS resistance on their own. Nonetheless, these findings are notable for two reasons. First, isoniazid (INH) has been shown to inhibit M. tuberculosis DHFR in vitro (1). Whether the same holds true for ethionamide, which shares a number of common resistance mechanisms with INH, was not tested (J. Blanchard, personal communication). In any case, the clinical relevance of DHFR-mediated INH resistance remains enigmatic. To date, only Ho et al. have addressed this question, but they did not identify any dfrA mutations in a screen of 127 INH-resistant clinical isolates (8). Consequently, Mathys et al. remain the first to describe mutations in this target (12). However, given that isolates with mutated DHFR are members of a cluster with baseline INH resistance, the importance of these mutations with respect to INH resistance remains unclear. Irrespective of their relevance in INH resistance, these dfrA mutations are noteworthy for a second reason. Contrary to previous wisdom, Forgacs et al. recently showed that M. tuberculosis is sensitive to the drug combination trimethoprim-sulfamethoxazole (TMP-SMX) (6, 18). DHFR is competitively inhibited by TMP, and consequently, mutations therein lead to resistance in a variety of organisms (9, 16, 19). The crystal structures of the wild-type M. tuberculosis DHFR in complex with

  12. Computer graphic modeling in drug design--conformational analysis of antifolate binding to avian dihydrofolate reductase: crystal and molecular structures of 2,4-diamino-5-cyclohexyl-6-methylpyrimidine and 5-cyclohexyl-6-methyluracil.

    Science.gov (United States)

    Cody, V; Ciszak, E

    1991-05-01

    The results of crystal structure determinations of the antifolate 2,4-diamino-5-cyclohexyl-6-methylprimidine (I), and its uracil derivative (II), show that the 5-cyclohexyl ring is gauche to the planar pyrimidine ring with torsion angles 82.4 (3) degrees and 63.7 (3) degrees for (I) and (II), respectively. Hydrogen bond patterns observed for these free base pyrimidines indicate a preference for N...N or N...O dimer formation around inversion centers, as observed in other antifolate structures. Computer graphic modeling studies were carried out comparing the avian dihydrofolate reductase active site interactions of the cyclohexyl antifolate (I) with the more potent 5-adamantyl analog and the less potent 5-hexyl and 5-heptyl antifolates. These data showed that although the cyclohexyl ring fits into the same conformational space as adamantyl, it makes fewer hydrophobic contacts. Similarly, cyclohexyl fills the active site better than either the 5-n-hexyl or heptyl side chains. These data are consistent with the increased potency of the adamantyl and cyclohexyl antifolates compared to n-alkyl analogs with similar hydrophobicities. These data indicate that the rigid structure of these ring systems increases their hydrophobic interactions, thus enhancing their biochemical activity.

  13. Vacuum-Ultraviolet Circular Dichroism Spectra of Escherichia coli Dihydrofolate Reductase and Its Mutants: Contributions of Phenylalanine and Tyrosine Side Chains and Exciton Coupling of Two Tryptophan Side Chains.

    Science.gov (United States)

    Ohmae, Eiji; Tanaka, Suguru; Miyashita, Yurina; Katayanagi, Katsuo; Matsuo, Koichi

    2015-10-15

    Vacuum-ultraviolet (VUV) circular dichroism (CD) spectroscopy has recently been used for secondary structure analysis of proteins; however, the contribution of aromatic side chains to protein VUV CD spectra is unresolved. In this report, VUV CD spectra of 10 Escherichia coli dihydrofolate reductase (DHFR) mutants, in which each phenylalanine or tyrosine residue was mutated to leucine, were measured down to 175 nm at 25 °C and pH 8.0 to elucidate the contributions of these aromatic side chains to the high-energy transitions of peptide bonds. The VUV CD spectra of these mutants were different from the spectrum of the wild-type protein, indicating that the contribution of the phenylalanine and tyrosine side chains of DHFR extends to the VUV region. Furthermore, the VUV CD spectrum and the folate- or NADP(+)-induced spectral change of F103L mutant DHFR indicated a modification and regeneration of exciton coupling between the Trp47 and Trp74 side chains, respectively, suggesting that exciton coupling may also contribute to the CD spectrum of DHFR in the VUV region. These results should be useful for theoretically characterizing the contribution of aromatic side chains to protein CD spectra, leading to the improvement of protein secondary-structure analysis by VUV CD spectroscopy.

  14. Molecular epidemiology of malaria in Cameroon. XXX. sequence analysis of Plasmodium falciparum ATPase 6, dihydrofolate reductase, and dihydropteroate synthase resistance markers in clinical isolates from children treated with an artesunate-sulfadoxine-pyrimethamine combination.

    Science.gov (United States)

    Menemedengue, Virginie; Sahnouni, Khalifa; Basco, Leonardo; Tahar, Rachida

    2011-07-01

    Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes are reliable molecular markers for antifolate resistance. The P. falciparum ATPase 6 (pfatp6) gene has been proposed to be a potential marker for artemisinin resistance. In our previous clinical study, we showed that artesunate-sulfadoxine-pyrimethamine is highly effective against uncomplicated malaria in Yaoundé, Cameroon. In the present study, dhfr, dhps, and pfatp6 mutations in P. falciparum isolates obtained from children treated with artesunate-sulfadoxine-pyrimethamine were determined. All 61 isolates had wild-type Pfatp6 263, 623, and 769 alleles, and 11 (18%) had a single E431K substitution. Three additional mutations, E643Q, E432K, and E641Q, were detected. The results did not indicate any warning signal of serious concern (i.e., no parasites were seen with quintuple dhfr-dhps, DHFR Ile164Leu, or pfatp6 mutations), as confirmed by the high clinical efficacy of artesunate-sulfadoxine-pyrimethamine. Further studies are required to identify a molecular marker that reliably predicts artemisinin resistance.

  15. Dihydrofolate-Reductase Mutations in Plasmodium knowlesi Appear Unrelated to Selective Drug Pressure from Putative Human-To-Human Transmission in Sabah, Malaysia.

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    Matthew J Grigg

    Full Text Available Malaria caused by zoonotic Plasmodium knowlesi is an emerging threat in Eastern Malaysia. Despite demonstrated vector competency, it is unknown whether human-to-human (H-H transmission is occurring naturally. We sought evidence of drug selection pressure from the antimalarial sulfadoxine-pyrimethamine (SP as a potential marker of H-H transmission.The P. knowlesi dihdyrofolate-reductase (pkdhfr gene was sequenced from 449 P. knowlesi malaria cases from Sabah (Malaysian Borneo and genotypes evaluated for association with clinical and epidemiological factors. Homology modelling using the pvdhfr template was used to assess the effect of pkdhfr mutations on the pyrimethamine binding pocket.Fourteen non-synonymous mutations were detected, with the most common being at codon T91P (10.2% and R34L (10.0%, resulting in 21 different genotypes, including the wild-type, 14 single mutants, and six double mutants. One third of the P. knowlesi infections were with pkdhfr mutants; 145 (32% patients had single mutants and 14 (3% had double-mutants. In contrast, among the 47 P. falciparum isolates sequenced, three pfdhfr genotypes were found, with the double mutant 108N+59R being fixed and the triple mutants 108N+59R+51I and 108N+59R+164L occurring with frequencies of 4% and 8%, respectively. Two non-random spatio-temporal clusters were identified with pkdhfr genotypes. There was no association between pkdhfr mutations and hyperparasitaemia or malaria severity, both hypothesized to be indicators of H-H transmission. The orthologous loci associated with resistance in P. falciparum were not mutated in pkdhfr. Subsequent homology modelling of pkdhfr revealed gene loci 13, 53, 120, and 173 as being critical for pyrimethamine binding, however, there were no mutations at these sites among the 449 P. knowlesi isolates.Although moderate diversity was observed in pkdhfr in Sabah, there was no evidence this reflected selective antifolate drug pressure in humans.

  16. Contributions of tryptophan 24 and glutamate 30 to binding long-lived water molecules in the ternary complex of human dihydrofolate reductase with methotrexate and NADPH studied by site-directed mutagenesis and nuclear magnetic resonance spectroscopy.

    Science.gov (United States)

    Meiering, E M; Li, H; Delcamp, T J; Freisheim, J H; Wagner, G

    1995-03-24

    Previous NMR studies on the ternary complex of human dihydrofolate reductase (hDHFR) with methotrexate (MTX) and NADPH detected six long-lived bound water molecules. Two of the water molecules, WatA and WatB, stabilize the structure of the protein while the other four, WatC, WatD, WatE and WatF, are involved in substrate binding and specificity. WatE may also act as a proton shuttle during catalysis. Here, the contributions of individual residues to the binding of these water molecules are investigated by performing NMR experiments on ternary complexes of mutant enzymes, W24F, E30A and E30Q. W24 and E30 are conserved residues that form hydrogen bonds with WatE in crystal structures of DHFR. Nuclear Overhauser effects (NOEs) are detected between WatE and the protein in all the mutant complexes, hence WatE still has a long lifetime bound to the complex when one of its hydrogen-bonding partners is deleted or altered by mutagenesis. The NOEs for WatE are much weaker, however, in the mutants than in wild-type. The NOEs for the other water molecules in and near the active site, WatA, WatC, WatD and WatF, also tend to be weaker in the mutant complexes. Little or no change is apparent in the NOEs for WatB, which is located outside the active site, farthest from the mutated residues. The decreased NOE intensities for the bound water molecules could be caused by changes in the positions and/or lifetimes of the water molecules. Chemical shift and NOE data indicate that the mutants have structures very similar to that of wild-type hDHFR, with possible conformational changes occurring only near the mutated residues. Based on the lack of structural change in the protein and evidence for increased structural fluctuations in the active sites of the mutant enzymes, it is likely that the NOE changes are caused, at least in part, by decreases in the lifetimes of the bound water molecules.

  17. Bacillus anthracis

    OpenAIRE

    2003-01-01

    The events of 11 September 2001 and the subsequent anthrax outbreaks have shown that the West needs to be prepared for an increasing number of terrorist attacks, which may include the use of biological warfare. Bacillus anthracis has long been considered a potential biological warfare agent, and this review will discuss the history of its use as such. It will also cover the biology of this organism and the clinical features of the three disease forms that it can produce: cutaneous, gastrointe...

  18. Bacillus anthracis

    OpenAIRE

    BOSERET, GÉRALDINE; Linden, Annick; Mainil, Jacques

    2002-01-01

    The literature describes several methods for detection of Bacillus anthracis based on application of specific bacteriophages. The following methods of pahoinpitely are used to identify the causative agent of anthrax: the reaction of bacteriophage titer growth (RBTG), the reaction of phage adsorption (RPA), fagoterapii method (FTM) and fluorescentserological method (FSM). The essence of RBTG consists in the following: if there is the researchform of bacteria presents in the test material, then...

  19. 双突变二氢叶酸还原酶基因对小鼠骨髓细胞化疗保护作用的体外研究%Research of chemoprotection through transferring double-mutant dihydrofolate reductase gene into mice bone marrow cells in vitro

    Institute of Scientific and Technical Information of China (English)

    路平; 鲁阳; 徐惠绵; 王舒宝; 陈峻青; 赵实诚

    2005-01-01

    目的:探讨将人双突变的二氢叶酸还原酶基因(dihydrofolate reductase,DHFR)导入小鼠骨髓细胞中,观察小鼠骨髓细胞对大剂量甲氨蝶呤(methotrexate,MTX)的耐受性,研究骨髓耐受大剂量化疗的可行性.方法:以反转录病毒为载体,将DHFR通过共培养转染入小鼠骨髓干细胞,观察共培养后的骨髓细胞耐MTX CFU-GM生成情况;小鼠骨髓细胞提取的DNA,用PCR检测小鼠骨髓细胞耐药基因的表达.结果:含有耐药基因(SFG-F/S)的骨髓细胞耐药克隆为16%,对照组为0, χ2=47.88, P<0.005;转基因小鼠骨髓细胞经PCR检测,显示有F/S基因条带;耐药基因转染后小鼠骨髓细胞对MTX 的耐受明显增加.结论:耐药基因可以进入小鼠骨髓细胞并且获得共表达,提高了造血细胞对MTX的耐药性.

  20. 氨甲蝶呤对映体获得性耐药A549细胞株二氢叶酸还原酶基因表达分析%Analysis for different expression of dihydrofolate reductase gene in methotrexate enantiomers-resistant lung cancer A549 cell lines

    Institute of Scientific and Technical Information of China (English)

    李道静; 何晓东; 孙余婕; 凡任芝; 许维东; 孙利; 张永娟; 张白银; 沈佐君

    2011-01-01

    Objective To study the relationship between methotrexate (MTX) enantiomers resistance and levels of dihydrofolate reductase (DHFR) mRNA. Methods AS49 cells were exposed to intermittenfiy and progressively increasing dose of the two enantiomers of MTX. The expression of DHFR gene was assayed by real-time fluorescence quantitative polymerase chain reaction ( FQ-PCR ). Resuits The resistant indexes of cell lines were different for L-( +)-MTX and D-(-)-MTX enantiomer. D-(-)-MTX resistance cell lines showed higher resistant index than L-( + )-MTX resistant cell lines. The expression level of DHFR mRNA in the resistant cell lines was less than that of parent cells at the concentration of 15 μ mol/L of beth L-( + )- and D-(-)-MTX enantiomer (P > 0.05 ). The expression level of DHFR mRNA in resistant cell lines was relatively high at increasing concentration of 35 μmol/L and 45 μ mol/L of D-(-) MTX. The results of the FQ-PCR revealed that the MTX resistance was associated with increased expression of DHFR mRNA. Conclusion The expression of DHFR gene was inhibited after the cell lines induced by 15 μmol/L of D-(-) -MTX enantoimers in MTX resistant cell line. The expression of DHFR gene showed significant difference in chirality. DHFR mRNA should be examined during MTX treatment, which could be helpful to prognosticate the resistant status of cell line.%目的 研究氨甲蝶呤(MTX)对映体[L-(+)-MTX和D-(-)-MTX]耐药与二氢叶酸还原酶(DHFR)基因表达的关系.方法 用浓度递增结合低剂量持续诱导法获得A549细胞对不同构型及不同浓度的MTX对映体的耐药细胞株,荧光定量PCR检测耐药细胞株中DHFR基因的相对含量.结果 对两种不同对映体的获得性耐药存在差异,D型耐药细胞耐药指数高于L型;对映体各浓度耐药细胞间耐药指数也有差异.15 μmol/L L型、D型MTX首次诱导耐药细胞的DHFR相对含量低于亲本细胞,对该浓度对映体耐药的各细胞组间没有差别(P>0

  1. Application of in vivo induced antigen technology (IVIAT to Bacillus anthracis.

    Directory of Open Access Journals (Sweden)

    Sean M Rollins

    Full Text Available In vivo induced antigen technology (IVIAT is an immuno-screening technique that identifies bacterial antigens expressed during infection and not during standard in vitro culturing conditions. We applied IVIAT to Bacillus anthracis and identified PagA, seven members of a N-acetylmuramoyl-L-alanine amidase autolysin family, three P60 family lipoproteins, two transporters, spore cortex lytic protein SleB, a penicillin binding protein, a putative prophage holin, respiratory nitrate reductase NarG, and three proteins of unknown function. Using quantitative real-time PCR comparing RNA isolated from in vitro cultured B. anthracis to RNA isolated from BALB/c mice infected with virulent Ames strain B. anthracis, we confirmed induced expression in vivo for a subset of B. anthracis genes identified by IVIAT, including L-alanine amidases BA3767, BA4073, and amiA (pXO2-42; the bacteriophage holin gene BA4074; and pagA (pXO1-110. The exogenous addition of two purified putative autolysins identified by IVIAT, N-acetylmuramoyl-L-alanine amidases BA0485 and BA2446, to vegetative B. anthracis cell suspensions induced a species-specific change in bacterial morphology and reduction in viable bacterial cells. Many of the proteins identified in our screen are predicted to affect peptidoglycan re-modeling, and our results support significant cell wall structural remodeling activity during B. anthracis infection. Identification of L-alanine amidases with B. anthracis specificity may suggest new potential therapeutic targets.

  2. Application of In Vivo Induced Antigen Technology (IVIAT) to Bacillus anthracis

    OpenAIRE

    Peppercorn, Amanda; Young, John S; Drysdale, Melissa; Baresch, Andrea; Bikowski, Margaret V.; Ashford, David A.; Quinn, Conrad P.; Handfield, Martin; Hillman, Jeffrey D.; Lyons, C. Rick; Koehler, Theresa M.; Sonenshein, Abraham L.; Rollins, Sean McKenzie; Calderwood, Stephen Beaven; Ryan, Edward Thomas

    2008-01-01

    In vivo induced antigen technology (IVIAT) is an immuno-screening technique that identifies bacterial antigens expressed during infection and not during standard in vitro culturing conditions. We applied IVIAT to Bacillus anthracis and identified PagA, seven members of a N-acetylmuramoyl-L-alanine amidase autolysin family, three P60 family lipoproteins, two transporters, spore cortex lytic protein SleB, a penicillin binding protein, a putative prophage holin, respiratory nitrate reductase Nar...

  3. Purification and properties of the dihydrofolate synthetase from Serratia indica

    International Nuclear Information System (INIS)

    The dihydrofolate synthetase (EC6.3.2.12) responsible for catalyzing the synthesis of dihydrofolic acid from dihydropteroic acid and L-glutamic acid was purified about 130-fold from extracts of Serratia indica IFO 3759 by ammonium sulfate fractionation, DEAE-Sephadex column chromatography, Sephadex G-200 gel filtration, and DEAE-cellulose column chromatography. The enzyme preparation obtained was shown to be homogeneous by DEAE-cellulose column chromatography and ultracentrifugal analysis. The sedimentation coefficient of this enzyme was 3.9 S, and the molecular weight was determined to be about 47,000 by Sephadex G-100. The optimum pH for the reaction was 9.0. The enzymatic reaction required dihydropteroate, L-glutamate and ATP as substrates, and Mg2+ and K+ as cofactors. γ-L-Glutamyl-L-glutamic acid cannot replace L-glutamic acid as the substrate. Neither pteroic acid nor tetrahydropteroic acid can be used as the substrate. ATP was partially replaced by ITP or GTP. The enzyme reaction was inhibited by the addition of ADP, but not by AMP. One mole of dihydrofolate, 1 mole of ADP and 1 mole of orthophosphate were produced from each 1 mole of dihydropteroic acid, L-glutamic acid, and ATP. These results suggest that the systematic name for the dihydrofolate synthetase is 7,8-dihydropteroate: L-glutamate ligase (ADP). (auth.)

  4. Quantitative immunofluorescence studies of the serology of Bacillus anthracis spores.

    OpenAIRE

    Phillips, A. P.; Martin, K L

    1983-01-01

    A fluorescein-conjugated antibody against formalin-inactivated spores of Bacillus anthracis Vollum reacted only weakly with a variety of Bacillus species in microfluorometric immunofluorescence assays. A conjugated antibody against spores of B. anthracis Sterne showed little affinity for spores of several B. anthracis isolates including B. anthracis Vollum, indicating that more than one anthrax spore serotype exists.

  5. Prevalence of Dihydrofolate reductase gene mutations in Plasmodium falciparum isolate from pregnant women in Nigeria

    Directory of Open Access Journals (Sweden)

    Olusola Ojurongbe

    2011-12-01

    Full Text Available We assessed the prevalence of Plasmodium falciparum and the frequency of the dhfr triple mutation that is associated with antifolate drug resistance among P. falciparumisolates obtained from pregnant women in Ilorin, Nigeria. The study included 179 women in the second and third trimester of pregnancy who have been exposed to intermittent preventive treatment in pregnancy (IPTp with sulfadoxinepyrimethamine. Thick and thin blood films and PCR were used for malaria parasite detection. Blood group and hemoglobin concentration were also determined. Mutations in P. falciparum dhfr were analyzed by sequencing DNA obtained from blood spots on filter paper. Prevalence of P. falciparum in the population (PCR corrected was 44.1% (79/179 with 66.7% and 33.3% in the second and third trimester, respectively. Primigravide (51.3% were more infected than multigravide (48.7% but the difference was not statistically significant. Women in blood group A had the highest P. falciparum malaria infection (30.8%. The mean hemoglobin concentration was lower among those infected with malaria parasite. Also, more women with the malaria parasite (38.4% had anemia compare to those without (21.4%. The prevalence of the P. falciparum dhfr mutant alleles was 64.1%, 61.5%, 38.5%, and 12.8% for I51, R59, N108 and T108, respectively. None of the samples had the L164 mutation. The combined triple dhfr mutation (51 + 59 + 108 in the population was 17.9% (7 of 39. Also, the prevalence of the triple mutant alleles was not significantly associated to the number of doses of SP taken by the women. These findings highlight the need for a regular assessment of IPTp/SP efficacy, and evaluation of possible alternative drugs.

  6. Bacillus anthracis diversity in Kruger National Park.

    Science.gov (United States)

    Smith, K L; DeVos, V; Bryden, H; Price, L B; Hugh-Jones, M E; Keim, P

    2000-10-01

    The Kruger National Park (KNP), South Africa, has a recorded history of periodic anthrax epidemics causing widespread disease among wild animals. Bacillus anthracis is the causative agent of anthrax, a disease primarily affecting ungulate herbivores. Worldwide there is little diversity among B. anthracis isolates, but examination of variable-number tandem repeat (VNTR) loci has identified six major clones, with the most dissimilar types split into the A and B branches. Both the A and B types are found in southern Africa, giving this region the greatest genetic diversity of B. anthracis worldwide. Consequently, southern Africa has been hypothesized to be the geographic origin of B. anthracis. In this study, we identify the genotypic types of 98 KNP B. anthracis isolates using multiple-locus VNTR analysis. Two major types are evident, the A branch and the B branch. The spatial and temporal distribution of the different genotypes indicates that anthrax epidemic foci are independent, though correlated through environmental cues. Kruger B isolates were found on significantly higher-calcium and higher-pH soils than were Kruger type A. This relationship between genotype and soil chemistry may be due to adaptive differences among divergent anthrax strains. While this association may be simply fortuitous, adaptation of A types to diverse environmental conditions is consistent with their greater geographic dispersal and genetic dissimilarity.

  7. Characterization of Bacillus anthracis persistence in vivo.

    Directory of Open Access Journals (Sweden)

    Sarah A Jenkins

    Full Text Available Pulmonary exposure to Bacillus anthracis spores initiates inhalational anthrax, a life-threatening infection. It is known that dormant spores can be recovered from the lungs of infected animals months after the initial spore exposure. Consequently, a 60-day course antibiotic treatment is recommended for exposed individuals. However, there has been little information regarding details or mechanisms of spore persistence in vivo. In this study, we investigated spore persistence in a mouse model. The results indicated that weeks after intranasal inoculation with B. anthracis spores, substantial amounts of spores could be recovered from the mouse lung. Moreover, spores of B. anthracis were significantly better at persisting in the lung than spores of a non-pathogenic Bacillus subtilis strain. The majority of B. anthracis spores in the lung were tightly associated with the lung tissue, as they could not be readily removed by lavage. Immunofluorescence staining of lung sections showed that spores associated with the alveolar and airway epithelium. Confocal analysis indicated that some of the spores were inside epithelial cells. This was further confirmed by differential immunofluorescence staining of lung cells harvested from the infected lungs, suggesting that association with lung epithelial cells may provide an advantage to spore persistence in the lung. There was no or very mild inflammation in the infected lungs. Furthermore, spores were present in the lung tissue as single spores rather than in clusters. We also showed that the anthrax toxins did not play a role in persistence. Together, the results suggest that B. anthracis spores have special properties that promote their persistence in the lung, and that there may be multiple mechanisms contributing to spore persistence.

  8. Differential toxicity of mitomycin C and porfiromycin to aerobic and hypoxic Chinese hamster ovary cells overexpressing human NADPH:cytochrome c (P-450) reductase.

    OpenAIRE

    Belcourt, M F; Hodnick, W F; Rockwell, S; Sartorelli, A C

    1996-01-01

    Purified NADPH:cytochrome c (P-450) reductase (FpT; NADPH-ferrihemoprotein oxidoreductase, EC 1.6.2.4) can reductively activate mitomycin antibiotics through a one-electron reduction to species that alkylate DNA. To assess the involvement of FpT in the intracellular activation of the mitomycins, transfectants overexpressing a human FpT cDNA were established from a Chinese hamster ovary cell line deficient in dihydrofolate reductase (CHO-K1/dhfr-). The parental cell line was equisensitive to t...

  9. Genetic Characterization of Bacillus anthracis 17 JB strain

    Directory of Open Access Journals (Sweden)

    Sakineh Seyed-Mohamadi

    2015-11-01

    Full Text Available Background and Objectives: Bacillus anthracis is one of the most homogenous bacteria ever described. Bacillus anthracis 17JB is a laboratory strain. It is broadly used as a challenge strain in guinea pigs for potency test of anthrax vaccine.Material and Methods: This work describes genetic characterization of B. anthracis 17 JB strain using the SNPs and MLVA genotyping.Results and Conclusion: In SNPs typing, the originally French 17JB strain represented the A. Br. 008/009 subgroup. In Levy's genotyping method, 843, 451 and 864 bp long fragments were identified at AA03, AJ03 and AA07 loci, respectively. In the vaccine manufacturer perspective these findings are much valuable on their own account, but similar research is required to extend molecular knowledge of B. anthracis epidemiology in Persia.Keywords: Bacillus anthracis 17JB, Genetic characterization, SNPs typing

  10. Detection of Bacillus anthracis DNA by LightCycler PCR

    OpenAIRE

    Bell, Constance A.; Uhl, James R.; Hadfield, Ted L.; David, John C.; Meyer, Richard F.; Smith, Thomas F.; Cockerill III, Franklin R.

    2002-01-01

    Anthrax is a zoonotic disease that is also well recognized as a potential agent of bioterrorism. Routine culture and biochemical testing methods are useful for the identification of Bacillus anthracis, but a definitive identification may take 24 to 48 h or longer and may require that specimens be referred to another laboratory. Virulent isolates of B. anthracis contain two plasmids (pX01 and pX02) with unique targets that allow the rapid and specific identification of B. anthracis by PCR. We ...

  11. Immunological analysis of cell-associated antigens of Bacillus anthracis.

    OpenAIRE

    Ezzell, J W; Abshire, T. G.

    1988-01-01

    Sera from Hartley guinea pigs vaccinated with a veterinary live spore anthrax vaccine were compared with sera from guinea pigs vaccinated with the human anthrax vaccine, which consists of aluminum hydroxide-adsorbed culture proteins of Bacillus anthracis V770-NP-1R. Sera from animals vaccinated with the spore vaccine recognized two major B. anthracis vegetative cell-associated proteins that were either not recognized or poorly recognized by sera from animals that received the human vaccine. T...

  12. Functional Comparison of the Two Bacillus anthracis Glutamate Racemases▿

    OpenAIRE

    Dodd, Dylan; Reese, Joseph G.; Louer, Craig R.; Ballard, Jimmy D.; Spies, M. Ashley; Blanke, Steven R.

    2007-01-01

    Glutamate racemase activity in Bacillus anthracis is of significant interest with respect to chemotherapeutic drug design, because l-glutamate stereoisomerization to d-glutamate is predicted to be closely associated with peptidoglycan and capsule biosynthesis, which are important for growth and virulence, respectively. In contrast to most bacteria, which harbor a single glutamate racemase gene, the genomic sequence of B. anthracis predicts two genes encoding glutamate racemases, racE1 and rac...

  13. Molecular Epidemiology of Bacillus anthracis: Determining the Correct Origin▿

    OpenAIRE

    Pilo, Paola; Perreten, Vincent; Frey, Joachim

    2008-01-01

    We analyzed and compared strains of Bacillus anthracis isolated from husbandry and industrial anthrax cases in Switzerland between 1952 and 1981 with published data using multiple-locus variable-number tandem repeat analysis. Strains isolated from autochthonous cases of anthrax in cattle belong to genotype B2, together with strains from continental Europe, while human B. anthracis strains clustered with genotype A4. These strains could be traced back to outbreaks of human anthrax that occurre...

  14. Natural Dissemination of Bacillus anthracis Spores in Northern Canada

    OpenAIRE

    Dragon, D C; Bader, D. E.; Mitchell, J.; Woollen, N.

    2005-01-01

    Soil samples were collected from around fresh and year-old bison carcasses and areas not associated with known carcasses in Wood Buffalo National Park during an active anthrax outbreak in the summer of 2001. Sample selection with a grid provided the most complete coverage of a site. Soil samples were screened for viable Bacillus anthracis spores via selective culture, phenotypic analysis, and PCR. Bacillus anthracis spores were isolated from 28.4% of the samples. The highest concentrations of...

  15. Production and purification of Bacillus anthracis protective antigen

    OpenAIRE

    2005-01-01

    Protective antigen (PA) plays crucial roles in the pathogenicity and virulence of Bacillus anthracis. Animals or human immunised with the protein acquire a complete protection against the disease. In addition to vaccine, PA can also be developed into a sensitive diagnostic test for anthrax. The purpose of this study was to produce PA using a culture medium easily obtained, and to develop a simple and effective technique for purification of the protein. To produce PA, B. anthracis Sterne 34F2 ...

  16. Bacillus anthracis IsdG, a Heme-Degrading Monooxygenase

    OpenAIRE

    Skaar, Eric P.; Gaspar, Andrew H.; Schneewind, Olaf

    2006-01-01

    Bacillus anthracis, the causative agent of anthrax, utilizes hemin and hemoglobin for growth in culture, suggesting that these host molecules serve as sources for the nutrient iron during bacterial infection. Bioinformatic analyses of the B. anthracis genome revealed genes with similarity to the iron-regulated surface determinant (isd) system responsible for heme uptake in Staphylococcus aureus. We show that the protein product of one of these genes, isdG, binds hemin in a manner resembling t...

  17. Genetic analysis of petrobactin transport in Bacillus anthracis

    OpenAIRE

    Carlson, Paul E.; Dixon, Shandee D.; Janes, Brian K.; Carr, Katherine A.; Nusca, Tyler D.; Anderson, Erica C.; Keene, Sarra E.; Sherman, David H.; Hanna, Philip C.

    2010-01-01

    Iron acquisition mechanisms play an important role in the pathogenesis of many infectious microbes. In Bacillus anthracis, the siderophore petrobactin is required for both growth in iron depleted conditions and for full virulence of the bacterium. Here we demonstrate the roles of two putative petrobactin binding proteins FatB and FpuA (encoded by GBAA5330 and GBAA4766, respectively) in Bacillus anthracis iron acquisition and pathogenesis. Markerless deletion mutants were created using allelic...

  18. Response of Plasmodium falciparum to cotrimoxazole therapy: relationship with plasma drug concentrations and dihydrofolate reductase and dihydropteroate synthase genotypes

    DEFF Research Database (Denmark)

    Khalil, Insaf F; Rønn, Anita M; Alifrangis, Michael;

    2005-01-01

    , MSP-1, MSP-2, and glutamate-rich protein before treatment and upon recrudescence showed that all recurring parasites were recrudescences. The plasma levels of TRM, AcSMX, and SMX indicated adequate drug absorption in all patients. This suggests parasite resistance as a cause of treatment failure...... treatment and at days 3, 7, and 14 or upon recrudescence to ascertain drug absorption. Forty patients (89%) had an adequate clinical response, one patient (2%) had an early treatment failure response, while four patients (8%) showed late treatment failure responses. Genotyping of merozoite surface protein 1....... The presence of dhfr Ile 51 and Asn 108 alone or coupled with dhps Ala-436 among parasites that were cleared after treatment indicates that these alleles alone are insufficient to cause in vivo resistance. However, the presence of the triple mutant dhfr (Ile-51/Arg-59/Asn-108) with the dhps Gly-437 genotype...

  19. Novel giant siphovirus from Bacillus anthracis features unusual genome characteristics.

    Directory of Open Access Journals (Sweden)

    Holly H Ganz

    Full Text Available Here we present vB_BanS-Tsamsa, a novel temperate phage isolated from Bacillus anthracis, the agent responsible for anthrax infections in wildlife, livestock and humans. Tsamsa phage is a giant siphovirus (order Caudovirales, featuring a long, flexible and non-contractile tail of 440 nm (not including baseplate structure and an isometric head of 82 nm in diameter. We induced Tsamsa phage in samples from two different carcass sites in Etosha National Park, Namibia. The Tsamsa phage genome is the largest sequenced Bacillus siphovirus, containing 168,876 bp and 272 ORFs. The genome features an integrase/recombinase enzyme, indicative of a temperate lifestyle. Among bacterial strains tested, the phage infected only certain members of the Bacillus cereus sensu lato group (B. anthracis, B. cereus and B. thuringiensis and exhibited moderate specificity for B. anthracis. Tsamsa lysed seven out of 25 B. cereus strains, two out of five B. thuringiensis strains and six out of seven B. anthracis strains tested. It did not lyse B. anthracis PAK-1, an atypical strain that is also resistant to both gamma phage and cherry phage. The Tsamsa endolysin features a broader lytic spectrum than the phage host range, indicating possible use of the enzyme in Bacillus biocontrol.

  20. Bacillus anthracis Factors for Phagosomal Escape

    Directory of Open Access Journals (Sweden)

    Irene Zornetta

    2012-07-01

    Full Text Available The mechanism of phagosome escape by intracellular pathogens is an important step in the infectious cycle. During the establishment of anthrax, Bacillus anthracis undergoes a transient intracellular phase in which spores are engulfed by local phagocytes. Spores germinate inside phagosomes and grow to vegetative bacilli, which emerge from their resident intracellular compartments, replicate and eventually exit from the plasma membrane. During germination, B. anthracis secretes multiple factors that can help its resistance to the phagocytes. Here the possible role of B. anthracis toxins, phospholipases, antioxidant enzymes and capsules in the phagosomal escape and survival, is analyzed and compared with that of factors of other microbial pathogens involved in the same type of process.

  1. Bacillus anthracis secretes proteins that mediate heme acquisition from hemoglobin.

    Directory of Open Access Journals (Sweden)

    Anthony W Maresso

    Full Text Available Acquisition of iron is necessary for the replication of nearly all bacterial pathogens; however, iron of vertebrate hosts is mostly sequestered by heme and bound to hemoglobin within red blood cells. In Bacillus anthracis, the spore-forming agent of anthrax, the mechanisms of iron scavenging from hemoglobin are unknown. We report here that B. anthracis secretes IsdX1 and IsdX2, two NEAT domain proteins, to remove heme from hemoglobin, thereby retrieving iron for bacterial growth. Unlike other Gram-positive bacteria, which rely on cell wall anchored Isd proteins for heme scavenging, B. anthracis seems to have also evolved NEAT domain proteins in the extracellular milieu and in the bacterial envelope to provide for the passage of heme.

  2. Secretion Genes as Determinants of Bacillus anthracis Chain Length

    OpenAIRE

    Nguyen-Mau, Sao-Mai; Oh, So-Young; Kern, Valerie J.; Missiakas, Dominique M.; Schneewind, Olaf

    2012-01-01

    Bacillus anthracis grows in chains of rod-shaped cells, a trait that contributes to its escape from phagocytic clearance in host tissues. Using a genetic approach to search for determinants of B. anthracis chain length, we identified mutants with insertional lesions in secA2. All isolated secA2 mutants exhibited an exaggerated chain length, whereas the dimensions of individual cells were not changed. Complementation studies revealed that slaP (S-layer assembly protein), a gene immediately dow...

  3. Killed but Metabolically Active Bacillus anthracis Vaccines Induce Broad and Protective Immunity against Anthrax▿

    OpenAIRE

    Skoble, Justin; Beaber, John W.; Gao, Yi; Lovchik, Julie A.; Sower, Laurie E.; Liu, Weiqun; Luckett, William; Johnny W. Peterson; Calendar, Richard; Daniel A Portnoy; Lyons, C. Rick; Dubensky, Thomas W

    2009-01-01

    Bacillus anthracis is the causative agent of anthrax. We have developed a novel whole-bacterial-cell anthrax vaccine utilizing B. anthracis that is killed but metabolically active (KBMA). Vaccine strains that are asporogenic and nucleotide excision repair deficient were engineered by deleting the spoIIE and uvrAB genes, rendering B. anthracis extremely sensitive to photochemical inactivation with S-59 psoralen and UV light. We also introduced point mutations into the lef and cya genes, which ...

  4. Bacillus anthracis HssRS signaling to HrtAB regulates heme resistance during infection

    OpenAIRE

    Stauff, Devin L; Skaar, Eric P.

    2009-01-01

    Bacillus anthracis proliferates to high levels within vertebrate tissues during the pathogenesis of anthrax. This growth is facilitated by the acquisition of nutrient iron from host heme. However, heme acquisition can lead to the accumulation of toxic amounts of heme within B. anthracis. Here, we show that B. anthracis resists heme toxicity by sensing heme through the HssRS two-component system, which regulates expression of the heme-detoxifying transporter HrtAB. In addition, we demonstrate ...

  5. Historical distribution and molecular diversity of Bacillus anthracis, Kazakhstan.

    Science.gov (United States)

    Aikembayev, Alim M; Lukhnova, Larissa; Temiraliyeva, Gulnara; Meka-Mechenko, Tatyana; Pazylov, Yerlan; Zakaryan, Sarkis; Denissov, Georgiy; Easterday, W Ryan; Van Ert, Matthew N; Keim, Paul; Francesconi, Stephen C; Blackburn, Jason K; Hugh-Jones, Martin; Hadfield, Ted

    2010-05-01

    To map the distribution of anthrax outbreaks and strain subtypes in Kazakhstan during 1937-2005, we combined geographic information system technology and genetic analysis by using archived cultures and data. Biochemical and genetic tests confirmed the identity of 93 archived cultures in the Kazakhstan National Culture Collection as Bacillus anthracis. Multilocus variable number tandem repeat analysis genotyping identified 12 genotypes. Cluster analysis comparing these genotypes with previously published genotypes indicated that most (n = 78) isolates belonged to the previously described A1.a genetic cluster, 6 isolates belonged to the A3.b cluster, and 2 belonged to the A4 cluster. Two genotypes in the collection appeared to represent novel genetic sublineages; 1 of these isolates was from Krygystan. Our data provide a description of the historical, geographic, and genetic diversity of B. anthracis in this Central Asian region. PMID:20409368

  6. Bacillus anthracis infections – new possibilities of treatment

    OpenAIRE

    Dorota Żakowska; Michał Bartoszcze; Marcin Niemcewicz; Agata Bielawska-Drózd; Józef Knap; Piotr Cieślik; Krzysztof Chomiczewski; Janusz Kocik

    2015-01-01

    [b]Introduction and objective[/b]. [i]Bacillus anthracis[/i] is one of biological agents which may be used in bioterrorism attacks. The aim of this study a review of the new treatment possibilities of anthrax, with particular emphasis on the treatment of pulmonary anthrax. [b]Abbreviated description of the state of knowledge[/b]. Pulmonary anthrax, as the most dangerous clinical form of the disease, is also extremely difficult to treat. Recently, considerable progress in finding new dru...

  7. Genotype Analysis of Bacillus anthracis Strains Circulating in Bangladesh.

    Science.gov (United States)

    Rume, Farzana Islam; Affuso, Alessia; Serrecchia, Luigina; Rondinone, Valeria; Manzulli, Viviana; Campese, Emanuele; Di Taranto, Pietro; Biswas, Paritosh Kumar; Ahsan, Chowdhury Rafiqul; Yasmin, Mahmuda; Fasanella, Antonio; Hugh-Jones, Martin

    2016-01-01

    In Bangladesh, anthrax, caused by the bacterium Bacillus anthracis, is considered an endemic disease affecting ruminants with sporadic zoonotic occurrences in humans. Due to the lack of knowledge about risks from an incorrect removal of infected carcasses, the disease is not properly monitored, and because of the socio-economic conditions, the situation is under-reported and under-diagnosed. For sensitive species, anthrax represents a fatal outcome with sudden death and sometimes bleeding from natural orifices. The most common source of infection for ruminants is ingestion of spores during grazing in contaminated pastures or through grass and water contaminated with anthrax spores. Domestic cattle, sheep and goats can also become infected through contaminated bone meal (used as feed) originating from anthrax-infected carcasses. The present investigation was conducted to isolate B. anthracis organisms from 169 samples (73 soil, 1 tissue, 4 bone and 91 bone meal samples) collected from 12 different districts of Bangladesh. The sampling was carried out from 2012 to 2015. Twelve samples resulted positive for B. anthracis. Biomolecular analyses were conducted starting from the Canonical Single Nucleotide Polymorphism (CanSNP) to analyze the phylogenetic origin of strains. The analysis of genotype, obtained through the Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) with the analysis of 15 Variable Number Tandem Repeats (VNTR), demonstrated four different genotypes: two of them were previously identified in the district of Sirajganj. The sub-genotyping, conducted with Single Nucleotide Repeats analysis, revealed the presence of eight subgenotypes. The data of the present study concluded that there was no observed correlation between imported cattle feed and anthrax occurrence in Bangladesh and that the remarkable genetic variations of B. anthracis were found in the soil of numerous outbreaks in this country. PMID:27082248

  8. Defensive strategies of Bacillus anthracis that promote a fatal disease

    OpenAIRE

    Mogridge, Jeremy

    2007-01-01

    Bacillus anthracis is a Gram-positive bacterium that causes anthrax. Bacterial spores that enter the host germinate into metabolically active bacilli that disseminate throughout the body and replicate to high numbers. Two virulence factors are essential for this unrestrained growth. The first is a weakly immunogenic poly γ-D-glutamic acid capsule that surrounds the bacilli and confers resistance to phagocytosis. The second virulence factor, anthrax toxin, disrupts multiple host functions to d...

  9. Cytokine Response to Infection with Bacillus anthracis Spores

    OpenAIRE

    Pickering, Alison K.; Osorio, Manuel; Lee, Gloria M.; Grippe, Vanessa K.; Bray, Mechelle; Merkel, Tod J.

    2004-01-01

    Bacillus anthracis, the etiological agent of anthrax, is a gram-positive, spore-forming bacterium. The inhalational form of anthrax is the most severe and is associated with rapid progression of the disease and the outcome is frequently fatal. Transfer from the respiratory epithelium to regional lymph nodes appears to be an essential early step in the establishment of infection. This transfer is believed to occur by means of carriage within alveolar macrophages following phagocytosis. Therefo...

  10. Historical Distribution and Molecular Diversity of Bacillus anthracis, Kazakhstan

    OpenAIRE

    Aikembayev, Alim M.; Lukhnova, Larissa; Temiraliyeva, Gulnara; Meka-Mechenko, Tatyana; Pazylov, Yerlan; Zakaryan, Sarkis; Denissov, Georgiy; Easterday, W. Ryan; Matthew N. Van Ert; Keim, Paul; Francesconi, Stephen C.; Jason K Blackburn; Hugh-Jones, Martin; Hadfield, Ted

    2010-01-01

    To map the distribution of anthrax outbreaks and strain subtypes in Kazakhstan during 1937–2005, we combined geographic information system technology and genetic analysis by using archived cultures and data. Biochemical and genetic tests confirmed the identity of 93 archived cultures in the Kazakhstan National Culture Collection as Bacillus anthracis. Multilocus variable number tandem repeat analysis genotyping identified 12 genotypes. Cluster analysis comparing these genotypes with previousl...

  11. Glycerol Monolaurate Inhibits Virulence Factor Production in Bacillus anthracis

    OpenAIRE

    Vetter, Sara M; Schlievert, Patrick M.

    2005-01-01

    Anthrax, caused by Bacillus anthracis, has been brought to the public's attention because of the 2001 bioterrorism attacks. However, anthrax is a disease that poses agricultural threats in the United States as well as human populations in Europe, China, Africa, and Australia. Glycerol monolaurate (GML) is a compound that has been shown to inhibit exotoxin production by Staphylococcus aureus and other gram-positive bacteria. Here, we study the effects of GML on growth and toxin production in B...

  12. DECONTAMINATION ASSESSMENT OF BACILLUS ANTHRACIS, BACILLUS SUBTILIS, AND GEOBACILLUS STEAROTHERMOPHILUS SPORES ON INDOOR SURFACTS USING A HYDROGEN PERIOXIDE GAS GENERATOR

    Science.gov (United States)

    Aims: To evaluate the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials using hydrogen peroxide gas. Methods and Results: B. anthracis, B. subtilis, and G. Stearothermophilus spores were dried on seven...

  13. The Bacillus anthracis Exosporium: What's the Big "Hairy" Deal?

    Science.gov (United States)

    Bozue, Joel A; Welkos, Susan; Cote, Christopher K

    2015-10-01

    In some Bacillus species, including Bacillus subtilis, the coat is the outermost layer of the spore. In others, such as the Bacillus cereus family, there is an additional layer that envelops the coat, called the exosporium. In the case of Bacillus anthracis, a series of fine hair-like projections, also referred to as a "hairy" nap, extends from the exosporium basal layer. The exact role of the exosporium in B. anthracis, or for any of the Bacillus species possessing this structure, remains unclear. However, it has been assumed that the exosporium would play some role in infection for B. anthracis, because it is the outermost structure of the spore and would make initial contact with host and immune cells during infection. Therefore, the exosporium has been a topic of great interest, and over the past decade much progress has been made to understand its composition, biosynthesis, and potential roles. Several key aspects of this spore structure, however, are still debated and remain undetermined. Although insights have been gained on the interaction of exosporium with the host during infection, the exact role and significance of this complex structure remain to be determined. Furthermore, because the exosporium is a highly antigenic structure, future strategies for the next-generation anthrax vaccine should pursue its inclusion as a component to provide protection against the spore itself during the initial stages of anthrax. PMID:26542035

  14. Setting risk-informed environmental standards for Bacillus anthracis spores.

    Science.gov (United States)

    Hong, Tao; Gurian, Patrick L; Ward, Nicholas F Dudley

    2010-10-01

    In many cases, human health risk from biological agents is associated with aerosol exposures. Because air concentrations decline rapidly after a release, it may be necessary to use concentrations found in other environmental media to infer future or past aerosol exposures. This article presents an approach for linking environmental concentrations of Bacillus. anthracis (B. anthracis) spores on walls, floors, ventilation system filters, and in human nasal passages with human health risk from exposure to B. anthracis spores. This approach is then used to calculate example values of risk-informed concentration standards for both retrospective risk mitigation (e.g., prophylactic antibiotics) and prospective risk mitigation (e.g., environmental clean up and reoccupancy). A large number of assumptions are required to calculate these values, and the resulting values have large uncertainties associated with them. The values calculated here suggest that documenting compliance with risks in the range of 10(-4) to 10(-6) would be challenging for small diameter (respirable) spore particles. For less stringent risk targets and for releases of larger diameter particles (which are less respirable and hence less hazardous), environmental sampling would be more promising.

  15. Molecular characterization of the circulating Bacillus anthracis in Jordan.

    Science.gov (United States)

    Aqel, Amin Abdelfattah; Hailat, Ekhlas; Serrecchia, Luigina; Aqel, Suad; Campese, Emanuele; Vicari, Nadia; Fasanella, Antonio

    2015-12-01

    To understand the biomolecular charcteristics of Bacillus anthracis in Jordan, 20 blood smear slides from dead animals with suspected anthrax were analyzed using conventional and molecular approaches. All slides were positive for B. anthracis by conventional staining but no growth of the organism on selective media was detected. However, of the 20 samples, 16 were B. anthracis DNA-positive using polymerase chain reaction (PCR). Seven samples provided enough quantity and quality of DNA, and their multilocus variable tandem repeat analysis (MLVA)-15 loci analysis revealed two different genotypes. All genotypes were belonging to A.B..r. 008/009 which is very common in Asia and Europe. Single nucleotide repeat (SNR) analysis revealed that there were no sub genotypes. Molecular diagnosis of animal anthrax in Jordan is not used routinely; henceforth, official diagnosis of anthrax is based on the observation of the slides by optical microscope and this can often cause reading errors. Therefore, the prevalence of the disease in Jordan might be slightly lower than that reported by the official bodies. PMID:26156620

  16. Multigeneration Cross-Contamination of Mail with Bacillus anthracis Spores.

    Science.gov (United States)

    Edmonds, Jason; Lindquist, H D Alan; Sabol, Jonathan; Martinez, Kenneth; Shadomy, Sean; Cymet, Tyler; Emanuel, Peter

    2016-01-01

    The release of biological agents, including those which could be used in biowarfare or bioterrorism in large urban areas, has been a concern for governments for nearly three decades. Previous incidents from Sverdlosk and the postal anthrax attack of 2001 have raised questions on the mechanism of spread of Bacillus anthracis spores as an aerosol or contaminant. Prior studies have demonstrated that Bacillus atrophaeus is easily transferred through simulated mail handing, but no reports have demonstrated this ability with Bacillus anthracis spores, which have morphological differences that may affect adhesion properties between spore and formite. In this study, equipment developed to simulate interactions across three generations of envelopes subjected to tumbling and mixing was used to evaluate the potential for cross-contamination of B. anthracis spores in simulated mail handling. In these experiments, we found that the potential for cross-contamination through letter tumbling from one generation to the next varied between generations while the presence of a fluidizer had no statistical impact on the transfer of material. Likewise, the presence or absence of a fluidizer had no statistically significant impact on cross-contamination levels or reaerosolization from letter opening. PMID:27123934

  17. Multigeneration Cross-Contamination of Mail with Bacillus anthracis Spores.

    Directory of Open Access Journals (Sweden)

    Jason Edmonds

    Full Text Available The release of biological agents, including those which could be used in biowarfare or bioterrorism in large urban areas, has been a concern for governments for nearly three decades. Previous incidents from Sverdlosk and the postal anthrax attack of 2001 have raised questions on the mechanism of spread of Bacillus anthracis spores as an aerosol or contaminant. Prior studies have demonstrated that Bacillus atrophaeus is easily transferred through simulated mail handing, but no reports have demonstrated this ability with Bacillus anthracis spores, which have morphological differences that may affect adhesion properties between spore and formite. In this study, equipment developed to simulate interactions across three generations of envelopes subjected to tumbling and mixing was used to evaluate the potential for cross-contamination of B. anthracis spores in simulated mail handling. In these experiments, we found that the potential for cross-contamination through letter tumbling from one generation to the next varied between generations while the presence of a fluidizer had no statistical impact on the transfer of material. Likewise, the presence or absence of a fluidizer had no statistically significant impact on cross-contamination levels or reaerosolization from letter opening.

  18. Multigeneration Cross-Contamination of Mail with Bacillus anthracis Spores

    Science.gov (United States)

    Edmonds, Jason; Lindquist, H. D. Alan; Sabol, Jonathan; Martinez, Kenneth; Shadomy, Sean; Cymet, Tyler; Emanuel, Peter

    2016-01-01

    The release of biological agents, including those which could be used in biowarfare or bioterrorism in large urban areas, has been a concern for governments for nearly three decades. Previous incidents from Sverdlosk and the postal anthrax attack of 2001 have raised questions on the mechanism of spread of Bacillus anthracis spores as an aerosol or contaminant. Prior studies have demonstrated that Bacillus atrophaeus is easily transferred through simulated mail handing, but no reports have demonstrated this ability with Bacillus anthracis spores, which have morphological differences that may affect adhesion properties between spore and formite. In this study, equipment developed to simulate interactions across three generations of envelopes subjected to tumbling and mixing was used to evaluate the potential for cross-contamination of B. anthracis spores in simulated mail handling. In these experiments, we found that the potential for cross-contamination through letter tumbling from one generation to the next varied between generations while the presence of a fluidizer had no statistical impact on the transfer of material. Likewise, the presence or absence of a fluidizer had no statistically significant impact on cross-contamination levels or reaerosolization from letter opening. PMID:27123934

  19. Inactivation of Spores of Bacillus anthracis Sterne, Bacillus cereus, and Bacillus thuringiensis subsp. israelensis by Chlorination

    OpenAIRE

    Rice, E W; Adcock, N. J.; Sivaganesan, M; Rose, L. J.

    2005-01-01

    Three species of Bacillus were evaluated as potential surrogates for Bacillus anthracis for determining the sporicidal activity of chlorination as commonly used in drinking water treatment. Spores of Bacillus thuringiensis subsp. israelensis were found to be an appropriate surrogate for spores of B. anthracis for use in chlorine inactivation studies.

  20. The Pathogenomic Sequence Analysis of B. cereus and B. Thuringiensis isolates closely related to Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Han, C S; Xie, G; Challacombe, J F; Altherr, M R; Bhotika, S S; Bruce, D; Campbell, C S; Campbell, M L; Chen, J; Chertkov, O; Cleland, C; Dimitrijevic-Bussod, M; Doggett, N A; Fawcett, J J; Glavina, T; Goodwin, L A; Hill, K K; Hitchcock, P; Jackson, P J; Keim, P; Kewalramani, A R; Longmire, J; Lucas, S; Malfatti, S; McMurry, K; Meincke, L J; Misra, M; Moseman, B L; Mundt, M; Munk, A C; Okinaka, R T; Parson-Quintana, B; Reilly, L P; Richardson, P; Robinson, D L; Rubin, E; Saunders, E; Tapia, R; Tesmer, J G; Thayer, N; Thompson, L S; Tice, H; Ticknor, L O; Wills, P L; Gilna, P; Brettin, T S

    2005-10-12

    The sequencing and analysis of two close relatives of Bacillus anthracis are reported. AFLP analysis of over 300 isolates of B. cereus, B. thuringiensis and B. anthracis identified two isolates as being very closely related to B. anthracis. One, a B. cereus, BcE33L, was isolated from a zebra carcass in Nambia; the second, a B. thuringiensis, 97-27, was isolated from a necrotic human wound. The B. cereus appears to be the closest anthracis relative sequenced to date. A core genome of over 3,900 genes was compiled for the Bacillus cereus group, including B anthracis. Comparative analysis of these two genomes with other members of the B. cereus group provides insight into the evolutionary relationships among these organisms. Evidence is presented that differential regulation modulates virulence, rather than simple acquisition of virulence factors. These genome sequences provide insight into the molecular mechanisms contributing to the host range and virulence of this group of organisms.

  1. A novel multiplex PCR discriminates Bacillus anthracis and its genetically related strains from other Bacillus cereus group species.

    Directory of Open Access Journals (Sweden)

    Hirohito Ogawa

    Full Text Available Anthrax is an important zoonotic disease worldwide that is caused by Bacillus anthracis, a spore-forming pathogenic bacterium. A rapid and sensitive method to detect B. anthracis is important for anthrax risk management and control in animal cases to address public health issues. However, it has recently become difficult to identify B. anthracis by using previously reported molecular-based methods because of the emergence of B. cereus, which causes severe extra-intestinal infection, as well as the human pathogenic B. thuringiensis, both of which are genetically related to B. anthracis. The close genetic relation of chromosomal backgrounds has led to complexity of molecular-based diagnosis. In this study, we established a B. anthracis multiplex PCR that can screen for the presence of B. anthracis virulent plasmids and differentiate B. anthracis and its genetically related strains from other B. cereus group species. Six sets of primers targeting a chromosome of B. anthracis and B. anthracis-like strains, two virulent plasmids, pXO1 and pXO2, a bacterial gene, 16S rRNA gene, and a mammalian gene, actin-beta gene, were designed. The multiplex PCR detected approximately 3.0 CFU of B. anthracis DNA per PCR reaction and was sensitive to B. anthracis. The internal control primers also detected all bacterial and mammalian DNAs examined, indicating the practical applicability of this assay as it enables monitoring of appropriate amplification. The assay was also applied for detection of clinical strains genetically related to B. anthracis, which were B. cereus strains isolated from outbreaks of hospital infections in Japan, and field strains isolated in Zambia, and the assay differentiated B. anthracis and its genetically related strains from other B. cereus group strains. Taken together, the results indicate that the newly developed multiplex PCR is a sensitive and practical method for detecting B. anthracis.

  2. Identification of Bacillus anthracis specific chromosomal sequences by suppressive subtractive hybridization

    Directory of Open Access Journals (Sweden)

    Redkar Rajendra

    2004-02-01

    Full Text Available Abstract Background Bacillus anthracis, Bacillus thuringiensis and Bacillus cereus are closely related members of the B. cereus-group of bacilli. Suppressive subtractive hybridization (SSH was used to identify specific chromosomal sequences unique to B. anthracis. Results Two SSH libraries were generated. Genomic DNA from plasmid-cured B. anthracis was used as the tester DNA in both libraries, while genomic DNA from either B. cereus or B. thuringiensis served as the driver DNA. Progressive screening of the libraries by colony filter and Southern blot analyses identified 29 different clones that were specific for the B. anthracis chromosome relative not only to the respective driver DNAs, but also to seven other different strains of B. cereus and B. thuringiensis included in the process. The nucleotide sequences of the clones were compared with those found in genomic databases, revealing that over half of the clones were located into 2 regions on the B. anthracis chromosome. Conclusions Genes encoding potential cell wall synthesis proteins dominated one region, while bacteriophage-related sequences dominated the other region. The latter supports the hypothesis that acquisition of these bacteriophage sequences occurred during or after speciation of B. anthracis relative to B. cereus and B. thuringiensis. This study provides insight into the chromosomal differences between B. anthracis and its closest phylogenetic relatives.

  3. Genetics Home Reference: sepiapterin reductase deficiency

    Science.gov (United States)

    Skip to main content Your Guide to Understanding Genetic Conditions Enable Javascript for addthis links to activate. ... Conditions Genes Chromosomes & mtDNA Resources Help Me Understand Genetics Home Health Conditions sepiapterin reductase deficiency sepiapterin reductase ...

  4. Characterization of the sortase repertoire in Bacillus anthracis.

    Directory of Open Access Journals (Sweden)

    Willy Aucher

    Full Text Available LPXTG proteins, present in most if not all Gram-positive bacteria, are known to be anchored by sortases to the bacterial peptidoglycan. More than one sortase gene is often encoded in a bacterial species, and each sortase is supposed to specifically anchor given LPXTG proteins, depending of the sequence of the C-terminal cell wall sorting signal (cwss, bearing an LPXTG motif or another recognition sequence. B. anthracis possesses three sortase genes. B. anthracis sortase deleted mutant strains are not affected in their virulence. To determine the sortase repertoires, we developed a genetic screen using the property of the gamma phage to lyse bacteria only when its receptor, GamR, an LPXTG protein, is exposed at the surface. We identified 10 proteins that contain a cell wall sorting signal and are covalently anchored to the peptidoglycan. Some chimeric proteins yielded phage lysis in all sortase mutant strains, suggesting that cwss proteins remained surface accessible in absence of their anchoring sortase, probably as a consequence of membrane localization of yet uncleaved precursor proteins. For definite assignment of the sortase repertoires, we consequently relied on a complementary test, using a biochemical approach, namely immunoblot experiments. The sortase anchoring nine of these proteins has thus been determined. The absence of virulence defect of the sortase mutants could be a consequence of the membrane localization of the cwss proteins.

  5. Impact of Gastrointestinal Bacillus anthracis Infection on Hepatic B Cells

    Directory of Open Access Journals (Sweden)

    Natacha Colliou

    2015-09-01

    Full Text Available Ingestion of Bacillus anthracis results in rapid gastrointestinal (GI infection, known as GI anthrax. We previously showed that during GI anthrax, there is swift deterioration of intestinal barrier function leading to translocation of gut-associated bacteria into systemic circulation. Additionally, we described dysfunction in colonic B cells. In concordance with our previous studies, here, we report early migration of the Sterne strain of B. anthracis along with other gut-resident bacteria into the infected murine liver. Additionally, despite a global decrease in the B cell population, we observed an increase in both B-1a and marginal zone (MZ-like B cells. Both of these cell types are capable of producing immunoglobulins against common pathogens and commensals, which act as a general antibody barrier before an antigen-specific antibody response. Accumulation of these cells in the liver was associated with an increase in chemokine expression. These data suggest that the presence of Sterne and other commensals in the liver trigger migration of MZ-like B cells from the spleen to the liver to neutralize systemic spread. Further research is required to evaluate the possible cause of their failure to clear the infection within the liver, including the potential role of dysfunctional mitogen-activated protein kinase (MAPK signaling.

  6. Bacillus anthracis genome organization in light of whole transcriptome sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Martin, Jeffrey; Zhu, Wenhan; Passalacqua, Karla D.; Bergman, Nicholas; Borodovsky, Mark

    2010-03-22

    Emerging knowledge of whole prokaryotic transcriptomes could validate a number of theoretical concepts introduced in the early days of genomics. What are the rules connecting gene expression levels with sequence determinants such as quantitative scores of promoters and terminators? Are translation efficiency measures, e.g. codon adaptation index and RBS score related to gene expression? We used the whole transcriptome shotgun sequencing of a bacterial pathogen Bacillus anthracis to assess correlation of gene expression level with promoter, terminator and RBS scores, codon adaptation index, as well as with a new measure of gene translational efficiency, average translation speed. We compared computational predictions of operon topologies with the transcript borders inferred from RNA-Seq reads. Transcriptome mapping may also improve existing gene annotation. Upon assessment of accuracy of current annotation of protein-coding genes in the B. anthracis genome we have shown that the transcriptome data indicate existence of more than a hundred genes missing in the annotation though predicted by an ab initio gene finder. Interestingly, we observed that many pseudogenes possess not only a sequence with detectable coding potential but also promoters that maintain transcriptional activity.

  7. Crystal structure of Bacillus anthracis transpeptidase enzyme CapD.

    Energy Technology Data Exchange (ETDEWEB)

    Wu, R.; Richter, S.; Zhang, R.; Anderson, V. J.; Missiakas, D.; Joachimiak, A.; Biosciences Division; Univ. of Chicago

    2009-09-04

    Bacillus anthracis elaborates a poly-{gamma}-d-glutamic acid capsule that protects bacilli from phagocytic killing during infection. The enzyme CapD generates amide bonds with peptidoglycan cross-bridges to anchor capsular material within the cell wall envelope of B. anthracis. The capsular biosynthetic pathway is essential for virulence during anthrax infections and can be targeted for anti-infective inhibition with small molecules. Here, we present the crystal structures of the {gamma}-glutamyltranspeptidase CapD with and without {alpha}-l-Glu-l-Glu dipeptide, a non-hydrolyzable analog of poly-{gamma}-d-glutamic acid, in the active site. Purified CapD displays transpeptidation activity in vitro, and its structure reveals an active site broadly accessible for poly-{gamma}-glutamate binding and processing. Using structural and biochemical information, we derive a mechanistic model for CapD catalysis whereby Pro{sup 427}, Gly{sup 428}, and Gly{sup 429} activate the catalytic residue of the enzyme, Thr{sup 352}, and stabilize an oxyanion hole via main chain amide hydrogen bonds.

  8. Genetic analysis of petrobactin transport in Bacillus anthracis.

    Science.gov (United States)

    Carlson, Paul E; Dixon, Shandee D; Janes, Brian K; Carr, Katherine A; Nusca, Tyler D; Anderson, Erica C; Keene, Sarra E; Sherman, David H; Hanna, Philip C

    2010-02-01

    Iron acquisition mechanisms play an important role in the pathogenesis of many infectious microbes. In Bacillus anthracis, the siderophore petrobactin is required for both growth in iron-depleted conditions and for full virulence of the bacterium. Here we demonstrate the roles of two putative petrobactin binding proteins FatB and FpuA (encoded by GBAA5330 and GBAA4766 respectively) in B. anthracis iron acquisition and pathogenesis. Markerless deletion mutants were created using allelic exchange. The Delta fatB strain was capable of wild-type levels of growth in iron-depleted conditions, indicating that FatB does not play an essential role in petrobactin uptake. In contrast, Delta fpuA bacteria exhibited a significant decrease in growth under low-iron conditions when compared with wild-type bacteria. This mutant could not be rescued by the addition of exogenous purified petrobactin. Further examination of this strain demonstrated increased levels of petrobactin accumulation in the culture supernatants, suggesting no defect in siderophore synthesis or export but, instead, an inability of Delta fpuA to import this siderophore. Delta fpuA spores were also significantly attenuated in a murine model of inhalational anthrax. These results provide the first genetic evidence demonstrating the role of FpuA in petrobactin uptake. PMID:20487286

  9. Environmental Persistence of Bacillus anthracis and Bacillus subtilis Spores

    Science.gov (United States)

    Wood, Joseph P.; Meyer, Kathryn M.; Kelly, Thomas J.; Choi, Young W.; Rogers, James V.; Riggs, Karen B.; Willenberg, Zachary J.

    2015-01-01

    There is a lack of data for how the viability of biological agents may degrade over time in different environments. In this study, experiments were conducted to determine the persistence of Bacillus anthracis and Bacillus subtilis spores on outdoor materials with and without exposure to simulated sunlight, using ultraviolet (UV)-A/B radiation. Spores were inoculated onto glass, wood, concrete, and topsoil and recovered after periods of 2, 14, 28, and 56 days. Recovery and inactivation kinetics for the two species were assessed for each surface material and UV exposure condition. Results suggest that with exposure to UV, decay of spore viability for both Bacillus species occurs in two phases, with an initial rapid decay, followed by a slower inactivation period. The exception was with topsoil, in which there was minimal loss of spore viability in soil over 56 days, with or without UV exposure. The greatest loss in viable spore recovery occurred on glass with UV exposure, with nearly a four log10 reduction after just two days. In most cases, B. subtilis had a slower rate of decay than B. anthracis, although less B. subtilis was recovered initially. PMID:26372011

  10. Mechanisms of DNA Binding and Regulation of Bacillus anthracis DNA Primase

    OpenAIRE

    Biswas, Subhasis B; Wydra, Eric; Biswas, Esther E.

    2009-01-01

    DNA primases are pivotal enzymes in chromosomal DNA replication in all organisms. In this article, we report unique mechanistic characteristics of recombinant DNA primase from Bacillus anthracis (B. anthracis). The mechanism of action of B. anthracis DNA primase (DnaGBA) may be described in several distinct steps as follows. Its mechanism of action is initiated when it binds to single-stranded DNA (ssDNA) in the form of a trimer. Although DnaGBA binds to different DNA sequences with moderate ...

  11. Colonic immune suppression, barrier dysfunction, and dysbiosis by gastrointestinal bacillus anthracis Infection.

    Directory of Open Access Journals (Sweden)

    Yaíma L Lightfoot

    Full Text Available Gastrointestinal (GI anthrax results from the ingestion of Bacillus anthracis. Herein, we investigated the pathogenesis of GI anthrax in animals orally infected with toxigenic non-encapsulated B. anthracis Sterne strain (pXO1+ pXO2- spores that resulted in rapid animal death. B. anthracis Sterne induced significant breakdown of intestinal barrier function and led to gut dysbiosis, resulting in systemic dissemination of not only B. anthracis, but also of commensals. Disease progression significantly correlated with the deterioration of innate and T cell functions. Our studies provide critical immunologic and physiologic insights into the pathogenesis of GI anthrax infection, whereupon cleavage of mitogen-activated protein kinases (MAPKs in immune cells may play a central role in promoting dysfunctional immune responses against this deadly pathogen.

  12. FORMALDEHYDE GAS INACTIVATION OF BACILLUS ANTHRACIS, BACILLUS SUBTILIS AND GEOBACILLUS STEAROTHERMOPHILUS SPORES ON INDOOR SURFACE MATERIALS.

    Science.gov (United States)

    Research evaluated the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface material using formaldehyde gas. Spores were dried on seven types of indoor surfaces and exposed to 1100 ppm formaldehyde gas for 10 hr. Fo...

  13. Structures of two superoxide dismutases from Bacillus anthracis reveal a novel active centre

    International Nuclear Information System (INIS)

    The crystal structures of two manganese superoxide dismutases from B. anthracis were solved by X-ray crystallography using molecular replacement. The BA4499 and BA5696 genes of Bacillus anthracis encode proteins homologous to manganese superoxide dismutase, suggesting that this organism has an expanded repertoire of antioxidant proteins. Differences in metal specificity and quaternary structure between the dismutases of prokaryotes and higher eukaryotes may be exploited in the development of therapeutic antibacterial compounds. Here, the crystal structure of two Mn superoxide dismutases from B. anthracis solved to high resolution are reported. Comparison of their structures reveals that a highly conserved residue near the active centre is substituted in one of the proteins and that this is a characteristic feature of superoxide dismutases from the B. cereus/B. anthracis/B. thuringiensis group of organisms

  14. Genome Sequence of Bacillus anthracis Strain Stendal, Isolated from an Anthrax Outbreak in Cattle in Germany

    OpenAIRE

    Antwerpen, Markus; Elschner, Mandy; Gaede, Wolfgang; Schliephake, Annette; Grass, Gregor; Tomaso, Herbert

    2016-01-01

    In July 2012, an anthrax outbreak occurred among cattle in northern Germany resulting in ten losses. Here, we report the draft genome sequence of Bacillus anthracis strain Stendal, isolated from one of the diseased cows.

  15. Genome Differences That Distinguish Bacillus anthracis from Bacillus cereus and Bacillus thuringiensis

    OpenAIRE

    Radnedge, Lyndsay; Agron, Peter G.; Hill, Karen K.; Jackson, Paul J.; Ticknor, Lawrence O; Keim, Paul; Andersen, Gary L.

    2003-01-01

    The three species of the group 1 bacilli, Bacillus anthracis, B. cereus, and B. thuringiensis, are genetically very closely related. All inhabit soil habitats but exhibit different phenotypes. B. anthracis is the causative agent of anthrax and is phylogenetically monomorphic, while B. cereus and B. thuringiensis are genetically more diverse. An amplified fragment length polymorphism analysis described here demonstrates genetic diversity among a collection of non-anthrax-causing Bacillus speci...

  16. Decontamination Efficacy and Skin Toxicity of Two Decontaminants against Bacillus anthracis

    OpenAIRE

    Chad W Stratilo; Crichton, Melissa K. F.; Sawyer, Thomas W.

    2015-01-01

    Decontamination of bacterial endospores such as Bacillus anthracis has traditionally required the use of harsh or caustic chemicals. The aim of this study was to evaluate the efficacy of a chlorine dioxide decontaminant in killing Bacillus anthracis spores in solution and on a human skin simulant (porcine cadaver skin), compared to that of commonly used sodium hypochlorite or soapy water decontamination procedures. In addition, the relative toxicities of these decontaminants were compared in ...

  17. In Vivo Demonstration and Quantification of Intracellular Bacillus anthracis in Lung Epithelial Cells▿

    OpenAIRE

    Russell, Brooke H.; Liu, Qing; Sarah A Jenkins; Tuvim, Michael J.; Dickey, Burton F.; Xu, Yi

    2008-01-01

    Inhalational anthrax is initiated by the entry of Bacillus anthracis spores into the lung. A critical early event in the establishment of an infection is the dissemination of spores from the lung. Using in vitro cell culture assays, we previously demonstrated that B. anthracis spores are capable of entering into epithelial cells of the lung and crossing a barrier of lung epithelial cells without apparent disruption of the barrier integrity, suggesting a novel portal for spores to disseminate ...

  18. A recombinant Bacillus anthracis strain producing the Clostridium perfringens Ib component induces protection against iota toxins.

    OpenAIRE

    Sirard, J C; Weber, M.; Duflot, E; Popoff, M R; Mock, M

    1997-01-01

    The Bacillus anthracis toxinogenic Sterne strain is currently used as a live veterinary vaccine against anthrax. The capacity of a toxin-deficient derivative strain to produce a heterologous antigen by using the strong inducible promoter of the B. anthracis pag gene was investigated. The expression of the foreign gene ibp, encoding the Ib component of iota toxin from Clostridium perfringens, was analyzed. A pag-ibp fusion was introduced by allelic exchange into a toxin-deficient Sterne strain...

  19. Rapid identification of Bacillus anthracis by cell wall and capsule components direct fluorescent antibody assay

    OpenAIRE

    Lily Natalia; Rahmat Setya AdjI

    2008-01-01

    During the outbreak of anthrax, early diagnosis is critical for effective treatment. Numerous attempts have been made to design antigen based detection tests and to rapidly identify truly anthrax specific antigens for B. anthracis. In Indonesia, standard identification of B. anthracis relies on a combination of time consuming steps including bacterial culture and Ascoli precipitin test, which can take several days to provide a diagnosis. In this study, two component (cell wall and capsule) di...

  20. Inflammatory Cytokine Response to Bacillus anthracis Peptidoglycan Requires Phagocytosis and Lysosomal Trafficking▿

    OpenAIRE

    Iyer, Janaki K.; Khurana, Taruna; Langer, Marybeth; West, Christopher M.; Ballard, Jimmy D.; Metcalf, Jordan P.; Merkel, Tod J.; Coggeshall, K. Mark

    2010-01-01

    During advanced stages of inhalation anthrax, Bacillus anthracis accumulates at high levels in the bloodstream of the infected host. This bacteremia leads to sepsis during late-stage anthrax; however, the mechanisms through which B. anthracis-derived factors contribute to the pathology of infected hosts are poorly defined. Peptidoglycan, a major component of the cell wall of Gram-positive bacteria, can provoke symptoms of sepsis in animal models. We have previously shown that peptidoglycan of...

  1. Aerosolized Bacillus anthracis Infection in New Zealand White Rabbits: Natural History and Intravenous Levofloxacin Treatment

    OpenAIRE

    Yee, Steven B.; Hatkin, Joshua M; Dyer, David N; Orr, Steven A.; Pitt, M. Louise M.

    2010-01-01

    The natural history for inhalational Bacillus anthracis (Ames strain) exposure in New Zealand white rabbits was investigated to better identify potential, early biomarkers of anthrax. Twelve SPF Bordetella-free rabbits were exposed to 150 LD50 aerosolized B. anthracis spores, and clinical signs, body temperature, complete blood count, bacteremia, and presence of protective antigen in the blood (that is, antigenemia) were examined. The development of antigenemia and bacteremia coincided and pr...

  2. Modulation of the Bacillus anthracis Secretome by the Immune Inhibitor A1 Protease

    OpenAIRE

    Pflughoeft, Kathryn J.; Swick, Michelle C.; Engler, David A.; Yeo, Hye-Jeong; Koehler, Theresa M.

    2014-01-01

    The Bacillus anthracis secretome includes protective antigen, lethal factor, and edema factor, which are the components of anthrax toxin, and other proteins with known or potential roles in anthrax disease. Immune inhibitor A1 (InhA1) is a secreted metalloprotease that is unique to pathogenic members of the Bacillus genus and has been associated with cleavage of host proteins during infection. Here, we report the effect of InhA1 on the B. anthracis secretome. Differential in-gel electrophores...

  3. Bridging the gap between detection and confirmation of B. anthracis in blood cultures

    OpenAIRE

    Hawkey, Suzanna

    2015-01-01

    The spore forming bacterium, Bacillus anthracis is the aetiological agent of anthrax. The 2001 US anthrax letter attacks and the 2009‐2010 outbreak of injectional anthrax in the UK highlighted the importance of early detection and confirmation of this agent, both for patient outcome and forensic investigations. A reliable and consistent method was used in this study to safely simulate blood cultures with B. anthracis and used to determine the time to positive detection. This was performed...

  4. The Bacillus anthracis chromosome contains four conserved, excision-proficient, putative prophages

    Directory of Open Access Journals (Sweden)

    Sozhamannan Shanmuga

    2006-04-01

    Full Text Available Abstract Background Bacillus anthracis is considered to be a recently emerged clone within the Bacillus cereus sensu lato group. The B. anthracis genome sequence contains four putative lambdoid prophages. We undertook this study in order to understand whether the four prophages are unique to B. anthracis and whether they produce active phages. Results More than 300 geographically and temporally divergent isolates of B. anthracis and its near neighbors were screened by PCR for the presence of specific DNA sequences from each prophage region. Every isolate of B. anthracis screened by PCR was found to produce all four phage-specific amplicons whereas none of the non-B. anthracis isolates, produced more than one phage-specific amplicon. Excision of prophages could be detected by a PCR based assay for attP sites on extra-chromosomal phage circles and for attB sites on phage-excised chromosomes. SYBR-green real-time PCR assays indicated that prophage excision occurs at very low frequencies (2 × 10-5 - 8 × 10-8/cell. Induction with mitomycin C increased the frequency of excision of one of the prophages by approximately 250 fold. All four prophages appear to be defective since, mitomycin C induced culture did not release any viable phage particle or lyse the cells or reveal any phage particle under electron microscopic examination. Conclusion The retention of all four putative prophage regions across all tested strains of B. anthracis is further evidence of the very recent emergence of this lineage and the prophage regions may be useful for differentiating the B. anthracis chromosome from that of its neighbors. All four prophages can excise at low frequencies, but are apparently defective in phage production.

  5. Inflammatory cytokine response to Bacillus anthracis peptidoglycan requires phagocytosis and lysosomal trafficking.

    Science.gov (United States)

    Iyer, Janaki K; Khurana, Taruna; Langer, Marybeth; West, Christopher M; Ballard, Jimmy D; Metcalf, Jordan P; Merkel, Tod J; Coggeshall, K Mark

    2010-06-01

    During advanced stages of inhalation anthrax, Bacillus anthracis accumulates at high levels in the bloodstream of the infected host. This bacteremia leads to sepsis during late-stage anthrax; however, the mechanisms through which B. anthracis-derived factors contribute to the pathology of infected hosts are poorly defined. Peptidoglycan, a major component of the cell wall of Gram-positive bacteria, can provoke symptoms of sepsis in animal models. We have previously shown that peptidoglycan of B. anthracis can induce the production of proinflammatory cytokines by cells in human blood. Here, we show that biologically active peptidoglycan is shed from an active culture of encapsulated B. anthracis strain Ames in blood. Peptidoglycan is able to bind to surfaces of responding cells, and internalization of peptidoglycan is required for the production of inflammatory cytokines. We also show that the peptidoglycan traffics to lysosomes, and lysosomal function is required for cytokine production. We conclude that peptidoglycan of B. anthracis is initially bound by an unknown extracellular receptor, is phagocytosed, and traffics to lysosomes, where it is degraded to a product recognized by an intracellular receptor. Binding of the peptidoglycan product to the intracellular receptor causes a proinflammatory response. These findings provide new insight into the mechanism by which B. anthracis triggers sepsis during a critical stage of anthrax disease. PMID:20308305

  6. Circulating lethal toxin decreases the ability of neutrophils to respond to Bacillus anthracis.

    Science.gov (United States)

    Weiner, Zachary P; Ernst, Stephen M; Boyer, Anne E; Gallegos-Candela, Maribel; Barr, John R; Glomski, Ian J

    2014-04-01

    Polymorphonuclear leucocytes (PMNs) play a protective role during Bacillus anthracis infection. However, B. anthracis is able to subvert the PMN response effectively as evidenced by the high mortality rates of anthrax. One major virulence factor produced by B. anthracis, lethal toxin (LT), is necessary for dissemination in the BSL2 model of mouse infection. While human and mouse PMNs kill vegetative B. anthracis, short in vitro half-lives of PMNs have made it difficult to determine how or if LT alters their bactericidal function. Additionally, the role of LT intoxication on PMN's ability to migrate to inflammatory signals remains controversial. LF concentrations in both serum and major organs were determined from mice infected with B. anthracis Sterne strain at defined stages of infection to guide subsequent administration of purified toxin. Bactericidal activity of PMNs assessed using ex vivo cell culture assays showed significant defects in killing B. anthracis. In vivo PMN recruitment to inflammatory stimuli was significantly impaired at 24 h as assessed by real-time analysis of light-producing PMNs within the mouse. The observations described above suggest that LT serves dual functions; it both attenuates accumulation of PMNs at sites of inflammation and impairs PMNs bactericidal activity against vegetative B. anthracis. PMID:24152301

  7. Identification of anthrax-specific signature sequence from Bacillus anthracis

    Science.gov (United States)

    Rastogi, Vipin K.; Cheng, Tu-chen

    2001-08-01

    The primary objective was to identify and clone novel chromosomal DNA fragments for use as B. anthracis-specific markers. Towards this goal, 300 random primers (RAPD technology, randomly amplified polymorphic DNA) were screened to identify polymorphic loci on the anthrax chromosome. Five such DNA fragments uniquely amplifying from anthrax chromosome were identified and isolated. These fragments were cloned in pCR vector and sequenced. Database (genebank) analysis of one of the cloned probe, VRTC899, revealed the presence of specific chromosomal DNA probe, Ba813 from anthrax. This prove also contains flanking DNA with no homology to known sequences. Availability of signature DNA probes for detection of antrax-causing agent in environmental samples is critical for field application of DNA-based sensor technologies. In conclusion, we have demonstrated application of RAPD technology for identification of anthrax-specific signature sequences. This strategy can be extended to identify signature sequences from other BW agents.

  8. Noncapsulated Toxinogenic Bacillus anthracis Presents a Specific Growth and Dissemination Pattern in Naive and Protective Antigen-Immune Mice▿

    OpenAIRE

    Glomski, Ian J.; Corre, Jean-Philippe; Mock, Michèle; Goossens, Pierre L

    2007-01-01

    Bacillus anthracis is a spore-forming bacterium that causes anthrax. B. anthracis has three major virulence factors, namely, lethal toxin, edema toxin, and a poly-γ-d-glutamic acid capsule. The toxins modulate host immune responses, and the capsule inhibits phagocytosis. With the goal of increasing safety, decreasing security concerns, and taking advantage of mammalian genetic tools and reagents, mouse models of B. anthracis infection have been developed using attenuated bacteria that produce...

  9. Bacillus anthracis Virulent Plasmid pX02 Genes Found in Large Plasmids of Two Other Bacillus Species

    OpenAIRE

    Luna, Vicki A.; King, Debra S.; Peak, K. Kealy; Reeves, Frank; Heberlein-Larson, Lea; Veguilla, William; Heller, L.; Duncan, Kathleen E; Cannons, Andrew C.; Amuso, Philip; Cattani, Jacqueline

    2006-01-01

    In order to cause the disease anthrax, Bacillus anthracis requires two plasmids, pX01 and pX02, which carry toxin and capsule genes, respectively, that are used as genetic targets in the laboratory detection of the bacterium. Clinical, forensic, and environmental samples that test positive by PCR protocols established by the Centers for Disease Control and Prevention for B. anthracis are considered to be potentially B. anthracis until confirmed by culture and a secondary battery of tests. We ...

  10. Decontamination Options for Drinking Water Contaminated with Bacillus anthracis Spores

    Energy Technology Data Exchange (ETDEWEB)

    Raber, E; Burklund, A

    2010-02-16

    Five parameters were evaluated with surrogates of Bacillus anthracis spores to determine effective decontamination options for use in a contaminated drinking water supply. The parameters were: (1) type of Bacillus spore surrogate (B. thuringiensis or B. atrophaeus); (2) spore concentration in suspension (10{sup 2} to 10{sup 6} spores/ml); (3) chemical characteristics of decontaminant [sodium dicholor-s-triazinetrione dihydrate (Dichlor), hydrogen peroxide, potassium peroxymonosulfate (Oxone), sodium hypochlorite, and VirkonS{reg_sign}]; (4) decontaminant concentration (0.01% to 5%); and (5) decontaminant exposure time (10 min to 24 hr). Results from 162 suspension tests with appropriate controls are reported. Hydrogen peroxide at a concentration of 5%, and Dichlor and sodium hypochlorite at a concentration of 2%, were effective at spore inactivation regardless of spore type tested, spore exposure time, or spore concentration evaluated. This is the first reported study of Dichlor as an effective decontaminant for B. anthracis spore surrogates. Dichlor's desirable characteristics of high oxidation potential, high level of free chlorine, and more neutral pH than that of other oxidizers evaluated appear to make it an excellent alternative. All three oxidizers were effective against B. atrophaeus spores in meeting EPA's biocide standard of greater than a 6 log kill after a 10-minute exposure time and at lower concentrations than typically reported for biocide use. Solutions of 5% VirkonS{reg_sign} and Oxone were less effective decontaminants than other options evaluated in this study and did not meet the EPA's efficacy standard for biocides. Differences in methods and procedures reported by other investigators make quantitative comparisons among studies difficult.

  11. Capsules, toxins and AtxA as virulence factors of emerging Bacillus cereus biovar anthracis.

    Science.gov (United States)

    Brézillon, Christophe; Haustant, Michel; Dupke, Susann; Corre, Jean-Philippe; Lander, Angelika; Franz, Tatjana; Monot, Marc; Couture-Tosi, Evelyne; Jouvion, Gregory; Leendertz, Fabian H; Grunow, Roland; Mock, Michèle E; Klee, Silke R; Goossens, Pierre L

    2015-04-01

    Emerging B. cereus strains that cause anthrax-like disease have been isolated in Cameroon (CA strain) and Côte d'Ivoire (CI strain). These strains are unusual, because their genomic characterisation shows that they belong to the B. cereus species, although they harbour two plasmids, pBCXO1 and pBCXO2, that are highly similar to the pXO1 and pXO2 plasmids of B. anthracis that encode the toxins and the polyglutamate capsule respectively. The virulence factors implicated in the pathogenicity of these B. cereus bv anthracis strains remain to be characterised. We tested their virulence by cutaneous and intranasal delivery in mice and guinea pigs; they were as virulent as wild-type B. anthracis. Unlike as described for pXO2-cured B. anthracis, the CA strain cured of the pBCXO2 plasmid was still highly virulent, showing the existence of other virulence factors. Indeed, these strains concomitantly expressed a hyaluronic acid (HA) capsule and the B. anthracis polyglutamate (PDGA) capsule. The HA capsule was encoded by the hasACB operon on pBCXO1, and its expression was regulated by the global transcription regulator AtxA, which controls anthrax toxins and PDGA capsule in B. anthracis. Thus, the HA and PDGA capsules and toxins were co-regulated by AtxA. We explored the respective effect of the virulence factors on colonisation and dissemination of CA within its host by constructing bioluminescent mutants. Expression of the HA capsule by itself led to local multiplication and, during intranasal infection, to local dissemination to the adjacent brain tissue. Co-expression of either toxins or PDGA capsule with HA capsule enabled systemic dissemination, thus providing a clear evolutionary advantage. Protection against infection by B. cereus bv anthracis required the same vaccination formulation as that used against B. anthracis. Thus, these strains, at the frontier between B. anthracis and B. cereus, provide insight into how the monomorphic B. anthracis may have emerged.

  12. Capsules, toxins and AtxA as virulence factors of emerging Bacillus cereus biovar anthracis.

    Directory of Open Access Journals (Sweden)

    Christophe Brézillon

    2015-04-01

    Full Text Available Emerging B. cereus strains that cause anthrax-like disease have been isolated in Cameroon (CA strain and Côte d'Ivoire (CI strain. These strains are unusual, because their genomic characterisation shows that they belong to the B. cereus species, although they harbour two plasmids, pBCXO1 and pBCXO2, that are highly similar to the pXO1 and pXO2 plasmids of B. anthracis that encode the toxins and the polyglutamate capsule respectively. The virulence factors implicated in the pathogenicity of these B. cereus bv anthracis strains remain to be characterised. We tested their virulence by cutaneous and intranasal delivery in mice and guinea pigs; they were as virulent as wild-type B. anthracis. Unlike as described for pXO2-cured B. anthracis, the CA strain cured of the pBCXO2 plasmid was still highly virulent, showing the existence of other virulence factors. Indeed, these strains concomitantly expressed a hyaluronic acid (HA capsule and the B. anthracis polyglutamate (PDGA capsule. The HA capsule was encoded by the hasACB operon on pBCXO1, and its expression was regulated by the global transcription regulator AtxA, which controls anthrax toxins and PDGA capsule in B. anthracis. Thus, the HA and PDGA capsules and toxins were co-regulated by AtxA. We explored the respective effect of the virulence factors on colonisation and dissemination of CA within its host by constructing bioluminescent mutants. Expression of the HA capsule by itself led to local multiplication and, during intranasal infection, to local dissemination to the adjacent brain tissue. Co-expression of either toxins or PDGA capsule with HA capsule enabled systemic dissemination, thus providing a clear evolutionary advantage. Protection against infection by B. cereus bv anthracis required the same vaccination formulation as that used against B. anthracis. Thus, these strains, at the frontier between B. anthracis and B. cereus, provide insight into how the monomorphic B. anthracis may have

  13. Pyrimethamine/sulfadoxine combination in the treatment of uncomplicated falciparum malaria: relation between dihydropteroate synthase/dihydrofolate reductase genotypes, sulfadoxine plasma levels, and treatment outcome

    DEFF Research Database (Denmark)

    Khalil, Insaf; Alifrangis, Michael; Rønn, Anita M;

    2002-01-01

    in relation to the frequency of dhfr and dhps mutations in 37 Plasmodium falciparum isolates sampled before treatment. Plasma levels of SDX measured at days 0, 3, 7, and 14 ascertained drug absorption. Point mutations were detected only at codons 51 and 108 of dhfr and codon 436 of dhps. The frequency of dhfr...... 51/108 and dhps 436 mutations was 79% and 8%. The plasma levels of SDX indicated adequate drug absorption by all patients. The presence of Ile 51 and Asn 108 mutations among parasites that cleared after treatment indicates that these mutations alone are insufficient to cause in vivo resistance......Several in vitro studies have shown the correlation between mutations in dhfr and dhps genes and resistance to pyrimethamine/sulfadoxine (PYR/SDX) combination, but the in vivo correlates of these mutations with PYR/ SDX efficacy have not been investigated fully. We assessed PYR/SDX efficacy...

  14. Fatty acyl-CoA reductase

    Energy Technology Data Exchange (ETDEWEB)

    Reiser, Steven E.; Somerville, Chris R.

    1998-12-01

    The present invention relates to bacterial enzymes, in particular to an acyl-CoA reductase and a gene encoding an acyl-CoA reductase, the amino acid and nucleic acid sequences corresponding to the reductase polypeptide and gene, respectively, and to methods of obtaining such enzymes, amino acid sequences and nucleic acid sequences. The invention also relates to the use of such sequences to provide transgenic host cells capable of producing fatty alcohols and fatty aldehydes.

  15. Protection of inactive intranasal ántrax vaccine to Bacillus anthracis infection

    Directory of Open Access Journals (Sweden)

    Adin Priadi

    2010-06-01

    Full Text Available Ánthrax is an endemic zoonotic disease distributed in many parts of Indonesia. Although vaccination program has been implemented in many areas, cases are still frequently reported. Farmers are reluctant to vaccinate their livestock since spore vaccine used in the field often cause side effects and death of the animals. To overcome this problem, an inactive vaccine composes of Bacillus anthracis toxins, cell wall and capsule subunits was developed. B. anthracis Sterne strain (34F2 was selected to produce toxins and cell walls. Local Bacillus anthracis isolated from Citaringgul was used to produce capsule as the Polymerase Chain Reaction (PCR revealed that this isolate poses cap gene encoding for capsule. Two vaccines compose of 15 μg toxoid, 30 μg of capsule, 15 μg of cell wall and 30 μg toxoid, 60 μg of capsule, 15 μg of cell walls were designated as vaccine I and vaccine II respectively. For each experiment, 10 mice were nasally immunized by placing 5 μl of vaccine into each nare 3 times at 2-week intervals. A group of 10 mice were unvaccinated and used as control. Blood was collected fortnightly to monitor antibody responses. All mice were challenged with 2 x 105 B. anthracis Sterne spores injected subcutaneously two weeks after the last vaccination. Two weeks after vaccination of antibodies to B. anthracis toxin, capsule and cell wall were detected in dot-blot assay. Mice that were immunised intranasally with chitosan adjuvanted vaccine developed high IgG responses in sera as detected by ELISA, and the response was dose dependent. Vaccine II gave better response than vaccine I. Vaccine I and II protected mice from challenge at a rate of 60 and 80% respectively. This results showed that intranasal B. anthracis vaccine composes of toxin, capsule and cell wall with chitosan as an adjuvant gave a good protection against B. anthracis Sterne spores challenge in mice.

  16. Detection of Bacillus anthracis DNA in complex soil and air samples using next-generation sequencing.

    Directory of Open Access Journals (Sweden)

    Nicholas A Be

    Full Text Available Bacillus anthracis is the potentially lethal etiologic agent of anthrax disease, and is a significant concern in the realm of biodefense. One of the cornerstones of an effective biodefense strategy is the ability to detect infectious agents with a high degree of sensitivity and specificity in the context of a complex sample background. The nature of the B. anthracis genome, however, renders specific detection difficult, due to close homology with B. cereus and B. thuringiensis. We therefore elected to determine the efficacy of next-generation sequencing analysis and microarrays for detection of B. anthracis in an environmental background. We applied next-generation sequencing to titrated genome copy numbers of B. anthracis in the presence of background nucleic acid extracted from aerosol and soil samples. We found next-generation sequencing to be capable of detecting as few as 10 genomic equivalents of B. anthracis DNA per nanogram of background nucleic acid. Detection was accomplished by mapping reads to either a defined subset of reference genomes or to the full GenBank database. Moreover, sequence data obtained from B. anthracis could be reliably distinguished from sequence data mapping to either B. cereus or B. thuringiensis. We also demonstrated the efficacy of a microbial census microarray in detecting B. anthracis in the same samples, representing a cost-effective and high-throughput approach, complementary to next-generation sequencing. Our results, in combination with the capacity of sequencing for providing insights into the genomic characteristics of complex and novel organisms, suggest that these platforms should be considered important components of a biosurveillance strategy.

  17. Co-purification of dihydrofolate synthetase and N10formyltetrahydropteroyldiglutamate synthetase from E. coli

    International Nuclear Information System (INIS)

    The enzymatic activities which add a single glutamate to dihydropteroate (H2Pte) and to N10formyltetrahydropteroylglutamate (N10formylH4PteGlu) remained at a constant ratio when various purification techniques were used, including ammonium sulfate fractionation, isoelectric focusing, polyacrylamide gel electrophoresis, and column chromatography on Sephadex G-100, DEAE-Sephadex, CM-Sephadex, ADP-Sepharose, N10formylfolate-agarose or matrix Gel Red-A. The best combination of these methods yielded 900-fold purified enzyme. However, the kinetic properties were dependent upon the substrate used. H2Pte was a noncompetitive inhibitor (Kii . 1.1 microM) of the utilization of N10formylH4PteGlu, but no inhibition was detected in the reciprocal experiment. Aminopterin was a competitive inhibitor (Kis . 370 microM) of the reaction with N10formylH4PteGlu but was not inhibitory with H2Pte as substrate. In the dihydrofolate synthetase reaction, in Tris-HCl, pH 8.9 with 50 mM KCl, the apparent Km values for glutamate and MgATP were 3.5 mM and 8.1 microM respectively. With N10formylH4PteGlu as substrate, these Km values were 1.2 mM and 80 microM. Since the two activities were not separated by protein purification procedures but exhibited different kinetic properties (including lack of reciprocal inhibition), the data suggest these reactions are catalyzed on independent sites of a common protein

  18. Bacillus anthracis in China and its relationship to worldwide lineages

    Directory of Open Access Journals (Sweden)

    Schupp James M

    2009-04-01

    Full Text Available Abstract Background The global pattern of distribution of 1033 B. anthracis isolates has previously been defined by a set of 12 conserved canonical single nucleotide polymorphisms (canSNP. These studies reinforced the presence of three major lineages and 12 sub-lineages and sub-groups of this anthrax-causing pathogen. Isolates that form the A lineage (unlike the B and C lineages have become widely dispersed throughout the world and form the basis for the geographical disposition of "modern" anthrax. An archival collection of 191 different B. anthracis isolates from China provides a glimpse into the possible role of Chinese trade and commerce in the spread of certain sub-lineages of this pathogen. Canonical single nucleotide polymorphism (canSNP and multiple locus VNTR analysis (MLVA typing has been used to examine this archival collection of isolates. Results The canSNP study indicates that there are 5 different sub-lineages/sub-groups in China out of 12 previously described world-wide canSNP genotypes. Three of these canSNP genotypes were only found in the western-most province of China, Xinjiang. These genotypes were A.Br.008/009, a sub-group that is spread across most of Europe and Asia; A.Br.Aust 94, a sub-lineage that is present in Europe and India, and A.Br.Vollum, a lineage that is also present in Europe. The remaining two canSNP genotypes are spread across the whole of China and belong to sub-group A.Br.001/002 and the A.Br.Ames sub-lineage, two closely related genotypes. MLVA typing adds resolution to the isolates in each canSNP genotype and diversity indices for the A.Br.008/009 and A.Br.001/002 sub-groups suggest that these represent older and established clades in China. Conclusion B. anthracis isolates were recovered from three canSNP sub-groups (A.Br.008/009, A.Br.Aust94, and A.Br.Vollum in the western most portion of the large Chinese province of Xinjiang. The city of Kashi in this province appears to have served as a crossroads

  19. Bacillus anthracis-like bacteria and other B. cereus group members in a microbial community within the international space station: a challenge for rapid and easy molecular detection of virulent B. anthracis.

    NARCIS (Netherlands)

    Tongeren, van S.P.; Roest, H.I.J.; Degener, J.E.; Harmsen, H.J.M.

    2014-01-01

    For some microbial species, such as Bacillus anthracis, the etiologic agent of the disease anthrax, correct detection and identification by molecular methods can be problematic. The detection of virulent B. anthracis is challenging due to multiple virulence markers that need to be present in order f

  20. Bacillus anthracis-Like Bacteria and Other B. cereus Group Members in a Microbial Community Within the International Space Station : A Challenge for Rapid and Easy Molecular Detection of Virulent B. anthracis

    NARCIS (Netherlands)

    van Tongeren, Sandra P.; Roest, Hendrik I. J.; Degener, John E.; Harmsen, Hermie J. M.

    2014-01-01

    For some microbial species, such as Bacillus anthracis, the etiologic agent of the disease anthrax, correct detection and identification by molecular methods can be problematic. The detection of virulent B. anthracis is challenging due to multiple virulence markers that need to be present in order f

  1. Curing of plasmid pXO1 from Bacillus anthracis using plasmid incompatibility.

    Directory of Open Access Journals (Sweden)

    Xiankai Liu

    Full Text Available The large plasmid pXO1 encoding the anthrax toxin is important for the virulence of Bacillus anthracis. It is essential to cure pXO1 from B. anthracis to evaluate its role in the pathogenesis of anthrax infection. Because conventional methods for curing plasmids (e.g., curing agents or growth at elevated temperatures can induce mutations in the host chromosomal DNA, we developed a specific and reliable method to eliminate pXO1 from B. anthracis using plasmid incompatibility. Three putative replication origins of pXO1 were inserted into a temperature-sensitive plasmid to generate three incompatible plasmids. One of the three plasmids successfully eliminated the large plasmid pXO1 from B. anthracis vaccine strain A16R and wild type strain A16. These findings provided additional information about the replication/partitioning of pXO1 and demonstrated that introducing a small incompatible plasmid can generate plasmid-cured strains of B. anthracis without inducing spontaneous mutations in the host chromosome.

  2. Bacillus anthracis interacts with plasmin(ogen to evade C3b-dependent innate immunity.

    Directory of Open Access Journals (Sweden)

    Myung-Chul Chung

    Full Text Available The causative agent of anthrax, Bacillus anthracis, is capable of circumventing the humoral and innate immune defense of the host and modulating the blood chemistry in circulation to initiate a productive infection. It has been shown that the pathogen employs a number of strategies against immune cells using secreted pathogenic factors such as toxins. However, interference of B. anthracis with the innate immune system through specific interaction of the spore surface with host proteins such as the complement system has heretofore attracted little attention. In order to assess the mechanisms by which B. anthracis evades the defense system, we employed a proteomic analysis to identify human serum proteins interacting with B. anthracis spores, and found that plasminogen (PLG is a major surface-bound protein. PLG efficiently bound to spores in a lysine- and exosporium-dependent manner. We identified α-enolase and elongation factor tu as PLG receptors. PLG-bound spores were capable of exhibiting anti-opsonic properties by cleaving C3b molecules in vitro and in rabbit bronchoalveolar lavage fluid, resulting in a decrease in macrophage phagocytosis. Our findings represent a step forward in understanding the mechanisms involved in the evasion of innate immunity by B. anthracis through recruitment of PLG resulting in the enhancement of anti-complement and anti-opsonization properties of the pathogen.

  3. Decontamination Efficacy and Skin Toxicity of Two Decontaminants against Bacillus anthracis.

    Directory of Open Access Journals (Sweden)

    Chad W Stratilo

    Full Text Available Decontamination of bacterial endospores such as Bacillus anthracis has traditionally required the use of harsh or caustic chemicals. The aim of this study was to evaluate the efficacy of a chlorine dioxide decontaminant in killing Bacillus anthracis spores in solution and on a human skin simulant (porcine cadaver skin, compared to that of commonly used sodium hypochlorite or soapy water decontamination procedures. In addition, the relative toxicities of these decontaminants were compared in human skin keratinocyte primary cultures. The chlorine dioxide decontaminant was similarly effective to sodium hypochlorite in reducing spore numbers of Bacillus anthracis Ames in liquid suspension after a 10 minute exposure. After five minutes, the chlorine dioxide product was significantly more efficacious. Decontamination of isolated swine skin contaminated with Bacillus anthracis Sterne with the chlorine dioxide product resulted in no viable spores sampled. The toxicity of the chlorine dioxide decontaminant was up to two orders of magnitude less than that of sodium hypochlorite in human skin keratinocyte cultures. In summary, the chlorine dioxide based decontaminant efficiently killed Bacillus anthracis spores in liquid suspension, as well as on isolated swine skin, and was less toxic than sodium hypochlorite in cultures of human skin keratinocytes.

  4. Rapid detection of Bacillus anthracis by γ phage amplification and lateral flow immunochromatography.

    Science.gov (United States)

    Cox, Christopher R; Jensen, Kirk R; Mondesire, Roy R; Voorhees, Kent J

    2015-11-01

    New, rapid point-of-need diagnostic methods for Bacillus anthracis detection can enhance civil and military responses to accidental or deliberate dispersal of anthrax as a biological weapon. Current laboratory-based methods for clinical identification of B. anthracis require 12 to 120h, and are confirmed by plaque assay using the well-characterized γ typing phage, which requires an additional minimum of 24h for bacterial culture. To reduce testing time, the natural specificity of γ phage amplification was investigated in combination with lateral flow immunochromatography (LFI) for rapid, point-of-need B. anthracis detection. Phage-based LFI detection of B. anthracis Sterne was validated over a range of bacterial and phage concentrations with optimal detection achieved in as little as 2h from the onset of amplification with a threshold sensitivity of 2.5×10(4)cfu/mL. The novel use of γ phage amplification detected with a simple, inexpensive LFI assay provides a rapid, sensitive, highly accurate, and field-deployable method for diagnostic ID of B. anthracis in a fraction of the time required by conventional techniques, and without the need for extensive laboratory culture. PMID:26310605

  5. [Species-specific sera against surface antigens of Bacillus anthracis strains].

    Science.gov (United States)

    Barkova, I A; Barkov, A M; Alekseev, V V; Lipnitskiĭ, A V

    2010-11-01

    The species-related specificity of sera against 94-KD proteins isolated from culture filtrates of B. anthracis strains with different levels of virulence plasmids was studied to determine whether they might be used to identify the pathogen of anthrax. Sera against fractions 1 of culture filtrates of B. anthracis strains CTI (pXO1+ pXO2-), 81/1TR (pXO1- pXO2-), Davies (pXO1- pXO) separated by gel chromatography on Sephacryl S-300 were examined. In the gel immunodiffusion test with growing cultures, the sera exhibited non-identical antigens and differed in the presence of antibodies to antigens of related bacilli. The sera against fractions 1 of culture filtrates of toxin-producing and plasmidless strains displayed antigens produced only by B. anthracis strains into nutrient agar. Electroimmunotransblotting revealed that they contained antibodies mainly to 94-kD proteins and failed to react with B. cereus proteins with a molecular weight of 94 kD and with B. thuringiensis proteins with a molecular weight of 97 kD, which were extracted from autonomous cells. In the immunofluorescence test, immunoglobulins of sera against fractions 1 of culture filtrates of three strains stained autonomous cells and spores of 23 B. anthracis strains with different levels of virulence plasmids. In working dilutions, they did not react with antigens of 18 strains of related bacilli, which presents a possibility of using them for species identification of B. anthracis. PMID:21319392

  6. Rapid detection methods for Bacillus anthracis in environmental samples: a review.

    Science.gov (United States)

    Irenge, Léonid M; Gala, Jean-Luc

    2012-02-01

    Bacillus anthracis is a Gram-positive, spore-forming bacterium, which causes anthrax, an often lethal disease of animals and humans. Although the disease has been well studied since the nineteenth century, it has witnessed a renewed interest during the past decade, due to its use as a bioterrorist agent in the fall of 2001 in the USA. A number of techniques aimed at rapidly detecting B. anthracis, in environmental samples as well as in point-of-care settings for humans suspected of exposure to the pathogen, are now available. These technologies range from culture-based methods to portable DNA amplification devices. Despite recent developments, specific identification of B. anthracis still remains difficult because of its phenotypic and genotypic similarities with other Bacillus species. Accordingly, many efforts are being made to improve the specificity of B. anthracis identification. This mini-review discusses the current challenges around B. anthracis identification, not only in reach-back laboratories but also in the field (in operational conditions). PMID:22262227

  7. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences

    NARCIS (Netherlands)

    Agren, J.; Hamidjaja, R.A.; Hansen, T.; Ruuls, R.C.; Thierry, S.; Vigre, H.; Janse, I.; Sundström, A.; Segerman, B.; Koene, M.G.J.; Löfström, Ch.; Rotterdam, van B.; Derzelle, S.

    2013-01-01

    Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely o

  8. Whole Genome Sequencing and Phylogenetic Analysis of a Historical Collection of Bacillus anthracis Strains from Danish Cattle

    DEFF Research Database (Denmark)

    Derzelle, Sylviane; Girault, Guillaume; Kokotovic, Branko;

    2015-01-01

    Bacillus anthracis, the causative agent of anthrax, is known as one of the most genetically monomorphic species. Canonical single-nucleotide polymorphism (SNP) typing and whole-genome sequencing were used to investigate the molecular diversity of eleven B. anthracis strains isolated from cattle...

  9. Genome Sequence of Bacillus anthracis Isolated from an Anthrax Burial Site in Pollino National Park, Basilicata Region (Southern Italy)

    OpenAIRE

    Fasanella, Antonio; Braun, Peter; Grass, Gregor; Hanczaruk, Matthias; Aceti, Angela; Serrecchia, Luigina; Leonzio, Giuseppe; Tolve, Francesco; Georgi, Enrico; Antwerpen, Markus

    2015-01-01

    A Bacillus anthracis strain was isolated from a burial-site in Pollino National Park where a bovine died of anthrax and was buried in 2004. We report the first genome sequence of B. anthracis isolated in the Basilicata region (southern Italy), which is the highest risk area of anthrax infection in Italy.

  10. Testing nucleoside analogues as inhibitors of Bacillus anthracis spore germination in vitro and in macrophage cell culture.

    Science.gov (United States)

    Alvarez, Zadkiel; Lee, Kyungae; Abel-Santos, Ernesto

    2010-12-01

    Bacillus anthracis, the etiological agent of anthrax, has a dormant stage in its life cycle known as the endospore. When conditions become favorable, spores germinate and transform into vegetative bacteria. In inhalational anthrax, the most fatal manifestation of the disease, spores enter the organism through the respiratory tract and germinate in phagosomes of alveolar macrophages. Germinated cells can then produce toxins and establish infection. Thus, germination is a crucial step for the initiation of pathogenesis. B. anthracis spore germination is activated by a wide variety of amino acids and purine nucleosides. Inosine and l-alanine are the two most potent nutrient germinants in vitro. Recent studies have shown that germination can be hindered by isomers or structural analogues of germinants. 6-Thioguanosine (6-TG), a guanosine analogue, is able to inhibit germination and prevent B. anthracis toxin-mediated necrosis in murine macrophages. In this study, we screened 46 different nucleoside analogues as activators or inhibitors of B. anthracis spore germination in vitro. These compounds were also tested for their ability to protect the macrophage cell line J774a.1 from B. anthracis cytotoxicity. Structure-activity relationship analysis of activators and inhibitors clarified the binding mechanisms of nucleosides to B. anthracis spores. In contrast, no structure-activity relationships were apparent for compounds that protected macrophages from B. anthracis-mediated killing. However, multiple inhibitors additively protected macrophages from B. anthracis. PMID:20921305

  11. Glyconanobiotics: Novel carbohydrated nanoparticle antibiotics for MRSA and Bacillus anthracis.

    Science.gov (United States)

    Abeylath, Sampath C; Turos, Edward; Dickey, Sonja; Lim, Daniel V

    2008-03-01

    This report describes the synthesis and evaluation of glycosylated polyacrylate nanoparticles that have covalently-bound antibiotics within their framework. The requisite glycosylated drug monomers were prepared from one of three known antibiotics, an N-sec-butylthio beta-lactam, ciprofloxacin, and a penicillin, by acylation with 3-O-acryloyl-1,2-O-isopropylidene-5,6 bis((chlorosuccinyl)oxy)-d-glucofuranose (7) or 6-O-acetyl-3-O-acryloyl-1,2-O-isopropylidene-5-(chlorosuccinyl)oxy-alpha-d-glucofuranose (10). These acrylated monomers were subjected to emulsion polymerization in a 7:3 (w:w) mixture of butyl acrylate-styrene in the presence of sodium dodecyl sulfate as surfactant (3 weight %) and potassium persulfate as a radical initiator (1 weight %). The resulting nanoparticle emulsions were characterized by dynamic light scattering and found to have similar diameters ( approximately 40 nm) and size distributions to those of our previously studied systems. Microbiological testing showed that the N-sec-butylthio beta-lactam and ciprofloxacin nanoparticles both have powerful in vitro activities against methicillin-resistant Staphylococcus aureus and Bacillus anthracis, while the penicillin-bound nanoparticles have no antimicrobial activity. This indicates the need for matching a suitable antibiotic with the nanoparticle carrier. Overall, the study shows that even relatively large, polar acrylate monomers (MW>1000 amu) can be efficiently incorporated into the nanoparticle matrix by emulsion polymerization, providing opportunities for further advances in nanomedicine. PMID:18063370

  12. Microbial forensics: fiber optic microarray subtyping of Bacillus anthracis

    Science.gov (United States)

    Shepard, Jason R. E.

    2009-05-01

    The past decade has seen increased development and subsequent adoption of rapid molecular techniques involving DNA analysis for detection of pathogenic microorganisms, also termed microbial forensics. The continued accumulation of microbial sequence information in genomic databases now better positions the field of high-throughput DNA analysis to proceed in a more manageable fashion. The potential to build off of these databases exists as technology continues to develop, which will enable more rapid, cost effective analyses. This wealth of genetic information, along with new technologies, has the potential to better address some of the current problems and solve the key issues involved in DNA analysis of pathogenic microorganisms. To this end, a high density fiber optic microarray has been employed, housing numerous DNA sequences simultaneously for detection of various pathogenic microorganisms, including Bacillus anthracis, among others. Each organism is analyzed with multiple sequences and can be sub-typed against other closely related organisms. For public health labs, real-time PCR methods have been developed as an initial preliminary screen, but culture and growth are still considered the gold standard. Technologies employing higher throughput than these standard methods are better suited to capitalize on the limitless potential garnered from the sequence information. Microarray analyses are one such format positioned to exploit this potential, and our array platform is reusable, allowing repetitive tests on a single array, providing an increase in throughput and decrease in cost, along with a certainty of detection, down to the individual strain level.

  13. Scalable purification of Bacillus anthracis protective antigen from Escherichia coli.

    Science.gov (United States)

    Gwinn, William; Zhang, Mei; Mon, Sandii; Sampey, Darryl; Zukauskas, David; Kassebaum, Corby; Zmuda, Jonathan F; Tsai, Amos; Laird, Michael W

    2006-01-01

    The anthrax toxin consists of three proteins, protective antigen (PA), lethal factor, and edema factor that are produced by the Gram-positive bacterium, Bacillus anthracis. Current vaccines against anthrax use PA as their primary component. In this study, we developed a scalable process to produce and purify multi-gram quantities of highly pure, recombinant PA (rPA) from Escherichia coli. The rPA protein was produced in a 50-L fermentor and purified to >99% purity using anion-exchange, hydrophobic interaction, and hydroxyapatite chromatography. The final yield of purified rPA from medium cell density fermentations resulted in approximately 2.7 g of rPA per kg of cell paste (approximately 270 mg/L) of highly pure, biologically active rPA protein. The results presented here exhibit the ability to generate multi-gram quantities of rPA from E. coli that may be used for the development of new anthrax vaccines and anthrax therapeutics. PMID:15935696

  14. Detection of Bacillus anthracis Spores Using Peptide Functionalized SERS-Active Substrates

    Directory of Open Access Journals (Sweden)

    Atanu Sengupta

    2012-01-01

    Full Text Available The need for portable technologies that can rapidly identify biological warfare agents (BWAs in the field remains an international priority as expressed at the 2011 Biological Weapons Convention. In recent years, the ability of surface-enhanced Raman spectroscopy (SERS to rapidly detect various BWAs at very low concentrations has been demonstrated. However, in the specific case of Bacillus anthracis, differentiation at the species level is required since other bacilli are common in the environment, representing potential false-positive responses. To overcome this limitation, we describe the use of a peptide attached to the SERS-active metal that selectively binds Bacillus anthracis-Sterne as the target analyte. Using this approach, 109  B. anthracis-Sterne spores/mL produced an intense dipicolinic acid spectrum upon the addition of acetic acid, while the same concentration and treatment of B. cereus and B. subtilis did not.

  15. Structure of purine nucleoside phosphorylase (DeoD) from Bacillus anthracis

    International Nuclear Information System (INIS)

    The crystal structure of purine nucleoside phosphorylase (DeoD) from B. anthracis was solved by X-ray crystallography using molecular replacement and refined at a resolution of 2.24 Å. Protein structures from the causative agent of anthrax (Bacillus anthracis) are being determined as part of a structural genomics programme. Amongst initial candidates for crystallographic analysis are enzymes involved in nucleotide biosynthesis, since these are recognized as potential targets in antibacterial therapy. Purine nucleoside phosphorylase is a key enzyme in the purine-salvage pathway. The crystal structure of purine nucleoside phosphorylase (DeoD) from B. anthracis has been solved by molecular replacement at 2.24 Å resolution and refined to an R factor of 18.4%. This is the first report of a DeoD structure from a Gram-positive bacterium

  16. Structure of 5-formyltetrahydrofolate cyclo-ligase from Bacillus anthracis (BA4489)

    International Nuclear Information System (INIS)

    The structure of 5-formyltetrahydrofolate cyclo-ligase from B. anthracis determined by X-ray crystallography at a resolution of 1.6 Å is described. Bacillus anthracis is a spore-forming bacterium and the causative agent of the disease anthrax. The Oxford Protein Production Facility has been targeting proteins from B. anthracis in order to develop high-throughput technologies within the Structural Proteomics in Europe project. As part of this work, the structure of 5-formyltetrahydrofolate cyclo-ligase (BA4489) has been determined by X-ray crystallography to 1.6 Å resolution. The structure, solved in complex with magnesium-ion-bound ADP and phosphate, gives a detailed picture of the proposed catalytic mechanism of the enzyme. Chemical differences from other cyclo-ligase structures close to the active site that could be exploited to design specific inhibitors are also highlighted

  17. Alveolar macrophages infected with Ames or Sterne strain of Bacillus anthracis elicit differential molecular expression patterns.

    Directory of Open Access Journals (Sweden)

    Felicia D Langel

    Full Text Available Alveolar macrophages (AMs phagocytose Bacillus anthracis following inhalation and induce the production of pro-inflammatory cytokines and chemokines to mediate the activation of innate immunity. Ames, the virulent strain of B. anthracis, contains two plasmids that encode the antiphagocytic poly-γ-d-glutamic acid capsule and the lethal toxin. The attenuated Sterne strain of B. anthracis, which lacks the plasmid encoding capsule, is widely adapted as a vaccine strain. Although differences in the outcome of infection with the two strains may have originated from the presence or absence of an anti-phagocytic capsule, the disease pathogenesis following infection will be manifested via the host responses, which is not well understood. To gain understanding of the host responses at cellular level, a microarray analysis was performed using primary rhesus macaque AMs infected with either Ames or Sterne spores. Notably, 528 human orthologs were identified to be differentially expressed in AMs infected with either strain of the B. anthracis. Meta-analyses revealed genes differentially expressed in response to B. anthracis infection were also induced upon infections with multiple pathogens such as Francisella Novicida or Staphylococcus aureus. This suggests the existence of a common molecular signature in response to pathogen infections. Importantly, the microarray and protein expression data for certain cytokines, chemokines and host factors provide further insights on how cellular processes such as innate immune sensing pathways, anti-apoptosis versus apoptosis may be differentially modulated in response to the virulent or vaccine strain of B. anthracis. The reported differences may account for the marked difference in pathogenicity between these two strains.

  18. Whole genome protein microarrays for serum profiling of immunodominant antigens of Bacillus anthracis

    Directory of Open Access Journals (Sweden)

    Karen Elizabeth Kempsell

    2015-08-01

    Full Text Available A commercial Bacillus anthracis (Anthrax whole genome protein microarray has been used to identify immunogenic Anthrax proteins using sera from groups of donors with (a confirmed B. anthracis naturally acquired cutaneous infection, (b confirmed B. anthracis intravenous drug use-acquired infection (c occupational exposure in a wool-sorters factory (d humans and rabbits vaccinated with the UK Anthrax protein vaccine and compared to naïve unexposed controls. Anti-IAP responses were observed for both IgG and IgA in the challenged groups; however the anti-IAP IgG response was more evident in the vaccinated group and the anti-IAP IgA response more evident in the B. anthracis-infected groups. Infected individuals appeared somewhat suppressed for their general IgG response, compared with other challenged groups.Immunogenic protein antigens were identified in all groups, some of which were shared between groups whilst others were specific for individual groups. The toxin proteins were immunodominant in all vaccinated, infected or other challenged groups. However a number of other chromosomally-located and plasmid encoded open reading frames were also recognised by infected or exposed groups in comparison to controls. Some of these antigens e.g. BA4182 are not recognised by vaccinated individuals, suggesting that there are proteins more specifically expressed by live Anthrax spores in vivo and are not currently found in the UK licensed Anthrax Vaccine (AVP. These may perhaps be preferentially expressed during infection and represent expression of alternative pathways in the B. anthracis ‘infectome’. These may make highly attractive candidates for diagnostic and vaccine biomarker development as they may be more specifically associated with the infectious phase of the pathogen. A number of B. anthracis small hypothetical protein targets have been synthesised, tested in mouse immunogenicity studies and validated in parallel using human sera from the

  19. Transient lipopolysaccharide-induced resistance to aerosolized Bacillus anthracis in New Zealand white rabbits.

    Science.gov (United States)

    Yee, Steven B; Dyer, David N; Twenhafel, Nancy A; Pitt, M Louise M

    2013-06-01

    Previous studies have demonstrated that prior infection by various bacterial pathogens induces nonspecific resistance to subsequent infection by other gram-negative and gram-positive bacterial pathogens. In the present study, we evaluated whether underlying inflammation enhanced host resistance to inhalational Bacillus anthracis infection in New Zealand White rabbits (SPF; Bordetella- and Pasteurella-free). Accordingly, rabbits were pretreated with either the inflammagen bacterial LPS (60,000 EU/kg), a component of the outer membrane of gram-negative bacteria, or saline (vehicle). Administration of LPS resulted in brief pyrexia and a significant increase in the proinflammatory cytokine TNFα, thus confirming LPS-induced inflammation. At 24 h after LPS treatment, rabbits were exposed to aerosolized B. anthracis spores (Ames strain; approximately 300 LD50). Blood samples collected at various times after challenge were cultured. Compared with their saline-pretreated counterparts, LPS-pretreated, B. anthracis challenged rabbits exhibited delays in 2 biomarkers of B. anthracis infection-anthrax-induced pyrexia (25 h versus 66 h after challenge, respectively) and bacteremia (26 h versus 63 h, respectively)-and survived longer (41 h versus 90 h, respectively). Similar to control animals, all LPS-pretreated, B. anthracis-challenged rabbits exhibited pathology consistent with inhalational anthrax. Taken together, these results suggest that prior or underlying stimulation of the innate immune system induces transient host resistance to subsequent B. anthracis infection in SPF New Zealand white rabbits. In particular, our results emphasize the importance of using animals that are free of underlying infections to prevent confounding data in studies for inhalational anthrax characterization and medical countermeasure evaluation.

  20. Untersuchung der Virulenz Bacillus anthracis-ähnlicher Isolate aus West- und Zentralafrika

    OpenAIRE

    Dupke, Susann

    2011-01-01

    In 2001 and 2004 several great apes died of an Anthrax-like disease in Cameroon and the Côte d´Ivoire on the African continent. PCR analysis and histological studies of carcasses led to the assumption that the animals died due to infection with a new strain of B. anthracis. Further molecular genetic methods and sequencing of one of the isolates from Côte d´Ivoire revealed a close relationship of the new strains to B. cereus rather than B. anthracis, even though both characteristic virulence p...

  1. Confirmation of Bacillus anthracis from flesh-eating flies collected during a West Texas anthrax season.

    Science.gov (United States)

    Blackburn, Jason K; Curtis, Andrew; Hadfield, Ted L; O'Shea, Bob; Mitchell, Mark A; Hugh-Jones, Martin E

    2010-07-01

    This case study confirms the interaction between necrophilic flies and white-tailed deer, Odocoileus virginianus, during an anthrax outbreak in West Texas (summer 2005). Bacillus anthracis was identified by culture and PCR from one of eight pooled fly collections from deer carcasses on a deer ranch with a well-documented history of anthrax. These results provide the first known isolation of B. anthracis from flesh-eating flies associated with a wildlife anthrax outbreak in North America and are discussed in the context of wildlife ecology and anthrax epizootics. PMID:20688697

  2. Rapid detection methods for Bacillus anthracis in environmental samples: a review.

    OpenAIRE

    Irenge, Léonid; Gala, Jean-Luc

    2012-01-01

    Bacillus anthracis is a Gram-positive, spore-forming bacterium, which causes anthrax, an often lethal disease of animals and humans. Although the disease has been well studied since the nineteenth century, it has witnessed a renewed interest during the past decade, due to its use as a bioterrorist agent in the fall of 2001 in the USA. A number of techniques aimed at rapidly detecting B. anthracis, in environmental samples as well as in point-of-care settings for humans suspected of exposure t...

  3. Sequencing of 16S rRNA Gene: A Rapid Tool for Identification of Bacillus anthracis

    OpenAIRE

    Sacchi, Claudio T.; Whitney, Anne M.; Mayer, Leonard W.; Morey, Roger; Steigerwalt, Arnold; Boras, Ariana; Weyant, Robin S.; Popovic, Tanja

    2002-01-01

    In a bioterrorism event, a tool is needed to rapidly differentiate Bacillus anthracis from other closely related spore-forming Bacillus species. During the recent outbreak of bioterrorism-associated anthrax, we sequenced the 16S rRNA generom these species to evaluate the potential of 16S rRNA gene sequencing as a diagnostic tool. We found eight distinct 16S types among all 107 16S rRNA gene seqs fuences that differed from each other at 1 to 8 positions (0.06% to 0.5%). All 86 B. anthracis had...

  4. Activation of the latent PlcR regulon in Bacillus anthracis

    OpenAIRE

    Sastalla, Inka; Maltese, Lauren M.; Pomerantseva, Olga M.; Pomerantsev, Andrei P; Keane-Myers, Andrea; Stephen H Leppla

    2010-01-01

    Many genes in Bacillus cereus and Bacillus thuringiensis are under the control of the transcriptional regulator PlcR and its regulatory peptide, PapR. In Bacillus anthracis, the causative agent of anthrax, PlcR is inactivated by truncation, and consequently genes having PlcR binding sites are expressed at very low levels when compared with B. cereus. We found that activation of the PlcR regulon in B. anthracis by expression of a PlcR–PapR fusion protein does not alter sporulation in strains c...

  5. Bacillus anthracis infections – new possibilities of treatment

    Directory of Open Access Journals (Sweden)

    Dorota Żakowska

    2015-05-01

    Full Text Available [b]Introduction and objective[/b]. [i]Bacillus anthracis[/i] is one of biological agents which may be used in bioterrorism attacks. The aim of this study a review of the new treatment possibilities of anthrax, with particular emphasis on the treatment of pulmonary anthrax. [b]Abbreviated description of the state of knowledge[/b]. Pulmonary anthrax, as the most dangerous clinical form of the disease, is also extremely difficult to treat. Recently, considerable progress in finding new drugs and suitable therapy for anthrax has been achieved, for example, new antibiotics worth to mentioning, levofloxacin, daptomycin, gatifloxacin and dalbavancin. However, alternative therapeutic options should also be considered, among them the antimicrobial peptides, characterized by lack of inducible mechanisms of pathogen resistance. Very promising research considers bacteriophages lytic enzymes against selected bacteria species, including antibiotic-resistant strains. [b]Results[/b]. Interesting results were obtained using monoclonal antibodies: raxibacumab, cAb29 or cocktails of antibodies. The application of CpG oligodeoxynucleotides to boost the immune response elicited by Anthrax Vaccine Adsorbed and CMG2 protein complexes, also produced satisfying therapy results. Furthermore, the IFN-α and IFN-β, PA-dominant negative mutant, human inter-alpha inhibitor proteins and LF inhibitors in combination with ciprofloxacin, also showed very promising results. [b]Conclusions[/b]. Recently, progress has been achieved in inhalation anthrax treatment. The most promising new possibilities include: new antibiotics, peptides and bacteriophages enzymes, monoclonal antibodies, antigen PA mutants, and inter alpha inhibitors applications. In the case of the possibility of bioterrorist attacks, the examination of inhalation anthrax treatment should be intensively continued.

  6. Differential toxicity of mitomycin C and porfiromycin to aerobic and hypoxic Chinese hamster ovary cells overexpressing human NADPH:cytochrome c (P-450) reductase.

    Science.gov (United States)

    Belcourt, M F; Hodnick, W F; Rockwell, S; Sartorelli, A C

    1996-01-01

    Purified NADPH:cytochrome c (P-450) reductase (FpT; NADPH-ferrihemoprotein oxidoreductase, EC 1.6.2.4) can reductively activate mitomycin antibiotics through a one-electron reduction to species that alkylate DNA. To assess the involvement of FpT in the intracellular activation of the mitomycins, transfectants overexpressing a human FpT cDNA were established from a Chinese hamster ovary cell line deficient in dihydrofolate reductase (CHO-K1/dhfr-). The parental cell line was equisensitive to the cytotoxic action of mitomycin C under oxygenated and hypoxic conditions. In contrast, porfiromycin was considerably less cytotoxic to wild-type parental cells than was mitomycin C in air and markedly more cytotoxic under hypoxia. Two FpT-transfected clones were selected that expressed 19- and 27-fold more FpT activity than the parental line. Levels of other oxidoreductases implicated in the activation of the mitomycins were unchanged. Significant increases in sensitivity to mitomycin C and porfiromycin in the two FpT-transfected clones were seen under both oxygenated and hypoxic conditions, with the increases in toxicity being greater under hypoxia than in air. These findings demonstrate that FpT can bioreductively activate the mitomycins in living cells and implicate FpT in the differential aerobic/hypoxic toxicity of the mitomycins. PMID:8552660

  7. Quantitative Determination of Lethal Toxin Proteins in Culture Supernatant of Human Live Anthrax Vaccine Bacillus anthracis A16R

    OpenAIRE

    Zai, Xiaodong; Zhang, Jun; Liu, Ju; Liu,Jie; Li, Liangliang; Yin, Ying; Fu, Ling; Xu, Junjie; Chen, Wei

    2016-01-01

    Bacillus anthracis (B. anthracis) is the etiological agent of anthrax affecting both humans and animals. Anthrax toxin (AT) plays a major role in pathogenesis. It includes lethal toxin (LT) and edema toxin (ET), which are formed by the combination of protective antigen (PA) and lethal factor (LF) or edema factor (EF), respectively. The currently used human anthrax vaccine in China utilizes live-attenuated B. anthracis spores (A16R; pXO1+, pXO2−) that produce anthrax toxin but cannot produce t...

  8. A Bacillus anthracis strain deleted for six proteases serves as an effective host for production of recombinant proteins

    OpenAIRE

    Pomerantsev, Andrei P.; Pomerantseva, Olga M.; Moayeri, Mahtab; Fattah, Rasem; Tallant, Cynthia; Leppla, Stephen H.

    2011-01-01

    Bacillus anthracis produces a number of extracellular proteases that impact the integrity and yield of other proteins in the B. anthracis secretome. In this study we show that anthrolysin O (ALO) and the three anthrax toxin proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF), produced from the B. anthracis Ames 35 strain (pXO1+, pXO2−), are completely degraded at the onset of stationary phase due to the action of proteases. An improved Cre-loxP gene knockout system wa...

  9. Comparison of Growth and Toxin Production in Two Vaccine Strains of Bacillus anthracis

    OpenAIRE

    Johnson, Anna D; Spero, Leonard

    1981-01-01

    Two vaccine strains of Bacillus anthracis were monitored in a 10-liter fermentor to compare growth patterns and toxin production. Under identical conditions, the Sterne strain produced all three components of anthrax toxin, whereas strain V770 produced only the protective antigen.

  10. Macrophage-Enhanced Germination of Bacillus anthracis Endospores Requires gerS

    OpenAIRE

    Ireland, John A. W.; Hanna, Philip C.

    2002-01-01

    Germination of Bacillus anthracis Sterne and plasmidless Δ-Sterne endospores was dramatically enhanced in RAW264.7 macrophage-like cells, while germination of nonpathogenic Bacillus endospores was not. Elimination of gerS, a germinant receptor locus, caused a complete loss of cell-enhanced germination, implicating gerS in the breaking of endospore dormancy in vivo.

  11. Feeding Anthrax: The Crystal Structure of Bacillus anthracis InhA Protease.

    Science.gov (United States)

    Schacherl, Magdalena; Baumann, Ulrich

    2016-01-01

    Pathogenic bacteria secrete proteases to evade host defense and to acquire nutrients. In this issue of Structure, Arolas et al. (2016) describe the structural basis of activation and latency of InhA, a major secreted protease of Bacillus anthracis. PMID:26745525

  12. Anthrax Toxins in Context of Bacillus anthracis Spores and Spore Germination

    OpenAIRE

    Cote, Christopher K.; Susan L. Welkos

    2015-01-01

    The interaction of anthrax toxin or toxin components with B. anthracis spores has been demonstrated. Germinating spores can produce significant amounts of toxin components very soon after the initiation of germination. In this review, we will summarize the work performed that has led to our understanding of toxin and spore interactions and discuss the complexities associated with these interactions.

  13. Functional characterization of WalRK: A two-component signal transduction system from Bacillus anthracis

    Directory of Open Access Journals (Sweden)

    Alisha Dhiman

    2014-01-01

    Full Text Available Two-component signal transduction systems (TCS, consisting of a sensor histidine protein kinase and its cognate response regulator, are an important mode of environmental sensing in bacteria. Additionally, they have been found to regulate virulence determinants in several pathogens. Bacillus anthracis, the causative agent of anthrax and a bioterrorism agent, harbours 41 pairs of TCS. However, their role in its pathogenicity has remained largely unexplored. Here, we show that WalRK of B. anthracis forms a functional TCS which exhibits some species-specific functions. Biochemical studies showed that domain variants of WalK, the histidine kinase, exhibit classical properties of autophosphorylation and phosphotransfer to its cognate response regulator WalR. Interestingly, these domain variants also show phosphatase activity towards phosphorylated WalR, thereby making WalK a bifunctional histidine kinase/phosphatase. An in silico regulon determination approach, using a consensus binding sequence from Bacillus subtilis, provided a list of 30 genes that could form a putative WalR regulon in B. anthracis. Further, electrophoretic mobility shift assay was used to show direct binding of purified WalR to the upstream regions of three putative regulon candidates, an S-layer protein EA1, a cell division ABC transporter FtsE and a sporulation histidine kinase KinB3. Our work lends insight into the species-specific functions and mode of action of B. anthracis WalRK.

  14. Crossing of the epithelial barriers by Bacillus anthracis: the Known and the Unknown

    Directory of Open Access Journals (Sweden)

    Pierre L Goossens

    2015-10-01

    Full Text Available Anthrax, caused by Bacillus anthracis, a Gram-positive spore-forming bacterium, is initiated by the entry of spores into the host body. There are three types of human infection: cutaneous, inhalational, and gastrointestinal. For each form, B. anthracis spores need to cross the cutaneous, respiratory or digestive epithelial barriers, respectively, as a first obligate step to establish infection. Anthrax is a toxi-infection: an association of toxemia and rapidly spreading infection progressing to septicemia. The pathogenicity of Bacillus anthracis mainly depends on two toxins and a capsule. The capsule protects bacilli from the immune system, thus promoting systemic dissemination. The toxins alter host cell signalling, thereby paralysing the immune response of the host and perturbing the endocrine and endothelial systems.In this review, we will mainly focus on the events and mechanisms leading to crossing of the respiratory epithelial barrier, as the majority of studies have addressed inhalational infection. We will discuss the critical gaps of knowledge that need to be addressed to gain a comprehensive view of the initial steps of inhalational anthrax. We will then discuss the few data available on B. anthracis crossing the cutaneous and digestive epithelia.

  15. Sepiapterin Reductase Deficiency: Mimic of Cerebral Palsy

    OpenAIRE

    J Gordon Millichap

    2012-01-01

    Researchers at University of California at San Diego, and 22 other US national and international centers studied the clinical, biochemical, and molecular findings in a cohort of 38 patients with sepiapterin reductase deficiency (SRD).

  16. BAC Library Construction and Physical Mapping of Bacillus anthracis A16R

    Institute of Scientific and Technical Information of China (English)

    Zhang Da; Zhu Houchu; Huang Liuyu

    2013-01-01

    Bacillus anthracis is an endospore-forming bacterium that causes severe inhalational anthrax, and bacillus anthracis A16R is an attenuated strain derived from Bacillus anthracis A16. The development of bacterial artificial chromosome (BAC) system has allowed the construction of large insert-size DNA libraries, and the bacterial artificial chromosomes (BACs) have become the preferred large insert cloning system for genomic analysis because such libraries are characteristically stable, high in ifdelity and easy to handle. To facilitate genome studies of this bacterium, a bacterial artiifcial chromosome library (BAC) has been established from genome DNA of Bacillus anthracis A16R. This library consisted of 9 600 clones randomly selected from more than 15 000 recombinant clones carrying inserts in the plindigoBAC-5 vectors. The mean insert size was 56 kbp, representing an approximate 12-fold genome coverage, while end sequences were obtained from 700 randomly selected clones. Sequences were compared with Bacillus anthracis Ames and Bacillus cereus ATCC 14579 Genome Project databases using the NCBI BLASTN search project. And most BLASTN results showed high identities and that the sequences’ sites could be used as STSs. To construct this physical map, Excel was used for the array of STSs and some gaps of the map were iflled up by PCR walking. Artemis-V4 was used in the construction of a genome-wide physical map with 93%genome coverage. The A16R BAC library proved to be a vital tool for the generation of a map that would not only allow the subsequent sequencing of defined areas of genome, but also provide immediate access to clones that were stable and convenient for functional genomic researches.

  17. The Bacillus anthracis cholesterol-dependent cytolysin, Anthrolysin O, kills human neutrophils, monocytes and macrophages

    Directory of Open Access Journals (Sweden)

    Rest Richard F

    2006-06-01

    Full Text Available Abstract Background Bacillus anthracis is an animal and human pathogen whose virulence is characterized by lethal and edema toxin, as well as a poly-glutamic acid capsule. In addition to these well characterized toxins, B. anthracis secretes several proteases and phospholipases, and a newly described toxin of the cholesterol-dependent cytolysin (CDC family, Anthrolysin O (ALO. Results In the present studies we show that recombinant ALO (rALO or native ALO, secreted by viable B. anthracis, is lethal to human primary polymorphonuclear leukocytes (PMNs, monocytes, monocyte-derived macrophages (MDMs, lymphocytes, THP-1 monocytic human cell line and ME-180, Detroit 562, and A549 epithelial cells by trypan blue exclusion or lactate dehydrogenase (LDH release viability assays. ALO cytotoxicity is dose and time dependent and susceptibility to ALO-mediated lysis differs between cell types. In addition, the viability of monocytes and hMDMs was assayed in the presence of vegetative Sterne strains 7702 (ALO+, UT231 (ALO-, and a complemented strain expressing ALO, UT231 (pUTE544, and was dependent upon the expression of ALO. Cytotoxicity of rALO is seen as low as 0.070 nM in the absence of serum. All direct cytotoxic activity is inhibited by the addition of cholesterol or serum concentration as low as 10%. Conclusion The lethality of rALO and native ALO on human monocytes, neutrophils, macrophages and lymphocytes supports the idea that ALO may represent a previously unidentified virulence factor of B. anthracis. The study of other factors produced by B. anthracis, along with the major anthrax toxins, will lead to a better understanding of this bacterium's pathogenesis, as well as provide information for the development of antitoxin vaccines for treating and preventing anthrax.

  18. The search and identification of the new immunodiagnostic targets of bacillus anthracis spore

    International Nuclear Information System (INIS)

    Spores of Bacillus anthracis have been used as bio warfare agent to bio terrorize purposes. As efficiency of anti-epidemic measures included urgent prevention and treatment is determined by terms within which the bio agent is identified. Direct and rapid spore detection by antibodies based detection system is very attractive alternative to current PCR-based assays or routine phenotyping which are the most accurate but are also complex, time-consumption and expensive. The main difficulty with respect to such kind of anthrax spores detection is a cross-reaction with spores of closely related bacteria. For development of species-specific antibodies to anthrax spores recombinant scFvs or hybridoma technique were used. In both case surface spore antigens contained species-specific epitopes are need. Among exosporium proteins only ExsF(BxpB), ExsK and SoaA are specific to B.cereus group. On the surface of B. anthracis spores, a unique tetrasaccharides containing an novel monosaccharide - anthrose, was discovered. It was shown that anthrose can be serving as species-specific target for B. anthracis spores detection. We have revealed that EA1 isolated from spore of Russians strain STI-1 contain carbohydrate which formed species-specific epitopes and determine immunogenicity of this antigen. Antibodies to this antigen specifically recognized the surface target of B. anthracis spores and do not reacted with others Bacillus spore. Based on these antibodies we developed the test-systems in different formats for rapid direct detection and identification of B. anthracis spores. The results of trial these test-systems with using more than 50 different Bacillus strains were indicated that carbohydrate of EA1 isolated from spore is effective immunodiagnostic target for anthrax spores bio detection.(author)

  19. The Use of Germinants to Potentiate the Sensitivity of Bacillus anthracis Spores to Peracetic Acid.

    Science.gov (United States)

    Celebi, Ozgur; Buyuk, Fatih; Pottage, Tom; Crook, Ant; Hawkey, Suzanna; Cooper, Callum; Bennett, Allan; Sahin, Mitat; Baillie, Leslie

    2016-01-01

    Elimination of Bacillus anthracis spores from the environment is a difficult and costly process due in part to the toxicity of current sporicidal agents. For this reason we investigated the ability of the spore germinants L-alanine (100 mM) and inosine (5 mM) to reduce the concentration of peracetic acid (PAA) required to inactivate B. anthracis spores. While L-alanine significantly enhanced (p = 0.0085) the bactericidal activity of 500 ppm PAA the same was not true for inosine suggesting some form of negative interaction. In contrast the germinant combination proved most effective at 100 ppm PAA (p = 0.0009). To determine if we could achieve similar results in soil we treated soil collected from the burial site of an anthrax infected animal which had been supplemented with spores of the Sterne strain of B. anthracis to increase the level of contamination to 10(4) spores/g. Treatment with germinants followed 1 h later by 5000 ppm PAA eliminated all of the spores. In contrast direct treatment of the animal burial site using this approach delivered using a back pack sprayer had no detectable effect on the level of B. anthracis contamination or on total culturable bacterial numbers over the course of the experiment. It did trigger a significant, but temporary, reduction (p < 0.0001) in the total spore count suggesting that germination had been triggered under real world conditions. In conclusion, we have shown that the application of germinants increase the sensitivity of bacterial spores to PAA. While the results of the single field trial were inconclusive, the study highlighted the potential of this approach and the challenges faced when attempting to perform real world studies on B. anthracis spores contaminated sites. PMID:26858699

  20. Inactivation of Bacillus anthracis Spores during Laboratory-Scale Composting of Feedlot Cattle Manure.

    Science.gov (United States)

    Xu, Shanwei; Harvey, Amanda; Barbieri, Ruth; Reuter, Tim; Stanford, Kim; Amoako, Kingsley K; Selinger, Leonard B; McAllister, Tim A

    2016-01-01

    Anthrax outbreaks in livestock have social, economic and health implications, altering farmer's livelihoods, impacting trade and posing a zoonotic risk. Our study investigated the survival of Bacillus thuringiensis and B. anthracis spores sporulated at 15, 20, or 37°C, over 33 days of composting. Spores (∼7.5 log10 CFU g(-1)) were mixed with manure and composted in laboratory scale composters. After 15 days, the compost was mixed and returned to the composter for a second cycle. Temperatures peaked at 71°C on day 2 and remained ≥55°C for an average of 7 days in the first cycle, but did not exceed 55°C in the second. For B. thuringiensis, spores generated at 15 and 21°C exhibited reduced (P < 0.05) viability of 2.7 and 2.6 log10 CFU g(-1) respectively, as compared to a 0.6 log10 CFU g(-1) reduction for those generated at 37°C. For B. anthracis, sporulation temperature did not impact spore survival as there was a 2.5, 2.2, and 2.8 log10 CFU g(-1) reduction after composting for spores generated at 15, 21, and 37°C, respectively. For both species, spore viability declined more rapidly (P < 0.05) in the first as compared to the second composting cycle. Our findings suggest that the duration of thermophilic exposure (≥55°C) is the main factor influencing survival of B. anthracis spores in compost. As sporulation temperature did not influence survival of B. anthracis, composting may lower the viability of spores associated with carcasses infected with B. anthracis over a range of sporulation temperatures. PMID:27303388

  1. The Use of Germinants to Potentiate the Sensitivity of Bacillus anthracis Spores to Peracetic Acid

    Science.gov (United States)

    Celebi, Ozgur; Buyuk, Fatih; Pottage, Tom; Crook, Ant; Hawkey, Suzanna; Cooper, Callum; Bennett, Allan; Sahin, Mitat; Baillie, Leslie

    2016-01-01

    Elimination of Bacillus anthracis spores from the environment is a difficult and costly process due in part to the toxicity of current sporicidal agents. For this reason we investigated the ability of the spore germinants L-alanine (100 mM) and inosine (5 mM) to reduce the concentration of peracetic acid (PAA) required to inactivate B. anthracis spores. While L-alanine significantly enhanced (p = 0.0085) the bactericidal activity of 500 ppm PAA the same was not true for inosine suggesting some form of negative interaction. In contrast the germinant combination proved most effective at 100 ppm PAA (p = 0.0009). To determine if we could achieve similar results in soil we treated soil collected from the burial site of an anthrax infected animal which had been supplemented with spores of the Sterne strain of B. anthracis to increase the level of contamination to 104 spores/g. Treatment with germinants followed 1 h later by 5000 ppm PAA eliminated all of the spores. In contrast direct treatment of the animal burial site using this approach delivered using a back pack sprayer had no detectable effect on the level of B. anthracis contamination or on total culturable bacterial numbers over the course of the experiment. It did trigger a significant, but temporary, reduction (p < 0.0001) in the total spore count suggesting that germination had been triggered under real world conditions. In conclusion, we have shown that the application of germinants increase the sensitivity of bacterial spores to PAA. While the results of the single field trial were inconclusive, the study highlighted the potential of this approach and the challenges faced when attempting to perform real world studies on B. anthracis spores contaminated sites. PMID:26858699

  2. Modulation of the Bacillus anthracis secretome by the immune inhibitor A1 protease.

    Science.gov (United States)

    Pflughoeft, Kathryn J; Swick, Michelle C; Engler, David A; Yeo, Hye-Jeong; Koehler, Theresa M

    2014-01-01

    The Bacillus anthracis secretome includes protective antigen, lethal factor, and edema factor, which are the components of anthrax toxin, and other proteins with known or potential roles in anthrax disease. Immune inhibitor A1 (InhA1) is a secreted metalloprotease that is unique to pathogenic members of the Bacillus genus and has been associated with cleavage of host proteins during infection. Here, we report the effect of InhA1 on the B. anthracis secretome. Differential in-gel electrophoresis of proteins present in culture supernatants from a parent strain and an isogenic inhA1-null mutant revealed multiple differences. Of the 1,340 protein spots observed, approximately one-third were less abundant and one-third were more abundant in the inhA1 secretome than in the parent strain secretome. Proteases were strongly represented among those proteins exhibiting a 9-fold or greater change. InhA1 purified from a B. anthracis culture supernatant directly cleaved each of the anthrax toxin proteins as well as an additional secreted protease, Npr599. The conserved zinc binding motif HEXXH of InhA1 (HEYGH) was critical for its proteolytic activity. Our data reveal that InhA1 directly and indirectly modulates the form and/or abundance of over half of all the secreted proteins of B. anthracis. The proteolytic activity of InhA1 on established secreted virulence factors, additional proteases, and other secreted proteins suggests that this major protease plays an important role in virulence not only by cleaving mammalian substrates but also by modulating the B. anthracis secretome itself. PMID:24214942

  3. DNA probe functionalized QCM biosensor based on gold nanoparticle amplification for Bacillus anthracis detection.

    Science.gov (United States)

    Hao, Rong-Zhang; Song, Hong-Bin; Zuo, Guo-Min; Yang, Rui-Fu; Wei, Hong-Ping; Wang, Dian-Bing; Cui, Zong-Qiang; Zhang, ZhiPing; Cheng, Zhen-Xing; Zhang, Xian-En

    2011-04-15

    The rapid detection of Bacillus anthracis, the causative agent of anthrax disease, has gained much attention since the anthrax spore bioterrorism attacks in the United States in 2001. In this work, a DNA probe functionalized quartz crystal microbalance (QCM) biosensor was developed to detect B. anthracis based on the recognition of its specific DNA sequences, i.e., the 168 bp fragment of the Ba813 gene in chromosomes and the 340 bp fragment of the pag gene in plasmid pXO1. A thiol DNA probe was immobilized onto the QCM gold surface through self-assembly via Au-S bond formation to hybridize with the target ss-DNA sequence obtained by asymmetric PCR. Hybridization between the target DNA and the DNA probe resulted in an increase in mass and a decrease in the resonance frequency of the QCM biosensor. Moreover, to amplify the signal, a thiol-DNA fragment complementary to the other end of the target DNA was functionalized with gold nanoparticles. The results indicate that the DNA probe functionalized QCM biosensor could specifically recognize the target DNA fragment of B. anthracis from that of its closest species, such as Bacillus thuringiensis, and that the limit of detection (LOD) reached 3.5 × 10(2)CFU/ml of B. anthracis vegetative cells just after asymmetric PCR amplification, but without culture enrichment. The DNA probe functionalized QCM biosensor demonstrated stable, pollution-free, real-time sensing, and could find application in the rapid detection of B. anthracis. PMID:21315574

  4. Inactivation of Bacillus anthracis Spores during Laboratory-Scale Composting of Feedlot Cattle Manure

    Science.gov (United States)

    Xu, Shanwei; Harvey, Amanda; Barbieri, Ruth; Reuter, Tim; Stanford, Kim; Amoako, Kingsley K.; Selinger, Leonard B.; McAllister, Tim A.

    2016-01-01

    Anthrax outbreaks in livestock have social, economic and health implications, altering farmer’s livelihoods, impacting trade and posing a zoonotic risk. Our study investigated the survival of Bacillus thuringiensis and B. anthracis spores sporulated at 15, 20, or 37°C, over 33 days of composting. Spores (∼7.5 log10 CFU g-1) were mixed with manure and composted in laboratory scale composters. After 15 days, the compost was mixed and returned to the composter for a second cycle. Temperatures peaked at 71°C on day 2 and remained ≥55°C for an average of 7 days in the first cycle, but did not exceed 55°C in the second. For B. thuringiensis, spores generated at 15 and 21°C exhibited reduced (P < 0.05) viability of 2.7 and 2.6 log10 CFU g-1 respectively, as compared to a 0.6 log10 CFU g-1 reduction for those generated at 37°C. For B. anthracis, sporulation temperature did not impact spore survival as there was a 2.5, 2.2, and 2.8 log10 CFU g-1 reduction after composting for spores generated at 15, 21, and 37°C, respectively. For both species, spore viability declined more rapidly (P < 0.05) in the first as compared to the second composting cycle. Our findings suggest that the duration of thermophilic exposure (≥55°C) is the main factor influencing survival of B. anthracis spores in compost. As sporulation temperature did not influence survival of B. anthracis, composting may lower the viability of spores associated with carcasses infected with B. anthracis over a range of sporulation temperatures. PMID:27303388

  5. BAC Library Construction and Physical Mapping of Bacillus anthracis A16R

    Directory of Open Access Journals (Sweden)

    Da Zhang

    2013-12-01

    Full Text Available Bacillus anthracis is an endospore-forming bacterium that causes severe inhalational anthrax, and bacillus anthracis A16R is an attenuated strain derived from Bacillus anthracis A16. The development of bacterial artificial chromosome (BAC system has allowed the construction of large insert-size DNA libraries, and the bacterial artificial chromosomes (BACs have become the preferred large insert cloning system for genomic analysis because such libraries are characteristically stable, high in fidelity and easy to handle. To facilitate genome studies of this bacterium, a bacterial artificial chromosome library (BAC has been established from genome DNA of Bacillus anthracis A16R. This library consisted of 9 600 clones randomly selected from more than 15 000 recombinant clones carrying inserts in the plindigoBAC-5 vectors. The mean insert size was 56 kbp, representing an approximate 12-fold genome coverage, while end sequences were obtained from 700 randomly selected clones. Sequences were compared with Bacillus anthracis Ames and Bacillus cereus ATCC 14579 Genome Project databases using the NCBI BLASTN search project. And most BLASTN results showed high identities and that the sequences’ sites could be used as STSs. To construct this physical map, Excel was used for the array of STSs and some gaps of the map were filled up by PCR walking. Artemis-V4 was used in the construction of a genome-wide physical map with 93% genome coverage. The A16R BAC library proved to be a vital tool for the generation of a map that would not only allow the subsequent sequencing of defined areas of genome, but also provide immediate access to clones that were stable and convenient for functional genomic researches.

  6. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences

    DEFF Research Database (Denmark)

    Ågren, Joakim; Hamidjaja, Raditijo A.; Hansen, Trine;

    2013-01-01

    Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely......-layer, and prophage-lambda. Following a review of the literature, an in silico analysis of all signature sequences reported for identification of B. anthracis was conducted. Published primer and probe sequences were compared for specificity against 134 available Bacillus spp. genomes. Although many of the chromosomal...... on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. For the detection of chromosomal DNA, different genes have been used, such as BA813, rpoB, gyrA, plcR, S...

  7. The Pathogenomic Sequence Analysis of B. cereus and B.thuringiensis Isolates Closely Related to Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Han, Cliff S.; Xie, Gary; Challacombe, Jean F.; Altherr, MichaelR.; Smriti, B.; Bruce, David; Campbell, Connie S.; Campbell, Mary L.; Chen, Jin; Chertkov, Olga; Cleland, Cathy; Dimitrijevic-Bussod, M.; Doggett, Norman A.; Fawcett, John J.; Glavina, Tijana; Goodwin, Lynne A.; Hill, Karen K.; Hitchcock, Penny; Jackson, Paul J.; Keim, Paul; Kewalramani, Avinash Ramesh; Longmire, Jon; Lucas, Susan; Malfatti,Stephanie; McMurry, Kim; Meincke, Linda J.; Misra, Monica; Moseman,Bernice L.; Mundt, Mark; Munk, A. Christine; Okinaka, Richard T.; Parson-Quintana, B.; Reilly, Lee P.; Richardson, Paul; Robinson, DonnaL.; Rubin, Eddy; Saunders, Elizabeth; Tapia, Roxanne; Tesmer, Judith G.; Thayer, Nina; Thompson, Linda S.; Tice, Hope; Ticknor, Lawrence O.; Wills, Patti L.; Gilna, Payl; Brettin, Thomas S.

    2005-08-18

    The sequencing and analysis of two close relatives of Bacillus anthracis are reported. AFLP analysis of over 300 isolates of B.cereus, B. thuringiensis and B. anthracis identified two isolates as being very closely related to B. anthracis. One, a B. cereus, BcE33L, was isolated from a zebra carcass in Nambia; the second, a B. thuringiensis, 97-27, was isolated from a necrotic human wound. The B. cereus appears to be the closest anthracis relative sequenced to date. A core genome of over 3,900 genes was compiled for the Bacillus cereus group, including Banthracis. Comparative analysis of these two genomes with other members of the B. cereus group provides insight into the evolutionary relationships among these organisms. Evidence is presented that differential regulation modulates virulence, rather than simple acquisition of virulence factors. These genome sequences provide insight into the molecular mechanisms contributing to the host range and virulence of this group of organisms.

  8. BslA, the S-layer adhesin of B. anthracis, is a virulence factor for anthrax pathogenesis

    OpenAIRE

    Kern, Justin; Schneewind, Olaf

    2009-01-01

    Microbial pathogens use adhesive surface proteins to bind to and interact with host tissues, events that are universal for the pathogenesis of infectious diseases. A surface adhesin of Bacillus anthracis, the causative agent of anthrax, required to mediate these steps has not been discovered. Previous work identified BslA, an S-layer protein, to be necessary and sufficient for adhesion of the anthrax vaccine strain, Bacillus anthracis Sterne, to host cells. Here we asked whether encapsulated ...

  9. Comparative efficacy of Bacillus anthracis live spore vaccine and protective antigen vaccine against anthrax in the guinea pig.

    OpenAIRE

    Little, S F; Knudson, G B

    1986-01-01

    Several strains of Bacillus anthracis have been reported previously to cause fatal infection in immunized guinea pigs. In this study, guinea pigs were immunized with either a protective antigen vaccine or a live Sterne strain spore vaccine, then challenged with virulent B. anthracis strains isolated from various host species from the United States and foreign sources. Confirmation of previously reported studies (which used only protective antigen vaccines) was made with the identification of ...

  10. Differential Effects of Linezolid and Ciprofloxacin on Toxin Production by Bacillus anthracis in an In Vitro Pharmacodynamic System

    OpenAIRE

    Louie, Arnold; VanScoy, Brian D.; Heine, Henry S.; Liu, Weiguo; Abshire, Terry; Holman, Kari; Kulawy, Robert; Brown, David L.; Drusano, George L.

    2012-01-01

    Bacillus anthracis causes anthrax. Ciprofloxacin is a gold standard for the treatment of anthrax. Previously, using the non-toxin-producing ΔSterne strain of B. anthracis, we demonstrated that linezolid was equivalent to ciprofloxacin for reducing the total (vegetative and spore) bacterial population. With ciprofloxacin therapy, the total population consisted of spores. With linezolid therapy, the population consisted primarily of vegetative bacteria. Linezolid is a protein synthesis inhibito...

  11. Bacillus anthracis Phospholipases C Facilitate Macrophage-Associated Growth and Contribute to Virulence in a Murine Model of Inhalation Anthrax

    OpenAIRE

    Heffernan, Brian J.; Thomason, Brendan; Herring-Palmer, Amy; Shaughnessy, Lee; McDonald, Rod; Fisher, Nathan; Huffnagle, Gary B.; Hanna, Philip

    2006-01-01

    Several models of anthrax pathogenesis suggest that early in the infectious process Bacillus anthracis endospores germinate and outgrow into vegetative bacilli within phagocytes before being released into the blood. Here, we define the respective contributions of three phospholipases C (PLCs) to the pathogenesis of B. anthracis. Genetic deletions of the PLCs were made in the Sterne 7702 background, resulting in the respective loss of their activities. The PLCs were redundant both in tissue cu...

  12. Herstellung monoklonaler Antikörper gegen thermostabile Antigene von Bacillus anthracis zur Anwendung in der Anthraxdiagnostik

    OpenAIRE

    Hilss, Karen

    2012-01-01

    Bei dem Ascoli Präzipitin Test (ASCOLI, 1911) handelt es sich um eine schnelle und kostengünstige Diagnostikmethode, bei der polyklonales Serum gegen thermostabile Antigene von B. anthracis eingesetzt wird. Allerdings ist dieser Test ungeeignet für Umweltproben, da Kreuzreaktionen mit anderen Bacillus Spezies auftreten. Durch die Verwendung monoklonaler Antikörper gegen spezifische thermostabile Antigene von B. anthracis könnte jedoch die Kreuzreaktivität mit anderen Bacillus Spezies eliminie...

  13. Anthrolysin O and fermentation products mediate the toxicity of Bacillus anthracis to lung epithelial cells under microaerobic conditions

    OpenAIRE

    Popova, Taissia G.; Millis, Bryan; Chung, Myung-Chul; Bailey, Charles; Popov, Serguei G

    2010-01-01

    Bacillus anthracis generates virulence factors such as lethal and edema toxins, capsule, and hemolytic proteins under conditions of reduced oxygenation. Here, we report on the acute cytotoxicity of culture supernatants (Sups) of six nonencapsulated B. anthracis strains grown till the stationary phase under static microaerobic conditions. Human small airway epithelial, umbilical vein endothelial, Caco-2, and Hep-G2 cells were found to be susceptible. Sups displayed a reduction of pH to 5.3–5.5...

  14. Implications of Limits of Detection of Various Methods for Bacillus anthracis in Computing Risks to Human Health▿ †

    OpenAIRE

    Herzog, Amanda B.; McLennan, S. Devin; Pandey, Alok K.; Gerba, Charles P.; Haas, Charles N.; Joan B. Rose; Hashsham, Syed A.

    2009-01-01

    Used for decades for biological warfare, Bacillus anthracis (category A agent) has proven to be highly stable and lethal. Quantitative risk assessment modeling requires descriptive statistics of the limit of detection to assist in defining the exposure. Furthermore, the sensitivities of various detection methods in environmental matrices are vital information for first responders. A literature review of peer-reviewed journal articles related to methods for detection of B. anthracis was undert...

  15. Roles of Macrophages and Neutrophils in the Early Host Response to Bacillus anthracis Spores in a Mouse Model of Infection

    OpenAIRE

    Cote, Christopher K.; Van Rooijen, Nico; Welkos, Susan L.

    2006-01-01

    The development of new approaches to combat anthrax requires that the pathogenesis and host response to Bacillus anthracis spores be better understood. We investigated the roles that macrophages and neutrophils play in the progression of infection by B. anthracis in a mouse model. Mice were treated with a macrophage depletion agent (liposome-encapsulated clodronate) or with a neutrophil depletion agent (cyclophosphamide or the rat anti-mouse granulocyte monoclonal antibody RB6-8C5), and the a...

  16. Discerning Viable from Nonviable Yersinia pestis pgm- and Bacillus anthracis Sterne using Propidium Monoazide in the Presence of White Powders

    Energy Technology Data Exchange (ETDEWEB)

    Hess, Becky M.; Kaiser, Brooke LD; Sydor, Michael A.; Wunschel, David S.; Bruckner-Lea, Cindy J.; Hutchison, Janine R.

    2015-12-23

    ABSTRACT Aims To develop and optimize an assay to determine viability status of Bacillus anthracis Sterne and Yersinia pestis pgm- strains in the presence of white powders by coupling propidium monoazide (PMA) treatment with real-time PCR (qPCR) analysis. Methods and Results PMA selectively enters nonviable cells and binds DNA, thereby increasing qPCR assay cycle threshold (CT) values compared to untreated samples. Dye concentration, cell number and fitness, incubation time, inactivation methods, and assay buffer were optimized for B. anthracis Sterne and Y. pestis pgm-. Differences in CT values in nonviable cells compared to untreated samples were consistently > 9 for both B. anthracis Sterne vegetative cells and Y. pestis pgm- in the presence and absence of three different white powders. Our method eliminates the need for a DNA extraction step prior to detection by qPCR. Conclusions The developed assay enables simultaneous identification and viability assessment for B. anthracis Sterne and Y. pestis pgm- under laboratory conditions, even in the presence of white powders. Eliminating the DNA extraction step that is typically used reduces total assay time and labor requirements for sample analysis. Significance and Impact of the Study The method developed for simultaneous detection and viability assessment for B. anthracis and Y. pestis can be employed in forming decisions about the severity of a biothreat event or the safety of food. Keywords Bacillus anthracis, Yersinia pestis, Propidium Monoazide, qPCR, White Powders, Rapid Viability Detection

  17. Respiratory arsenate reductase as a bidirectional enzyme

    Science.gov (United States)

    Richey, C.; Chovanec, P.; Hoeft, S.E.; Oremland, R.S.; Basu, P.; Stolz, J.F.

    2009-01-01

    The haloalkaliphilic bacterium Alkalilimnicola ehrlichii is capable of anaerobic chemolithoautotrophic growth by coupling the oxidation of arsenite (As(III)) to the reduction of nitrate and carbon dioxide. Analysis of its complete genome indicates that it lacks a conventional arsenite oxidase (Aox), but instead possesses two operons that each encode a putative respiratory arsenate reductase (Arr). Here we show that one homolog is expressed under chemolithoautotrophic conditions and exhibits both arsenite oxidase and arsenate reductase activity. We also demonstrate that Arr from two arsenate respiring bacteria, Alkaliphilus oremlandii and Shewanella sp. strain ANA-3, is also biochemically reversible. Thus Arr can function as a reductase or oxidase. Its physiological role in a specific organism, however, may depend on the electron potentials of the molybdenum center and [Fe–S] clusters, additional subunits, or constitution of the electron transfer chain. This versatility further underscores the ubiquity and antiquity of microbial arsenic metabolism.

  18. Transcriptional profiling of Bacillus anthracis Sterne (34F2 during iron starvation.

    Directory of Open Access Journals (Sweden)

    Paul E Carlson

    Full Text Available Lack of available iron is one of many environmental challenges that a bacterium encounters during infection and adaptation to iron starvation is important for the pathogen to efficiently replicate within the host. Here we define the transcriptional response of B. anthracis Sterne (34F(2 to iron depleted conditions. Genome-wide transcript analysis showed that B. anthracis undergoes considerable changes in gene expression during growth in iron-depleted media, including the regulation of known and candidate virulence factors. Two genes encoding putative internalin proteins were chosen for further study. Deletion of either gene (GBAA0552 or GBAA1340 resulted in attenuation in a murine model of infection. This attenuation was amplified in a double mutant strain. These data define the transcriptional changes induced during growth in low iron conditions and illustrate the potential of this dataset in the identification of putative virulence determinants for future study.

  19. Variable Lymphocyte Receptor Recognition of the Immunodominant Glycoprotein of Bacillus anthracis Spores

    Energy Technology Data Exchange (ETDEWEB)

    Kirchdoerfer, Robert N.; Herrin, Brantley R.; Han, Byung Woo; Turnbough, Jr., Charles L.; Cooper, Max D.; Wilson, Ian A. (SNU); (Scripps); (Emory); (UAB); (Emory Vaccine)

    2012-07-25

    Variable lymphocyte receptors (VLRs) are the adaptive immune receptors of jawless fish, which evolved adaptive immunity independent of other vertebrates. In lieu of the immunoglobulin fold-based T and B cell receptors, lymphocyte-like cells of jawless fish express VLRs (VLRA, VLRB, or VLRC) composed of leucine-rich repeats and are similar to toll-like receptors (TLRs) in structure, but antibodies (VLRB) and T cell receptors (VLRA and VLRC) in function. Here, we present the structural and biochemical characterization of VLR4, a VLRB, in complex with BclA, the immunodominant glycoprotein of Bacillus anthracis spores. Using a combination of crystallography, mutagenesis, and binding studies, we delineate the mode of antigen recognition and binding between VLR4 and BclA, examine commonalities in VLRB recognition of antigens, and demonstrate the potential of VLR4 as a diagnostic tool for the identification of B. anthracis spores.

  20. Protection of rhesus macaques against inhalational anthrax with a Bacillus anthracis capsule conjugate vaccine.

    Science.gov (United States)

    Chabot, Donald J; Ribot, Wilson J; Joyce, Joseph; Cook, James; Hepler, Robert; Nahas, Debbie; Chua, Jennifer; Friedlander, Arthur M

    2016-07-25

    The efficacy of currently licensed anthrax vaccines is largely attributable to a single Bacillus anthracis immunogen, protective antigen. To broaden protection against possible strains resistant to protective antigen-based vaccines, we previously developed a vaccine in which the anthrax polyglutamic acid capsule was covalently conjugated to the outer membrane protein complex of Neisseria meningitidis serotype B and demonstrated that two doses of 2.5μg of this vaccine conferred partial protection of rhesus macaques against inhalational anthrax . Here, we demonstrate complete protection of rhesus macaques against inhalational anthrax with a higher 50μg dose of the same capsule conjugate vaccine. These results indicate that B. anthracis capsule is a highly effective vaccine component that should be considered for incorporation in future generation anthrax vaccines. PMID:27329184

  1. Laboratory Studies on Surface Sampling of Bacillus anthracis Contamination: Summary, Gaps, and Recommendations

    Energy Technology Data Exchange (ETDEWEB)

    Piepel, Gregory F.; Amidan, Brett G.; Hu, Rebecca

    2011-11-28

    This report summarizes previous laboratory studies to characterize the performance of methods for collecting, storing/transporting, processing, and analyzing samples from surfaces contaminated by Bacillus anthracis or related surrogates. The focus is on plate culture and count estimates of surface contamination for swab, wipe, and vacuum samples of porous and nonporous surfaces. Summaries of the previous studies and their results were assessed to identify gaps in information needed as inputs to calculate key parameters critical to risk management in biothreat incidents. One key parameter is the number of samples needed to make characterization or clearance decisions with specified statistical confidence. Other key parameters include the ability to calculate, following contamination incidents, the (1) estimates of Bacillus anthracis contamination, as well as the bias and uncertainties in the estimates, and (2) confidence in characterization and clearance decisions for contaminated or decontaminated buildings. Gaps in knowledge and understanding identified during the summary of the studies are discussed and recommendations are given for future studies.

  2. Evolution of plant defense mechanisms. Relationships of phenylcoumaran benzylic ether reductases to pinoresinol-lariciresinol and isoflavone reductases.

    Science.gov (United States)

    Gang, D R; Kasahara, H; Xia, Z Q; Vander Mijnsbrugge, K; Bauw, G; Boerjan, W; Van Montagu, M; Davin, L B; Lewis, N G

    1999-03-12

    Pinoresinol-lariciresinol and isoflavone reductase classes are phylogenetically related, as is a third, the so-called "isoflavone reductase homologs." This study establishes the first known catalytic function for the latter, as being able to engender the NADPH-dependent reduction of phenylcoumaran benzylic ethers. Accordingly, all three reductase classes are involved in the biosynthesis of important and related phenylpropanoid-derived plant defense compounds. In this investigation, the phenylcoumaran benzylic ether reductase from the gymnosperm, Pinus taeda, was cloned, with the recombinant protein heterologously expressed in Escherichia coli. The purified enzyme reduces the benzylic ether functionalities of both dehydrodiconiferyl alcohol and dihydrodehydrodiconiferyl alcohol, with a higher affinity for the former, as measured by apparent Km and Vmax values and observed kinetic 3H-isotope effects. It abstracts the 4R-hydride of the required NADPH cofactor in a manner analogous to that of the pinoresinol-lariciresinol reductases and isoflavone reductases. A similar catalytic function was observed for the corresponding recombinant reductase whose gene was cloned from the angiosperm, Populus trichocarpa. Interestingly, both pinoresinol-lariciresinol reductases and isoflavone reductases catalyze enantiospecific conversions, whereas the phenylcoumaran benzylic ether reductase only shows regiospecific discrimination. A possible evolutionary relationship among the three reductase classes is proposed, based on the supposition that phenylcoumaran benzylic ether reductases represent the progenitors of pinoresinol-lariciresinol and isoflavone reductases.

  3. Recombinant expression and purification of a tumor-targeted toxin in Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Bachran, Christopher; Abdelazim, Suzanne; Fattah, Rasem J.; Liu, Shihui [National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 (United States); Leppla, Stephen H., E-mail: sleppla@niaid.nih.gov [National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 (United States)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Non-infectious and protease-deficient Bacillus anthracis protein expression system. Black-Right-Pointing-Pointer Successful expression and purification of a tumor-targeted fusion protein drug. Black-Right-Pointing-Pointer Very low endotoxin contamination of purified protein. Black-Right-Pointing-Pointer Efficient protein secretion simplifies purification. Black-Right-Pointing-Pointer Functional anti-tumor fusion protein purified. -- Abstract: Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression host offers a solution to circumvent these problems. Using the multiple protease-deficient strain BH460, we expressed a fusion of the N-terminal 254 amino acids of anthrax lethal factor (LFn), the N-terminal 389 amino acids of diphtheria toxin (DT389) and human transforming growth factor alpha (TGF{alpha}). The resulting fusion protein was constitutively expressed and successfully secreted by B. anthracis into the culture supernatant. Purification was achieved by anion exchange chromatography and proteolytic cleavage removed LFn from the desired fusion protein (DT389 fused to TGF{alpha}). The fusion protein showed the intended specific cytotoxicity to epidermal growth factor receptor-expressing human head and neck cancer cells. Final analyses showed low levels of lipopolysaccharides, originating most likely from contamination during the purification process. Thus, the fusion to LFn for protein secretion and expression in B. anthracis BH460 provides an elegant tool to obtain high levels of lipopolysaccharide-free recombinant protein.

  4. Allelic variation on murine chromosome 11 modifies host inflammatory responses and resistance to Bacillus anthracis.

    Directory of Open Access Journals (Sweden)

    Jill K Terra

    2011-12-01

    Full Text Available Anthrax is a potentially fatal disease resulting from infection with Bacillus anthracis. The outcome of infection is influenced by pathogen-encoded virulence factors such as lethal toxin (LT, as well as by genetic variation within the host. To identify host genes controlling susceptibility to anthrax, a library of congenic mice consisting of strains with homozygous chromosomal segments from the LT-responsive CAST/Ei strain introgressed on a LT-resistant C57BL/6 (B6 background was screened for response to LT. Three congenic strains containing CAST/Ei regions of chromosome 11 were identified that displayed a rapid inflammatory response to LT similar to, but more severe than that driven by a LT-responsive allele of the inflammasome constituent NRLP1B. Importantly, increased response to LT in congenic mice correlated with greater resistance to infection by the Sterne strain of B. anthracis. The genomic region controlling the inflammatory response to LT was mapped to 66.36-74.67 Mb on chromosome 11, a region that encodes the LT-responsive CAST/Ei allele of Nlrp1b. However, known downstream effects of NLRP1B activation, including macrophage pyroptosis, cytokine release, and leukocyte infiltration could not fully explain the response to LT or the resistance to B. anthracis Sterne in congenic mice. Further, the exacerbated response in congenic mice is inherited in a recessive manner while the Nlrp1b-mediated response to LT is dominant. Finally, congenic mice displayed increased responsiveness in a model of sepsis compared with B6 mice. In total, these data suggest that allelic variation of one or more chromosome 11 genes in addition to Nlrp1b controls the severity of host response to multiple inflammatory stimuli and contributes to resistance to B. anthracis Sterne. Expression quantitative trait locus analysis revealed 25 genes within this region as high priority candidates for contributing to the host response to LT.

  5. Automated thermochemolysis reactor for detection of Bacillus anthracis endospores by gas chromatography–mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Li, Dan [Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602 (United States); Rands, Anthony D.; Losee, Scott C. [Torion Technologies, American Fork, UT 84003 (United States); Holt, Brian C. [Department of Statistics, Brigham Young University, Provo, UT 84602 (United States); Williams, John R. [Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602 (United States); Lammert, Stephen A. [Torion Technologies, American Fork, UT 84003 (United States); Robison, Richard A. [Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602 (United States); Tolley, H. Dennis [Department of Statistics, Brigham Young University, Provo, UT 84602 (United States); Lee, Milton L., E-mail: milton_lee@byu.edu [Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602 (United States)

    2013-05-02

    Graphical abstract: -- Highlights: •An automated sample preparation system for Bacillus anthracis endospores was developed. •A thermochemolysis method was applied to produce and derivatize biomarkers for Bacillus anthracis detection. •The autoreactor controlled the precise delivery of reagents, and TCM reaction times and temperatures. •Solid phase microextraction was used to extract biomarkers, and GC–MS was used for final identification. •This autoreactor was successfully applied to the identification of Bacillus anthracis endospores. -- Abstract: An automated sample preparation system was developed and tested for the rapid detection of Bacillus anthracis endospores by gas chromatography–mass spectrometry (GC–MS) for eventual use in the field. This reactor is capable of automatically processing suspected bio-threat agents to release and derivatize unique chemical biomarkers by thermochemolysis (TCM). The system automatically controls the movement of sample vials from one position to another, crimping of septum caps onto the vials, precise delivery of reagents, and TCM reaction times and temperatures. The specific operations of introduction of sample vials, solid phase microextraction (SPME) sampling, injection into the GC–MS system, and ejection of used vials from the system were performed manually in this study, although they can be integrated into the automated system. Manual SPME sampling is performed by following visual and audible signal prompts for inserting the fiber into and retracting it from the sampling port. A rotating carousel design allows for simultaneous sample collection, reaction, biomarker extraction and analysis of sequential samples. Dipicolinic acid methyl ester (DPAME), 3-methyl-2-butenoic acid methyl ester (a fragment of anthrose) and two methylated sugars were used to compare the performance of the autoreactor with manual TCM. Statistical algorithms were used to construct reliable bacterial endospore signatures, and 24

  6. Modeling the potential distribution of Bacillus anthracis under multiple climate change scenarios for Kazakhstan.

    Directory of Open Access Journals (Sweden)

    Timothy Andrew Joyner

    Full Text Available Anthrax, caused by the bacterium Bacillus anthracis, is a zoonotic disease that persists throughout much of the world in livestock, wildlife, and secondarily infects humans. This is true across much of Central Asia, and particularly the Steppe region, including Kazakhstan. This study employed the Genetic Algorithm for Rule-set Prediction (GARP to model the current and future geographic distribution of Bacillus anthracis in Kazakhstan based on the A2 and B2 IPCC SRES climate change scenarios using a 5-variable data set at 55 km(2 and 8 km(2 and a 6-variable BioClim data set at 8 km(2. Future models suggest large areas predicted under current conditions may be reduced by 2050 with the A2 model predicting approximately 14-16% loss across the three spatial resolutions. There was greater variability in the B2 models across scenarios predicting approximately 15% loss at 55 km(2, approximately 34% loss at 8 km(2, and approximately 30% loss with the BioClim variables. Only very small areas of habitat expansion into new areas were predicted by either A2 or B2 in any models. Greater areas of habitat loss are predicted in the southern regions of Kazakhstan by A2 and B2 models, while moderate habitat loss is also predicted in the northern regions by either B2 model at 8 km(2. Anthrax disease control relies mainly on livestock vaccination and proper carcass disposal, both of which require adequate surveillance. In many situations, including that of Kazakhstan, vaccine resources are limited, and understanding the geographic distribution of the organism, in tandem with current data on livestock population dynamics, can aid in properly allocating doses. While speculative, contemplating future changes in livestock distributions and B. anthracis spore promoting environments can be useful for establishing future surveillance priorities. This study may also have broader applications to global public health surveillance relating to other diseases in addition to B

  7. Study of Immunization against Anthrax with the Purified Recombinant Protective Antigen of Bacillus anthracis

    OpenAIRE

    Singh,Yogendra; Ivins, Bruce E.; Leppla, Stephen H.

    1998-01-01

    Protective antigen (PA) of anthrax toxin is the major component of human anthrax vaccine. Currently available human vaccines in the United States and Europe consist of alum-precipitated supernatant material from cultures of toxigenic, nonencapsulated strains of Bacillus anthracis. Immunization with these vaccines requires several boosters and occasionally causes local pain and edema. We previously described the biological activity of a nontoxic mutant of PA expressed in Bacillus subtilis. In ...

  8. New Developments in Vaccines, Inhibitors of Anthrax Toxins, and Antibiotic Therapeutics for Bacillus anthracis

    OpenAIRE

    Beierlein, J.M.; Anderson, A. C.

    2011-01-01

    Bacillus anthracis, the causative agent responsible for anthrax infections, poses a significant biodefense threat. There is a high mortality rate associated with untreated anthrax infections; specifically, inhalation anthrax is a particularly virulent form of infection with mortality rates close to 100%, even with aggressive treatment. Currently, a vaccine is not available to the general public and few antibiotics have been approved by the FDA for the treatment of inhalation anthrax. With the...

  9. Identification of the pXO1 plasmid in attenuated Bacillus anthracis vaccine strains.

    Science.gov (United States)

    Liang, Xudong; Zhang, Huijuan; Zhang, Enmin; Wei, Jianchun; Li, Wei; Wang, Bingxiang; Dong, Shulin; Zhu, Jin

    2016-07-01

    Anthrax toxins and capsule are the major virulence factors of Bacillus anthracis. They are encoded by genes located on the plasmids pXO1 and pXO2, respectively. The vaccine strain Pasteur II was produced from high temperature subcultures of B. anthracis, which resulted in virulence attenuation through the loss of the plasmid pXO1. However, it is unclear whether the high temperature culture completely abolishes the plasmid DNA or affects the replication of the plasmid pXO1. In this study, we tested 3 B. anthracis vaccine strains, including Pasteur II from France, Qiankefusiji II from Russia, and Rentian II from Japan, which were all generated from subcultures at high temperatures. Surprisingly, we detected the presence of pXO1 plasmid DNA using overlap PCR in all these vaccine strains. DNA sequencing analysis of overlap PCR products further confirmed the presence of pXO1. Moreover, the expression of the protective antigen (PA) encoded on pXO1 was determined by using SDS-PAGE and western blotting. In addition, we mimicked Pasteur's method and exposed the A16R vaccine strain, which lacks the pXO2 plasmid, to high temperature, and identified the pXO1 plasmid in the subcultures at high temperatures. This indicated that the high temperature treatment at 42.5°C was unable to eliminate pXO1 plasmid DNA from B. anthracis. Our results suggest that the attenuation of the Pasteur II vaccine strain is likely due to the impact of high temperature stress on plasmid replication, which in turn limits the copy number of pXO1. Our data provide new insights into the mechanisms of the remaining immunogenicity and toxicity of the vaccine strains. PMID:27029580

  10. Passive protection by polyclonal antibodies against Bacillus anthracis infection in guinea pigs.

    OpenAIRE

    Little, S F; Ivins, B E; Fellows, P F; Friedlander, A M

    1997-01-01

    The protective effects of polyclonal antisera produced by injecting guinea pigs with protective antigen (PA), the chemical anthrax vaccine AVA, or Sterne spore vaccine, as well as those of toxin-neutralizing monoclonal antibodies (MAbs) produced against PA, lethal factor, and edema factor, were examined in animals infected with Bacillus anthracis spores. Only the anti-PA polyclonal serum significantly protected the guinea pigs from death, with 67% of infected animals surviving. Although none ...

  11. Development of an Inhalational Bacillus anthracis Exposure Therapeutic Model in Cynomolgus Macaques

    OpenAIRE

    Henning, Lisa N.; Comer, Jason E.; Stark, Gregory V.; Ray, Bryan D.; Tordoff, Kevin P.; Knostman, Katherine A. B.; Meister, Gabriel T.

    2012-01-01

    Appropriate animal models are required to test medical countermeasures to bioterrorist threats. To that end, we characterized a nonhuman primate (NHP) inhalational anthrax therapeutic model for use in testing anthrax therapeutic medical countermeasures according to the U.S. Food and Drug Administration Animal Rule. A clinical profile was recorded for each NHP exposed to a lethal dose of Bacillus anthracis Ames spores. Specific diagnostic parameters were detected relatively early in disease pr...

  12. Rapid Detection of Viable Bacillus anthracis Spores in Environmental Samples by Using Engineered Reporter Phages.

    Science.gov (United States)

    Sharp, Natasha J; Molineux, Ian J; Page, Martin A; Schofield, David A

    2016-04-01

    Bacillus anthracis, the causative agent of anthrax, was utilized as a bioterrorism agent in 2001 when spores were distributed via the U.S. postal system. In responding to this event, the Federal Bureau of Investigation used traditional bacterial culture viability assays to ascertain the extent of contamination of the postal facilities within 24 to 48 h of environmental sample acquisition. Here, we describe a low-complexity, second-generation reporter phage assay for the rapid detection of viableB. anthracis spores in environmental samples. The assay uses an engineered B. anthracis reporter phage (Wβ::luxAB-2) which transduces bioluminescence to infected cells. To facilitate low-level environmental detection and maximize the signal response, expression of luxABin an earlier version of the reporter phage (Wβ::luxAB-1) was optimized. These alterations prolonged signal kinetics, increased light output, and improved assay sensitivity. Using Wβ::luxAB-2, detection of B. anthracis spores was 1 CFU in 8 h from pure cultures and as low as 10 CFU/g in sterile soil but increased to 10(5)CFU/g in unprocessed soil due to an unstable signal and the presence of competing bacteria. Inclusion of semiselective medium, mediated by a phage-expressed antibiotic resistance gene, maintained signal stability and enabled the detection of 10(4)CFU/g in 6 h. The assay does not require spore extraction and relies on the phage infecting germinating cells directly in the soil sample. This reporter phage displays promise for the rapid detection of low levels of spores on clean surfaces and also in grossly contaminated environmental samples from complex matrices such as soils. PMID:26873316

  13. A Bacillus anthracis Genome Sequence from the Sverdlovsk 1979 Autopsy Specimens

    Science.gov (United States)

    Sahl, Jason W.; Pearson, Talima; Okinaka, Richard; Schupp, James M.; Gillece, John D.; Heaton, Hannah; Birdsell, Dawn; Hepp, Crystal; Fofanov, Viacheslav; Noseda, Ramón; Fasanella, Antonio; Hoffmaster, Alex; Wagner, David M.

    2016-01-01

    ABSTRACT Anthrax is a zoonotic disease that occurs naturally in wild and domestic animals but has been used by both state-sponsored programs and terrorists as a biological weapon. A Soviet industrial production facility in Sverdlovsk, USSR, proved deficient in 1979 when a plume of spores was accidentally released and resulted in one of the largest known human anthrax outbreaks. In order to understand this outbreak and others, we generated a Bacillus anthracis population genetic database based upon whole-genome analysis to identify all single-nucleotide polymorphisms (SNPs) across a reference genome. Phylogenetic analysis has defined three major clades (A, B, and C), B and C being relatively rare compared to A. The A clade has numerous subclades, including a major polytomy named the trans-Eurasian (TEA) group. The TEA radiation is a dominant evolutionary feature of B. anthracis, with many contemporary populations having resulted from a large spatial dispersal of spores from a single source. Two autopsy specimens from the Sverdlovsk outbreak were deep sequenced to produce draft B. anthracis genomes. This allowed the phylogenetic placement of the Sverdlovsk strain into a clade with two Asian live vaccine strains, including the Russian Tsiankovskii strain. The genome was examined for evidence of drug resistance manipulation or other genetic engineering, but none was found. The Soviet Sverdlovsk strain genome is consistent with a wild-type strain from Russia that had no evidence of genetic manipulation during its industrial production. This work provides insights into the world’s largest biological weapons program and provides an extensive B. anthracis phylogenetic reference. PMID:27677796

  14. Analysis of the sporicidal activity of chlorine dioxide disinfectant against Bacillus anthracis (Sterne strain)

    OpenAIRE

    Chatuev, B.A.; Peterson, J W

    2010-01-01

    Routine surface decontamination is an essential hospital and laboratory procedure, but the list of effective, noncorrosive disinfectants that kill spores is limited. We investigated the sporicidal potential of an aqueous chlorine dioxide solution and encountered some unanticipated problems. Quantitative bacteriological culture methods were used to determine the log10 reduction of Bacillus anthracis (Sterne strain) spores following 3 min exposure to various concentrations of aqueous chlorine d...

  15. Fluorescent Amplified Fragment Length Polymorphism Analysis of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis Isolates

    OpenAIRE

    Hill, Karen K.; Ticknor, Lawrence O.; Okinaka, Richard T.; Asay, Michelle; Blair, Heather; Bliss, Katherine A.; Laker, Mariam; Pardington, Paige E.; Richardson, Amber P.; Tonks, Melinda; Beecher, Douglas J.; Kemp, John D.; Kolstø, Anne-Brit; Wong, Amy C. Lee; Keim, Paul

    2004-01-01

    DNA from over 300 Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis isolates was analyzed by fluorescent amplified fragment length polymorphism (AFLP). B. thuringiensis and B. cereus isolates were from diverse sources and locations, including soil, clinical isolates and food products causing diarrheal and emetic outbreaks, and type strains from the American Type Culture Collection, and over 200 B. thuringiensis isolates representing 36 serovars or subspecies were from the U.S. D...

  16. Differentiation of Bacillus anthracis from Bacillus cereus by gas chromatographic whole-cell fatty acid analysis.

    OpenAIRE

    Lawrence, D; Heitefuss, S; Seifert, H S

    1991-01-01

    Three strains of Bacillus anthracis and seven strains of Bacillus cereus were grown on complex medium and on synthetic medium. Gas chromatographic analysis of whole-cell fatty acids of strains grown on complex medium gave nearly identical fatty acid patterns. Fatty acid patterns of strains grown on synthetic medium showed a high content of branched-chain fatty acids. Significant differences between the fatty acid patterns of the two species were found. Odd iso/anteiso fatty acid ratios were a...

  17. The Early Humoral Immune Response to Bacillus anthracis Toxins in Patients Infected with Cutaneous Anthrax

    OpenAIRE

    Doganay, Mehmet; Brenneman, Karen E.; Akmal, Arya; Goldman, Stanley; Galloway, Darrell R.; Mateczun, Alfred J; Cross, Alan S.; Baillie, Leslie W.

    2011-01-01

    Bacillus anthracis, the causative agent of anthrax, elaborates a tripartite toxin composed of two enzymatically active subunits, lethal factor (LF) and edema factor (EF) which, when associated with a cell-binding component, protective antigen (PA), form lethal toxin (LT) and edema toxin (ET), respectively. In this preliminary study we characterised the toxin-specific antibody responses observed in 17 individuals infected with cutaneous anthrax. The majority of the toxin-specific antibody resp...

  18. PCR Assay To Detect Bacillus anthracis Spores in Heat-Treated Specimens

    OpenAIRE

    Fasanella, A.; Losito, S.; Adone, R.; Ciuchini, F.; Trotta, T.; Altamura, S. A.; D. Chiocco; Ippolito, G

    2003-01-01

    Recent interest in anthrax is due to its potential use in bioterrorism and as a biowarfare agent against civilian populations. The development of rapid and sensitive techniques to detect anthrax spores in suspicious specimens is the most important aim for public health. With a view to preventing exposure of laboratory workers to viable Bacillus anthracis spores, this study evaluated the suitability of PCR assays for detecting anthrax spores previously inactivated at 121°C for 45 min. The resu...

  19. GcoGSA-BA: A Global Core Genome SNP Analysis for Bacillus anthracis

    OpenAIRE

    Yamashita, Akifmi; Sekizuka, Tsuyoshi; Kuroda, Makoto

    2015-01-01

    As an issue of biosecurity, it is important to identify the origin of a suspected sample to distinguish whether it originated from the release of a bioterrorism agent or from environmental contamination with a virulent agent. Here we have developed an analytical pipeline that can infer the phylogenetic position of Bacillus cereus group species, including B. anthracis, from next-generation sequencing reads without extensive genomics skills. GcoGSA-BA can also detect the existence of anthrax pl...

  20. Forensic Application of Microbiological Culture Analysis To Identify Mail Intentionally Contaminated with Bacillus anthracis Spores†

    OpenAIRE

    Beecher, Douglas J.

    2006-01-01

    The discovery of a letter intentionally filled with dried Bacillus anthracis spores in the office of a United States senator prompted the collection and quarantine of all mail in congressional buildings. This mail was subsequently searched for additional intentionally contaminated letters. A microbiological sampling strategy was used to locate heavy contamination within the 642 separate plastic bags containing the mail. Swab sampling identified 20 bags for manual and visual examination. Air s...

  1. Activation of the latent PlcR regulon in Bacillus anthracis.

    Science.gov (United States)

    Sastalla, Inka; Maltese, Lauren M; Pomerantseva, Olga M; Pomerantsev, Andrei P; Keane-Myers, Andrea; Leppla, Stephen H

    2010-10-01

    Many genes in Bacillus cereus and Bacillus thuringiensis are under the control of the transcriptional regulator PlcR and its regulatory peptide, PapR. In Bacillus anthracis, the causative agent of anthrax, PlcR is inactivated by truncation, and consequently genes having PlcR binding sites are expressed at very low levels when compared with B. cereus. We found that activation of the PlcR regulon in B. anthracis by expression of a PlcR-PapR fusion protein does not alter sporulation in strains containing the virulence plasmid pXO1 and thereby the global regulator AtxA. Using comparative 2D gel electrophoresis, we showed that activation of the PlcR regulon in B. anthracis leads to upregulation of many proteins found in the secretome of B. cereus, including phospholipases and proteases, such as the putative protease BA1995. Transcriptional analysis demonstrated expression of BA1995 to be dependent on PlcR-PapR, even though the putative PlcR recognition site of the BA1995 gene does not exactly match the PlcR consensus sequence, explaining why this protein had escaped recognition as belonging to the PlcR regulon. Additionally, while transcription of major PlcR-dependent haemolysins, sphingomyelinase and anthrolysin O is enhanced in response to PlcR activation in B. anthracis, only anthrolysin O contributes significantly to lysis of human erythrocytes. In contrast, the toxicity of bacterial culture supernatants from a PlcR-positive strain towards murine macrophages occurred independently of anthrolysin O expression in vitro and in vivo. PMID:20688829

  2. Soluble Expression and Characterization of Biologically Active Bacillus anthracis Protective Antigen in Escherichia coli

    OpenAIRE

    Nagendra Suryanarayana; Vanlalhmuaka,; Bharti Mankere; Monika Verma; Kulanthaivel Thavachelvam; Urmil Tuteja

    2016-01-01

    Bacillus anthracis secretory protein protective antigen (PA) is primary candidate for subunit vaccine against anthrax. Attempts to obtain large quantity of PA from Escherichia coli expression system often result in the formation of insoluble inclusion bodies. Therefore, it is always better to produce recombinant proteins in a soluble form. In the present study, we have obtained biologically active recombinant PA in small scale E. coli shake culture system using three different expression cons...

  3. EXPRESSION OF BACILLUS ANTHRACIS PROTECTIVE ANTIGEN IN VACCINE STRAIN BRUCELLA ABORTUS RB51

    OpenAIRE

    Poff, Sherry Ann

    1997-01-01

    Bacillus anthracis is a facultative intracellular bacterial pathogen that can cause cutaneous, gastrointestinal or respiratory disease in many vertebrates, including humans. Commercially available anthrax vaccines for immunization of humans are of limited duration and do not protect against the respiratory form of the disease. Brucella abortus is a facultative intracellular bacterium that causes chronic infection in animals and humans. As with other intracellular pathogens, cell mediated im...

  4. Effective Antimicrobial Regimens for Use in Humans for Therapy of Bacillus anthracis Infections and Postexposure Prophylaxis†

    OpenAIRE

    Deziel, Mark R.; Heine, Henry; Louie, Arnold; Kao, Mark; Byrne, William R.; Basset, Jennifer; Miller, Lynda; Bush, Karen; Kelly, Michael; Drusano, G L

    2005-01-01

    Expanded options for treatments directed against pathogens that can be used for bioterrorism are urgently needed. Treatment regimens directed against such pathogens can be identified only by using data derived from in vitro and animal studies. It is crucial that these studies reliably predict the efficacy of proposed treatments in humans. The objective of this study was to identify a levofloxacin treatment regimen that will serve as an effective therapy for Bacillus anthracis infections and p...

  5. Recombinant Expression and Purification of a Tumor-Targeted Toxin in Bacillus anthracis

    OpenAIRE

    Bachran, Christopher; Abdelazim, Suzanne; Fattah, Rasem J.; Liu, Shihui; Leppla, Stephen H.

    2012-01-01

    Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression host offers a solution to circumvent these problems. Using the multiple pr...

  6. Aerosolized Bacillus anthracis infection in New Zealand white rabbits: natural history and intravenous levofloxacin treatment.

    Science.gov (United States)

    Yee, Steven B; Hatkin, Joshua M; Dyer, David N; Orr, Steven A; Pitt, M Louise M

    2010-12-01

    The natural history for inhalational Bacillus anthracis (Ames strain) exposure in New Zealand white rabbits was investigated to better identify potential, early biomarkers of anthrax. Twelve SPF Bordetella-free rabbits were exposed to 150 LD(50) aerosolized B. anthracis spores, and clinical signs, body temperature, complete blood count, bacteremia, and presence of protective antigen in the blood (that is, antigenemia) were examined. The development of antigenemia and bacteremia coincided and preceded both pyrexia and inversion of the heterophil:lymphocyte ratio, an indicator of infection. Antigenemia was determined within 1 h by electrochemiluminescence immunoassay, compared with the 24-h traditional culture needed for bacteremia determination. Rabbits appeared clinically normal until shortly before succumbing to anthrax approximately 47 h after challenge or approximately 22 h after antigenemia, which suggests a relatively narrow therapeutic window of opportunity. To evaluate the therapeutic rabbit model, B. anthracis-exposed rabbits were treated (after determination of antigenemia and later confirmed to be bacteremic) intravenously with the fluoroquinolone antibiotic levofloxacin for 5 d at a total daily dose of 25 or 12.5 mg/kg, resulting in nearly 90% and 70% survival, respectively, to the study end (28 d after challenge). The peak level for 12.5 mg/kg was equivalent to that observed for a 500-mg daily levofloxacin dose in humans. These results suggest that intravenous levofloxacin is an effective therapeutic against inhalational anthrax. Taken together, our findings indicate that antigenemia is a viable and early biomarker for B. anthracis infection that can be used as a treatment trigger to allow for timely intervention against this highly pathogenic disease. PMID:21262133

  7. The role of anthrolysin O in gut epithelial barrier disruption during Bacillus anthracis infection

    OpenAIRE

    Bishop, Brian L.; Lodolce, James P.; Kolodziej, Lauren; Boone, David L.; Tang, Wei Jen

    2010-01-01

    Gastrointestinal (GI) anthrax, caused by the bacterial infection of Bacillus anthracis, posts a significant bioterrorism threat by its relatively high mortality rate in humans. Different from inhalational anthrax by the route of infection, accumulating evidence indicates the bypass of vegetative bacteria across GI epithelium is required to initiate GI anthrax. Previously, we reported that purified anthrolysin O (ALO), instead of tripartite anthrax edema and lethal toxins, is capable of disrup...

  8. Rapid Detection of Viable Bacillus anthracis Spores in Environmental Samples by Using Engineered Reporter Phages

    Science.gov (United States)

    Sharp, Natasha J.; Molineux, Ian J.; Page, Martin A.

    2016-01-01

    Bacillus anthracis, the causative agent of anthrax, was utilized as a bioterrorism agent in 2001 when spores were distributed via the U.S. postal system. In responding to this event, the Federal Bureau of Investigation used traditional bacterial culture viability assays to ascertain the extent of contamination of the postal facilities within 24 to 48 h of environmental sample acquisition. Here, we describe a low-complexity, second-generation reporter phage assay for the rapid detection of viable B. anthracis spores in environmental samples. The assay uses an engineered B. anthracis reporter phage (Wβ::luxAB-2) which transduces bioluminescence to infected cells. To facilitate low-level environmental detection and maximize the signal response, expression of luxAB in an earlier version of the reporter phage (Wβ::luxAB-1) was optimized. These alterations prolonged signal kinetics, increased light output, and improved assay sensitivity. Using Wβ::luxAB-2, detection of B. anthracis spores was 1 CFU in 8 h from pure cultures and as low as 10 CFU/g in sterile soil but increased to 105 CFU/g in unprocessed soil due to an unstable signal and the presence of competing bacteria. Inclusion of semiselective medium, mediated by a phage-expressed antibiotic resistance gene, maintained signal stability and enabled the detection of 104 CFU/g in 6 h. The assay does not require spore extraction and relies on the phage infecting germinating cells directly in the soil sample. This reporter phage displays promise for the rapid detection of low levels of spores on clean surfaces and also in grossly contaminated environmental samples from complex matrices such as soils. PMID:26873316

  9. Recombinant expression and purification of a tumor-targeted toxin in Bacillus anthracis

    International Nuclear Information System (INIS)

    Highlights: ► Non-infectious and protease-deficient Bacillus anthracis protein expression system. ► Successful expression and purification of a tumor-targeted fusion protein drug. ► Very low endotoxin contamination of purified protein. ► Efficient protein secretion simplifies purification. ► Functional anti-tumor fusion protein purified. -- Abstract: Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression host offers a solution to circumvent these problems. Using the multiple protease-deficient strain BH460, we expressed a fusion of the N-terminal 254 amino acids of anthrax lethal factor (LFn), the N-terminal 389 amino acids of diphtheria toxin (DT389) and human transforming growth factor alpha (TGFα). The resulting fusion protein was constitutively expressed and successfully secreted by B. anthracis into the culture supernatant. Purification was achieved by anion exchange chromatography and proteolytic cleavage removed LFn from the desired fusion protein (DT389 fused to TGFα). The fusion protein showed the intended specific cytotoxicity to epidermal growth factor receptor-expressing human head and neck cancer cells. Final analyses showed low levels of lipopolysaccharides, originating most likely from contamination during the purification process. Thus, the fusion to LFn for protein secretion and expression in B. anthracis BH460 provides an elegant tool to obtain high levels of lipopolysaccharide-free recombinant protein.

  10. Automated thermochemolysis reactor for detection of Bacillus anthracis endospores by gas chromatography–mass spectrometry

    International Nuclear Information System (INIS)

    Graphical abstract: -- Highlights: •An automated sample preparation system for Bacillus anthracis endospores was developed. •A thermochemolysis method was applied to produce and derivatize biomarkers for Bacillus anthracis detection. •The autoreactor controlled the precise delivery of reagents, and TCM reaction times and temperatures. •Solid phase microextraction was used to extract biomarkers, and GC–MS was used for final identification. •This autoreactor was successfully applied to the identification of Bacillus anthracis endospores. -- Abstract: An automated sample preparation system was developed and tested for the rapid detection of Bacillus anthracis endospores by gas chromatography–mass spectrometry (GC–MS) for eventual use in the field. This reactor is capable of automatically processing suspected bio-threat agents to release and derivatize unique chemical biomarkers by thermochemolysis (TCM). The system automatically controls the movement of sample vials from one position to another, crimping of septum caps onto the vials, precise delivery of reagents, and TCM reaction times and temperatures. The specific operations of introduction of sample vials, solid phase microextraction (SPME) sampling, injection into the GC–MS system, and ejection of used vials from the system were performed manually in this study, although they can be integrated into the automated system. Manual SPME sampling is performed by following visual and audible signal prompts for inserting the fiber into and retracting it from the sampling port. A rotating carousel design allows for simultaneous sample collection, reaction, biomarker extraction and analysis of sequential samples. Dipicolinic acid methyl ester (DPAME), 3-methyl-2-butenoic acid methyl ester (a fragment of anthrose) and two methylated sugars were used to compare the performance of the autoreactor with manual TCM. Statistical algorithms were used to construct reliable bacterial endospore signatures, and 24

  11. Fieldable genotyping of Bacillus anthracis and Yersinia pestis based on 25-loci Multi Locus VNTR Analysis

    Directory of Open Access Journals (Sweden)

    Carattoli Alessandra

    2008-01-01

    Full Text Available Abstract Background Anthrax and plague are diseases caused by Bacillus anthracis and Yersinia pestis respectively. These bacteria are etiological agents for worldwide zoonotic diseases and are considered among the most feared potential bioterror agents. Strain differentiation is difficult for these microorganisms because of their high intraspecies genome homogeneity. Moreover, fast strain identification and comparison with known genotypes may be crucial for naturally occurring outbreaks versus bioterrorist events discrimination. Results Thirty-nine B. anthracis and ten Y. pestis strains, representative of the species genetic diversity, were genotyped by Agilent 2100 Bioanalyzer using previously described Multiple Locus VNTR Analysis assays (MLVA. Results were compared to previous data obtained by standard genotyping system (capillary electrophoresis on automatic sequencer and, when necessary, direct amplicon sequencing. A reference comparison table containing actual fragment sizes, sequencer sizes and Agilent sizes was produced. Conclusion In this report an automated DNA electrophoresis apparatus which provides a cheaper alternative compared to capillary electrophoresis approaches was applied for genotyping of B. anthracis and Y. pestis. This equipment, uses pre-cast gels and provides easy transportation, low maintenance and overall general logistic requirements and costs, is easy to set up and provides rapid analysis. This platform is a candidate for on-site MLVA genotyping of biothreat agents as well as other bacterial pathogens. It is an alternative to the more expensive and demanding capillary electrophoresis methods, and to the less expensive but more time-consuming classical gel electrophoresis approach.

  12. Ca-asp bound X-ray structure and inhibition of Bacillus anthracis dihydroorotase (DHOase).

    Science.gov (United States)

    Rice, Amy J; Lei, Hao; Santarsiero, Bernard D; Lee, Hyun; Johnson, Michael E

    2016-10-01

    Dihydroorotase (DHOase) is the third enzyme in the de novo pyrimidine synthesis pathway and is responsible for the reversible cyclization of carbamyl-aspartate (Ca-asp) to dihydroorotate (DHO). DHOase is further divided into two classes based on several structural characteristics, one of which is the length of the flexible catalytic loop that interacts with the substrate, Ca-asp, regulating the enzyme activity. Here, we present the crystal structure of Class I Bacillus anthracis DHOase with Ca-asp in the active site, which shows the peptide backbone of glycine in the shorter loop forming the necessary hydrogen bonds with the substrate, in place of the two threonines found in Class II DHOases. Despite the differences in the catalytic loop, the structure confirms that the key interactions between the substrate and active site residues are similar between Class I and Class II DHOase enzymes, which we further validated by mutagenesis studies. B. anthracis DHOase is also a potential antibacterial drug target. In order to identify prospective inhibitors, we performed high-throughput screening against several libraries using a colorimetric enzymatic assay and an orthogonal fluorescence thermal binding assay. Surface plasmon resonance was used for determining binding affinity (KD) and competition analysis with Ca-asp. Our results highlight that the primary difference between Class I and Class II DHOase is the catalytic loop. We also identify several compounds that can potentially be further optimized as potential B. anthracis inhibitors.

  13. Sample collection of virulent and non-virulent B. anthracis and Y. pestis for bioforensics analysis

    Energy Technology Data Exchange (ETDEWEB)

    Hong-geller, Elizabeth [Los Alamos National Laboratory; Valdez, Yolanda E [Los Alamos National Laboratory; Shou, Yulin [Los Alamos National Laboratory; Yoshida, Thomas M [Los Alamos National Laboratory; Marrone, Babetta L [Los Alamos National Laboratory; Dunbar, John [Los Alamos National Laboratory

    2009-01-01

    Validated sample collection methods are needed for recovery of microbial evidence in the event of accidental or intentional release of biological agents into the environment. To address this need, we evaluated the sample recovery efficiencies of two collection methods -- swabs and wipes -- for both non-virulent and virulent strains of B. anthracis and Y. pestis from four types of non-porous surfaces: two hydrophilic surfaces, stainless steel and glass, and two hydrophobic surfaces, vinyl and plastic. Sample recovery was quantified using Real-time qPCR to assay for intact DNA signatures. We found no consistent difference in collection efficiency between swabs or wipes. Furthermore, collection efficiency was more surface-dependent for virulent strains than non-virulent strains. For the two non-virulent strains, B. anthracis Sterne and Y. pestis A1122, collection efficiency was approximately 100% and 1 %, respectively, from all four surfaces. In contrast, recovery of B. anthracis Ames spores and Y. pestis C092 from vinyl and plastic was generally lower compared to collection from glass or stainless steel, suggesting that surface hydrophobicity may playa role in the strength of pathogen adhesion. The surface-dependent collection efficiencies observed with the virulent strains may arise from strain-specific expression of capsular material or other cell surface receptors that alter cell adhesion to specific surfaces. These findings contribute to validation of standard bioforensics procedures and emphasize the importance of specific strain and surface interactions in pathogen detection.

  14. Structural study and thermodynamic characterization of inhibitor binding to lumazine synthase from Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Morgunova, Ekaterina [Karolinska Institutet NOVUM, Center of Structural Biochemistry, Hälsovägen 7-9, 141 57 Huddinge (Sweden); Illarionov, Boris; Saller, Sabine [Institut für Lebensmittelchemie, Universität Hamburg, Grindelallee 117, 20146 Hamburg (Germany); Popov, Aleksander [European Synchrotron Radiation Facility, BP 220, F-38043 Grenoble CEDEX 09 (France); Sambaiah, Thota [Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University (United States); Bacher, Adelbert [Chemistry Department, Technical University of Munich, 85747 Garching (Germany); Cushman, Mark [Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University (United States); Fischer, Markus [Institut für Lebensmittelchemie, Universität Hamburg, Grindelallee 117, 20146 Hamburg (Germany); Ladenstein, Rudolf, E-mail: rudolf.ladenstein@ki.se [Karolinska Institutet NOVUM, Center of Structural Biochemistry, Hälsovägen 7-9, 141 57 Huddinge (Sweden)

    2010-09-01

    Crystallographic studies of lumazine synthase, the penultimate enzyme of the riboflavin-biosynthetic pathway in B. anthracis, provide a structural framework for the design of antibiotic inhibitors, together with calorimetric and kinetic investigations of inhibitor binding. The crystal structure of lumazine synthase from Bacillus anthracis was solved by molecular replacement and refined to R{sub cryst} = 23.7% (R{sub free} = 28.4%) at a resolution of 3.5 Å. The structure reveals the icosahedral symmetry of the enzyme and specific features of the active site that are unique in comparison with previously determined orthologues. The application of isothermal titration calorimetry in combination with enzyme kinetics showed that three designed pyrimidine derivatives bind to lumazine synthase with micromolar dissociation constants and competitively inhibit the catalytic reaction. Structure-based modelling suggested the binding modes of the inhibitors in the active site and allowed an estimation of the possible contacts formed upon binding. The results provide a structural framework for the design of antibiotics active against B. anthracis.

  15. Potential role of autophagy in the bactericidal activity of human PMNs for Bacillus anthracis.

    Science.gov (United States)

    Ramachandran, Girish; Gade, Padmaja; Tsai, Pei; Lu, Wuyuan; Kalvakolanu, Dhananjaya V; Rosen, Gerald M; Cross, Alan S

    2015-12-01

    Bacillus anthracis, the causative agent of anthrax, is acquired by mammalian hosts from the environment, as quiescent endospores. These endospores must germinate inside host cells, forming vegetative bacilli, before they can express the virulence factors that enable them to evade host defenses and disseminate throughout the body. While the role of macrophages and dendritic cells in this initial interaction has been established, the role of polymorphonuclear leukocytes (PMNs) has not been adequately defined. We discovered that while B. anthracis 34F2 Sterne endospores germinate poorly within non-activated human PMNs, these phagocytes exhibit rapid microbicidal activity toward the outgrown vegetative bacilli, independent of superoxide and nitric oxide. These findings suggest that a non-free radical pathway kills B. anthracis bacilli. We also find in PMNs an autophagic mechanism of bacterial killing based on the rapid induction of LC-3 conversion, beclin-1 expression, sequestosome 1 (SQSTM1) degradation and inhibition of bactericidal activity by the inhibitor, 3-methyladenine. These findings extend to PMNs an autophagic bactericidal mechanism previously described for other phagocytes. PMID:26424808

  16. Bacillus anthracis TIR Domain-Containing Protein Localises to Cellular Microtubule Structures and Induces Autophagy.

    Science.gov (United States)

    Carlsson, Emil; Thwaite, Joanne E; Jenner, Dominic C; Spear, Abigail M; Flick-Smith, Helen; Atkins, Helen S; Byrne, Bernadette; Ding, Jeak Ling

    2016-01-01

    Toll-like receptors (TLRs) recognise invading pathogens and mediate downstream immune signalling via Toll/IL-1 receptor (TIR) domains. TIR domain proteins (Tdps) have been identified in multiple pathogenic bacteria and have recently been implicated as negative regulators of host innate immune activation. A Tdp has been identified in Bacillus anthracis, the causative agent of anthrax. Here we present the first study of this protein, designated BaTdp. Recombinantly expressed and purified BaTdp TIR domain interacted with several human TIR domains, including that of the key TLR adaptor MyD88, although BaTdp expression in cultured HEK293 cells had no effect on TLR4- or TLR2- mediated immune activation. During expression in mammalian cells, BaTdp localised to microtubular networks and caused an increase in lipidated cytosolic microtubule-associated protein 1A/1B-light chain 3 (LC3), indicative of autophagosome formation. In vivo intra-nasal infection experiments in mice showed that a BaTdp knockout strain colonised host tissue faster with higher bacterial load within 4 days post-infection compared to the wild type B. anthracis. Taken together, these findings indicate that BaTdp does not play an immune suppressive role, but rather, its absence increases virulence. BaTdp present in wild type B. anthracis plausibly interact with the infected host cell, which undergoes autophagy in self-defence. PMID:27391310

  17. Ecological niche modeling of Bacillus anthracis on three continents: evidence for genetic-ecological divergence?

    Directory of Open Access Journals (Sweden)

    Jocelyn C Mullins

    Full Text Available We modeled the ecological niche of a globally successful Bacillus anthracis sublineage in the United States, Italy and Kazakhstan to better understand the geographic distribution of anthrax and potential associations between regional populations and ecology. Country-specific ecological-niche models were developed and reciprocally transferred to the other countries to determine if pathogen presence could be accurately predicted on novel landscapes. Native models accurately predicted endemic areas within each country, but transferred models failed to predict known occurrences in the outside countries. While the effects of variable selection and limitations of the genetic data should be considered, results suggest differing ecological associations for the B. anthracis populations within each country and may reflect niche specialization within the sublineage. Our findings provide guidance for developing accurate ecological niche models for this pathogen; models should be developed regionally, on the native landscape, and with consideration to population genetics. Further genomic analysis will improve our understanding of the genetic-ecological dynamics of B. anthracis across these countries and may lead to more refined predictive models for surveillance and proactive vaccination programs. Further studies should evaluate the impact of variable selection of native and transferred models.

  18. Global metabolomic analysis of a mammalian host infected with Bacillus anthracis.

    Science.gov (United States)

    Nguyen, Chinh T Q; Shetty, Vivekananda; Maresso, Anthony W

    2015-12-01

    Whereas DNA provides the information to design life and proteins provide the materials to construct it, the metabolome can be viewed as the physiology that powers it. As such, metabolomics, the field charged with the study of the dynamic small-molecule fluctuations that occur in response to changing biology, is now being used to study the basis of disease. Here, we describe a comprehensive metabolomic analysis of a systemic bacterial infection using Bacillus anthracis, the etiological agent of anthrax disease, as the model pathogen. An organ and blood analysis identified approximately 400 metabolites, including several key classes of lipids involved in inflammation, as being suppressed by B. anthracis. Metabolite changes were detected as early as 1 day postinfection, well before the onset of disease or the spread of bacteria to organs, which testifies to the sensitivity of this methodology. Functional studies using pharmacologic inhibition of host phospholipases support the idea of a role of these key enzymes and lipid mediators in host survival during anthrax disease. Finally, the results are integrated to provide a comprehensive picture of how B. anthracis alters host physiology. Collectively, the results of this study provide a blueprint for using metabolomics as a platform to identify and study novel host-pathogen interactions that shape the outcome of an infection. PMID:26438791

  19. Recombinant expression and purification of a tumor-targeted toxin in Bacillus anthracis.

    Science.gov (United States)

    Bachran, Christopher; Abdelazim, Suzanne; Fattah, Rasem J; Liu, Shihui; Leppla, Stephen H

    2013-01-01

    Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression host offers a solution to circumvent these problems. Using the multiple protease-deficient strain BH460, we expressed a fusion of the N-terminal 254 amino acids of anthrax lethal factor (LFn), the N-terminal 389 amino acids of diphtheria toxin (DT389) and human transforming growth factor alpha (TGFα). The resulting fusion protein was constitutively expressed and successfully secreted by B. anthracis into the culture supernatant. Purification was achieved by anion exchange chromatography and proteolytic cleavage removed LFn from the desired fusion protein (DT389 fused to TGFα). The fusion protein showed the intended specific cytotoxicity to epidermal growth factor receptor-expressing human head and neck cancer cells. Final analyses showed low levels of lipopolysaccharides, originating most likely from contamination during the purification process. Thus, the fusion to LFn for protein secretion and expression in B. anthracis BH460 provides an elegant tool to obtain high levels of lipopolysaccharide-free recombinant protein. PMID:23200832

  20. The role of anthrolysin O in gut epithelial barrier disruption during Bacillus anthracis infection.

    Science.gov (United States)

    Bishop, Brian L; Lodolce, James P; Kolodziej, Lauren E; Boone, David L; Tang, Wei Jen

    2010-04-01

    Gastrointestinal (GI) anthrax, caused by the bacterial infection of Bacillus anthracis, posts a significant bioterrorism threat by its relatively high mortality rate in humans. Different from inhalational anthrax by the route of infection, accumulating evidence indicates the bypass of vegetative bacteria across GI epithelium is required to initiate GI anthrax. Previously, we reported that purified anthrolysin O (ALO), instead of tripartite anthrax edema and lethal toxins, is capable of disrupting gut epithelial tight junctions and barrier function in cultured cells. Here, we show that ALO can disrupt intestinal tissue barrier function in an ex vivo mouse model. To explore the effects of ALO in a cell culture model of B. anthracis infection, we showed that anthrax bacteria can effectively reduce the monolayer integrity of human Caco-2 brush-border expressor (C2BBE) cells based on the reduced transepithelial resistance and the increased leakage of fluorescent dye. This disruption is likely caused by tight junction dysfunction observed by the reorganization of the tight junction protein occludin. Consequently, we observe significant passage of vegetative anthrax bacteria across C2BBE cells. This barrier disruption and bacterial crossover requires ALO since ALO-deficient B. anthracis strains fail to induce monolayer dysfunction and allow the passage of anthrax bacteria. Together these findings point to a pivotal role for ALO within the establishment of GI anthrax infection and the initial bypass of the epithelial barrier. PMID:20188700

  1. Over-expression, purification, and confirmation of Bacillus anthracis transcriptional regulator NprR.

    Science.gov (United States)

    Rice, Amy J; Woo, Jerry K; Khan, Attiya; Szypulinski, Michael Z; Johnson, Michael E; Lee, Hyunwoo; Lee, Hyun

    2016-09-01

    Quorum sensing (QS) has been recognized as an important biological phenomenon in which bacterial cells communicate and coordinate their gene expression and cellular processes with respect to population density. Bacillus anthracis is the etiological agent of fatal pulmonary anthrax infections, and the NprR/NprX QS system may be involved in its pathogenesis. NprR, renamed as aqsR for anthrax quorum sensing Regulator, is a transcriptional regulator that may control the expression of genes required for proliferation and survival. Currently, there is no protocol reported to over-express and purify B. anthracis AqsR. In this study, we describe cloning, purification, and confirmation of functional full-length B. anthracis AqsR protein. The AqsR gene was cloned into the pQE-30 vector with an HRV 3C protease recognition site between AqsR and the N-terminal His6-tag in order to yield near native AqsR after the His-tag cleavage, leaving only two additional amino acid residues at the N-terminus. PMID:26344899

  2. Optimization Studies on Cellulase Production from Bacillus Anthracis and Ochrobactrum Anthropic (YZ1 Isolated from Soil

    Directory of Open Access Journals (Sweden)

    Mohammad Badrud Duza

    2015-06-01

    Full Text Available The present study was carried out to demonstrate the optimization of growth conditions of bacteria with high cellulase activity. Cellulose degrading bacteria were isolated from soil samples collected from different areas of Guntur district, A.P. The bacteria were isolated using serial dilution and pour plate methods. The isolated bacteria were identified by morphological, biochemical and molecular procedures. The isolated bacterial species were screened for cellulase production in sub-merged fermentation process. The two tested bacterial species showed maximum yield for cellulase production. These two bacteria were identified as Bacillus anthracis and Ochrobactrum anthropi (YZ1. Supplementation of glucose, peptone, tyrosine and EDTA to the fermentation medium is favoured enzyme secretion. The optimum pH and temperature for the activity of crude enzyme was 8 and 45°C, respectively for Ochrobactrum anthropi (YZ1 while for Bacillus anthracis, it was 8 and 4°C, respectively.14% of inoculum level and 96 h of incubation period showed the maximum yield by both the species bacteria for cellulase production. The results of present study indicated that favorable fermentation conditions and the selection of a suitable growth medium played a key role in the production of cellulase from newly isolated Bacillus anthracis and Ochrobactrum anthropi (YZ1.

  3. Characterization of the chlorate reductase from Pseudomonas chloritidismutans

    NARCIS (Netherlands)

    Wolterink, A.F.W.M.; Schiltz, E.; Hagedoorn, P.L.; Hagen, W.R.; Kengen, S.W.M.; Stams, A.J.M.

    2003-01-01

    A chlorate reductase has been purified from the chlorate-reducing strain Pseudomonas chloritidismutans. Comparison with the periplasmic (per)chlorate reductase of strain GR-1 showed that the cytoplasmic chlorate reductase of P. chloritidismutans reduced only chlorate and bromate. Differences were al

  4. 21 CFR 864.7375 - Glutathione reductase assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glutathione reductase assay. 864.7375 Section 864... reductase assay. (a) Identification. A glutathione reductase assay is a device used to determine the... fluorescence and photometry. The results of this assay are used in the diagnosis of liver disease,...

  5. Bacillus anthracis-like bacteria and other B. cereus group members in a microbial community within the International Space Station: a challenge for rapid and easy molecular detection of virulent B. anthracis.

    Directory of Open Access Journals (Sweden)

    Sandra P van Tongeren

    Full Text Available For some microbial species, such as Bacillus anthracis, the etiologic agent of the disease anthrax, correct detection and identification by molecular methods can be problematic. The detection of virulent B. anthracis is challenging due to multiple virulence markers that need to be present in order for B. anthracis to be virulent and its close relationship to Bacillus cereus and other members of the B. cereus group. This is especially the case in environments where build-up of Bacillus spores can occur and several representatives of the B. cereus group may be present, which increases the chance for false-positives. In this study we show the presence of B. anthracis-like bacteria and other members of the B. cereus group in a microbial community within the human environment of the International Space Station and their preliminary identification by using conventional culturing as well as molecular techniques including 16S rDNA sequencing, PCR and real-time PCR. Our study shows that when monitoring the microbial hygiene in a given human environment, health risk assessment is troublesome in the case of virulent B. anthracis, especially if this should be done with rapid, easy to apply and on-site molecular methods.

  6. Bacillus anthracis-like bacteria and other B. cereus group members in a microbial community within the International Space Station: a challenge for rapid and easy molecular detection of virulent B. anthracis.

    Science.gov (United States)

    van Tongeren, Sandra P; Roest, Hendrik I J; Degener, John E; Harmsen, Hermie J M

    2014-01-01

    For some microbial species, such as Bacillus anthracis, the etiologic agent of the disease anthrax, correct detection and identification by molecular methods can be problematic. The detection of virulent B. anthracis is challenging due to multiple virulence markers that need to be present in order for B. anthracis to be virulent and its close relationship to Bacillus cereus and other members of the B. cereus group. This is especially the case in environments where build-up of Bacillus spores can occur and several representatives of the B. cereus group may be present, which increases the chance for false-positives. In this study we show the presence of B. anthracis-like bacteria and other members of the B. cereus group in a microbial community within the human environment of the International Space Station and their preliminary identification by using conventional culturing as well as molecular techniques including 16S rDNA sequencing, PCR and real-time PCR. Our study shows that when monitoring the microbial hygiene in a given human environment, health risk assessment is troublesome in the case of virulent B. anthracis, especially if this should be done with rapid, easy to apply and on-site molecular methods.

  7. Redefining the Australian Anthrax Belt: Modeling the Ecological Niche and Predicting the Geographic Distribution of Bacillus anthracis.

    Directory of Open Access Journals (Sweden)

    Alassane S Barro

    2016-06-01

    Full Text Available The ecology and distribution of B. anthracis in Australia is not well understood, despite the continued occurrence of anthrax outbreaks in the eastern states of the country. Efforts to estimate the spatial extent of the risk of disease have been limited to a qualitative definition of an anthrax belt extending from southeast Queensland through the centre of New South Wales and into northern Victoria. This definition of the anthrax belt does not consider the role of environmental conditions in the distribution of B. anthracis. Here, we used the genetic algorithm for rule-set prediction model system (GARP, historical anthrax outbreaks and environmental data to model the ecological niche of B. anthracis and predict its potential geographic distribution in Australia. Our models reveal the niche of B. anthracis in Australia is characterized by a narrow range of ecological conditions concentrated in two disjunct corridors. The most dominant corridor, used to redefine a new anthrax belt, parallels the Eastern Highlands and runs from north Victoria to central east Queensland through the centre of New South Wales. This study has redefined the anthrax belt in eastern Australia and provides insights about the ecological factors that limit the distribution of B. anthracis at the continental scale for Australia. The geographic distributions identified can help inform anthrax surveillance strategies by public and veterinary health agencies.

  8. Redefining the Australian Anthrax Belt: Modeling the Ecological Niche and Predicting the Geographic Distribution of Bacillus anthracis.

    Science.gov (United States)

    Barro, Alassane S; Fegan, Mark; Moloney, Barbara; Porter, Kelly; Muller, Janine; Warner, Simone; Blackburn, Jason K

    2016-06-01

    The ecology and distribution of B. anthracis in Australia is not well understood, despite the continued occurrence of anthrax outbreaks in the eastern states of the country. Efforts to estimate the spatial extent of the risk of disease have been limited to a qualitative definition of an anthrax belt extending from southeast Queensland through the centre of New South Wales and into northern Victoria. This definition of the anthrax belt does not consider the role of environmental conditions in the distribution of B. anthracis. Here, we used the genetic algorithm for rule-set prediction model system (GARP), historical anthrax outbreaks and environmental data to model the ecological niche of B. anthracis and predict its potential geographic distribution in Australia. Our models reveal the niche of B. anthracis in Australia is characterized by a narrow range of ecological conditions concentrated in two disjunct corridors. The most dominant corridor, used to redefine a new anthrax belt, parallels the Eastern Highlands and runs from north Victoria to central east Queensland through the centre of New South Wales. This study has redefined the anthrax belt in eastern Australia and provides insights about the ecological factors that limit the distribution of B. anthracis at the continental scale for Australia. The geographic distributions identified can help inform anthrax surveillance strategies by public and veterinary health agencies. PMID:27280981

  9. Bacillus cereus Biovar Anthracis Causing Anthrax in Sub-Saharan Africa—Chromosomal Monophyly and Broad Geographic Distribution

    Science.gov (United States)

    Mabon, Philip; Zimmermann, Fee; Lankester, Felix; Peller, Tianna; Feistner, Anna; Todd, Angelique; Herbinger, Ilka; de Nys, Hélène M.; Muyembe-Tamfun, Jean-Jacques; Karhemere, Stomy; Wittig, Roman M.; Couacy-Hymann, Emmanuel; Grunow, Roland; Calvignac-Spencer, Sébastien; Corbett, Cindi R.; Klee, Silke R.; Leendertz, Fabian H.

    2016-01-01

    Through full genome analyses of four atypical Bacillus cereus isolates, designated B. cereus biovar anthracis, we describe a distinct clade within the B. cereus group that presents with anthrax-like disease, carrying virulence plasmids similar to those of classic Bacillus anthracis. We have isolated members of this clade from different mammals (wild chimpanzees, gorillas, an elephant and goats) in West and Central Africa (Côte d’Ivoire, Cameroon, Central African Republic and Democratic Republic of Congo). The isolates shared several phenotypic features of both B. anthracis and B. cereus, but differed amongst each other in motility and their resistance or sensitivity to penicillin. They all possessed the same mutation in the regulator gene plcR, different from the one found in B. anthracis, and in addition, carry genes which enable them to produce a second capsule composed of hyaluronic acid. Our findings show the existence of a discrete clade of the B. cereus group capable of causing anthrax-like disease, found in areas of high biodiversity, which are possibly also the origin of the worldwide distributed B. anthracis. Establishing the impact of these pathogenic bacteria on threatened wildlife species will require systematic investigation. Furthermore, the consumption of wildlife found dead by the local population and presence in a domestic animal reveal potential sources of exposure to humans. PMID:27607836

  10. Redefining the Australian Anthrax Belt: Modeling the Ecological Niche and Predicting the Geographic Distribution of Bacillus anthracis

    Science.gov (United States)

    Barro, Alassane S.; Fegan, Mark; Moloney, Barbara; Porter, Kelly; Muller, Janine; Warner, Simone; Blackburn, Jason K.

    2016-01-01

    The ecology and distribution of B. anthracis in Australia is not well understood, despite the continued occurrence of anthrax outbreaks in the eastern states of the country. Efforts to estimate the spatial extent of the risk of disease have been limited to a qualitative definition of an anthrax belt extending from southeast Queensland through the centre of New South Wales and into northern Victoria. This definition of the anthrax belt does not consider the role of environmental conditions in the distribution of B. anthracis. Here, we used the genetic algorithm for rule-set prediction model system (GARP), historical anthrax outbreaks and environmental data to model the ecological niche of B. anthracis and predict its potential geographic distribution in Australia. Our models reveal the niche of B. anthracis in Australia is characterized by a narrow range of ecological conditions concentrated in two disjunct corridors. The most dominant corridor, used to redefine a new anthrax belt, parallels the Eastern Highlands and runs from north Victoria to central east Queensland through the centre of New South Wales. This study has redefined the anthrax belt in eastern Australia and provides insights about the ecological factors that limit the distribution of B. anthracis at the continental scale for Australia. The geographic distributions identified can help inform anthrax surveillance strategies by public and veterinary health agencies. PMID:27280981

  11. A New Generation Microarray for the Simultaneous Detection and Identification of Yersinia pestis and Bacillus anthracis in Food

    Directory of Open Access Journals (Sweden)

    Noriko Goji

    2012-01-01

    Full Text Available The use of microarrays as a multiple analytic system has generated increased interest and provided a powerful analytical tool for the simultaneous detection of pathogens in a single experiment. A wide array of applications for this technology has been reported. A low density oligonucleotide microarray was generated from the genetic sequences of Y. pestis and B. anthracis and used to fabricate a microarray chip. The new generation chip, consisting of 2,240 spots in 4 quadrants with the capability of stripping/rehybridization, was designated as “Y-PESTIS/B-ANTHRACIS 4x2K Array.” The chip was tested for specificity using DNA from a panel of bacteria that may be potentially present in food. In all, 37 unique Y. pestis-specific and 83 B. anthracis-specific probes were identified. The microarray assay distinguished Y. pestis and B. anthracis from the other bacterial species tested and correctly identified the Y. pestis-specific oligonucleotide probes using DNA extracted from experimentally inoculated milk samples. Using a whole genome amplification method, the assay was able to detect as low as 1 ng genomic DNA as the start sample. The results suggest that oligonucleotide microarray can specifically detect and identify Y. pestis and B. anthracis and may be a potentially useful diagnostic tool for detecting and confirming the organisms in food during a bioterrorism event.

  12. Molecular diversity of Bacillus anthracis in the Netherlands: investigating the relationship to the wordwide population using whole-genome SNP discovery

    NARCIS (Netherlands)

    Derzelle, S.; Girault, G.; Roest, H.I.J.; Koene, M.G.J.

    2015-01-01

    Bacillus anthracis, the causative agent of anthrax, has been widely described as a clonal species. Here we report the use of both canonical SNP analysis and whole-genome sequencing to characterize the phylogenetic lineages of B. anthracis from the Netherlands. Eleven strains isolated over a 25-years

  13. Gamma-phage lysin PlyG sequence-based synthetic peptides coupled with Qdot-nanocrystals are useful for developing detection methods for Bacillus anthracis by using its surrogates, B. anthracis-Sterne and B. cereus-4342

    Directory of Open Access Journals (Sweden)

    Atreya Chintamani

    2009-07-01

    Full Text Available Abstract Background Previous reports of site-directed deletion analysis on gamma (γ-phage lysin protein (PlyG have demonstrated that removal of a short amino acid sequence in the C-terminal region encompassing a 10-amino acid motif (190LKMTADFILQ199 abrogates its binding activity specific to the cell wall of Bacillus anthracis. Whether short synthetic peptides representing the10-amino acid PlyG putative binding motif flanked by surrounding N- and C-terminal residues also selectively bind to the bacterial cell wall has not been evaluated. If such peptides do demonstrate selective binding to the cell wall, they could serve as bio-probes towards developing detection technologies for B. anthracis. Furthermore, by using B. anthracis (Sterne, 34F2, an animal vaccine and B. cereus-4342, a γ-phage susceptible rare strain as surrogates of B. anthracis, development of proof-of-concepts for B. anthracis are feasible. Results Using four different methods, we evaluated six synthetic peptides representing the putative binding motif including flanking sequences (PlyG-P1 through P6 for the bacterial cell wall binding capacity. Our analysis identified PlyG-P1, PlyG-P3 and PlyG-P5 to have binding capability to both B. anthracis (Sterne, 34F2 and B. cereus-4342. The peptides however did not bind to B. cereus-11778, B. thuringiensis, and B. cereus-10876 suggesting their specificity for B. anthracis-Sterne and B. cereus-4342. PlyG-P3 in combination with fluorescent light microscopy detected even a single bacterium in plasma spiked with the bacteria. Conclusion Overall, these studies illustrate that the short 10-amino acid sequence 'LKMTADFILQ' in fact is a stand-alone bacterial cell wall-binding motif of PlyG. In principle, synthetic peptides PlyG-P1, PlyG-P3 and PlyG-P5, especially PlyG-P3 coupled with Qdot-nanocrystals are useful as high-sensitivity bio-probes in developing detection technologies for B. anthracis.

  14. A Bacillus anthracis strain deleted for six proteases serves as an effective host for production of recombinant proteins.

    Science.gov (United States)

    Pomerantsev, Andrei P; Pomerantseva, Olga M; Moayeri, Mahtab; Fattah, Rasem; Tallant, Cynthia; Leppla, Stephen H

    2011-11-01

    Bacillus anthracis produces a number of extracellular proteases that impact the integrity and yield of other proteins in the B. anthracis secretome. In this study we show that anthrolysin O (ALO) and the three anthrax toxin proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF), produced from the B. anthracis Ames 35 strain (pXO1⁺, pXO2⁻), are completely degraded at the onset of stationary phase due to the action of proteases. An improved Cre-loxP gene knockout system was used to sequentially delete the genes encoding six proteases (InhA1, InhA2, camelysin, TasA, NprB, and MmpZ). The role of each protease in degradation of the B. anthracis toxin components and ALO was demonstrated. Levels of the anthrax toxin components and ALO in the supernatant of the sporulation defective, pXO1⁺ A35HMS mutant strain deleted for the six proteases were significantly increased and remained stable over 24 h. A pXO1-free variant of this six-protease mutant strain, designated BH460, provides an improved host strain for the preparation of recombinant proteins. As an example, BH460 was used to produce recombinant EF, which previously has been difficult to obtain from B. anthracis. The EF protein produced from BH460 had the highest in vivo potency of any EF previously purified from B. anthracis or Escherichia coli hosts. BH460 is recommended as an effective host strain for recombinant protein production, typically yielding greater than 10mg pure protein per liter of culture. PMID:21827967

  15. Fate of Bacillus anthracis during production of laboratory-scale cream cheese and homemade-style yoghurt.

    Science.gov (United States)

    Mertens, Katja; Schneider, Oda; Schmoock, Gernot; Melzer, Falk; Elschner, Mandy C

    2015-04-01

    The viability of Bacillus anthracis during production and storage of cream cheese and yoghurt was evaluated. Experimental cheeses were manufactured from whole milk inoculated with a suspension of B. anthracis vegetative cells and spores at a final concentration of 10(4) cfu/ml. Lactic acid bacteria (LAB) and lab ferment were used to induce milk ripening and milk coagulation. The pH-value of the contaminated milk dropped below 4.5 within the first 6 h and the amount of LAB increased by approximately 2-logs. During cheese production and storage at 5-9 °C for 24 days no growth of B. anthracis was observed. The amount of vegetative cells and spores fluctuated by 1-log. Inoculation of whole milk with heat-treated spores at 10(4) cfu/ml resulted in a slight increase of vegetative cell counts during the first 6 h. This indicated that germination occurred, but replication of vegetative cells was still inhibited in the produced cheese. Incubation of cheeses at room temperature or heating after milk coagulation strongly reduced the amount of LAB but had no effect on the growth behaviour of B. anthracis. The vegetative cell and spore content remained steady at 10(4) cfu/100 mg. During yoghurt production the pH-value decreased within 5 h below 5 and growth of B. anthracis was inhibited throughout storage. A pH-value of 5 or less is likely a critical factor to control the growth of B. anthracis. However, spores remained viable in experimental cream cheeses and yoghurts and are a potential risk of infection. PMID:25475304

  16. Discovery of pinoresinol reductase genes in sphingomonads.

    Science.gov (United States)

    Fukuhara, Y; Kamimura, N; Nakajima, M; Hishiyama, S; Hara, H; Kasai, D; Tsuji, Y; Narita-Yamada, S; Nakamura, S; Katano, Y; Fujita, N; Katayama, Y; Fukuda, M; Kajita, S; Masai, E

    2013-01-10

    Bacterial genes for the degradation of major dilignols produced in lignifying xylem are expected to be useful tools for the structural modification of lignin in plants. For this purpose, we isolated pinZ involved in the conversion of pinoresinol from Sphingobium sp. strain SYK-6. pinZ showed 43-77% identity at amino acid level with bacterial NmrA-like proteins of unknown function, a subgroup of atypical short chain dehydrogenases/reductases, but revealed only 15-21% identity with plant pinoresinol/lariciresinol reductases. PinZ completely converted racemic pinoresinol to lariciresinol, showing a specific activity of 46±3 U/mg in the presence of NADPH at 30°C. In contrast, the activity for lariciresinol was negligible. This substrate preference is similar to a pinoresinol reductase, AtPrR1, of Arabidopsis thaliana; however, the specific activity of PinZ toward (±)-pinoresinol was significantly higher than that of AtPrR1. The role of pinZ and a pinZ ortholog of Novosphingobium aromaticivorans DSM 12444 were also characterized.

  17. A multiplex bead-based suspension array assay for interrogation of phylogenetically informative single nucleotide polymorphisms for Bacillus anthracis

    DEFF Research Database (Denmark)

    Thierry, Simon; Hamidjaja, Raditijo A.; Girault, Guillaume;

    2013-01-01

    need. By using the versatile Luminex® xTAG technology, we developed an efficient multiplexed SNP genotyping assay to score 13 phylogenetically informative SNPs within the genome of Bacillus anthracis. The Multiplex Oligonucleotide Ligation-PCR procedure (MOL-PCR) described by Deshpande et al., 2010 has...... laboratories. While cost-effective compared to other singleplex methods, the present MOL-PCR method offers a high degree of flexibility and scalability. It can easily accommodate newly identified SNPs to increase resolving power to the canSNP typing of B. anthracis....

  18. Mechanical transmission of Bacillus anthracis by stable flies (Stomoxys calcitrans) and mosquitoes (Aedes aegypti and Aedes taeniorhynchus).

    OpenAIRE

    Turell, M J; Knudson, G B

    1987-01-01

    We evaluated the potential of stable flies, Stomoxys calcitrans, and two species of mosquitoes, Aedes aegypti and Aedes taeniorhynchus, to transmit Bacillus anthracis Vollum 1B mechanically. After probing on Hartley guinea pigs with a bacteremia of ca. 10(8.6) CFU of B. anthracis per ml of blood, individual or pools of two to four stable flies or mosquitoes were allowed to continue feeding on either uninfected guinea pigs or A/J mice. All three insect species transmitted lethal anthrax infect...

  19. cis-Acting Elements That Control Expression of the Master Virulence Regulatory Gene atxA in Bacillus anthracis

    OpenAIRE

    Dale, Jennifer L.; Raynor, Malik J.; Dwivedi, Prabhat; Koehler, Theresa M.

    2012-01-01

    Transcription of the Bacillus anthracis structural genes for the anthrax toxin proteins and biosynthetic operon for capsule is positively regulated by AtxA, a transcription regulator with unique properties. Consistent with the role of atxA in virulence factor expression, a B. anthracis atxA-null mutant is avirulent in a murine model for anthrax. In culture, multiple signals impact atxA transcript levels, and the timing and steady-state level of atxA expression are critical for optimal toxin a...

  20. Molecular cloning and expression of the Bacillus anthracis edema factor toxin gene: a calmodulin-dependent adenylate cyclase.

    OpenAIRE

    Tippetts, M T; Robertson, D L

    1988-01-01

    The Bacillus anthracis exotoxin is composed of a lethal factor, a protective antigen, and an edema factor (EF). EF is a calmodulin-dependent adenylate cyclase which elevates cyclic AMP levels within cells. The entire EF gene (cya) has been cloned in Escherichia coli, but EF gene expression by its own B. anthracis promoter could not be detected in E. coli. However, when the EF gene was placed downstream from the lac or the T7 promoter, enzymatically active EF was produced. The EF gene, like th...

  1. Achieving consistent multiple daily low-dose Bacillus anthracis spore inhalation exposures in the rabbit model

    Directory of Open Access Journals (Sweden)

    Roy E Barnewall

    2012-06-01

    Full Text Available Repeated low-level exposures to Bacillus anthracis could occur before or after the remediation of an environmental release. This is especially true for persistent agents such as Bacillus anthracis spores, the causative agent of anthrax. Studies were conducted to examine aerosol methods needed for consistent daily low aerosol concentrations to deliver a low-dose (less than 106 colony forming units (CFU of B. anthracis spores and included a pilot feasibility characterization study, acute exposure study, and a multiple fifteen day exposure study. This manuscript focuses on the state-of-the-science aerosol methodologies used to generate and aerosolize consistent daily low aerosol concentrations and resultant low inhalation doses. The pilot feasibility characterization study determined that the aerosol system was consistent and capable of producing very low aerosol concentrations. In the acute, single day exposure experiment, targeted inhaled doses of 1 x 102, 1 x 103, 1 x 104, and 1 x 105 CFU were used. In the multiple daily exposure experiment, rabbits were exposed multiple days to targeted inhaled doses of 1 x 102, 1 x 103, and 1 x 104 CFU. In all studies, targeted inhaled doses remained fairly consistent from rabbit to rabbit and day to day. The aerosol system produced aerosolized spores within the optimal mass median aerodynamic diameter particle size range to reach deep lung alveoli. Consistency of the inhaled dose was aided by monitoring and recording respiratory parameters during the exposure with real-time plethysmography. Overall, the presented results show that the animal aerosol system was stable and highly reproducible between different studies and multiple exposure days.

  2. Isolation and chimerization of a highly neutralizing antibody conferring passive protection against lethal Bacillus anthracis infection.

    Directory of Open Access Journals (Sweden)

    Ronit Rosenfeld

    Full Text Available Several studies have demonstrated that the passive transfer of protective antigen (PA-neutralizing antibodies can protect animals against Bacillus anthracis infection. The standard protocol for the isolation of PA-neutralizing monoclonal antibodies is based upon a primary selection of the highest PA-binders by ELISA, and usually yields only few candidates antibodies. We demonstrated that by applying a PA-neutralization functionality-based screen as the primary criterion for positive clones, it was possible to isolate more than 100 PA-neutralizing antibodies, some of which exhibited no measurable anti-PA titers in ELISA. Among the large panel of neutralizing antibodies identified, mAb 29 demonstrated the most potent activity, and was therefore chimerized. The variable region genes of the mAb 29 were fused to human constant region genes, to form the chimeric 29 antibody (cAb 29. Guinea pigs were fully protected against infection by 40LD(50B. anthracis spores following two separate administrations with 10 mg/kg of cAb 29: the first administration was given before the challenge, and a second dose was administered on day 4 following exposure. Moreover, animals that survived the challenge and developed endogenous PA-neutralizing antibodies with neutralizing titers above 100 were fully protected against repeat challenges with 40LD(50 of B. anthracis spores. The data presented here emphasize the importance of toxin neutralization-based screens for the efficient isolation of protective antibodies that were probably overlooked in the standard screening protocol. The protective activity of the chimeric cAb 29 demonstrated in this study suggest that it may serve as an effective immunotherapeutic agent against anthrax.

  3. Purification, crystallization and preliminary structural analysis of nucleoside diphosphate kinase from Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Misra, Gauri [Molecular and Structural Biology Division, Central Drug Research Institute, PO Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow 226 001 (India); Aggarwal, Anita [Institute of Genomics and Integrative Biology, Mall Road, Delhi 110 007 (India); Mittal, Sonia [Molecular and Structural Biology Division, Central Drug Research Institute, PO Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow 226 001 (India); Singh, Yogendra [Institute of Genomics and Integrative Biology, Mall Road, Delhi 110 007 (India); Ramachandran, Ravishankar, E-mail: r-ravishankar@cdri.res.in [Molecular and Structural Biology Division, Central Drug Research Institute, PO Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow 226 001 (India)

    2007-12-01

    Nucleoside diphosphate kinase from B. anthracis has been crystallized. Preliminary crystallographic analysis shows that there is one monomer in the asymmetric unit of the crystal. Bacillus anthracis nucleoside diphosphate kinase (BaNdk) is an enzyme whose primary function is to maintain deoxynucleotide triphosphate (dNTP) pools by converting deoxynucleotide diphosphates to triphosphates using ATP as the major phosphate donor. Although the structures of Ndks from a variety of organisms have been elucidated, the enzyme from sporulating bacteria has not been structurally characterized to date. Crystals of the B. anthracis enzyme were grown using the vapour-diffusion method from a hanging drop consisting of 2 µl 10 mg ml{sup −1} protein in 50 mM Tris–HCl pH 8.0, 50 mM NaCl, 5 mM EDTA equilibrated against 500 µl reservoir solution consisting of 2.25 M ammonium formate and 0.1 M HEPES buffer pH 7.25. Diffraction data extending to 2.0 Å were collected at room temperature from a single crystal with unit-cell parameters a = b = 107.53, c = 52.3 Å. The crystals are hexagonal in shape and belong to space group P6{sub 3}22. The crystals contain a monomer in the asymmetric unit, which corresponds to a Matthews coefficient (V{sub M}) of 2.1 Å{sup 3} Da{sup −1} and a solvent content of about 36.9%.

  4. Protein profiles of field isolates ofBacillus anthracis from different endemic areas of Indonesia

    Directory of Open Access Journals (Sweden)

    M Bhakti Poerwadikarta

    1998-03-01

    Full Text Available Sonicated cell-free extract proteins of 14 field isolates ofBacillus anthracis from six different endemic areas of Indonesia were analyzed by the use of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE methods . The protein profiles of each field isolate tested demonstrated slightly different at the protein bands with molecular weights of 18, 37, 52, 65 and 70 kDa, and varied between the field isolates and vaccine strains. The variation could provide clues to the source of anthrax transmission whether it was originated from similar strain or not.

  5. Transcriptional Stimulation of Anthrax Toxin Receptors by Anthrax Edema Toxin and Bacillus anthracis Sterne Spore

    OpenAIRE

    Xu, Qingfu; Hesek, Eric D.; Zeng, Mingtao

    2007-01-01

    We used quantitative real-time RT-PCR to not only investigate the mRNA levels of anthrax toxin receptor 1 (ANTXR1) and 2 (ANTXR2) in the murine J774A.1 macrophage cells and different tissues of mice, but also evaluate the effect of anthrax edema toxin and Bacillus anthracis Sterne spores on the expression of mRNA of these receptors. The mRNA transcripts of both receptors was detected in J774A.1 cells and mouse tissues such as the lung, heart, kidney, spleen, stomach, jejunum, brain, skeleton ...

  6. An Essential DnaB Helicase of Bacillus anthracis: Identification, Characterization, and Mechanism of Action▿

    OpenAIRE

    Biswas, Esther E.; Barnes, Marjorie H.; Moir, Donald T.; Biswas, Subhasis B

    2008-01-01

    We have described a novel essential replicative DNA helicase from Bacillus anthracis, the identification of its gene, and the elucidation of its enzymatic characteristics. Anthrax DnaB helicase (DnaBBA) is a 453-amino-acid, 50-kDa polypeptide with ATPase and DNA helicase activities. DnaBBA displayed distinct enzymatic and kinetic properties. DnaBBA has low single-stranded DNA (ssDNA)-dependent ATPase activity but possesses a strong 5′→3′ DNA helicase activity. The stimulation of ATPase activi...

  7. CXCL10 Acts as a Bifunctional Antimicrobial Molecule against Bacillus anthracis

    Directory of Open Access Journals (Sweden)

    Katie R. Margulieux

    2016-05-01

    Full Text Available Bacillus anthracis is killed by the interferon-inducible, ELR(− CXC chemokine CXCL10. Previous studies showed that disruption of the gene encoding FtsX, a conserved membrane component of the ATP-binding cassette transporter-like complex FtsE/X, resulted in resistance to CXCL10. FtsX exhibits some sequence similarity to the mammalian CXCL10 receptor, CXCR3, suggesting that the CXCL10 N-terminal region that interacts with CXCR3 may also interact with FtsX. A C-terminal truncated CXCL10 was tested to determine if the FtsX-dependent antimicrobial activity is associated with the CXCR3-interacting N terminus. The truncated CXCL10 exhibited antimicrobial activity against the B. anthracis parent strain but not the ΔftsX mutant, which supports a key role for the CXCL10 N terminus. Mutations in FtsE, the conserved ATP-binding protein of the FtsE/X complex, resulted in resistance to both CXCL10 and truncated CXCL10, indicating that both FtsX and FtsE are important. Higher concentrations of CXCL10 overcame the resistance of the ΔftsX mutant to CXCL10, suggesting an FtsX-independent killing mechanism, likely involving its C-terminal α-helix, which resembles a cationic antimicrobial peptide. Membrane depolarization studies revealed that CXCL10 disrupted membranes of the B. anthracis parent strain and the ΔftsX mutant, but only the parent strain underwent depolarization with truncated CXCL10. These findings suggest that CXCL10 is a bifunctional molecule that kills B. anthracis by two mechanisms. FtsE/X-dependent killing is mediated through an N-terminal portion of CXCL10 and is not reliant upon the C-terminal α-helix. The FtsE/X-independent mechanism involves membrane depolarization by CXCL10, likely because of its α-helix. These findings present a new paradigm for understanding mechanisms by which CXCL10 and related chemokines kill bacteria.

  8. Physical Sequestration of Bacillus anthracis in the Pulmonary Capillaries in Terminal Infection.

    Science.gov (United States)

    Jouvion, Gregory; Corre, Jean-Philippe; Khun, Huot; Moya-Nilges, Marie; Roux, Pascal; Latroche, Claire; Tournier, Jean-Nicolas; Huerre, Michel; Chrétien, Fabrice; Goossens, Pierre L

    2016-07-15

    The lung is the terminal target of Bacillus anthracis before death, whatever the route of infection (cutaneous, inhalational, or digestive). During a cutaneous infection in absence of toxins, we observed encapsulated bacteria colonizing the alveolar capillary network, bacteria and hemorrhages in alveolar and bronchiolar spaces, and hypoxic foci in the lung (endothelial cells) and brain (neurons and neuropil). Circulating encapsulated bacteria were as chains of approximately 13 µm in length. Bacteria of such size were immediately trapped within the lung capillary network, but bacteria of shorter length were not. Controlling lung-targeted pathology would be beneficial for anthrax treatment. PMID:26977051

  9. Biochip for the Detection of Bacillus anthracis Lethal Factor and Therapeutic Agents against Anthrax Toxins.

    Science.gov (United States)

    Silin, Vitalii; Kasianowicz, John J; Michelman-Ribeiro, Ariel; Panchal, Rekha G; Bavari, Sina; Robertson, Joseph W F

    2016-01-01

    Tethered lipid bilayer membranes (tBLMs) have been used in many applications, including biosensing and membrane protein structure studies. This report describes a biosensor for anthrax toxins that was fabricated through the self-assembly of a tBLM with B. anthracis protective antigen ion channels that are both the recognition element and electrochemical transducer. We characterize the sensor and its properties with electrochemical impedance spectroscopy and surface plasmon resonance. The sensor shows a sensitivity similar to ELISA and can also be used to rapidly screen for molecules that bind to the toxins and potentially inhibit their lethal effects. PMID:27348008

  10. Efficacy of Daptomycin against Bacillus anthracis in a Murine Model of Anthrax Spore Inhalation▿

    OpenAIRE

    Heine, Henry S.; Bassett, Jennifer; Miller, Lynda; Purcell, Bret K.; Byrne, W. Russell

    2010-01-01

    Daptomycin demonstrated in vitro (MIC90, 4 μg/ml) and in vivo activities against Bacillus anthracis. Twice-daily treatment with a dose of 50 mg/kg of body weight was begun 24 h after challenge and continued for 14 or 21 days; results were compared to those for controls treated with phosphate-buffered saline or ciprofloxacin. Day 43 survival rates were 6/10 mice for the 14-day and 9/10 mice for the 21-day treatment groups, compared to survival with ciprofloxacin: 8/10 and 9/10 mice, respective...

  11. Importance of srtA and srtB for Growth of Bacillus anthracis in Macrophages

    OpenAIRE

    Zink, Steven D.; Burns, Drusilla L.

    2005-01-01

    We examined the effect of mutation of two sortase genes of Bacillus anthracis, srtA and srtB, on the ability of the bacterium to grow in J774A.1 cells, a mouse macrophage-like cell line. While disruption of either srtA or srtB had no effect on the ability of the bacteria to grow in rich culture media, mutations in each of these genes dramatically attenuated growth of the bacterium in J774A.1 cells. Complementation of the mutation restored the ability of bacteria to grow in the cells. Since th...

  12. Biochip for the Detection of Bacillus anthracis Lethal Factor and Therapeutic Agents against Anthrax Toxins

    Science.gov (United States)

    Silin, Vitalii; Kasianowicz, John J.; Michelman-Ribeiro, Ariel; Panchal, Rekha G.; Bavari, Sina; Robertson, Joseph W. F.

    2016-01-01

    Tethered lipid bilayer membranes (tBLMs) have been used in many applications, including biosensing and membrane protein structure studies. This report describes a biosensor for anthrax toxins that was fabricated through the self-assembly of a tBLM with B. anthracis protective antigen ion channels that are both the recognition element and electrochemical transducer. We characterize the sensor and its properties with electrochemical impedance spectroscopy and surface plasmon resonance. The sensor shows a sensitivity similar to ELISA and can also be used to rapidly screen for molecules that bind to the toxins and potentially inhibit their lethal effects. PMID:27348008

  13. Methionine sulfoxide reductase contributes to meeting dietary methionine requirements

    OpenAIRE

    Zhao, Hang; Kim, Geumsoo; Levine, Rodney L.

    2012-01-01

    Methionine sulfoxide reductases are present in all aerobic organisms. They contribute to antioxidant defenses by reducing methionine sulfoxide in proteins back to methionine. However, the actual in vivo roles of these reductases are not well defined. Since methionine is an essential amino acid in mammals, we hypothesized that methionine sulfoxide reductases may provide a portion of the dietary methionine requirement by recycling methionine sulfoxide. We used a classical bioassay, the growth o...

  14. Ultrasensitive electrochemical immunoassay for surface array protein, a Bacillus anthracis biomarker using Au-Pd nanocrystals loaded on boron-nitride nanosheets as catalytic labels.

    Science.gov (United States)

    Sharma, Mukesh Kumar; Narayanan, J; Pardasani, Deepak; Srivastava, Divesh N; Upadhyay, Sanjay; Goel, Ajay Kumar

    2016-06-15

    Bacillus anthracis, the causative agent of anthrax, is a well known bioterrorism agent. The determination of surface array protein (Sap), a unique biomarker for B. anthracis can offer an opportunity for specific detection of B. anthracis in culture broth. In this study, we designed a new catalytic bionanolabel and fabricated a novel electrochemical immunosensor for ultrasensitive detection of B. anthracis Sap antigen. Bimetallic gold-palladium nanoparticles were in-situ grown on poly (diallyldimethylammonium chloride) functionalized boron nitride nanosheets (Au-Pd NPs@BNNSs) and conjugated with the mouse anti-B. anthracis Sap antibodies (Ab2); named Au-Pd NPs@BNNSs/Ab2. The resulting Au-Pd NPs@BNNSs/Ab2 bionanolabel demonstrated high catalytic activity towards reduction of 4-nitrophenol. The sensitivity of the electrochemical immunosensor along with redox cycling of 4-aminophenol to 4-quinoneimine was improved to a great extent. Under optimal conditions, the proposed immunosensor exhibited a wide working range from 5 pg/mL to 100 ng/mL with a minimum detection limit of 1 pg/mL B. anthracis Sap antigen. The practical applicability of the immunosensor was demonstrated by specific detection of Sap secreted by the B. anthracis in culture broth just after 1h of growth. These labels open a new direction for the ultrasensitive detection of different biological warfare agents and their markers in different matrices. PMID:26874112

  15. Laboratory studies on surface sampling of Bacillus anthracis contamination: summary, gaps, and recommendations

    Energy Technology Data Exchange (ETDEWEB)

    Piepel, Gregory F.; Amidan, Brett G.; Hu, Rebecca

    2012-12-01

    This article summarizes previous laboratory studies to characterize the performance of methods for collecting, storing/transporting, processing, and analyzing samples from surfaces contaminated by Bacillus anthracis or related surrogates. The focus is on plate culture and count estimates of surface contamination for swab, wipe, and vacuum samples of porous and nonporous surfaces. Summaries of the previous studies and their results were assessed to identify gaps in information needed as inputs to calculate key parameters critical to risk management in biothreat incidents. One key parameter is the number of samples needed to make characterization or clearance decisions with specified statistical confidence. Other key parameters include the ability to calculate, following contamination incidents, the 1) estimates of Bacillus anthracis contamination, as well as the bias and uncertainties in the estimates, and 2) confidence in characterization and clearance decisions for contaminated or decontaminated buildings. Gaps in knowledge and understanding identified during the summary of the studies are discussed. Recommendations are given for future evaluations of data from existing studies and possible new studies.

  16. Identification of the immunogenic spore and vegetative proteins of Bacillus anthracis vaccine strain A16R.

    Directory of Open Access Journals (Sweden)

    Xiankai Liu

    Full Text Available Immunoproteomics was used to screen the immunogenic spore and vegetative proteins of Bacillus anthracis vaccine strain A16R. The spore and vegetative proteins were separated by 2D gel electrophoresis and transferred to polyvinylidene difluoride membranes, and then western blotting was performed with rabbit immune serum against B.anthracis live spores. Immunogenic spots were cut and digested by trypsin. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was performed to identify the proteins. As a result, 11 and 45 immunogenic proteins were identified in the spores and vegetative cells, respectively; 26 of which have not been reported previously. To verify their immunogenicity, 12 of the identified proteins were selected to be expressed, and the immune sera from the mice vaccinated by the 12 expressed proteins, except BA0887, had a specific western blot band with the A16R whole cellular lytic proteins. Some of these immunogenic proteins might be used as novel vaccine candidates themselves or for enhancing the protective efficacy of a protective-antigen-based vaccine.

  17. Improved Proteomic Analysis Following Trichloroacetic Acid Extraction of Bacillus anthracis Spore Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Kaiser, Brooke LD; Wunschel, David S.; Sydor, Michael A.; Warner, Marvin G.; Wahl, Karen L.; Hutchison, Janine R.

    2015-08-07

    Proteomic analysis of bacterial samples provides valuable information about cellular responses and functions under different environmental pressures. Proteomic analysis is dependent upon efficient extraction of proteins from bacterial samples without introducing bias toward extraction of particular protein classes. While no single method can recover 100% of the bacterial proteins, selected protocols can improve overall protein isolation, peptide recovery, or enrich for certain classes of proteins. The method presented here is technically simple and does not require specialized equipment such as a mechanical disrupter. Our data reveal that for particularly challenging samples, such as B. anthracis Sterne spores, trichloroacetic acid extraction improved the number of proteins identified within a sample compared to bead beating (714 vs 660, respectively). Further, TCA extraction enriched for 103 known spore specific proteins whereas bead beating resulted in 49 unique proteins. Analysis of C. botulinum samples grown to 5 days, composed of vegetative biomass and spores, showed a similar trend with improved protein yields and identification using our method compared to bead beating. Interestingly, easily lysed samples, such as B. anthracis vegetative cells, were equally as effectively processed via TCA and bead beating, but TCA extraction remains the easiest and most cost effective option. As with all assays, supplemental methods such as implementation of an alternative preparation method may provide additional insight to the protein biology of the bacteria being studied.

  18. A Study on molecular characterization of Razi Bacillus anthracis Sterne 34F2 substrain in Iran

    Directory of Open Access Journals (Sweden)

    Tadayon, K.

    2016-07-01

    Full Text Available Anthrax, a zoonotic disease caused by Bacillus anthracis, has affected humans since ancient times. For genomic characterization of Razi B. anthracis Sterne 34F2 substrain, single nucleotide polymorphism (SNP genotyping method developed by Van Erth, variable-number tandem-repeat (VNTR-8 analysis proposed by Keim, and multiple-locus VNTR analysis (MLVA-3 introduced by Levy were employed. In the SNPs typing system, where the nucleotide content of the genome at 13 evolutionary canonical loci was collectively analyzed, the originally South African 34F2 substrain was categorized in the A.Br.001/002 subgroup. In the VNTR-8 analysis, fragments with lengths of 314, 229, 162, 580, 532, 158, and 137 bp were identified at the following loci: vrrA, vrrB1, vrrB2, vrrC1, vrrC2, CG3, and pxO1, respectively. In addition, application of Levy's MLVA-3 genotyping method revealed that the genome of this strain carried 941, 451, and 864 bp fragments at AA03, AJ03, and AA07 loci, respectively. The present findings are undoubtedly helpful in meeting the requirements set by the World Organization for Animal Health (OIE and World Health Organization (WHO for anthrax vaccine manufacturers including Razi Institute. However, further similar studies are required to promote the current epidemiological knowledge of anthrax in Iran.

  19. The role of DNA restriction-modification systems in the biology of Bacillus anthracis

    Directory of Open Access Journals (Sweden)

    Ramakrishnan eSitaraman

    2016-01-01

    Full Text Available Restriction-modification (R-M systems are widespread among prokaryotes and, depending on their type, may be viewed as selfish genetic elements that persist as toxin-antitoxin modules or as cellular defense systems against phage infection. Studies in the last decade have made it amply clear that these two options do not exhaust the list of possible biological roles for R-M systems. Their presence in a cell may also have a bearing on other processes such as horizontal gene transfer and gene regulation. From genome sequencing and experimental data, we know that Bacillus anthracis encodes at least three methylation-dependent (typeIV restriction endonucleases, and an orphan DNA methyltransferase. In this article, we first present an outline of our current knowledge of R-M systems in Bacillus anthracis. Based on available DNA sequence data, and on our current understanding of the functions of similar genes in other systems, we conclude with hypotheses on the possible roles of the three restriction endonucleases and the orphan DNA methyltransferase.

  20. A Novel FtsZ-Like Protein Is Involved in Replication of the Anthrax Toxin-Encoding pXO1 Plasmid in Bacillus anthracis

    OpenAIRE

    Tinsley, Eowyn; Khan, Saleem A.

    2006-01-01

    Plasmid pXO1 encodes the tripartite anthrax toxin, which is the major virulence factor of Bacillus anthracis. In spite of the important role of pXO1 in anthrax pathogenesis, very little is known about its replication and maintenance in B. anthracis. We cloned a 5-kb region of the pXO1 plasmid into an Escherichia coli vector and showed that this plasmid can replicate when introduced into B. anthracis. Mutational analysis showed that open reading frame 45 (repX) of pXO1 was required for the rep...

  1. Literatuuronderzoek naar gegevens betreffende de betekenis van een aantal verwekkers van zoonosen in verband met de vleesconsumptie XIII Bacillus anthracis

    NARCIS (Netherlands)

    Bos JM; Engel HWB; Groothuis DG; Knapen F van; Noorle Jansen LM van; Weiss JW

    1989-01-01

    Bacillus anthracis, de verwekker van anthrax (miltvuur), komt mondiaal voor. Zowel mens als dier zijn in meer of mindere mate gevoelig voor infectie met deze bacterie. Bij dieren komen ziektebeelden voor varierend van peracute sterfte tot een meer chronisch beeld met locale laesis en aangetaste

  2. Bacillus anthracis Diversity and Geographic Potential across Nigeria, Cameroon and Chad: Further Support of a Novel West African Lineage.

    Directory of Open Access Journals (Sweden)

    Jason K Blackburn

    Full Text Available Zoonoses, diseases affecting both humans and animals, can exert tremendous pressures on human and veterinary health systems, particularly in resource limited countries. Anthrax is one such zoonosis of concern and is a disease requiring greater public health attention in Nigeria. Here we describe the genetic diversity of Bacillus anthracis in Nigeria and compare it to Chad, Cameroon and a broader global dataset based on the multiple locus variable number tandem repeat (MLVA-25 genetic typing system. Nigerian B. anthracis isolates had identical MLVA genotypes and could only be resolved by measuring highly mutable single nucleotide repeats (SNRs. The Nigerian MLVA genotype was identical or highly genetically similar to those in the neighboring countries, confirming the strains belong to this unique West African lineage. Interestingly, sequence data from a Nigerian isolate shares the anthrose deficient genotypes previously described for strains in this region, which may be associated with vaccine evasion. Strains in this study were isolated over six decades, indicating a high level of temporal strain stability regionally. Ecological niche models were used to predict the geographic distribution of the pathogen for all three countries. We describe a west-east habitat corridor through northern Nigeria extending into Chad and Cameroon. Ecological niche models and genetic results show B. anthracis to be ecologically established in Nigeria. These findings expand our understanding of the global B. anthracis population structure and can guide regional anthrax surveillance and control planning.

  3. Glycosylation of BclA Glycoprotein from Bacillus cereus and Bacillus anthracis Exosporium Is Domain-specific.

    Science.gov (United States)

    Maes, Emmanuel; Krzewinski, Frederic; Garenaux, Estelle; Lequette, Yannick; Coddeville, Bernadette; Trivelli, Xavier; Ronse, Annette; Faille, Christine; Guerardel, Yann

    2016-04-29

    The spores of the Bacillus cereus group (B. cereus, Bacillus anthracis, and Bacillus thuringiensis) are surrounded by a paracrystalline flexible yet resistant layer called exosporium that plays a major role in spore adhesion and virulence. The major constituent of its hairlike surface, the trimerized glycoprotein BclA, is attached to the basal layer through an N-terminal domain. It is then followed by a repetitive collagen-like neck bearing a globular head (C-terminal domain) that promotes glycoprotein trimerization. The collagen-like region of B. anthracis is known to be densely substituted by unusual O-glycans that may be used for developing species-specific diagnostics of B. anthracis spores and thus targeted therapeutic interventions. In the present study, we have explored the species and domain specificity of BclA glycosylation within the B. cereus group. First, we have established that the collagen-like regions of both B. anthracis and B. cereus are similarly substituted by short O-glycans that bear the species-specific deoxyhexose residues anthrose and the newly observed cereose, respectively. Second we have discovered that the C-terminal globular domains of BclA from both species are substituted by polysaccharide-like O-linked glycans whose structures are also species-specific. The presence of large carbohydrate polymers covering the surface of Bacillus spores may have a profound impact on the way that spores regulate their interactions with biotic and abiotic surfaces and represents potential new diagnostic targets. PMID:26921321

  4. Noncapsulated toxinogenic Bacillus anthracis presents a specific growth and dissemination pattern in naive and protective antigen-immune mice.

    Science.gov (United States)

    Glomski, Ian J; Corre, Jean-Philippe; Mock, Michèle; Goossens, Pierre L

    2007-10-01

    Bacillus anthracis is a spore-forming bacterium that causes anthrax. B. anthracis has three major virulence factors, namely, lethal toxin, edema toxin, and a poly-gamma-D-glutamic acid capsule. The toxins modulate host immune responses, and the capsule inhibits phagocytosis. With the goal of increasing safety, decreasing security concerns, and taking advantage of mammalian genetic tools and reagents, mouse models of B. anthracis infection have been developed using attenuated bacteria that produce toxins but no capsule. While these models have been useful in studying both toxinogenic infections and antitoxin vaccine efficacy, we questioned whether eliminating the capsule changed bacterial growth and dissemination characteristics. Thus, the progression of infection by toxinogenic noncapsulated B. anthracis was analyzed and compared to that by previously reported nontoxinogenic capsulated bacteria, using in vivo bioluminescence imaging. The influence of immunization with the toxin component protective antigen (PA) on the development of infection was also examined. The toxinogenic noncapsulated bacteria were initially confined to the cutaneous site of infection. Bacteria then progressed to the draining lymph nodes and, finally, late in the infection, to the lungs, kidneys, and frequently the gastrointestinal tract. There was minimal colonization of the spleen. PA immunization reduced bacterial growth from the outset and limited infection to the site of inoculation. These in vivo observations show that dissemination by toxinogenic noncapsulated strains differs markedly from that by nontoxinogenic capsulated strains. Additionally, PA immunization counters bacterial growth and dissemination in vivo from the onset of infection. PMID:17635863

  5. Reagent-free and portable detection of Bacillus anthracis spores using a microfluidic incubator and smartphone microscope

    Energy Technology Data Exchange (ETDEWEB)

    Hutchison, Janine R.; Erikson, Rebecca L.; Sheen, Allison M.; Ozanich, Richard M.; Kelly, Ryan T.

    2015-08-06

    Rapid, cost-effective bacterial detection systems are needed to respond to potential biothreat events. Here we report the use of smartphone-based microscopy in combination with a simple microfluidic incubation device to detect 5000 Bacillus anthracis spores in 3 hours. This field-deployable approach is compatible with real-time PCR for secondary confirmation.

  6. Pilot-scale crossflow-microfiltration and pasturization to remove spores of Bacillus anthracis (Sterne) from milk

    Science.gov (United States)

    HTST pasteurization of milk is generally ineffective against spore-forming bacteria such as Bacillus anthracis (BA) but is lethal to its vegetative cells. Crossflow microfiltration (MF), using ceramic membranes with a pore diameter of 1.4 um, has been shown to physically remove somatic cells, vegeta...

  7. Antimicrobial properties of CuO nanorods and multi-armed nanoparticles against B. anthracis vegetative cells and endospores

    Directory of Open Access Journals (Sweden)

    Pratibha Pandey

    2014-06-01

    Full Text Available Two different kinds of CuO nanoparticles (NPs namely CuO nanorods (PS2 and multi-armed nanoparticles (P5 were synthesized by wet and electrochemical routes, respectively. Their structure, morphology, size and compositions were characterized by SEM, EDX and XRD. The NPs demonstrated strong bactericidal potential against Bacillus anthracis cells and endospores. PS2 killed 92.17% of 4.5 × 104 CFU/mL B. anthracis cells within 1 h at a dose of 1 mg/mL. Whereas P5 showed a higher efficacy by killing 99.92% of 7 × 105 CFU/mL B. anthracis cells within 30 min at a dose of 0.5 mg/mL and 99.6% of 1.25 × 104 CFU/mL B. anthracis cells within 5 min at a dose of 2 mg/mL. More than 99% of spores were killed within 8 h with 2 mg/mL PS2 in LB media.

  8. Does Bacillus anthracis Lethal Toxin Directly Depress Myocardial Function? A Review of Clinical Cases and Preclinical Studies

    Directory of Open Access Journals (Sweden)

    Dante A. Suffredini

    2015-12-01

    Full Text Available The US outbreak of B.anthracis infection in 2001 and subsequent cases in the US and Europe demonstrate that anthrax is a continuing risk for the developed world. While several bacterial components contribute to the pathogenesis of B. anthracis, production of lethal toxin (LT is strongly associated with the development of hypotension and lethality. However, the mechanisms underlying the cardiovascular instability LT produces are unclear. Some evidence suggests that LT causes shock by impairing the peripheral vasculature, effects consistent with the substantial extravasation of fluid in patients dying with B. anthracis. Other data suggests that LT directly depresses myocardial function. However a clinical correlate for this latter possibility is less evident since functional studies and post-mortem examination in patients demonstrate absent or minimal cardiac changes. The purposes of this review were to first present clinical studies of cardiac functional and histologic pathology with B. anthracis infection and to then examine in vivo, in vitro, and ex vivo preclinical studies of LT’s myocardial effects. Together, these data suggest that it is unclear whether that LT directly depresses cardiac function. This question is important for the clinical management and development of new therapies for anthrax and efforts should continue to be made to answer it.

  9. Bacillus anthracis Spore Surface Protein BclA Mediates Complement Factor H Binding to Spores and Promotes Spore Persistence.

    Science.gov (United States)

    Wang, Yanyu; Jenkins, Sarah A; Gu, Chunfang; Shree, Ankita; Martinez-Moczygemba, Margarita; Herold, Jennifer; Botto, Marina; Wetsel, Rick A; Xu, Yi

    2016-06-01

    Spores of Bacillus anthracis, the causative agent of anthrax, are known to persist in the host lungs for prolonged periods of time, however the underlying mechanism is poorly understood. In this study, we demonstrated that BclA, a major surface protein of B. anthracis spores, mediated direct binding of complement factor H (CFH) to spores. The surface bound CFH retained its regulatory cofactor activity resulting in C3 degradation and inhibition of downstream complement activation. By comparing results from wild type C57BL/6 mice and complement deficient mice, we further showed that BclA significantly contributed to spore persistence in the mouse lungs and dampened antibody responses to spores in a complement C3-dependent manner. In addition, prior exposure to BclA deletion spores (ΔbclA) provided significant protection against lethal challenges by B. anthracis, whereas the isogenic parent spores did not, indicating that BclA may also impair protective immunity. These results describe for the first time an immune inhibition mechanism of B. anthracis mediated by BclA and CFH that promotes spore persistence in vivo. The findings also suggested an important role of complement in persistent infections and thus have broad implications. PMID:27304426

  10. Bacillus anthracis Spore Surface Protein BclA Mediates Complement Factor H Binding to Spores and Promotes Spore Persistence.

    Directory of Open Access Journals (Sweden)

    Yanyu Wang

    2016-06-01

    Full Text Available Spores of Bacillus anthracis, the causative agent of anthrax, are known to persist in the host lungs for prolonged periods of time, however the underlying mechanism is poorly understood. In this study, we demonstrated that BclA, a major surface protein of B. anthracis spores, mediated direct binding of complement factor H (CFH to spores. The surface bound CFH retained its regulatory cofactor activity resulting in C3 degradation and inhibition of downstream complement activation. By comparing results from wild type C57BL/6 mice and complement deficient mice, we further showed that BclA significantly contributed to spore persistence in the mouse lungs and dampened antibody responses to spores in a complement C3-dependent manner. In addition, prior exposure to BclA deletion spores (ΔbclA provided significant protection against lethal challenges by B. anthracis, whereas the isogenic parent spores did not, indicating that BclA may also impair protective immunity. These results describe for the first time an immune inhibition mechanism of B. anthracis mediated by BclA and CFH that promotes spore persistence in vivo. The findings also suggested an important role of complement in persistent infections and thus have broad implications.

  11. RAZOR EX Anthrax Air Detection System for detection of Bacillus anthracis spores from aerosol collection samples: collaborative study.

    Science.gov (United States)

    Hadfield, Ted; Ryan, Valorie; Spaulding, Usha K; Clemens, Kristine M; Ota, Irene M; Brunelle, Sharon L

    2013-01-01

    The RAZOR EX Anthrax Air Detection System was validated in a collaborative study for the detection of Bacillus anthracis in aerosol collection buffer. Phosphate-buffered saline was charged with 1 mg/mL standardized dust to simulate an authentic aerosol collection sample. The dust-charged buffer was spiked with either B. anthracis Ames at 2000 spores/mL or Bacillus cereus at 20 000 spores/mL. Twelve collaborators participated in the study, with four collaborators at each of three sites. Each collaborator tested 12 replicates of B. anthracis in dust-charged buffer and 12 replicates of B. cereus in dust-charged buffer. All samples sets were randomized and blind-coded. All collaborators produced valid data sets (no collaborators displayed systematic errors) and there was only one invalid data point. After unblinding, the analysis revealed a cross-collaborator probability of detection (CPOD) of 1.00 (144 positive results from 144 replicates, 95% confidence interval 0.975-1.00) for the B. anthracis samples and a CPOD of 0.00 (0 positive results from 143 replicates, 95% confidence interval 0.00-0.0262) for the B. cereus samples. These data meet the requirements of AOAC Standard Method Performance Requirement 2010.003, developed by the Stakeholder Panel on Agent Detection Assays. PMID:23767365

  12. Effect of pH on the Electrophoretic Mobility of Spores of Bacillus anthracis and Its Surrogates in Aqueous Solutions

    Science.gov (United States)

    Electrophoretic mobility (EPM) of endospores of Bacillus anthracis and surrogates were measured in aqueous solution across a broad pH range and several ionic strengths. EPM values trended around phylogenetic clustering based on the 16S rRNA gene. Measurements reported here prov...

  13. Technical Note: Simple, scalable, and sensitive protocol for retrieving Bacillus anthracis (and other live bacteria) from heroin.

    Science.gov (United States)

    Grass, Gregor; Ahrens, Bjoern; Schleenbecker, Uwe; Dobrzykowski, Linda; Wagner, Matthias; Krüger, Christian; Wölfel, Roman

    2016-02-01

    We describe a culture-based method suitable for isolating Bacillus anthracis and other live bacteria from heroin. This protocol was developed as a consequence of the bioforensic need to retrieve bacteria from batches of the drug associated with cases of injectional anthrax among heroin-consumers in Europe. This uncommon manifestation of infection with the notorious pathogen B. anthracis has resulted in 26 deaths between the years 2000 to 2013. Thus far, no life disease agent has been isolated from heroin during forensic investigations surrounding these incidences. Because of the conjectured very small number of disease-causing endospores in the contaminated drug it is likely that too few target sequences are available for molecular genetic analysis. Therefore, a direct culture-based approach was chosen here. Endospores of attenuated B. anthracis artificially spiked into heroin were successfully retrieved at 84-98% recovery rates using a wash solution consisting of 0.5% Tween 20 in water. Using this approach, 82 samples of un-cut heroin originating from the German Federal Criminal Police Office's heroin analysis program seized during the period between 2000 and 2014 were tested and found to be surprisingly poor in retrievable bacteria. Notably, while no B. anthracis was isolated from the drug batches, other bacteria were successfully cultured. The resulting methodical protocol is therefore suitable for analyzing un-cut heroin which can be anticipated to comprise the original microbiota from the drug's original source without interference from contaminations introduced by cutting. PMID:26734987

  14. Does Bacillus anthracis Lethal Toxin Directly Depress Myocardial Function? A Review of Clinical Cases and Preclinical Studies.

    Science.gov (United States)

    Suffredini, Dante A; Sampath-Kumar, Hanish; Li, Yan; Ohanjanian, Lernik; Remy, Kenneth E; Cui, Xizhong; Eichacker, Peter Q

    2015-12-01

    The US outbreak of B.anthracis infection in 2001 and subsequent cases in the US and Europe demonstrate that anthrax is a continuing risk for the developed world. While several bacterial components contribute to the pathogenesis of B. anthracis, production of lethal toxin (LT) is strongly associated with the development of hypotension and lethality. However, the mechanisms underlying the cardiovascular instability LT produces are unclear. Some evidence suggests that LT causes shock by impairing the peripheral vasculature, effects consistent with the substantial extravasation of fluid in patients dying with B. anthracis. Other data suggests that LT directly depresses myocardial function. However a clinical correlate for this latter possibility is less evident since functional studies and post-mortem examination in patients demonstrate absent or minimal cardiac changes. The purposes of this review were to first present clinical studies of cardiac functional and histologic pathology with B. anthracis infection and to then examine in vivo, in vitro, and ex vivo preclinical studies of LT's myocardial effects. Together, these data suggest that it is unclear whether that LT directly depresses cardiac function. This question is important for the clinical management and development of new therapies for anthrax and efforts should continue to be made to answer it. PMID:26703730

  15. Structure and mechanism of dimethylsulfoxide reductase, a molybdopterin-containing enzyme of DMSO reductase family

    International Nuclear Information System (INIS)

    Full text: Apart from nitrogenase, enzymes containing molybdenum are members of a superfamily, the molybdopterin-containing enzymes. Most of these enzymes catalyse an oxygen atom transfer and two electron transfer reaction. During catalysis the Mo at the active site cycles between the Mo(VI) and Mo(IV) states. The DMSO reductase family of molybdopterin-containing enzymes all contain a bis(molybdopterin guanine dinucleotide)Mo cofactor and over thirty examples have now been described. Over the last five years crystal structures of dimethylsulfoxide (DMSO) reductase and four other enzymes of the DMSO reductase family have revealed that enzymes of this family have a similar tertiary structure. The Mo atom at the active site is coordinated by four thiolate ligands provided by the dithiolene side chains of the two MGD molecules of the bis(MGD)Mo cofactor as well as a ligand provided by an amino acid side chain. In addition, an oxygen atom in the form of an oxo, hydroxo or aqua group is also coordinated to the Mo atom. In the case of dimethylsulfoxide reductase X-ray crystallography of the product-reduced species and Raman spectroscopy has demonstrated that the enzyme contains a single exchangeable oxo group that is H-bonded to W116

  16. Identification, Functional Characterization and Regulon Prediction of a Novel Two Component System Comprising BAS0540-BAS0541 of Bacillus anthracis

    Science.gov (United States)

    Gopalani, Monisha; Kandari, Divya; Bhatnagar, Rakesh

    2016-01-01

    Two component systems (TCSs) can be envisaged as complex molecular devices that help the bacteria to sense its environment and respond aptly. 41 TCSs are predicted in Bacillus anthracis, a potential bioterrorism agent, of which only four have been studied so far. Thus, the intricate signaling network contributed by TCSs remains largely unmapped in B. anthracis and needs comprehensive exploration. In this study, we functionally characterized one such system composed of BAS0540 (Response regulator) and BAS0541 (Histidine kinase). BAS0540-BAS0541, the closest homolog of CiaRH of Streptococcus in B. anthracis, forms a functional TCS with BAS0541 displaying autophosphorylation and subsequent phosphotransfer to BAS0540. BAS0540 was also found to accept phosphate from physiologically relevant small molecule phosphodonors like acetyl phosphate and carbamoyl phosphate. Results of qRT-PCR and immunoblotting demonstrated that BAS0540 exhibits a constitutive expression throughout the growth of B. anthracis. Regulon prediction for BAS0540 in B. anthracis was done in silico using the consensus DNA binding sequence of CiaR of Streptococcus. The predicted regulon of BAS0540 comprised of 23 genes, which could be classified into 8 functionally diverse categories. None of the proven virulence factors were a part of the predicted regulon, an observation contrasting with the regulon of CiaRH in Streptococci. Electrophoretic mobility shift assay was used to show direct binding of purified BAS0540 to the upstream regions of 5 putative regulon candidates- BAS0540 gene itself; a gene predicted to encode cell division protein FtsA; a self–immunity gene; a RND family transporter gene and a gene encoding stress (heat) responsive protein. A significant enhancement in the DNA binding ability of BAS0540 was observed upon phosphorylation. Overexpression of response regulator BAS0540 in B. anthracis led to a prodigious increase of ~6 folds in the cell length, thereby conferring it a filamentous

  17. Bacillus anthracis spore interactions with mammalian cells: Relationship between germination state and the outcome of in vitro

    Directory of Open Access Journals (Sweden)

    Stojkovic Bojana

    2011-02-01

    Full Text Available Abstract Background During inhalational anthrax, internalization of Bacillus anthracis spores by host cells within the lung is believed to be a key step for initiating the transition from the localized to disseminated stages of infection. Despite compelling in vivo evidence that spores remain dormant within the bronchioalveolar spaces of the lungs, and germinate only after uptake into host cells, most in vitro studies of infection have been conducted under conditions that promote rapid germination of spores within the culture medium. Results Using an in vitro model of infection, we evaluated the influence of the germination state of B. anthracis spores, as controlled by defined culture conditions, on the outcome of infection. Spores prepared from B. anthracis Sterne 7702 germinated in a variety of common cell culture media supplemented with fetal bovine serum (FBS while, in the absence of FBS, germination was strictly dependent on medium composition. RAW264.7 macrophage-like cells internalized spores to the same extent in either germinating or non-germinating media. However, significantly more viable, intracellular B. anthracis were recovered from cells infected under non-germinating conditions compared to germinating conditions. At the same time, RAW264.7 cells demonstrated a significant loss in viability when infected under non-germinating conditions. Conclusions These results suggest that the outcome of host cell infection is sensitive to the germination state of spores at the time of uptake. Moreover, this study demonstrates the efficacy of studying B. anthracis spore infection of host cells within a defined, non-germinating, in vitro environment.

  18. Iron-mediated effects on nitrate reductase in marine phytoplankton

    NARCIS (Netherlands)

    Timmermans, K.R.; Stolte, W.; Baar, H.J.W. de

    1994-01-01

    The potential activity of nitrate reductase was determined in uni-algal cultures in the laboratory and in natural marine phytoplankton assemblages. In the laboratory bioassays, distinct differences in nitrate reductase activity were observed in iron replete versus depleted cultures for Emiliania hux

  19. Membrane-associated chromate reductase activity from Enterobacter cloacae.

    OpenAIRE

    P. C. Wang; Mori, T.; Toda, K.; Ohtake, H

    1990-01-01

    Washed cells of Enterobacter cloacae HO1 reduced hexavalent chromium (chromate: CrO4(2-) anaerobically. Chromate reductase activity was preferentially associated with the membrane fraction of the cells. Right-side-out membrane vesicles prepared from E. cloacae cells showed high chromate reductase activities when ascorbate-reduced phenazine methosulfate was added as an electron donor.

  20. Biliverdin Reductase: a Target for Cancer Therapy?

    Directory of Open Access Journals (Sweden)

    Peter eGibbs

    2015-06-01

    Full Text Available Biliverdin reductase (BVR is a multifunctional protein that is the primary source of the potent antioxidant, bilirubin. BVR regulates activities/functions in the insulin/IGF-1/IRK/PI3K/MAPK pathways. Activation of certain kinases in these pathways is/are hallmark(s of cancerous cells. The protein is a scaffold/bridge and intracellular transporter of kinases that regulate growth and proliferation of cells, including PKCs, ERK and Akt, and their targets including NF-κB, Elk1, HO-1 and iNOS. The scaffold and transport functions enable activated BVR to relocate from the cytosol to the nucleus or to the plasma membrane, depending on the activating stimulus. This enables the reductase to function in diverse signaling pathways. And, its expression at the transcript and protein levels are increased in human tumors and the infiltrating T-cells, monocytes and circulating lymphocytes, as well as the circulating and infiltrating macrophages. These functions suggest that the cytoprotective role of BVR may be permissive for cancer/tumor growth. In this review, we summarize the recent developments that define the pro-growth activities of BVR, particularly with respect to its input into the MAPK signaling pathway and present evidence that BVR-based peptides inhibit activation of protein kinases, including MEK, PKCδ and ERK as well as downstream targets including Elk1 and iNOS, and thus offers a credible novel approach to reduce cancer cell proliferation.

  1. Bacillus anthracis secretome time course under host-simulated conditions and identification of immunogenic proteins

    Directory of Open Access Journals (Sweden)

    Whittington Jessica

    2007-07-01

    Full Text Available Abstract Background The secretion time course of Bacillus anthracis strain RA3R (pXO1+/pXO2- during early, mid, and late log phase were investigated under conditions that simulate those encountered in the host. All of the identified proteins were analyzed by different software algorithms to characterize their predicted mode of secretion and cellular localization. In addition, immunogenic proteins were identified using sera from humans with cutaneous anthrax. Results A total of 275 extracellular proteins were identified by a combination of LC MS/MS and MALDI-TOF MS. All of the identified proteins were analyzed by SignalP, SecretomeP, PSORT, LipoP, TMHMM, and PROSITE to characterize their predicted mode of secretion, cellular localization, and protein domains. Fifty-three proteins were predicted by SignalP to harbor the cleavable N-terminal signal peptides and were therefore secreted via the classical Sec pathway. Twenty-three proteins were predicted by SecretomeP for secretion by the alternative Sec pathway characterized by the lack of typical export signal. In contrast to SignalP and SecretomeP predictions, PSORT predicted 171 extracellular proteins, 7 cell wall-associated proteins, and 6 cytoplasmic proteins. Moreover, 51 proteins were predicted by LipoP to contain putative Sec signal peptides (38 have SpI sites, lipoprotein signal peptides (13 have SpII sites, and N-terminal membrane helices (9 have transmembrane helices. The TMHMM algorithm predicted 25 membrane-associated proteins with one to ten transmembrane helices. Immunogenic proteins were also identified using sera from patients who have recovered from anthrax. The charge variants (83 and 63 kDa of protective antigen (PA were the most immunodominant secreted antigens, followed by charge variants of enolase and transketolase. Conclusion This is the first description of the time course of protein secretion for the pathogen Bacillus anthracis. Time course studies of protein secretion and

  2. The genome of a Bacillus isolate causing anthrax in chimpanzees combines chromosomal properties of B. cereus with B. anthracis virulence plasmids.

    Directory of Open Access Journals (Sweden)

    Silke R Klee

    Full Text Available Anthrax is a fatal disease caused by strains of Bacillus anthracis. Members of this monophyletic species are non motile and are all characterized by the presence of four prophages and a nonsense mutation in the plcR regulator gene. Here we report the complete genome sequence of a Bacillus strain isolated from a chimpanzee that had died with clinical symptoms of anthrax. Unlike classic B. anthracis, this strain was motile and lacked the four prohages and the nonsense mutation. Four replicons were identified, a chromosome and three plasmids. Comparative genome analysis revealed that the chromosome resembles those of non-B. anthracis members of the Bacillus cereus group, whereas two plasmids were identical to the anthrax virulence plasmids pXO1 and pXO2. The function of the newly discovered third plasmid with a length of 14 kbp is unknown. A detailed comparison of genomic loci encoding key features confirmed a higher similarity to B. thuringiensis serovar konkukian strain 97-27 and B. cereus E33L than to B. anthracis strains. For the first time we describe the sequence of an anthrax causing bacterium possessing both anthrax plasmids that apparently does not belong to the monophyletic group of all so far known B. anthracis strains and that differs in important diagnostic features. The data suggest that this bacterium has evolved from a B. cereus strain independently from the classic B. anthracis strains and established a B. anthracis lifestyle. Therefore we suggest to designate this isolate as "B. cereus variety (var. anthracis".

  3. A plant-produced protective antigen vaccine confers protection in rabbits against a lethal aerosolized challenge with Bacillus anthracis Ames spores

    OpenAIRE

    Chichester, Jessica A.; Manceva, Slobodanka D; Rhee, Amy; Coffin, Megan V.; Musiychuk, Konstantin; Mett, Vadim; Shamloul, Moneim; Norikane, Joey; Streatfield, Stephen J.; Yusibov, Vidadi

    2013-01-01

    The potential use of Bacillus anthracis as a bioterrorism weapon threatens the security of populations globally, requiring the immediate availability of safe, efficient and easily delivered anthrax vaccine for mass vaccination. Extensive research efforts have been directed toward the development of recombinant subunit vaccines based on protective antigen (PA), the principal virulence factor of B. anthracis. Among the emerging technologies for the production of these vaccine antigens is our la...

  4. Observations on the Inactivation Efficacy of a MALDI-TOF MS Chemical Extraction Method on Bacillus anthracis Vegetative Cells and Spores.

    Directory of Open Access Journals (Sweden)

    Simon A Weller

    Full Text Available A chemical (ethanol; formic acid; acetonitrile protein extraction method for the preparation of bacterial samples for matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS identification was evaluated for its ability to inactivate bacterial species. Initial viability tests (with and without double filtration of the extract through 0.2 μM filters, indicated that the method could inactivate Escherichia coli MRE 162 and Klebsiella pneumoniae ATCC 35657, with or without filtration, but that filtration was required to exclude viable, avirulent, Bacillus anthracis UM23CL2 from extracts. Multiple, high stringency, viability experiments were then carried out on entire filtered extracts prepared from virulent B. anthracis Vollum vegetative cells and spores ranging in concentration from 10(6-10(8 cfu per extract. B. anthracis was recovered in 3/18 vegetative cell extracts and 10/18 spore extracts. From vegetative cell extracts B. anthracis was only recovered from extracts that had undergone prolonged Luria (L-broth (7 day and L-agar plate (a further 7 days incubations. We hypothesise that the recovery of B. anthracis in vegetative cell extracts is due to the escape of individual sub-lethally injured cells. We discuss our results in view of working practises in clinical laboratories and in the context of recent inadvertent releases of viable B. anthracis.

  5. Observations on the Inactivation Efficacy of a MALDI-TOF MS Chemical Extraction Method on Bacillus anthracis Vegetative Cells and Spores.

    Science.gov (United States)

    Weller, Simon A; Stokes, Margaret G M; Lukaszewski, Roman A

    2015-01-01

    A chemical (ethanol; formic acid; acetonitrile) protein extraction method for the preparation of bacterial samples for matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) identification was evaluated for its ability to inactivate bacterial species. Initial viability tests (with and without double filtration of the extract through 0.2 μM filters), indicated that the method could inactivate Escherichia coli MRE 162 and Klebsiella pneumoniae ATCC 35657, with or without filtration, but that filtration was required to exclude viable, avirulent, Bacillus anthracis UM23CL2 from extracts. Multiple, high stringency, viability experiments were then carried out on entire filtered extracts prepared from virulent B. anthracis Vollum vegetative cells and spores ranging in concentration from 10(6)-10(8) cfu per extract. B. anthracis was recovered in 3/18 vegetative cell extracts and 10/18 spore extracts. From vegetative cell extracts B. anthracis was only recovered from extracts that had undergone prolonged Luria (L)-broth (7 day) and L-agar plate (a further 7 days) incubations. We hypothesise that the recovery of B. anthracis in vegetative cell extracts is due to the escape of individual sub-lethally injured cells. We discuss our results in view of working practises in clinical laboratories and in the context of recent inadvertent releases of viable B. anthracis. PMID:26633884

  6. Phages Preying on Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis: Past, Present and Future

    Directory of Open Access Journals (Sweden)

    Annika Gillis

    2014-07-01

    Full Text Available Many bacteriophages (phages have been widely studied due to their major role in virulence evolution of bacterial pathogens. However, less attention has been paid to phages preying on bacteria from the Bacillus cereus group and their contribution to the bacterial genetic pool has been disregarded. Therefore, this review brings together the main information for the B. cereus group phages, from their discovery to their modern biotechnological applications. A special focus is given to phages infecting Bacillus anthracis, B. cereus and Bacillus thuringiensis. These phages belong to the Myoviridae, Siphoviridae, Podoviridae and Tectiviridae families. For the sake of clarity, several phage categories have been made according to significant characteristics such as lifestyles and lysogenic states. The main categories comprise the transducing phages, phages with a chromosomal or plasmidial prophage state, γ-like phages and jumbo-phages. The current genomic characterization of some of these phages is also addressed throughout this work and some promising applications are discussed here.

  7. Amperometric Detection of Bacillus anthracis Spores: A Portable, Low-Cost Approach to the ELISA

    Directory of Open Access Journals (Sweden)

    Gabriel D. Peckham

    2013-01-01

    Full Text Available Antibody-based detection assays are generally robust, a desirable characteristic for in-the-field use. However, to quantify the colorimetric or fluorescent signal, these assays require expensive and fragile instruments which are ill-suited to in-the-field use. Lateral flow devices (LFDs circumvent these barriers to portability but suffer from poor sensitivity and subjective interpretation. Here, an antibody-based method for detecting Bacillus anthracis spores via amperometric signal generation is compared to ELISA and LFDs. This amperometric immunoassay uses antibody conjugated to magnetic beads and glucose oxidase (GOX along with the electron mediator 2, 6-dichlorophenolindophenol (DCPIP for production of a measurable current from a 0.4 V bias voltage. With similar sensitivity to ELISA, the assay can be completed in about 75 minutes while being completely powered and operated from a laptop computer. Immunoassay amperometry holds promise for bringing low-cost, quantitative detection of hazardous agents to the field.

  8. Efficacy of Daptomycin against Bacillus anthracis in a murine model of anthrax spore inhalation.

    Science.gov (United States)

    Heine, Henry S; Bassett, Jennifer; Miller, Lynda; Purcell, Bret K; Byrne, W Russell

    2010-10-01

    Daptomycin demonstrated in vitro (MIC(90), 4 μg/ml) and in vivo activities against Bacillus anthracis. Twice-daily treatment with a dose of 50 mg/kg of body weight was begun 24 h after challenge and continued for 14 or 21 days; results were compared to those for controls treated with phosphate-buffered saline or ciprofloxacin. Day 43 survival rates were 6/10 mice for the 14-day and 9/10 mice for the 21-day treatment groups, compared to survival with ciprofloxacin: 8/10 and 9/10 mice, respectively. Culture results from tissues removed at the termination of the experiment were negative. PMID:20643899

  9. Production and purification of Bacillus anthracis protective antigen from Escherichia coli.

    Science.gov (United States)

    Laird, Michael W; Zukauskas, David; Johnson, Kelly; Sampey, Gavin C; Olsen, Henrik; Garcia, Andy; Karwoski, Jeffrey D; Cooksey, Bridget A; Choi, Gil H; Askins, Janine; Tsai, Amos; Pierre, Jennifer; Gwinn, William

    2004-11-01

    Anthrax is caused by the gram-positive, spore-forming bacterium, Bacillus anthracis. The anthrax toxin consists of three proteins, protective antigen (PA), lethal factor, and edema factor. Current vaccines against anthrax use PA as their primary component since it confers protective immunity. In this work, we expressed soluble, recombinant PA in relatively high amounts in the periplasm of E. coli from shake flasks and bioreactors. The PA protein was purified using Q-Sepharose-HP and hydroxyapatite chromatography, and routinely found to be 96-98% pure. Yields of purified PA varied depending on the method of production; however, medium cell density fermentations resulted in approximately 370 mg/L of highly pure biologically active PA protein. These results exhibit the ability to generate gram quantities of PA from E. coli. PMID:15477093

  10. Phages preying on Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis: past, present and future.

    Science.gov (United States)

    Gillis, Annika; Mahillon, Jacques

    2014-07-01

    Many bacteriophages (phages) have been widely studied due to their major role in virulence evolution of bacterial pathogens. However, less attention has been paid to phages preying on bacteria from the Bacillus cereus group and their contribution to the bacterial genetic pool has been disregarded. Therefore, this review brings together the main information for the B. cereus group phages, from their discovery to their modern biotechnological applications. A special focus is given to phages infecting Bacillus anthracis, B. cereus and Bacillus thuringiensis. These phages belong to the Myoviridae, Siphoviridae, Podoviridae and Tectiviridae families. For the sake of clarity, several phage categories have been made according to significant characteristics such as lifestyles and lysogenic states. The main categories comprise the transducing phages, phages with a chromosomal or plasmidial prophage state, γ-like phages and jumbo-phages. The current genomic characterization of some of these phages is also addressed throughout this work and some promising applications are discussed here. PMID:25010767

  11. Recovery efficiency and limit of detection of aerosolized Bacillus anthracis Sterne from environmental surface samples.

    Science.gov (United States)

    Estill, Cheryl Fairfield; Baron, Paul A; Beard, Jeremy K; Hein, Misty J; Larsen, Lloyd D; Rose, Laura; Schaefer, Frank W; Noble-Wang, Judith; Hodges, Lisa; Lindquist, H D Alan; Deye, Gregory J; Arduino, Matthew J

    2009-07-01

    After the 2001 anthrax incidents, surface sampling techniques for biological agents were found to be inadequately validated, especially at low surface loadings. We aerosolized Bacillus anthracis Sterne spores within a chamber to achieve very low surface loading (ca. 3, 30, and 200 CFU per 100 cm(2)). Steel and carpet coupons seeded in the chamber were sampled with swab (103 cm(2)) or wipe or vacuum (929 cm(2)) surface sampling methods and analyzed at three laboratories. Agar settle plates (60 cm(2)) were the reference for determining recovery efficiency (RE). The minimum estimated surface concentrations to achieve a 95% response rate based on probit regression were 190, 15, and 44 CFU/100 cm(2) for sampling steel surfaces and 40, 9.2, and 28 CFU/100 cm(2) for sampling carpet surfaces with swab, wipe, and vacuum methods, respectively; however, these results should be cautiously interpreted because of high observed variability. Mean REs at the highest surface loading were 5.0%, 18%, and 3.7% on steel and 12%, 23%, and 4.7% on carpet for the swab, wipe, and vacuum methods, respectively. Precision (coefficient of variation) was poor at the lower surface concentrations but improved with increasing surface concentration. The best precision was obtained with wipe samples on carpet, achieving 38% at the highest surface concentration. The wipe sampling method detected B. anthracis at lower estimated surface concentrations and had higher RE and better precision than the other methods. These results may guide investigators to more meaningfully conduct environmental sampling, quantify contamination levels, and conduct risk assessment for humans.

  12. Nanoscale structural and mechanical analysis of Bacillus anthracis spores inactivated with rapid dry heating.

    Science.gov (United States)

    Xing, Yun; Li, Alex; Felker, Daniel L; Burggraf, Larry W

    2014-03-01

    Effective killing of Bacillus anthracis spores is of paramount importance to antibioterrorism, food safety, environmental protection, and the medical device industry. Thus, a deeper understanding of the mechanisms of spore resistance and inactivation is highly desired for developing new strategies or improving the known methods for spore destruction. Previous studies have shown that spore inactivation mechanisms differ considerably depending upon the killing agents, such as heat (wet heat, dry heat), UV, ionizing radiation, and chemicals. It is believed that wet heat kills spores by inactivating critical enzymes, while dry heat kills spores by damaging their DNA. Many studies have focused on the biochemical aspects of spore inactivation by dry heat; few have investigated structural damages and changes in spore mechanical properties. In this study, we have inactivated Bacillus anthracis spores with rapid dry heating and performed nanoscale topographical and mechanical analysis of inactivated spores using atomic force microscopy (AFM). Our results revealed significant changes in spore morphology and nanomechanical properties after heat inactivation. In addition, we also found that these changes were different under different heating conditions that produced similar inactivation probabilities (high temperature for short exposure time versus low temperature for long exposure time). We attributed the differences to the differential thermal and mechanical stresses in the spore. The buildup of internal thermal and mechanical stresses may become prominent only in ultrafast, high-temperature heat inactivation when the experimental timescale is too short for heat-generated vapor to efficiently escape from the spore. Our results thus provide direct, visual evidences of the importance of thermal stresses and heat and mass transfer to spore inactivation by very rapid dry heating.

  13. Structure and expression of human dihydropteridine reductase

    International Nuclear Information System (INIS)

    Dihydropteridine reductase catalyzes the NADH-mediated reduction of quinonoid dihydrobiopterin and is an essential component of the pterindependent aromatic amino acid hydroxylating systems. A cDNA for human DHPR was isolated from a human liver cDNA library in the vector λgt11 using a monospecific antibody against sheep DHPR. The nucleic acid sequence and amino acid sequence of human DHPR were determined from a full-length clone. A 112 amino acid sequence of sheep DHPR was obtained by sequencing purified sheep DHPR. This sequence is highly homologous to the predicted amino acid sequence of the human protein. Gene transfer of the recombinant human DHPR into COS cells leads to expression of DHPR enzymatic activity. These results indicate that the cDNA clone identified by antibody screening is an authentic and full-length cDNA for human DHPR

  14. Rapid Antimicrobial Susceptibility Testing of Bacillus anthracis, Yersinia pestis, and Burkholderia pseudomallei by Use of Laser Light Scattering Technology.

    Science.gov (United States)

    Bugrysheva, Julia V; Lascols, Christine; Sue, David; Weigel, Linda M

    2016-06-01

    Rapid methods to determine antimicrobial susceptibility would assist in the timely distribution of effective treatment or postexposure prophylaxis in the aftermath of the release of bacterial biothreat agents such as Bacillus anthracis, Yersinia pestis, or Burkholderia pseudomallei Conventional susceptibility tests require 16 to 48 h of incubation, depending on the bacterial species. We evaluated a method that is based on laser light scattering technology that measures cell density in real time. We determined that it has the ability to rapidly differentiate between growth (resistant) and no growth (susceptible) of several bacterial threat agents in the presence of clinically relevant antimicrobials. Results were available in 10 h of incubation. Use of laser scattering technology decreased the time required to determine antimicrobial susceptibility by 50% to 75% for B. anthracis, Y. pestis, and B. pseudomallei compared to conventional methods. PMID:26984973

  15. Expression, purification, crystallization and preliminary X-ray studies of a prolyl-4-hydroxylase protein from Bacillus anthracis

    International Nuclear Information System (INIS)

    Prolyl-4-hydroxylase from B. anthracis has been cloned, expressed and crystallized. A complete MAD data set has been collected to 1.4 Å resolution. Collagen prolyl-4-hydroxylase (C-P4H) catalyzes the hydroxylation of specific proline residues in procollagen, which is an essential step in collagen biosynthesis. A new form of P4H from Bacillus anthracis (anthrax-P4H) that shares many characteristics with the type I C-P4H from human has recently been characterized. The structure of anthrax-P4H could provide important insight into the chemistry of C-P4Hs and into the function of this unique homodimeric P4H. X-ray diffraction data of selenomethionine-labeled anthrax-P4H recombinantly expressed in Escherichia coli have been collected to 1.4 Å resolution

  16. Anthrax Vaccine Antigen-Adjuvant Formulations Completely Protect New Zealand White Rabbits against Challenge with Bacillus anthracis Ames Strain Spores

    OpenAIRE

    Peachman, Kristina K.; Li, Qin; Matyas, Gary R.; Shivachandra, Sathish B.; Lovchik, Julie; Lyons, Rick C.; Alving, Carl R; Rao, Venigalla B.; Rao, Mangala

    2012-01-01

    In an effort to develop an improved anthrax vaccine that shows high potency, five different anthrax protective antigen (PA)-adjuvant vaccine formulations that were previously found to be efficacious in a nonhuman primate model were evaluated for their efficacy in a rabbit pulmonary challenge model using Bacillus anthracis Ames strain spores. The vaccine formulations include PA adsorbed to Alhydrogel, PA encapsulated in liposomes containing monophosphoryl lipid A, stable liposomal PA oil-in-wa...

  17. Detection of Anthrax Toxin in the Serum of Animals Infected with Bacillus anthracis by Using Engineered Immunoassays

    OpenAIRE

    Mabry, Robert; Brasky, Kathleen; Geiger, Robert; Carrion, Ricardo; Hubbard, Gene B; Leppla, Stephen; Patterson, Jean L.; Georgiou, George; Iverson, B L

    2006-01-01

    Several strategies that target anthrax toxin are being developed as therapies for infection by Bacillus anthracis. Although the action of the tripartite anthrax toxin has been extensively studied in vitro, relatively little is known about the presence of toxins during an infection in vivo. We developed a series of sensitive sandwich enzyme-linked immunosorbent assays (ELISAs) for detection of both the protective antigen (PA) and lethal factor (LF) components of the anthrax exotoxin in serum. ...

  18. Testing Nucleoside Analogues as Inhibitors of Bacillus anthracis Spore Germination In Vitro and in Macrophage Cell Culture ▿

    OpenAIRE

    Alvarez, Zadkiel; Lee, Kyungae; Abel-Santos, Ernesto

    2010-01-01

    Bacillus anthracis, the etiological agent of anthrax, has a dormant stage in its life cycle known as the endospore. When conditions become favorable, spores germinate and transform into vegetative bacteria. In inhalational anthrax, the most fatal manifestation of the disease, spores enter the organism through the respiratory tract and germinate in phagosomes of alveolar macrophages. Germinated cells can then produce toxins and establish infection. Thus, germination is a crucial step for the i...

  19. Baulamycins A and B, Broad-Spectrum Antibiotics Identified as Inhibitors of Siderophore Biosynthesis in Staphylococcus aureus and Bacillus anthracis

    OpenAIRE

    Tripathi, Ashootosh; Schofield, Michael M.; Chlipala, George E.; Schultz, Pamela J.; Yim, Isaiah; Newmister, Sean A.; Nusca, Tyler D.; Scaglione, Jamie B.; Hanna, Philip C.; Tamayo-Castillo, Giselle; Sherman, David H.

    2014-01-01

    Siderophores are high-affinity iron chelators produced by microorganisms and frequently contribute to the virulence of human pathogens. Targeted inhibition of the biosynthesis of siderophores staphyloferrin B of Staphylococcus aureus and petrobactin of Bacillus anthracis hold considerable potential as a single or combined treatment for methicillin-resistant S. aureus (MRSA) and anthrax infection, respectively. The biosynthetic pathways for both siderophores involve a nonribosomal peptide synt...

  20. Ground Anthrax Bacillus Refined Isolation (GABRI) method for analyzing environmental samples with low levels of Bacillus anthracis contamination

    OpenAIRE

    Fasanella, Antonio; Di Taranto, Pietro; Garofolo, Giuliano; Colao, Valeriana; Marino, Leonardo; Buonavoglia, Domenico; Pedarra, Carmine; Adone, Rosanna; Hugh-Jones, Martin

    2013-01-01

    Background In this work are reported the results of a qualitative analytical method capable of detecting Bacillus anthracis spores when they are present in very low concentration in the soil. The Ground Anthrax Bacillus Refined Isolation (GABRI) method, assessed in our laboratory, was compared with the classic method. The comparison involved artificially anthrax-contaminated soil samples (500 spores/7.5 grams soil) and naturally contaminated soil samples collected in Bangladesh during a field...

  1. Bacillus anthracis spore interactions with mammalian cells: Relationship between germination state and the outcome of in vitro

    OpenAIRE

    Stojkovic Bojana; Prouty Angela M; Tamilselvam Batcha; Gut Ian M; Czeschin Stephanie; van der Donk Wilfred A; Blanke Steven R

    2011-01-01

    Abstract Background During inhalational anthrax, internalization of Bacillus anthracis spores by host cells within the lung is believed to be a key step for initiating the transition from the localized to disseminated stages of infection. Despite compelling in vivo evidence that spores remain dormant within the bronchioalveolar spaces of the lungs, and germinate only after uptake into host cells, most in vitro studies of infection have been conducted under conditions that promote rapid germin...

  2. Immunoassay for Capsular Antigen of Bacillus anthracis Enables Rapid Diagnosis in a Rabbit Model of Inhalational Anthrax

    OpenAIRE

    Marcellene A Gates-Hollingsworth; Perry, Mark R.; Chen, Hongjing; Needham, James; Houghton, Raymond L.; Raychaudhuri, Syamal; Mark A Hubbard; Thomas R Kozel

    2015-01-01

    Inhalational anthrax is a serious biothreat. Effective antibiotic treatment of inhalational anthrax requires early diagnosis; the further the disease has progressed, the less the likelihood for cure. Current means for diagnosis such as blood culture require several days to a result and require advanced laboratory infrastructure. An alternative approach to diagnosis is detection of a Bacillus anthracis antigen that is shed into blood and can be detected by rapid immunoassay. The goal of the st...

  3. A simple, high-throughput method to detect Plasmodium falciparum single nucleotide polymorphisms in the dihydrofolate reductase, dihydropteroate synthase, and P. falciparum chloroquine resistance transporter genes using polymerase chain reaction- and enzyme-linked immunosorbent

    DEFF Research Database (Denmark)

    Alifrangis, Michael; Enosse, Sonia; Pearce, Richard;

    2005-01-01

    . However, to be a practical tool in the surveillance of drug resistance, simpler methods for high-throughput haplotyping are warranted. Here we describe a quick and simple technique that detects dhfr, dhps, and Pfcrt SNPs using polymerase chain reaction (PCR)- and enzyme-linked immunosorbent assay (ELISA......)-based technology. Biotinylated PCR products of dhfr, dhps, or Pfcrt were captured on streptavidin-coated microtiter plates and sequence-specific oligonucleotide probes (SSOPs) were hybridized with the PCR products. A stringent washing procedure enabled detection of remaining bound SSOPs and distinguished between...... the SNPs of dhfr, dhps, and Pfcrt with high specificity. The SSOP-ELISA compared well with a standard PCR-restriction fragment length polymorphism procedure, and gave identical positive results in more than 90% of the P. falciparum slide-positive samples tested. The SSOP-ELISA of all dhfr, dhps, or Pfcrt...

  4. Increasing prevalence of wildtypes in the dihydrofolate reductase gene of Plasmodium falciparum in an area with high levels of sulfadoxine/pyrimethamine resistance after introduction of treated bed nets

    DEFF Research Database (Denmark)

    Alifrangis, Michael; Lemnge, Martha M; Rønn, Anita M;

    2003-01-01

    responsible for resistance to S/P. In December 1998, Magoda received insecticide-treated nets (ITNs), whereas ITNs were introduced in Mpapayu in March 2001. We have studied the effect of ITNs on P. falciparum resistance genes by monitoring the prevalence of dhfr and dhps genotypes in children less than five...... years old living in the villages from 1998 to 2000. In 2000, after two years of bed net use, the prevalence of wild types in codon 51, 59, and 108 of dhfr increased significantly in Magoda compared with previous years. Furthermore, the prevalence of dhfr wild types was significantly higher in Magoda...

  5. DNA sequence conservation between the Bacillus anthracis pXO2 plasmid and genomic sequence from closely related bacteria

    Directory of Open Access Journals (Sweden)

    Sabin Robert

    2002-12-01

    Full Text Available Abstract Background Complete sequencing and annotation of the 96.2 kb Bacillus anthracis plasmid, pXO2, predicted 85 open reading frames (ORFs. Bacillus cereus and Bacillus thuringiensis isolates that ranged in genomic similarity to B. anthracis, as determined by amplified fragment length polymorphism (AFLP analysis, were examined by PCR for the presence of sequences similar to 47 pXO2 ORFs. Results The two most distantly related isolates examined, B. thuringiensis 33679 and B. thuringiensis AWO6, produced the greatest number of ORF sequences similar to pXO2; 10 detected in 33679 and 16 in AWO6. No more than two of the pXO2 ORFs were detected in any one of the remaining isolates. Dot-blot DNA hybridizations between pXO2 ORF fragments and total genomic DNA from AWO6 were consistent with the PCR assay results for this isolate and also revealed nine additional ORFs shared between these two bacteria. Sequences similar to the B. anthracis cap genes or their regulator, acpA, were not detected among any of the examined isolates. Conclusions The presence of pXO2 sequences in the other Bacillus isolates did not correlate with genomic relatedness established by AFLP analysis. The presence of pXO2 ORF sequences in other Bacillus species suggests the possibility that certain pXO2 plasmid gene functions may also be present in other closely related bacteria.

  6. Quantitative Determination of Lethal Toxin Proteins in Culture Supernatant of Human Live Anthrax Vaccine Bacillus anthracis A16R.

    Science.gov (United States)

    Zai, Xiaodong; Zhang, Jun; Liu, Ju; Liu, Jie; Li, Liangliang; Yin, Ying; Fu, Ling; Xu, Junjie; Chen, Wei

    2016-03-01

    Bacillus anthracis (B. anthracis) is the etiological agent of anthrax affecting both humans and animals. Anthrax toxin (AT) plays a major role in pathogenesis. It includes lethal toxin (LT) and edema toxin (ET), which are formed by the combination of protective antigen (PA) and lethal factor (LF) or edema factor (EF), respectively. The currently used human anthrax vaccine in China utilizes live-attenuated B. anthracis spores (A16R; pXO1+, pXO2-) that produce anthrax toxin but cannot produce the capsule. Anthrax toxins, especially LT, have key effects on both the immunogenicity and toxicity of human anthrax vaccines. Thus, determining quantities and biological activities of LT proteins expressed by the A16R strain is meaningful. Here, we explored LT expression patterns of the A16R strain in culture conditions using another vaccine strain Sterne as a control. We developed a sandwich ELISA and cytotoxicity-based method for quantitative detection of PA and LF. Expression and degradation of LT proteins were observed in culture supernatants over time. Additionally, LT proteins expressed by the A16R and Sterne strains were found to be monomeric and showed cytotoxic activity, which may be the main reason for side effects of live anthrax vaccines. Our work facilitates the characterization of anthrax vaccines components and establishment of a quality control standard for vaccine production which may ultimately help to ensure the efficacy and safety of the human anthrax vaccine A16R. PMID:26927174

  7. Measurement of 100 B. anthracis Ames spores within 15 minutes by SERS at the US Army Edgewood Chemical Biological Ctr.

    Science.gov (United States)

    Farquharson, Stuart; Shende, Chetan; Smith, Wayne; Huang, Hermes; Sperry, Jay; Sickler, Todd; Prugh, Amber; Guicheteau, Jason

    2014-05-01

    Since the distribution of Bacillus anthracis-Ames spores through the US Postal System, there has been a persistent fear that biological warfare agents will be used by terrorists against our military abroad and our civilians at home. While there has been substantial effort since the anthrax attack of 2001 to develop analyzers to detect this and other biological warfare agents, the analyzers remain either too slow, lack sensitivity, produce high false-positive rates, or cannot be fielded. In an effort to overcome these limitations we have been developing a surface-enhanced Raman spectroscopy system. Here we describe the use of silver nanoparticles functionalized with a short peptide to selectively capture Bacillus anthracis spores and produce SER scattering. Specifically, measurements of 100 B. anthracis-Ames spores/mL in ~25 minutes performed at the US Army's Edgewood Chemical Biological Center are presented. The measurements provide a basis for the development of systems that can detect spores collected from the air or water supplies with the potential of saving lives during a biological warfare attack.

  8. Anthrolysin O and fermentation products mediate the toxicity of Bacillus anthracis to lung epithelial cells under microaerobic conditions.

    Science.gov (United States)

    Popova, Taissia G; Millis, Bryan; Chung, Myung-Chul; Bailey, Charles; Popov, Serguei G

    2011-02-01

    Bacillus anthracis generates virulence factors such as lethal and edema toxins, capsule, and hemolytic proteins under conditions of reduced oxygenation. Here, we report on the acute cytotoxicity of culture supernatants (Sups) of six nonencapsulated B. anthracis strains grown till the stationary phase under static microaerobic conditions. Human small airway epithelial, umbilical vein endothelial, Caco-2, and Hep-G2 cells were found to be susceptible. Sups displayed a reduction of pH to 5.3-5.5, indicating the onset of acid anaerobic fermentation; however, low pH itself was not a major factor of toxicity. The pore-forming hemolysin, anthrolysin O (ALO), contributed to the toxicity in a concentration-dependent manner. Its effect was found to be synergistic with a metabolic product of B. anthracis, succinic acid. Cells exposed to Sups demonstrated cytoplasmic membrane blebbing, increased permeability, loss of ATP, mitochondrial membrane potential collapse, and arrest of cell respiration. The toxicity was reduced by inhibition of ALO by cholesterol, decomposition of reactive oxygen species, and inhibition of mitochondrial succinate dehydrogenase. Cell death appears to be caused by an acute primary membrane permeabilization by ALO, followed by a burst of reactive radicals from the mitochondria fuelled by the succinate, which is generated by bacteria in the hypoxic environment. This mechanism of metabolic toxicity is relevant to the late-stage conditions of hypoxia and acidosis found in anthrax patients and might operate at anatomical locations of the host deprived from oxygen supply. PMID:20946354

  9. Immunoassay for Capsular Antigen of Bacillus anthracis Enables Rapid Diagnosis in a Rabbit Model of Inhalational Anthrax.

    Directory of Open Access Journals (Sweden)

    Marcellene A Gates-Hollingsworth

    Full Text Available Inhalational anthrax is a serious biothreat. Effective antibiotic treatment of inhalational anthrax requires early diagnosis; the further the disease has progressed, the less the likelihood for cure. Current means for diagnosis such as blood culture require several days to a result and require advanced laboratory infrastructure. An alternative approach to diagnosis is detection of a Bacillus anthracis antigen that is shed into blood and can be detected by rapid immunoassay. The goal of the study was to evaluate detection of poly-γ-D-glutamic acid (PGA, the capsular antigen of B. anthracis, as a biomarker surrogate for blood culture in a rabbit model of inhalational anthrax. The mean time to a positive blood culture was 26 ± 5.7 h (mean ± standard deviation, whereas the mean time to a positive ELISA was 22 ± 4.2 h; P = 0.005 in comparison with blood culture. A lateral flow immunoassay was constructed for detection of PGA in plasma at concentrations of less than 1 ng PGA/ml. Use of the lateral flow immunoassay for detection of PGA in the rabbit model found that antigen was detected somewhat earlier than the earliest time point at which the blood culture became positive. The low cost, ease of use, and rapid time to result of the lateral flow immunoassay format make an immunoassay for PGA a viable surrogate for blood culture for detection of infection in individuals who have a likelihood of exposure to B. anthracis.

  10. Immunoassay for Capsular Antigen of Bacillus anthracis Enables Rapid Diagnosis in a Rabbit Model of Inhalational Anthrax.

    Science.gov (United States)

    Gates-Hollingsworth, Marcellene A; Perry, Mark R; Chen, Hongjing; Needham, James; Houghton, Raymond L; Raychaudhuri, Syamal; Hubbard, Mark A; Kozel, Thomas R

    2015-01-01

    Inhalational anthrax is a serious biothreat. Effective antibiotic treatment of inhalational anthrax requires early diagnosis; the further the disease has progressed, the less the likelihood for cure. Current means for diagnosis such as blood culture require several days to a result and require advanced laboratory infrastructure. An alternative approach to diagnosis is detection of a Bacillus anthracis antigen that is shed into blood and can be detected by rapid immunoassay. The goal of the study was to evaluate detection of poly-γ-D-glutamic acid (PGA), the capsular antigen of B. anthracis, as a biomarker surrogate for blood culture in a rabbit model of inhalational anthrax. The mean time to a positive blood culture was 26 ± 5.7 h (mean ± standard deviation), whereas the mean time to a positive ELISA was 22 ± 4.2 h; P = 0.005 in comparison with blood culture. A lateral flow immunoassay was constructed for detection of PGA in plasma at concentrations of less than 1 ng PGA/ml. Use of the lateral flow immunoassay for detection of PGA in the rabbit model found that antigen was detected somewhat earlier than the earliest time point at which the blood culture became positive. The low cost, ease of use, and rapid time to result of the lateral flow immunoassay format make an immunoassay for PGA a viable surrogate for blood culture for detection of infection in individuals who have a likelihood of exposure to B. anthracis. PMID:25942409

  11. Regulation of ribonucleotide reductase by Spd1 involves multiple mechanisms

    DEFF Research Database (Denmark)

    Nestoras, Konstantinos; Mohammed, Asma Hadi; Schreurs, Ann-Sofie;

    2010-01-01

    The correct levels of deoxyribonucleotide triphosphates and their relative abundance are important to maintain genomic integrity. Ribonucleotide reductase (RNR) regulation is complex and multifaceted. RNR is regulated allosterically by two nucleotide-binding sites, by transcriptional control, and...

  12. Structural features of the ribonucleotide reductase of Aujeszky's disease virus.

    Science.gov (United States)

    Kaliman, A V; Boldogköi, Z; Fodor, I

    1994-01-01

    A gene construct of the Aujeszky's disease virus (ADV) genome was prepared and the DNA fragment encoding the ribonucleotide reductase was structurally characterized. We determined the entire DNA sequence of two adjacent open reading frames of the ribonucleotide reductase genes with the intergenic sequence of nine base pairs. From the sequence analysis we predict that Aujeszky's disease virus encodes a ribonucleotide reductase which comprises two polypeptides--large and small subunits, with sizes of 835 and 303 amino acids, respectively. Nucleotide and amino acid sequences of the large and small subunits of the Aujeszky's disease virus ribonucleotide reductase have been compared with that of other herpesviruses, and structural features of both proteins have been characterized. PMID:7810419

  13. Aldose reductase inhibitory activity and antioxidant capacity of pomegranate extracts

    OpenAIRE

    Karasu, Çimen; CUMAOĞLU, Ahmet; Gürpinar, Ali Rifat; Kartal, Murat; Kovacikova, Lucia; Milackova, Ivana; Stefek, Milan

    2012-01-01

    The pomegranate, Punica granatum L., has been the subject of current interest as a medicinal agent with wide-ranging therapeutic indications. In the present study, pomegranate ethanolic seed and hull extracts were tested, in comparison with a commercial sample, for the inhibition of aldose reductase, an enzyme involved in the etiology of diabetic complications. In vitro inhibition of rat lens aldose reductase was determined by a conventional method. Pomegranate ethanolic hull extract and comm...

  14. An overview on 5alpha-reductase inhibitors.

    Science.gov (United States)

    Aggarwal, Saurabh; Thareja, Suresh; Verma, Abhilasha; Bhardwaj, Tilak Raj; Kumar, Manoj

    2010-02-01

    Benign prostatic hyperplasia (BPH) is the noncancerous proliferation of the prostate gland associated with benign prostatic obstruction and lower urinary tract symptoms (LUTS) such as frequency, hesitancy, urgency, etc. Its prevalence increases with age affecting around 70% by the age of 70 years. High activity of 5alpha-reductase enzyme in humans results in excessive dihydrotestosterone levels in peripheral tissues and hence suppression of androgen action by 5alpha-reductase inhibitors is a logical treatment for BPH as they inhibit the conversion of testosterone to dihydrotestosterone. Finasteride (13) was the first steroidal 5alpha-reductase inhibitor approved by U.S. Food and Drug Administration (USFDA). In human it decreases the prostatic DHT level by 70-90% and reduces the prostatic size. Dutasteride (27) another related analogue has been approved in 2002. Unlike Finasteride, Dutasteride is a competitive inhibitor of both 5alpha-reductase type I and type II isozymes, reduced DHT levels >90% following 1 year of oral administration. A number of classes of non-steroidal inhibitors of 5alpha-reductase have also been synthesized generally by removing one or more rings from the azasteroidal structure or by an early non-steroidal lead (ONO-3805) (261). In this review all categories of inhibitors of 5alpha-reductase have been covered. PMID:19879888

  15. Genetic Comparison of B. Anthracis and its Close Relatives Using AFLP and PCR Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Jackson, P.J.; Hill, K.K.; Laker, M.T.; Ticknor, L.O.; Keim, P.S.

    1999-02-01

    Amplified Fragment length Polymorphism (AFLP) analysis allows a rapid, relatively simple analysis of a large portion of a microbial genome, providing information about the species and its phylogenetic relationship to other microbes (Vos, et al., 1995). The method simply surveys the genome for length and sequence polymorphisms. The pattern identified can be used for comparison to the genomes of other species. Unlike other methods, it does not rely on analysis of a single genetic locus that may bias the interpretation of results and it does not require any prior knowledge of the targeted organism. Moreover, a standard set of reagents can be applied to any species without using species-specific information or molecular probes. The authors are using AFLP's to rapidly identify different bacterial species. A comparison of AFLP profiles generated from a large battery of B. anthracis strains shows very little variability among different isolates (Keim, et al., 1997). By contrast, there is a significant difference between AFLP profiles generated for any B. anthracis strain and even the most closely related Bacillus species. Sufficient variability is apparent among all known microbial species to allow phylogenetic analysis based on large numbers of genetically unlinked loci. These striking differences among AFLP profiles allow unambiguous identification of previously identified species and phylogenetic placement of newly characterized isolates relative to known species based on a large number of independent genetic loci. Data generated thus far show that the method provides phylogenetic analyses that are consistent with other widely accepted phylogenetic methods. However, AFLP analysis provides a more detailed analysis of the targets and samples a much larger portion of the genome. Consequently, it provides an inexpensive, rapid means of characterizing microbial isolates to further differentiate among strains and closely related microbial species. Such information

  16. Aldose reductase mediates retinal microglia activation.

    Science.gov (United States)

    Chang, Kun-Che; Shieh, Biehuoy; Petrash, J Mark

    2016-04-29

    Retinal microglia (RMG) are one of the major immune cells in charge of surveillance of inflammatory responses in the eye. In the absence of an inflammatory stimulus, RMG reside predominately in the ganglion layer and inner or outer plexiform layers. However, under stress RMG become activated and migrate into the inner nuclear layer (INL) or outer nuclear layer (ONL). Activated RMG in cell culture secrete pro-inflammatory cytokines in a manner sensitive to downregulation by aldose reductase inhibitors. In this study, we utilized CX3CR1(GFP) mice carrying AR mutant alleles to evaluate the role of AR on RMG activation and migration in vivo. When tested on an AR(WT) background, IP injection of LPS induced RMG activation and migration into the INL and ONL. However, this phenomenon was largely prevented by AR inhibitors or in AR null mice, or was exacerbated in transgenic mice that over-express AR. LPS-induced increases in ocular levels of TNF-α and CX3CL-1 in WT mice were substantially lower in AR null mice or were reduced by AR inhibitor treatment. These studies demonstrate that AR expression in RMG may contribute to the proinflammatory phenotypes common to various eye diseases such as uveitis and diabetic retinopathy.

  17. Aldose reductase, oxidative stress and diabetic mellitus

    Directory of Open Access Journals (Sweden)

    Waiho eTang

    2012-05-01

    Full Text Available Diabetes mellitus (DM is a complex metabolic disorder arising from lack of insulin production or insulin resistance 1. DM is a leading cause of morbidity and mortality in the developed world, particularly from vascular complications such as atherothrombosis in the coronary vessels. Aldose reductase (AR [ALR2; EC 1.1.1.21], a key enzyme in the polyol pathway, catalyzes NADPH-dependent reduction of glucose to sorbitol, leading to excessive accumulation of intracellular reactive oxygen species (ROS in various tissues of DM including the heart, vasculature, neurons, eyes and kidneys. As an example, hyperglycemia through such polyol pathway induced oxidative stress, may have dual heart actions, on coronary blood vessel (atherothrombosis and myocardium (heart failure leading to severe morbidity and mortality (reviewed in 2. In cells cultured under high glucose conditions, many studies have demonstrated similar AR-dependent increases in ROS production, confirming AR as an important factor for the pathogenesis of many diabetic complications. Moreover, recent studies have shown that AR inhibitors may be able to prevent or delay the onset of cardiovascular complications such as ischemia/reperfusion injury, atherosclerosis and atherothrombosis. In this review, we will focus on describing pivotal roles of AR in the pathogenesis of cardiovascular diseases as well as other diabetic complications, and the potential use of AR inhibitors as an emerging therapeutic strategy in preventing DM complications.

  18. Methylenetetrahydrofolate Reductase Activity and Folate Metabolism

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    Nursen Keser

    2014-04-01

    Full Text Available Folate is a vital B vitamin which is easily water-soluble. It is a natural source which is found in the herbal and animal foods. Folate has important duties in the human metabolism, one of them is the adjustment of the level of plasma homocysteine. Reduction in MTHFR (methylenetetrahydrofolate reductase,which is in charge of the metabolism of homocysteine activity affects the level of homocysteine. Therefore MTHFR is an important enzyme in folate metabolism. Some of the mutations occurring in the MTHFR gene is a risk factor for various diseases and may be caused the hyperhomocysteinemia or the homocystinuria, and they also may lead to metabolic problems. MTHFR is effective in the important pathways such as DNA synthesis, methylation reactions and synthesis of RNA. C677T and A1298C are the most commonly occurring polymorphisms in the gene of MTHFR. The frequency of these polymorphisms show differences in the populations. MTHFR, folate distribution, metabolism of homocysteine and S-adenosylmethionine, by the MTHFR methylation the genetic defects have the potential of affecting the risk of disease in the negative or positive way.

  19. Aldose reductase inhibitory compounds from Xanthium strumarium.

    Science.gov (United States)

    Yoon, Ha Na; Lee, Min Young; Kim, Jin-Kyu; Suh, Hong-Won; Lim, Soon Sung

    2013-09-01

    As part of our ongoing search for natural sources of therapeutic and preventive agents for diabetic complications, we evaluated the inhibitory effects of components of the fruit of Xanthium strumarium (X. strumarium) on aldose reductase (AR) and galactitol formation in rat lenses with high levels of glucose. To identify the bioactive components of X. strumarium, 7 caffeoylquinic acids and 3 phenolic compounds were isolated and their chemical structures were elucidated on the basis of spectroscopic evidence and comparison with published data. The abilities of 10 X. strumarium-derived components to counteract diabetic complications were investigated by means of inhibitory assays with rat lens AR (rAR) and recombinant human AR (rhAR). From the 10 isolated compounds, methyl-3,5-di-O-caffeoylquinate showed the most potent inhibition, with IC₅₀ values of 0.30 and 0.67 μM for rAR and rhAR, respectively. In the kinetic analyses using Lineweaver-Burk plots of 1/velocity and 1/substrate, methyl-3,5-di-O-caffeoylquinate showed competitive inhibition of rhAR. Furthermore, methyl-3,5-di-O-caffeoylquinate inhibited galactitol formation in the rat lens and in erythrocytes incubated with a high concentration of glucose, indicating that this compound may be effective in preventing diabetic complications. PMID:23604720

  20. Cloning, expression, and characterization of recombinant nitric oxide synthase-like protein from Bacillus anthracis

    International Nuclear Information System (INIS)

    Nitric oxide synthase (NOS) is amongst a family of evolutionarily conserved enzymes, involved in a multi-turnover process that results in NO as a product. The significant role of NO in various pathological and physiological processes has created an interest in this enzyme from several perspectives. This study describes for the first time, cloning and expression of a NOS-like protein, baNOS, from Bacillus anthracis, a pathogenic bacterium responsible for causing anthrax. baNOS was expressed in Escherichia coli as a soluble and catalytically active enzyme. Homology models generated for baNOS indicated that the key structural features that are involved in the substrate and active site interaction have been highly conserved. Further, the behavior of baNOS in terms of heme-substrate interactions and heme-transitions was studied in detail. The optical perturbation spectra of the heme domain demonstrated that the ligands perturb the heme site in a ligand specific manner. baNOS forms a five-coordinate, high-spin complex with L-arginine analogs and a six-coordinate low-spin complex with inhibitor imidazole. Studies indicated that the binding of L-arginine, N ω-hydroxy-L-arginine, and imidazole produces various spectroscopic species that closely correspond to the equivalent complexes of mammalian NOS. The values of spectral binding constants further corroborated these results. The overall conservation of the key structural features and the correlation of heme-substrate interactions in baNOS and mammalian NOS, thus, point towards an interesting phenomenon of convergent evolution. Importantly, the NO generated by NOS of mammalian macrophages plays a potent role in antimicrobicidal activity. Because of the existence of high structural and behavioral similarity between mammalian NOS and baNOS, we propose that NO produced by B. anthracis may also have a pivotal pathophysiological role in anthrax infection. Therefore, this first report of characterization of a NOS-like protein

  1. Panning of a phage display library against a synthetic capsule for peptide ligands that bind to the native capsule of Bacillus anthracis.

    Directory of Open Access Journals (Sweden)

    Michael Beer

    Full Text Available Bacillus anthracis is the causative agent of anthrax with the ability to not only produce a tripartite toxin, but also an enveloping capsule comprised primarily of γ-D-glutamic acid residues. The purpose of this study was to isolate peptide ligands capable of binding to the native capsule of B. anthracis from a commercial phage display peptide library using a synthetic form of the capsule consisting of 12 γ-D-glutamic acid residues. Following four rounds of selection, 80 clones were selected randomly and analysed by DNA sequencing. Four clones, each containing a unique consensus sequence, were identified by sequence alignment analysis. Phage particles were prepared and their derived 12-mer peptides were also chemically synthesized and conjugated to BSA. Both the phage particles and free peptide-BSA conjugates were evaluated by ELISA for binding to encapsulated cells of B. anthracis as well as a B. anthracis capsule extract. All the phage particles tested except one were able to bind to both the encapsulated cells and the capsule extract. However, the peptide-BSA conjugates could only bind to the encapsulated cells. One of the peptide-BSA conjugates, with the sequence DSSRIPMQWHPQ (termed G1, was fluorescently labelled and its binding to the encapsulated cells was further confirmed by confocal microscopy. The results demonstrated that the synthetic capsule was effective in isolating phage-displayed peptides with binding affinity for the native capsule of B. anthracis.

  2. Isolation and characterization of cDNAs encoding leucoanthocyanidin reductase and anthocyanidin reductase from Populus trichocarpa.

    Directory of Open Access Journals (Sweden)

    Lijun Wang

    Full Text Available Proanthocyanidins (PAs contribute to poplar defense mechanisms against biotic and abiotic stresses. Transcripts of PA biosynthetic genes accumulated rapidly in response to infection by the fungus Marssonina brunnea f.sp. multigermtubi, treatments of salicylic acid (SA and wounding, resulting in PA accumulation in poplar leaves. Anthocyanidin reductase (ANR and leucoanthocyanidin reductase (LAR are two key enzymes of the PA biosynthesis that produce the main subunits: (+-catechin and (--epicatechin required for formation of PA polymers. In Populus, ANR and LAR are encoded by at least two and three highly related genes, respectively. In this study, we isolated and functionally characterized genes PtrANR1 and PtrLAR1 from P. trichocarpa. Phylogenetic analysis shows that Populus ANR1 and LAR1 occurr in two distinct phylogenetic lineages, but both genes have little difference in their tissue distribution, preferentially expressed in roots. Overexpression of PtrANR1 in poplar resulted in a significant increase in PA levels but no impact on catechin levels. Antisense down-regulation of PtrANR1 showed reduced PA accumulation in transgenic lines, but increased levels of anthocyanin content. Ectopic expression of PtrLAR1 in poplar positively regulated the biosynthesis of PAs, whereas the accumulation of anthocyanin and flavonol was significantly reduced (P<0.05 in all transgenic plants compared to the control plants. These results suggest that both PtrANR1 and PtrLAR1 contribute to PA biosynthesis in Populus.

  3. Transcripts of Anthocyanidin Reductase and Leucoanthocyanidin Reductase and Measurement of Catechin and Epicatechin in Tartary Buckwheat

    Directory of Open Access Journals (Sweden)

    Yeon Bok Kim

    2014-01-01

    Full Text Available Anthocyanidin reductase (ANR and leucoanthocyanidin reductase (LAR play an important role in the monomeric units biosynthesis of proanthocyanidins (PAs such as catechin and epicatechin in several plants. The aim of this study was to clone ANR and LAR genes involved in PAs biosynthesis and examine the expression of these two genes in different organs under different growth conditions in two tartary buckwheat cultivars, Hokkai T8 and T10. Gene expression was carried out by quantitative real-time RT-PCR, and catechin and epicatechin content was analyzed by high performance liquid chromatography. The expression pattern of ANR and LAR did not match the accumulation pattern of PAs in different organs of two cultivars. Epicatechin content was the highest in the flowers of both cultivars and it was affected by light in only Hokkai T8 sprouts. ANR and LAR levels in tartary buckwheat might be regulated by different mechanisms for catechin and epicatechin biosynthesis under light and dark conditions.

  4. Equine 5α-reductase activity and expression in epididymis.

    Science.gov (United States)

    Corbin, C J; Legacki, E L; Ball, B A; Scoggin, K E; Stanley, S D; Conley, A J

    2016-10-01

    The 5α-reductase enzymes play an important role during male sexual differentiation, and in pregnant females, especially equine species where maintenance relies on 5α-reduced progesterone, 5α-dihydroprogesterone (DHP). Epididymis expresses 5α-reductases but was not studied elaborately in horses. Epididymis from younger and older postpubertal stallions was divided into caput, corpus and cauda and examined for 5α-reductase activity and expression of type 1 and 2 isoforms by quantitative real-time polymerase chain reaction (qPCR). Metabolism of progesterone and testosterone to DHP and dihydrotestosterone (DHT), respectively, by epididymal microsomal protein was examined by thin-layer chromatography and verified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Relative inhibitory potencies of finasteride and dutasteride toward equine 5α-reductase activity were investigated. Pregnenolone was investigated as an additional potential substrate for 5α-reductase, suggested previously from in vivo studies in mares but never directly examined. No regional gradient of 5α-reductase expression was observed by either enzyme activity or transcript analysis. Results of PCR experiments suggested that type 1 isoform predominates in equine epididymis. Primers for the type 2 isoform were unable to amplify product from any samples examined. Progesterone and testosterone were readily reduced to DHP and DHT, and activity was effectively inhibited by both inhibitors. Using epididymis as an enzyme source, no experimental evidence was obtained supporting the notion that pregnenolone could be directly metabolized by equine 5α-reductases as has been suggested by previous investigators speculating on alternative metabolic pathways leading to DHP synthesis in placenta during equine pregnancies. PMID:27466384

  5. Rugged single domain antibody detection elements for Bacillus anthracis spores and vegetative cells.

    Directory of Open Access Journals (Sweden)

    Scott A Walper

    Full Text Available Significant efforts to develop both laboratory and field-based detection assays for an array of potential biological threats started well before the anthrax attacks of 2001 and have continued with renewed urgency following. While numerous assays and methods have been explored that are suitable for laboratory utilization, detection in the field is often complicated by requirements for functionality in austere environments, where limited cold-chain facilities exist. In an effort to overcome these assay limitations for Bacillus anthracis, one of the most recognizable threats, a series of single domain antibodies (sdAbs were isolated from a phage display library prepared from immunized llamas. Characterization of target specificity, affinity, and thermal stability was conducted for six sdAb families isolated from rounds of selection against the bacterial spore. The protein target for all six sdAb families was determined to be the S-layer protein EA1, which is present in both vegetative cells and bacterial spores. All of the sdAbs examined exhibited a high degree of specificity for the target bacterium and its spore, with affinities in the nanomolar range, and the ability to refold into functional antigen-binding molecules following several rounds of thermal denaturation and refolding. This research demonstrates the capabilities of these sdAbs and their potential for integration into current and developing assays and biosensors.

  6. The Saccharomyces boulardii CNCM I-745 Strain Shows Protective Effects against the B. anthracis LT Toxin

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    Rodolphe Pontier-Bres

    2015-10-01

    Full Text Available The probiotic yeast Saccharomyces boulardii (S. boulardii has been prescribed for the prophylaxis and treatment of several infectious diarrheal diseases. Gastrointestinal anthrax causes fatal systemic disease. In the present study, we investigated the protective effects conferred by Saccharomyces boulardii CNCM I-745 strain on polarized T84 columnar epithelial cells intoxicated by the lethal toxin (LT of Bacillus anthracis. Exposure of polarized T84 cells to LT affected cell monolayer integrity, modified the morphology of tight junctions and induced the formation of actin stress fibers. Overnight treatment of cells with S. boulardii before incubation with LT maintained the integrity of the monolayers, prevented morphological modification of tight junctions, restricted the effects of LT on actin remodeling and delayed LT-induced MEK-2 cleavage. Mechanistically, we demonstrated that in the presence of S. boulardii, the medium is depleted of both LF and PA sub-units of LT and the appearance of a cleaved form of PA. Our study highlights the potential of the S. boulardii CNCM I-745 strain as a prophylactic agent against the gastrointestinal form of anthrax.

  7. Expression and Purification of the Bacillus anthracis Protective Antigen Receptor-binding Domain

    Institute of Scientific and Technical Information of China (English)

    葛猛; 徐俊杰; 李冰; 董大勇; 宋小红; 郭强; 赵剑; 陈薇

    2004-01-01

    The aim of this study is to express the receptor-binding domain of Bacillus anthracis protective antigen in E. coli. Signal sequence of the outer membrane protein A (OmpA) of E. coli was attached to the 5' end of the gene encoding protective antigen receptor-binding domain (the 4th domain of PA, PALM). The plasmid carrying the fusion gene was then transformed into E. coli and induced to express recombinant PAlM by IFFG. The recombinant protein was purified by chromatography and then identified by N-terrainal sequencing and Western blot. The recombinant protein, about 10% of the total bacterial protein in volume, was secreted to the periplasmic space of the cell. After a purification procedure including ionexchange chromatography and gel filtration, about 10 mg of homogenous recombinant PAD4 was obtained from 1 L culture. Data from N-terminal sequencing suggested that the amino acid sequence of recombinant PAD4 was identical with its natural counterpart. And the result of Western blot showed the recombinant protein could bind with anti-PA serum from rabbit. High level secreted expression of PAD4 was obtained in E. coli. The results reported here are parts of a continuing research to evaluate PAD4 as a potential drug for anthrax therapy or a candidate of new vaccine.

  8. Bacillus anthracis Co-Opts Nitric Oxide and Host Serum Albumin for Pathogenicity in Hypoxic Conditions

    Directory of Open Access Journals (Sweden)

    Stephen eSt John

    2013-05-01

    Full Text Available Bacillus anthracis is a dangerous pathogen of humans and many animal species. Its virulence has been mainly attributed to the production of Lethal and Edema toxins as well as the antiphagocytic capsule. Recent data indicate that the nitric oxide (NO synthase (baNOS plays an important pathogenic role at the early stage of disease by protecting bacteria from the host reactive species and S-nytrosylating the mitochondrial proteins in macrophages. In this study we for the first time present evidence that bacteria-derived NO participates in the generation of highly reactive oxidizing species which could be abolished by the NOS inhibitor L-NAME, free thiols, and superoxide dismutase but not catalase. The formation of toxicants is likely a result of the simultaneous formation of NO and superoxide leading to a labile peroxynitrite and its stable decomposition product, nitrogen dioxide. The toxicity of bacteria could be potentiated in the presence of bovine serum albumin. This effect is consistent with the property of serum albumin to serves as a trap of a volatile NO accelerating its reactions. Our data suggest that during infection in the hypoxic environment of pre-mortal host the accumulated NO is expected to have a broad toxic impact on host cell functions.

  9. In Silico Genomic Fingerprints of the Bacillus anthracis Group Obtained by Virtual Hybridization

    Directory of Open Access Journals (Sweden)

    Hueman Jaimes-Díaz

    2015-02-01

    Full Text Available In this study we evaluate the capacity of Virtual Hybridization to identify between highly related bacterial strains. Eight genomic fingerprints were obtained by virtual hybridization for the Bacillus anthracis genome set, and a set of 15,264 13-nucleotide short probes designed to produce genomic fingerprints unique for each organism. The data obtained from each genomic fingerprint were used to obtain hybridization patterns simulating a DNA microarray. Two virtual hybridization methods were used: the Direct and the Extended method to identify the number of potential hybridization sites and thus determine the minimum sensitivity value to discriminate between genomes with 99.9% similarity. Genomic fingerprints were compared using both methods and phylogenomic trees were constructed to verify that the minimum detection value is 0.000017. Results obtained from the genomic fingerprints suggest that the distribution in the trees is correct, as compared to other taxonomic methods. Specific virtual hybridization sites for each of the genomes studied were also identified.

  10. Soluble Expression and Characterization of Biologically Active Bacillus anthracis Protective Antigen in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Nagendra Suryanarayana

    2016-01-01

    Full Text Available Bacillus anthracis secretory protein protective antigen (PA is primary candidate for subunit vaccine against anthrax. Attempts to obtain large quantity of PA from Escherichia coli expression system often result in the formation of insoluble inclusion bodies. Therefore, it is always better to produce recombinant proteins in a soluble form. In the present study, we have obtained biologically active recombinant PA in small scale E. coli shake culture system using three different expression constructs. The PA gene was cloned in expression vectors bearing trc, T5, and T7 promoters and transformed into their respective E. coli hosts. The growth conditions were optimized to obtain maximum expression of PA in soluble form. The expression construct PA-pET32c in DE3-pLysS E. coli host resulted in a maximum production of soluble PA (15 mg L−1 compared to other combinations. Purified PA was subjected to trypsin digestion and binding assay with lethal factor to confirm the protein’s functionality. Biological activity was confirmed by cytotoxicity assay on J774.1 cells. Balb/c mice were immunized with PA and the immunogenicity was tested by ELISA and toxin neutralization assay. This study highlights the expression of soluble and biologically active recombinant PA in larger quantity using simpler E. coli production platform.

  11. Intranasal immunization with protective antigen of Bacillus anthracis induces a long-term immunological memory response.

    Science.gov (United States)

    Woo, Sun-Je; Kang, Seok-Seong; Park, Sung-Moo; Yang, Jae Seung; Song, Man Ki; Yun, Cheol-Heui; Han, Seung Hyun

    2015-10-01

    Although intranasal vaccination has been shown to be effective for the protection against inhalational anthrax, establishment of long-term immunity has yet to be achieved. Here, we investigated whether intranasal immunization with recombinant protective antigen (rPA) of Bacillus anthracis induces immunological memory responses in the mucosal and systemic compartments. Intranasal immunization with rPA plus cholera toxin (CT) sustained PA-specific antibody responses for 6 months in lung, nasal washes, and vaginal washes as well as serum. A significant induction of PA-specific memory B cells was observed in spleen, cervical lymph nodes (CLNs) and lung after booster immunization. Furthermore, intranasal immunization with rPA plus CT remarkably generated effector memory CD4(+) T cells in the lung. PA-specific CD4(+) T cells preferentially increased the expression of Th1- and Th17-type cytokines in lung, but not in spleen or CLNs. Collectively, the intranasal immunization with rPA plus CT promoted immunologic memory responses in the mucosal and systemic compartments, providing long-term immunity. PMID:26278659

  12. Soluble Expression and Characterization of Biologically Active Bacillus anthracis Protective Antigen in Escherichia coli.

    Science.gov (United States)

    Suryanarayana, Nagendra; Vanlalhmuaka; Mankere, Bharti; Verma, Monika; Thavachelvam, Kulanthaivel; Tuteja, Urmil

    2016-01-01

    Bacillus anthracis secretory protein protective antigen (PA) is primary candidate for subunit vaccine against anthrax. Attempts to obtain large quantity of PA from Escherichia coli expression system often result in the formation of insoluble inclusion bodies. Therefore, it is always better to produce recombinant proteins in a soluble form. In the present study, we have obtained biologically active recombinant PA in small scale E. coli shake culture system using three different expression constructs. The PA gene was cloned in expression vectors bearing trc, T5, and T7 promoters and transformed into their respective E. coli hosts. The growth conditions were optimized to obtain maximum expression of PA in soluble form. The expression construct PA-pET32c in DE3-pLysS E. coli host resulted in a maximum production of soluble PA (15 mg L(-1)) compared to other combinations. Purified PA was subjected to trypsin digestion and binding assay with lethal factor to confirm the protein's functionality. Biological activity was confirmed by cytotoxicity assay on J774.1 cells. Balb/c mice were immunized with PA and the immunogenicity was tested by ELISA and toxin neutralization assay. This study highlights the expression of soluble and biologically active recombinant PA in larger quantity using simpler E. coli production platform. PMID:26966576

  13. First detection of Bacillus anthracis in feces of free-ranging raptors from central Argentina.

    Science.gov (United States)

    Saggese, Miguel D; Noseda, Ramón P; Uhart, Marcela M; Deem, Sharon L; Ferreyra, Hebe; Romano, Marcelo C; Ferreyra-Armas, María C; Hugh-Jones, Martin

    2007-01-01

    Prevalence of anthrax spores in feces of raptors was determined from samples collected in November-December 2000 and April-May 2001 in an agricultural region of Santa Fé province, Argentina. Feces were tested from 48 birds of six raptor species. One of 14 chimango caracaras (Milvago chimango) and one of eight road-side hawks (Buteo magnirostris) tested positive. The prevalence of Bacillus anthracis spores in feces for the six species was 4% (n=48). The prevalence was 7% (n=14) for chimango caracaras, 13% for road-side hawks (n=8), and 0% for the remaining species (Burrowing owl [Speotyto cunicularia] [n=17], Swainson's hawk [Buteo swainsoni] [n=3], Aplomado falcon [Falco femoralis] [n=2], and American kestrel [Falco sparverius] [n=4]). Grouped by their feeding habits, prevalence for scavenger species was not significantly different than for predators (7% vs. 3%, P>0.999). This study provides evidence that in central Argentina scavenger and non-scavenger raptors may have a role in the epidemiology of anthrax. Long-term studies to determine the extent of this potential involvement in the epidemiology of anthrax in central Argentina are required.

  14. A poly-γ-(D)-glutamic acid depolymerase that degrades the protective capsule of Bacillus anthracis.

    Science.gov (United States)

    Negus, David; Taylor, Peter W

    2014-03-01

    A mixed culture of Pseudomonas fluorescens and Pusillimonas noertemanii, obtained by soil enrichment, elaborated an enzyme (EnvD) which rapidly hydrolysed poly-γ-d-glutamic acid (PDGA), the constituent of the anti-phagocytic capsule conferring virulence on Bacillus anthracis. The EnvD gene is carried on the P. noertemanii genome but co-culture is required for the elaboration of PDGA depolymerase activity. EnvD showed strong sequence homology to dienelactone hydrolases from other Gram-negative bacteria, possessed no general protease activity but cleaved γ-links in both d- and l-glutamic acid-containing polymers. The stability at 37°C was markedly superior to that of CapD, a γ-glutamyltranspeptidase with PDGA depolymerase activity. Recombinant EnvD was recovered from inclusion bodies in soluble form from an Escherichia coli expression vector and the enzyme stripped the PDGA capsule from the surface of B. anthracis Pasteur within 5 min. We conclude from this in vitro study that rEnvD shows promise as a potential therapeutic for the treatment of anthrax. PMID:24428662

  15. Measurements of the Ultraviolet Fluorescence Cross Sections and Spectra of Bacillus Anthracis Simulants

    Energy Technology Data Exchange (ETDEWEB)

    Stephens, J.R.

    1998-09-01

    Measurements of the ultraviolet autofluorescence spectra and absolute cross sections of the Bacillus anthracis (Ba) simulants Bacillus globigii (Bg), Bacillus megaterium (Bm), Bacillus subtilis (Bs), and Bacillus cereus (Bc) were measured. Fluorescence spectra and cross sections of pine pollen (Pina echinata) were measured for comparison. Both dried vegetative cells and spores separated from the sporulated vegetative material were studied. The spectra were obtained by suspending a small number (<10) of particles in air in our Single Particle Spectroscopy Apparatus (SPSA), illuminating the particles with light from a spectrally filtered arc lamp, and measuring the fluorescence spectra of the particles. The illumination was 280 nm (20 nm FWHM) and the fluorescence spectra was measured between 300 and 450 nm. The fluorescence cross section of vegetative Bg peaks at 320 nm with a maximum cross section of 5 X 10{sup -14} cm{sup 2}/sr-nm-particle while the Bg spore fluorescence peaks at 310 nm with peak fluorescence of 8 X 10{sup -15} cm{sup 2}/sr-nm-particle. Pine pollen particles showed a higher fluorescence peaking at 355 nm with a cross section of 1.7 X 10{sup -13} cm{sup 2}/sr-nm-particle. Integrated cross sections ranged from 3.0 X 10{sup -13} for the Bg spores through 2.25 X 10{sup -12} (cm{sup 2}/sr-particle) for the vegetative cells.

  16. Discrimination of Bacillus anthracis from closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microchips

    Science.gov (United States)

    Bavykin, Sergei G.; Mirzabekov, Andrei D.

    2007-10-30

    The present invention is directed to a novel method of discriminating a highly infectious bacterium Bacillus anthracis from a group of closely related microorganisms. Sequence variations in the 16S and 23S rRNA of the B. cereus subgroup including B. anthracis are utilized to construct an array that can detect these sequence variations through selective hybridizations. The identification and analysis of these sequence variations enables positive discrimination of isolates of the B. cereus group that includes B. anthracis. Discrimination of single base differences in rRNA was achieved with a microchip during analysis of B. cereus group isolates from both single and in mixed probes, as well as identification of polymorphic sites. Successful use of a microchip to determine the appropriate subgroup classification using eight reference microorganisms from the B. cereus group as a study set, was demonstrated.

  17. Possible Use of Bacteriophages Active against Bacillus anthracis and Other B. cereus Group Members in the Face of a Bioterrorism Threat

    Science.gov (United States)

    Weber-Dąbrowska, Beata; Borysowski, Jan; Górski, Andrzej

    2014-01-01

    Anthrax is an infectious fatal disease with epidemic potential. Nowadays, bioterrorism using Bacillus anthracis is a real possibility, and thus society needs an effective weapon to neutralize this threat. The pathogen may be easily transmitted to human populations. It is easy to store, transport, and disseminate and may survive for many decades. Recent data strongly support the effectiveness of bacteriophage in treating bacterial diseases. Moreover, it is clear that bacteriophages should be considered a potential incapacitative agent against bioterrorism using bacteria belonging to B. cereus group, especially B. anthracis. Therefore, we have reviewed the possibility of using bacteriophages active against Bacillus anthracis and other species of the B. cereus group in the face of a bioterrorism threat. PMID:25247187

  18. Possible Use of Bacteriophages Active against Bacillus anthracis and Other B. cereus Group Members in the Face of a Bioterrorism Threat

    Directory of Open Access Journals (Sweden)

    Ewa Jończyk-Matysiak

    2014-01-01

    Full Text Available Anthrax is an infectious fatal disease with epidemic potential. Nowadays, bioterrorism using Bacillus anthracis is a real possibility, and thus society needs an effective weapon to neutralize this threat. The pathogen may be easily transmitted to human populations. It is easy to store, transport, and disseminate and may survive for many decades. Recent data strongly support the effectiveness of bacteriophage in treating bacterial diseases. Moreover, it is clear that bacteriophages should be considered a potential incapacitative agent against bioterrorism using bacteria belonging to B. cereus group, especially B. anthracis. Therefore, we have reviewed the possibility of using bacteriophages active against Bacillus anthracis and other species of the B. cereus group in the face of a bioterrorism threat.

  19. Possible use of bacteriophages active against Bacillus anthracis and other B. cereus group members in the face of a bioterrorism threat.

    Science.gov (United States)

    Jończyk-Matysiak, Ewa; Kłak, Marlena; Weber-Dąbrowska, Beata; Borysowski, Jan; Górski, Andrzej

    2014-01-01

    Anthrax is an infectious fatal disease with epidemic potential. Nowadays, bioterrorism using Bacillus anthracis is a real possibility, and thus society needs an effective weapon to neutralize this threat. The pathogen may be easily transmitted to human populations. It is easy to store, transport, and disseminate and may survive for many decades. Recent data strongly support the effectiveness of bacteriophage in treating bacterial diseases. Moreover, it is clear that bacteriophages should be considered a potential incapacitative agent against bioterrorism using bacteria belonging to B. cereus group, especially B. anthracis. Therefore, we have reviewed the possibility of using bacteriophages active against Bacillus anthracis and other species of the B. cereus group in the face of a bioterrorism threat.

  20. 4-Dimethylaminoazobenzenes: carcinogenicities and reductive cleavage by microsomal azo reductase.

    Science.gov (United States)

    Lambooy, J P; Koffman, B M

    1985-01-01

    Twenty-four 4-dimethylaminoazobenzenes (DABs) in which systematic structural modifications have been made in the prime ring have been studied for substrate specificity for microsomal azo reductase. The DABs were also evaluated for carcinogenicity and it was found that there was no correlation between carcinogenicity and extent of azo bond cleavage by azo reductase. While any substituent in the prime ring reduces the rate of cleavage of the azo bond relative to the unsubstituted dye, there is a correlation between substituent size and susceptibility to the enzyme. Substituent size was also found to be a significant factor in the induction of hepatomas by the dyes. Preliminary studies have shown that there appears to be a positive correlation between microsomal riboflavin content and the activity of the azo reductase.

  1. Intramolecular electron transfer in Pseudomonas aeruginosa cd(1) nitrite reductase

    DEFF Research Database (Denmark)

    Farver, Ole; Brunori, Maurizio; Cutruzzolà, Francesca;

    2009-01-01

    The cd(1) nitrite reductases, which catalyze the reduction of nitrite to nitric oxide, are homodimers of 60 kDa subunits, each containing one heme-c and one heme-d(1). Heme-c is the electron entry site, whereas heme-d(1) constitutes the catalytic center. The 3D structure of Pseudomonas aeruginosa...... nitrite reductase has been determined in both fully oxidized and reduced states. Intramolecular electron transfer (ET), between c and d(1) hemes is an essential step in the catalytic cycle. In earlier studies of the Pseudomonas stutzeri enzyme, we observed that a marked negative cooperativity...... is controlling this internal ET step. In this study we have investigated the internal ET in the wild-type and His369Ala mutant of P. aeruginosa nitrite reductases and have observed similar cooperativity to that of the Pseudomonas stutzeri enzyme. Heme-c was initially reduced, in an essentially diffusion...

  2. Development and field testing of a mobile chlorine dioxide generation system for the decontamination of buildings contaminated with Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Wood, Joseph P., E-mail: wood.joe@epa.gov [U.S. Environmental Protection Agency, Office of Research and Development, National Homeland Security Research Center, MC-E343-06, Research Triangle Park, NC 27711 (United States); Blair Martin, G., E-mail: martin.blair@epa.gov [U.S. Environmental Protection Agency, Office of Research and Development, National Risk Management Research Laboratory, MC-E340-C, Research Triangle Park, NC 27711 (United States)

    2009-05-30

    The numerous buildings that became contaminated with Bacillus anthracis (the bacterium causing the disease anthrax) in 2001, and more recent B. anthracis - related events, point to the need to have effective decontamination technologies for buildings contaminated with biological threat agents. The U.S. Government developed a portable chlorine dioxide (ClO{sub 2}) generation system to decontaminate buildings contaminated with B. anthracis spores, and this so-called mobile decontamination trailer (MDT) prototype was tested through a series of three field trials. The first test of the MDT was conducted at Fort McClellan in Anniston, AL. during October 2004. Four test attempts occurred over two weekends; however, a number of system problems resulted in termination of the activity prior to any ClO{sub 2} introduction into the test building. After making several design enhancements and equipment changes, the MDT was subjected to a second test. During this test, extensive leak checks were made using argon and nitrogen in lieu of chlorine gas; each subsystem was checked for functionality, and the MDT was operated for 24 h. This second test demonstrated the MDT flow and control systems functioned satisfactorily, and thus it was decided to proceed to a third, more challenging field trial. In the last field test, ClO{sub 2} was generated and routed directly to the scrubber in a 12-h continuous run. Measurement of ClO{sub 2} levels at the generator outlet showed that the desired production rate was not achieved. Additionally, only one of the two scrubbers performed adequately with regard to maintaining ClO{sub 2} emissions below the limit. Numerous lessons were learned in the field trials of this ClO{sub 2} decontamination technology.

  3. Development and field testing of a mobile chlorine dioxide generation system for the decontamination of buildings contaminated with Bacillus anthracis

    International Nuclear Information System (INIS)

    The numerous buildings that became contaminated with Bacillus anthracis (the bacterium causing the disease anthrax) in 2001, and more recent B. anthracis - related events, point to the need to have effective decontamination technologies for buildings contaminated with biological threat agents. The U.S. Government developed a portable chlorine dioxide (ClO2) generation system to decontaminate buildings contaminated with B. anthracis spores, and this so-called mobile decontamination trailer (MDT) prototype was tested through a series of three field trials. The first test of the MDT was conducted at Fort McClellan in Anniston, AL. during October 2004. Four test attempts occurred over two weekends; however, a number of system problems resulted in termination of the activity prior to any ClO2 introduction into the test building. After making several design enhancements and equipment changes, the MDT was subjected to a second test. During this test, extensive leak checks were made using argon and nitrogen in lieu of chlorine gas; each subsystem was checked for functionality, and the MDT was operated for 24 h. This second test demonstrated the MDT flow and control systems functioned satisfactorily, and thus it was decided to proceed to a third, more challenging field trial. In the last field test, ClO2 was generated and routed directly to the scrubber in a 12-h continuous run. Measurement of ClO2 levels at the generator outlet showed that the desired production rate was not achieved. Additionally, only one of the two scrubbers performed adequately with regard to maintaining ClO2 emissions below the limit. Numerous lessons were learned in the field trials of this ClO2 decontamination technology.

  4. Rapid focused sequencing: a multiplexed assay for simultaneous detection and strain typing of Bacillus anthracis, Francisella tularensis, and Yersinia pestis.

    Directory of Open Access Journals (Sweden)

    Rosemary S Turingan

    Full Text Available 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

  5. Mitochondrial Thioredoxin-Glutathione Reductase from Larval Taenia crassiceps (Cysticerci

    Directory of Open Access Journals (Sweden)

    Alberto Guevara-Flores

    2010-01-01

    Full Text Available Mitochondrial thioredoxin-glutathione reductase was purified from larval Taenia crassiceps (cysticerci. The preparation showed NADPH-dependent reductase activity with either thioredoxin or GSSG, and was able to perform thiol/disulfide exchange reactions. At 25∘C specific activities were 437  ±  27 mU mg-1 and 840  ±  49 mU mg-1 with thioredoxin and GSSG, respectively. Apparent Km values were 0.87  ±  0.04  μM, 41  ±  6  μM and 19  ±  10  μM for thioredoxin, GSSG and NADPH, respectively. Thioredoxin from eukaryotic sources was accepted as substrate. The enzyme reduced H2O2 in a NADPH-dependent manner, although with low catalytic efficiency. In the presence of thioredoxin, mitochondrial TGR showed a thioredoxin peroxidase-like activity. All disulfide reductase activities were inhibited by auranofin, suggesting mTGR is dependent on selenocysteine. The reductase activity with GSSG showed a higher dependence on temperature as compared with the DTNB reductase activity. The variation of the GSSG- and DTNB reductase activities on pH was dependent on the disulfide substrate. Like the cytosolic isoform, mTGR showed a hysteretic kinetic behavior at moderate or high GSSG concentrations, but it was less sensitive to calcium. The enzyme was able to protect glutamine synthetase from oxidative inactivation, suggesting that mTGR is competent to contend with oxidative stress.

  6. 炭疽杆菌鞭毛的初步研究%The Preliminary Studies on Flagellum of Bacillus anthracis

    Institute of Scientific and Technical Information of China (English)

    梁旭东

    1999-01-01

    通过半固体扩散生长法发现中国炭疽杆菌(Bacillus anthracis)大多数具有鞭毛,并经各种经典方法和分子生物学方法证实了这一结果,提示具有鞭毛是中国炭疽杆菌的一种特征,且不排除国外也有这种特征的菌株.

  7. A tandem repeats database for bacterial genomes: application to the genotyping of Yersinia pestis and Bacillus anthracis

    Directory of Open Access Journals (Sweden)

    Denoeud France

    2001-03-01

    Full Text Available Abstract Background Some pathogenic bacteria are genetically very homogeneous, making strain discrimination difficult. In the last few years, tandem repeats have been increasingly recognized as markers of choice for genotyping a number of pathogens. The rapid evolution of these structures appears to contribute to the phenotypic flexibility of pathogens. The availability of whole-genome sequences has opened the way to the systematic evaluation of tandem repeats diversity and application to epidemiological studies. Results This report presents a database (http://minisatellites.u-psud.fr of tandem repeats from publicly available bacterial genomes which facilitates the identification and selection of tandem repeats. We illustrate the use of this database by the characterization of minisatellites from two important human pathogens, Yersinia pestis and Bacillus anthracis. In order to avoid simple sequence contingency loci which may be of limited value as epidemiological markers, and to provide genotyping tools amenable to ordinary agarose gel electrophoresis, only tandem repeats with repeat units at least 9 bp long were evaluated. Yersinia pestis contains 64 such minisatellites in which the unit is repeated at least 7 times. An additional collection of 12 loci with at least 6 units, and a high internal conservation were also evaluated. Forty-nine are polymorphic among five Yersinia strains (twenty-five among three Y. pestis strains. Bacillus anthracis contains 30 comparable structures in which the unit is repeated at least 10 times. Half of these tandem repeats show polymorphism among the strains tested. Conclusions Analysis of the currently available bacterial genome sequences classifies Bacillus anthracis and Yersinia pestis as having an average (approximately 30 per Mb density of tandem repeat arrays longer than 100 bp when compared to the other bacterial genomes analysed to date. In both cases, testing a fraction of these sequences for

  8. Enhanced Immune Response to DNA Vaccine Encoding Bacillus anthracis PA-D4 Protects Mice against Anthrax Spore Challenge

    OpenAIRE

    Kim, Na Young; Chang, Dong Suk; Kim, Yeonsu; Kim, Chang Hwan; Hur, Gyeung Haeng; Yang, Jai Myung; Shin, Sungho

    2015-01-01

    Anthrax has long been considered the most probable bioweapon-induced disease. The protective antigen (PA) of Bacillus anthracis plays a crucial role in the pathogenesis of anthrax. In the current study, we evaluated the efficiency of a genetic vaccination with the fourth domain (D4) of PA, which is responsible for initial binding of the anthrax toxin to the cellular receptor. The eukaryotic expression vector was designed with the immunoglobulin M (IgM) signal sequence encoding for PA-D4, whic...

  9. Genotyping of Bacillus anthracis strains based on automated capillary 25-loci Multiple Locus Variable-Number Tandem Repeats Analysis

    Directory of Open Access Journals (Sweden)

    Ciervo Alessandra

    2006-04-01

    Full Text Available Abstract Background The genome of Bacillus anthracis, the etiological agent of anthrax, is highly monomorphic which makes differentiation between strains difficult. A Multiple Locus Variable-number tandem repeats (VNTR Analysis (MLVA assay based on 20 markers was previously described. It has considerable discrimination power, reproducibility, and low cost, especially since the markers proposed can be typed by agarose-gel electrophoresis. However in an emergency situation, faster genotyping and access to representative databases is necessary. Results Genotyping of B. anthracis reference strains and isolates from France and Italy was done using a 25 loci MLVA assay combining 21 previously described loci and 4 new ones. DNA was amplified in 4 multiplex PCR reactions and the length of the resulting 25 amplicons was estimated by automated capillary electrophoresis. The results were reproducible and the data were consistent with other gel based methods once differences in mobility patterns were taken into account. Some alleles previously unresolved by agarose gel electrophoresis could be resolved by capillary electrophoresis, thus further increasing the assay resolution. One particular locus, Bams30, is the result of a recombination between a 27 bp tandem repeat and a 9 bp tandem repeat. The analysis of the array illustrates the evolution process of tandem repeats. Conclusion In a crisis situation of suspected bioterrorism, standardization, speed and accuracy, together with the availability of reference typing data are important issues, as illustrated by the 2001 anthrax letters event. In this report we describe an upgrade of the previously published MLVA method for genotyping of B. anthracis and apply the method to the typing of French and Italian B. anthracis strain collections. The increased number of markers studied compared to reports using only 8 loci greatly improves the discrimination power of the technique. An Italian strain belonging to the

  10. Effect of vanadium on nitrate reductase activity in tomato leaves

    OpenAIRE

    J. Buczek

    2015-01-01

    The activity of nitrate reductase in cell-free extracts from tomato leaves is completely inhibited by 100 μM NaVO3 or VOCl2. In experiments in vivo vanadium ions inhibit the activity of the enzyme in 50 to 60 per cent. Addition of l mM vanadium to the medium on which tomato seedlings are grown causes after 24 h almost complete inhibition of nitrate reductase activity in cell-free extracts of the enzyme. Inhibition with vanadium may be abolished in experiments in vitro if the extract is treate...

  11. The intramolecular electron transfer between copper sites of nitrite reductase

    DEFF Research Database (Denmark)

    Farver, O; Eady, R R; Abraham, Z H;

    1998-01-01

    The intramolecular electron transfer (ET) between the type 1 Cu(I) and the type 2 Cu(II) sites of Alcaligenes xylosoxidans dissimilatory nitrite reductase (AxNiR) has been studied in order to compare it with the analogous process taking place in ascorbate oxidase (AO). This internal process is......(I) and the trinuclear copper centre in ascorbate oxidase, and the characteristics of the internal ET processes of these enzymes are compared. The data are consistent with the faster ET observed in nitrite reductase arising from a more advantageous entropy of activation when compared with ascorbate...

  12. Revisiting the Concept of Targeting Only Bacillus anthracis Toxins as a Treatment for Anthrax.

    Science.gov (United States)

    Glinert, Itai; Bar-David, Elad; Sittner, Assa; Weiss, Shay; Schlomovitz, Josef; Ben-Shmuel, Amir; Mechaly, Adva; Altboum, Zeev; Kobiler, David; Levy, Haim

    2016-08-01

    Protective antigen (PA)-based vaccines are effective in preventing the development of fatal anthrax disease both in humans and in relevant animal models. The Bacillus anthracis toxins lethal toxin (lethal factor [LF] plus PA) and edema toxin (edema factor [EF] plus PA) are essential for the establishment of the infection, as inactivation of these toxins results in attenuation of the pathogen. Since the toxins reach high toxemia levels at the bacteremic stages of the disease, the CDC's recommendations include combining antibiotic treatment with antitoxin (anti-PA) immunotherapy. We demonstrate here that while treatment with a highly potent neutralizing monoclonal antibody was highly efficient as postexposure prophylaxis treatment, it failed to protect rabbits with any detectable bacteremia (≥10 CFU/ml). In addition, we show that while PA vaccination was effective against a subcutaneous spore challenge, it failed to protect rabbits against systemic challenges (intravenous injection of vegetative bacteria) with the wild-type Vollum strain or a toxin-deficient mutant. To test the possibility that additional proteins, which are secreted by the bacteria under pathogenicity-stimulating conditions in vitro, may contribute to the vaccine's potency, we immunized rabbits with a secreted protein fraction from a toxin-null mutant. The antiserum raised against the secreted fraction reacts with the bacteria in an immunofluorescence assay. Immunization with the secreted protein fraction did not protect the rabbits against a systemic challenge with the fully pathogenic bacteria. Full protection was obtained only by a combined vaccination with PA and the secreted protein fraction. Therefore, these results indicate that an effective antiserum treatment in advanced stages of anthrax must include toxin-neutralizing antibodies in combination with antibodies against bacterial cell targets.

  13. Composite sampling of a Bacillus anthracis surrogate with cellulose sponge surface samplers from a nonporous surface.

    Directory of Open Access Journals (Sweden)

    Jenia A M Tufts

    Full Text Available A series of experiments was conducted to explore the utility of composite-based collection of surface samples for the detection of a Bacillus anthracis surrogate using cellulose sponge samplers on a nonporous stainless steel surface. Two composite-based collection approaches were evaluated over a surface area of 3716 cm2 (four separate 929 cm2 areas, larger than the 645 cm2 prescribed by the standard Centers for Disease Control (CDC and Prevention cellulose sponge sampling protocol for use on nonporous surfaces. The CDC method was also compared to a modified protocol where only one surface of the sponge sampler was used for each of the four areas composited. Differences in collection efficiency compared to positive controls and the potential for contaminant transfer for each protocol were assessed. The impact of the loss of wetting buffer from the sponge sampler onto additional surface areas sampled was evaluated. Statistical tests of the results using ANOVA indicate that the collection of composite samples using the modified sampling protocol is comparable to the collection of composite samples using the standard CDC protocol (p  =  0.261. Most of the surface-bound spores are collected on the first sampling pass, suggesting that multiple passes with the sponge sampler over the same surface may be unnecessary. The effect of moisture loss from the sponge sampler on collection efficiency was not significant (p  =  0.720 for both methods. Contaminant transfer occurs with both sampling protocols, but the magnitude of transfer is significantly greater when using the standard protocol than when the modified protocol is used (p<0.001. The results of this study suggest that composite surface sampling, by either method presented here, could successfully be used to increase the surface area sampled per sponge sampler, resulting in reduced sampling times in the field and decreased laboratory processing cost and turn-around times.

  14. Activated protein C ameliorates Bacillus anthracis lethal toxin-induced lethal pathogenesis in rats

    Directory of Open Access Journals (Sweden)

    Kau Jyh-Hwa

    2012-11-01

    Full Text Available Abstract Background Lethal toxin (LT is a major virulence factor of Bacillus anthracis. Sprague Dawley rats manifest pronounced lung edema and shock after LT treatments, resulting in high mortality. The heart failure that is induced by LT has been suggested to be a principal mechanism of lung edema and mortality in rodents. Since LT-induced death occurs more rapidly in rats than in mice, suggesting that other mechanisms in addition to the heart dysfunction may be contributed to the fast progression of LT-induced pathogenesis in rats. Coagulopathy may contribute to circulatory failure and lung injury. However, the effect of LT on coagulation-induced lung dysfunction is unclear. Methods To investigate the involvement of coagulopathy in LT-mediated pathogenesis, the mortality, lung histology and coagulant levels of LT-treated rats were examined. The effects of activated protein C (aPC on LT-mediated pathogenesis were also evaluated. Results Fibrin depositions were detected in the lungs of LT-treated rats, indicating that coagulation was activated. Increased levels of plasma D-dimer and thrombomodulin, and the ameliorative effect of aPC further suggested that the activation of coagulation-fibrinolysis pathways plays a role in LT-mediated pathogenesis in rats. Reduced mortality was associated with decreased plasma levels of D-dimer and thrombomodulin following aPC treatments in rats with LT-mediated pathogenesis. Conclusions These findings suggest that the activation of coagulation in lung tissue contributes to mortality in LT-mediated pathogenesis in rats. In addition, anticoagulant aPC may help to develop a feasible therapeutic strategy.

  15. Revisiting the Concept of Targeting Only Bacillus anthracis Toxins as a Treatment for Anthrax.

    Science.gov (United States)

    Glinert, Itai; Bar-David, Elad; Sittner, Assa; Weiss, Shay; Schlomovitz, Josef; Ben-Shmuel, Amir; Mechaly, Adva; Altboum, Zeev; Kobiler, David; Levy, Haim

    2016-08-01

    Protective antigen (PA)-based vaccines are effective in preventing the development of fatal anthrax disease both in humans and in relevant animal models. The Bacillus anthracis toxins lethal toxin (lethal factor [LF] plus PA) and edema toxin (edema factor [EF] plus PA) are essential for the establishment of the infection, as inactivation of these toxins results in attenuation of the pathogen. Since the toxins reach high toxemia levels at the bacteremic stages of the disease, the CDC's recommendations include combining antibiotic treatment with antitoxin (anti-PA) immunotherapy. We demonstrate here that while treatment with a highly potent neutralizing monoclonal antibody was highly efficient as postexposure prophylaxis treatment, it failed to protect rabbits with any detectable bacteremia (≥10 CFU/ml). In addition, we show that while PA vaccination was effective against a subcutaneous spore challenge, it failed to protect rabbits against systemic challenges (intravenous injection of vegetative bacteria) with the wild-type Vollum strain or a toxin-deficient mutant. To test the possibility that additional proteins, which are secreted by the bacteria under pathogenicity-stimulating conditions in vitro, may contribute to the vaccine's potency, we immunized rabbits with a secreted protein fraction from a toxin-null mutant. The antiserum raised against the secreted fraction reacts with the bacteria in an immunofluorescence assay. Immunization with the secreted protein fraction did not protect the rabbits against a systemic challenge with the fully pathogenic bacteria. Full protection was obtained only by a combined vaccination with PA and the secreted protein fraction. Therefore, these results indicate that an effective antiserum treatment in advanced stages of anthrax must include toxin-neutralizing antibodies in combination with antibodies against bacterial cell targets. PMID:27270276

  16. Computational Fluid Dynamics Modeling of Bacillus anthracis Spore Deposition in Rabbit and Human Respiratory Airways

    Energy Technology Data Exchange (ETDEWEB)

    Kabilan, Senthil; Suffield, Sarah R.; Recknagle, Kurtis P.; Jacob, Rick E.; Einstein, Daniel R.; Kuprat, Andrew P.; Carson, James P.; Colby, Sean M.; Saunders, James H.; Hines, Stephanie; Teeguarden, Justin G.; Straub, Tim M.; Moe, M.; Taft, Sarah; Corley, Richard A.

    2016-09-30

    Three-dimensional computational fluid dynamics and Lagrangian particle deposition models were developed to compare the deposition of aerosolized Bacillus anthracis spores in the respiratory airways of a human with that of the rabbit, a species commonly used in the study of anthrax disease. The respiratory airway geometries for each species were derived from computed tomography (CT) or µCT images. Both models encompassed airways that extended from the external nose to the lung with a total of 272 outlets in the human model and 2878 outlets in the rabbit model. All simulations of spore deposition were conducted under transient, inhalation-exhalation breathing conditions using average species-specific minute volumes. The highest exposure concentration was modeled in the rabbit based upon prior acute inhalation studies. For comparison, human simulation was also conducted at the same concentration. Results demonstrated that regional spore deposition patterns were sensitive to airway geometry and ventilation profiles. Due to the complex airway geometries in the rabbit nose, higher spore deposition efficiency was predicted in the upper conducting airways compared to the human at the same air concentration of anthrax spores. As a result, higher particle deposition was predicted in the conducting airways and deep lung of the human compared to the rabbit lung due to differences in airway branching pattern. This information can be used to refine published and ongoing biokinetic models of inhalation anthrax spore exposures, which currently estimate deposited spore concentrations based solely upon exposure concentrations and inhaled doses that do not factor in species-specific anatomy and physiology.

  17. Examination of serological memory in rabbits injected with Bacillus anthracis protective antigen adsorbed to Alhydrogel

    Directory of Open Access Journals (Sweden)

    Stephen F. Little

    2015-01-01

    Full Text Available Serological memory after inoculation of protective antigen (PA combined with Alhydrogel adjuvant (PA/Alhydrogel was examined in New Zealand white rabbits, an animal model for anthrax. A threshold dose of 0.1 μg of PA/Alhydrogel was identified which resulted in an ELISA titer 2 weeks after a primary immunization of only 0.168 μg anti-PA IgG per ml and a toxin-neutralizing antibody titer (TNA ED50 of 1.8 (n = 40. A significant increase in anti-PA IgG and TNA ED50 titers were measured (p < 0.0001 2 weeks after a booster immunization with 0.1 μg of PA/Alhydrogel at 14 days (n = 10; 40.9 μg anti-PA IgG per ml; 522 TNA ED50 and 28 days (n = 10; 63.8 μg anti-PA IgG per ml; 501 TNA ED50. At this threshold dose of PA/Alhydrogel, protection against an aerosol exposure to Bacillus anthracis Ames spores improved as the booster immunization was administered from 4 days (40% survival, to 8 days (50% survival, and to 12 days (80% survival before challenge. The partial protection of rabbits, even in the absence of protective antibody titers (0.9 μg anti-PA IgG per ml and 26 TNA ED50 when the booster immunization was administered 4 days before challenge, suggested a protective potential for serologic memory.

  18. Development of an inhalational Bacillus anthracis exposure therapeutic model in cynomolgus macaques.

    Science.gov (United States)

    Henning, Lisa N; Comer, Jason E; Stark, Gregory V; Ray, Bryan D; Tordoff, Kevin P; Knostman, Katherine A B; Meister, Gabriel T

    2012-11-01

    Appropriate animal models are required to test medical countermeasures to bioterrorist threats. To that end, we characterized a nonhuman primate (NHP) inhalational anthrax therapeutic model for use in testing anthrax therapeutic medical countermeasures according to the U.S. Food and Drug Administration Animal Rule. A clinical profile was recorded for each NHP exposed to a lethal dose of Bacillus anthracis Ames spores. Specific diagnostic parameters were detected relatively early in disease progression, i.e., by blood culture (∼37 h postchallenge) and the presence of circulating protective antigen (PA) detected by electrochemiluminescence (ECL) ∼38 h postchallenge, whereas nonspecific clinical signs of disease, i.e., changes in body temperature, hematologic parameters (ca. 52 to 66 h), and clinical observations, were delayed. To determine whether the presentation of antigenemia (PA in the blood) was an appropriate trigger for therapeutic intervention, a monoclonal antibody specific for PA was administered to 12 additional animals after the circulating levels of PA were detected by ECL. Seventy-five percent of the monoclonal antibody-treated animals survived compared to 17% of the untreated controls, suggesting that intervention at the onset of antigenemia is an appropriate treatment trigger for this model. Moreover, the onset of antigenemia correlated with bacteremia, and NHPs were treated in a therapeutic manner. Interestingly, brain lesions were observed by histopathology in the treated nonsurviving animals, whereas this observation was absent from 90% of the nonsurviving untreated animals. Our results support the use of the cynomolgus macaque as an appropriate therapeutic animal model for assessing the efficacy of medical countermeasures developed against anthrax when administered after a confirmation of infection. PMID:22956657

  19. Artificial electron donors for nitrate and nitrite reductases usable as mediators in amperometric biosensors

    Energy Technology Data Exchange (ETDEWEB)

    Strehlitz, B. (Umweltforschungszentrum Leipzig-Halle GmbH, Leipzig (Germany)); Gruendig, B. (Institut fuer Chemo- und Biosensorik, Muenster-Roxel (Germany)); Vorlop, K.D. (Bundesforschungsanstalt fuer Landwirtschaft, Braunschweig (Germany). Inst. fuer Technologie); Bartholmes, P. (Witten-Herdecke Univ., Witten (Germany). Inst. fuer Biochemie); Kotte, H. (Umweltforschungszentrum Leipzig-Halle GmbH, Leipzig (Germany)); Stottmeister, U. (Umweltforschungszentrum Leipzig-Halle GmbH, Leipzig (Germany))

    1994-07-01

    Various nitrate and nitrite reductases are capable of accepting electrons from artificial donors. Combining these redox active donors with an amperometric redox electrode which is covered with an immobilized layer of such a nitrate or nitrite reductase, new enzyme sensors can be created for the detection of nitrate or nitrite, respectively. A range of suitable electron donors for nitrate reductases and nitrite reductase from different sources have been selected and characterized by electrochemical methods. (orig.)

  20. Inhibition of Albendazole and Oxfendazole on the Activity of Fumaric Reductase in Cysticercus cellulosae

    Institute of Scientific and Technical Information of China (English)

    GAO Xue-jun; LI Qing-zhang; LI Xia

    2004-01-01

    The activity of fumaric reductase in Cysticercus cellulosae tissue homogenate with albendazole and oxfendazole individually was detected. Results showed that the two kinds of drugs both could inhabite the activity of fumaric reductase. The results indicate that the mechanism of action of benzimidazole carbamate drugs is probably inhabiting the complex of fumaric reductase noncompetently, thus lead to the exhaostion of energy and death.

  1. Thioredoxin and NADP-thioredoxin reductase from cultured carrot cells

    Science.gov (United States)

    Johnson, T. C.; Cao, R. Q.; Kung, J. E.; Buchanan, B. B.

    1987-01-01

    Dark-grown carrot (Daucus carota L.) tissue cultures were found to contain both protein components of the NADP/thioredoxin system--NADP-thioredoxin reductase and the thioredoxin characteristic of heterotrophic systems, thioredoxin h. Thioredoxin h was purified to apparent homogeneity and, like typical bacterial counterparts, was a 12-kdalton (kDa) acidic protein capable of activating chloroplast NADP-malate dehydrogenase (EC 1.1.1.82) more effectively than fructose-1,6-bisphosphatase (EC 3.1.3.11). NADP-thioredoxin reductase (EC 1.6.4.5) was partially purified and found to be an arsenite-sensitive enzyme composed of two 34-kDa subunits. Carrot NADP-thioredoxin reductase resembled more closely its counterpart from bacteria rather than animal cells in acceptor (thioredoxin) specificity. Upon greening of the cells, the content of NADP-thioredoxin-reductase activity, and, to a lesser extent, thioredoxin h decreased. The results confirm the presence of a heterotrophic-type thioredoxin system in plant cells and raise the question of its physiological function.

  2. Bidirectional catalysis by copper-containing nitrite reductase

    NARCIS (Netherlands)

    Wijma, HJ; Canters, GW; de Vries, S; Verbeet, MP

    2004-01-01

    The copper-containing nitrite reductase from Alcaligenes faecalis S-6 was found to catalyze the oxidation of nitric oxide to nitrite, the reverse of its physiological reaction. Thermodynamic and kinetic constants with the physiological electron donor pseudoazurin were determined for both directions

  3. Fatty Acyl-CoA Reductase 1 Deficiency

    Directory of Open Access Journals (Sweden)

    Charles N Swisher

    2015-01-01

    Full Text Available Investigators from Erlangen, Germany; Calgary, CA; and Kafranbel, Syria, identified mutations in the gene, fatty acyl-CoA reductase 1 (FAR1 deficiency, adding to three other genes involved in plasmalogen biosynthesis, in two families affected by severe intellectual disability, early-onset epilepsy, microcephaly, congenital cataracts, growth retardation, and spasticity.

  4. Sepiapterin reductase deficiency an autosomal recessive DOPA-responsive dystonia

    NARCIS (Netherlands)

    N.G. Abeling; M. Duran; H.D. Bakker; L. Stroomer; B. Thony; N. Blau; J. Booij; B.T. Poll-The

    2006-01-01

    The diagnosis of a 14-year-old girl with a new homoallelic mutation in the sepiapterin reductase (SR) gene is reported. Initially she presented at the age of 2 with hypotonia and mild cognitive developmental delay, and was diagnosed as having mild methylmalonic aciduria, which was recently identifie

  5. Loss of σI affects heat-shock response and virulence gene expression in Bacillus anthracis.

    Science.gov (United States)

    Kim, Jenny Gi Yae; Wilson, Adam C

    2016-02-01

    The pathogenesis of Bacillus anthracis depends on several virulence factors, including the anthrax toxin. Loss of the alternative sigma factor σI results in a coordinate decrease in expression of all three toxin subunits. Our observations suggest that loss of σI alters the activity of the master virulence regulator AtxA, but atxA transcription is unaffected by loss of σI. σI-containing RNA polymerase does not appear to directly transcribe either atxA or the toxin gene pagA. As in Bacillus subtilis, loss of σI in B. anthracis results in increased sensitivity to heat shock and transcription of sigI, encoding σI, is induced by elevated temperature. Encoded immediately downstream of and part of a bicistronic message with sigI is an anti-sigma factor, RsgI, which controls σI activity. Loss of RsgI has no direct effect on virulence gene expression. sigI appears to be expressed from both the σI and σA promoters, and transcription from the σA promoter is likely more significant to virulence regulation. We propose a model in which σI can be induced in response to heat shock, whilst, independently, σI is produced under non-heat-shock, toxin-inducing conditions to indirectly regulate virulence gene expression. PMID:26744224

  6. Structural Basis for Latency and Function of Immune Inhibitor A Metallopeptidase, a Modulator of the Bacillus anthracis Secretome.

    Science.gov (United States)

    Arolas, Joan L; Goulas, Theodoros; Pomerantsev, Andrei P; Leppla, Stephen H; Gomis-Rüth, F Xavier

    2016-01-01

    Immune inhibitor A(InhA)-type metallopeptidases are potential virulence factors secreted by members of the Bacillus cereus group. Two paralogs from anthrax-causing Bacillus anthracis (BaInhA1 and BaInhA2) were shown to degrade host tissue proteins with broad substrate specificity. Analysis of their activation mechanism and the crystal structure of a zymogenic BaInhA2 variant revealed a ∼750-residue four-domain structure featuring a pro-peptide, a catalytic domain, a domain reminiscent of viral envelope glycoproteins, and a MAM domain grafted into the latter. This domain, previously found only in eukaryotes, is required for proper protein expression in B. anthracis and evinces certain flexibility. Latency is uniquely modulated by the N-terminal segment of the pro-peptide, which binds the catalytic zinc through its α-amino group and occupies the primed side of the active-site cleft. The present results further our understanding of the modus operandi of an anthrax secretome regulator. PMID:26745529

  7. Bacillus anthracis lethal toxin disrupts TCR signaling in CD1d-restricted NKT cells leading to functional anergy.

    Directory of Open Access Journals (Sweden)

    Sunil K Joshi

    2009-09-01

    Full Text Available Exogenous CD1d-binding glycolipid (alpha-Galactosylceramide, alpha-GC stimulates TCR signaling and activation of type-1 natural killer-like T (NKT cells. Activated NKT cells play a central role in the regulation of adaptive and protective immune responses against pathogens and tumors. In the present study, we tested the effect of Bacillus anthracis lethal toxin (LT on NKT cells both in vivo and in vitro. LT is a binary toxin known to suppress host immune responses during anthrax disease and intoxicates cells by protective antigen (PA-mediated intracellular delivery of lethal factor (LF, a potent metalloprotease. We observed that NKT cells expressed anthrax toxin receptors (CMG-2 and TEM-8 and bound more PA than other immune cell types. A sub-lethal dose of LT administered in vivo in C57BL/6 mice decreased expression of the activation receptor NKG2D by NKT cells but not by NK cells. The in vivo administration of LT led to decreased TCR-induced cytokine secretion but did not affect TCR expression. Further analysis revealed LT-dependent inhibition of TCR-stimulated MAP kinase signaling in NKT cells attributable to LT cleavage of the MAP kinase kinase MEK-2. We propose that Bacillus anthracis-derived LT causes a novel form of functional anergy in NKT cells and therefore has potential for contributing to immune evasion by the pathogen.

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

    Science.gov (United States)

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

    2010-01-01

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

  9. A Spontaneous Translational Fusion of Bacillus cereus PlcR and PapR Activates Transcription of PlcR-Dependent Genes in Bacillus anthracis via Binding with a Specific Palindromic Sequence

    OpenAIRE

    Pomerantsev, Andrei P; Pomerantseva, Olga M.; Stephen H Leppla

    2004-01-01

    Transformation of Bacillus anthracis with plasmid pUTE29-plcR-papR carrying the native Bacillus cereus plcR-papR gene cluster did not activate expression of B. anthracis hemolysin genes, even though these are expected to be responsive to activation by the global regulator PlcR. To further characterize the action of PlcR, we examined approximately 3,000 B. anthracis transformants containing pUTE29-plcR-papR and found a single hemolytic colony. The hemolytic strain contained a plasmid having a ...

  10. Rapid identification of bacillus anthracis spores in suspicious powder samples by using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)

    NARCIS (Netherlands)

    Dybwad, M.; Laaken, A.L. van der; Blatny, J.M.; Paauw, A.

    2013-01-01

    Rapid and reliable identification of Bacillus anthracis spores in suspicious powders is important to mitigate the safety risks and economic burdens associated with such incidents. The aim of this study was to develop and validate a rapid and reliable laboratory- based matrix-assisted laser desorptio

  11. Delineating the effect of host environmental signals on a fully virulent strain of Bacillus anthracis using an integrated transcriptomics and proteomics approach

    NARCIS (Netherlands)

    Panda, G.; Basak, T.; Tanwer, P.; Sengupta, S.; Martins dos Santos, V.A.P.; Bhatnagar, R.

    2014-01-01

    Pathogenic bacteria sense the host environment and regulate expression of virulence-related genes. Environmental signals like temperature, bicarbonate/CO2 and glucose induce toxin production in Bacillus anthracis, but the mechanisms by which these signals contribute to virulence and overall physiolo

  12. Human monoclonal antibodies against anthrax lethal factor and protective antigen act independently to protect against Bacillus anthracis infection and enhance endogenous immunity to anthrax

    NARCIS (Netherlands)

    Albrecht, Mark T.; Li, Han; Williamson, E. Diane; LeButt, Chris S.; Flick-Smith, Helen C.; Quinn, Conrad P.; Westra, Hans; Galloway, Darrell; Mateczun, Alfred; Goldman, Stanley; Groen, Herman; Baillie, Les W. J.

    2007-01-01

    The unpredictable nature of bioterrorism and the absence of real-time detection systems have highlighted the need for an efficient postexposure therapy for Bacillus anthracis infection. One approach is passive immunization through the administration of antibodies that mitigate the biological action

  13. 3-Oxoacyl-[ACP] reductase from oilseed rape (Brassica napus).

    Science.gov (United States)

    Sheldon, P S; Kekwick, R G; Smith, C G; Sidebottom, C; Slabas, A R

    1992-04-01

    3-Oxoacyl-[ACP] reductase (E.C. 1.1.1.100, alternatively known as beta-ketoacyl-[ACP] reductase), a component of fatty acid synthetase has been purified from seeds of rape by ammonium sulphate fractionation, Procion Red H-E3B chromatography, FPLC gel filtration and high performance hydroxyapatite chromatography. The purified enzyme appears on SDS-PAGE as a number of 20-30 kDa components and has a strong tendency to exist in a dimeric form, particularly when dithiothreitol is not present to reduce disulphide bonds. Cleveland mapping and cross-reactivity with antiserum raised against avocado 3-oxoacyl-[ACP] reductase both indicate that the multiple components have similar primary structures. On gel filtration the enzyme appears to have a molecular mass of 120 kDa suggesting that the native structure is tetrameric. The enzyme has a strong preference for the acetoacetyl ester of acyl carrier protein (Km = 3 microM) over the corresponding esters of the model substrates N-acetyl cysteamine (Km = 35 mM) and CoA (Km = 261 microM). It is inactivated by dilution but this can be partly prevented by the inclusion of NADPH. Using an antiserum prepared against avocado 3-oxoacyl-[ACP] reductase, the enzyme has been visualised inside the plastids of rape embryo and leaf tissues by immunoelectron microscopy. Amino acid sequencing of two peptides prepared by digestion of the purified enzyme with trypsin showed strong similarities with 3-oxoacyl-[ACP] reductase from avocado pear and the Nod G gene product from Rhizobium meliloti.

  14. (+)-Pinoresinol/(+)-lariciresinol reductase from Forsythia intermedia. Protein purification, cDNA cloning, heterologous expression and comparison to isoflavone reductase.

    Science.gov (United States)

    Dinkova-Kostova, A T; Gang, D R; Davin, L B; Bedgar, D L; Chu, A; Lewis, N G

    1996-11-15

    Lignans are a widely distributed class of natural products, whose functions and distribution suggest that they are one of the earliest forms of defense to have evolved in vascular plants; some, such as podophyllotoxin and enterodiol, have important roles in cancer chemotherapy and prevention, respectively. Entry into lignan enzymology has been gained by the approximately 3000-fold purification of two isoforms of (+)-pinoresinol/(+)-lariciresinol reductase, a pivotal branchpoint enzyme in lignan biosynthesis. Both have comparable ( approximately 34.9 kDa) molecular mass and kinetic (Vmax/Km) properties and catalyze sequential, NADPH-dependent, stereospecific, hydride transfers where the incoming hydride takes up the pro-R position. The gene encoding (+)-pinoresinol/(+)-lariciresinol reductase has been cloned and the recombinant protein heterologously expressed as a functional beta-galactosidase fusion protein. Its amino acid sequence reveals a strong homology to isoflavone reductase, a key branchpoint enzyme in isoflavonoid metabolism and primarily found in the Fabaceae (angiosperms). This is of great evolutionary significance since both lignans and isoflavonoids have comparable plant defense properties, as well as similar roles as phytoestrogens. Given that lignans are widespread from primitive plants onwards, whereas the isoflavone reductase-derived isoflavonoids are mainly restricted to the Fabaceae, it is tempting to speculate that this branch of the isoflavonoid pathway arose via evolutionary divergence from that giving the lignans.

  15. Crystal structures of pinoresinol-lariciresinol and phenylcoumaran benzylic ether reductases and their relationship to isoflavone reductases.

    Science.gov (United States)

    Min, Tongpil; Kasahara, Hiroyuki; Bedgar, Diana L; Youn, Buhyun; Lawrence, Paulraj K; Gang, David R; Halls, Steven C; Park, HaJeung; Hilsenbeck, Jacqueline L; Davin, Laurence B; Lewis, Norman G; Kang, ChulHee

    2003-12-12

    Despite the importance of plant lignans and isoflavonoids in human health protection (e.g. for both treatment and prevention of onset of various cancers) as well as in plant biology (e.g. in defense functions and in heartwood development), systematic studies on the enzymes involved in their biosynthesis have only recently begun. In this investigation, three NADPH-dependent aromatic alcohol reductases were comprehensively studied, namely pinoresinol-lariciresinol reductase (PLR), phenylcoumaran benzylic ether reductase (PCBER), and isoflavone reductase (IFR), which are involved in central steps to the various important bioactive lignans and isoflavonoids. Of particular interest was in determining how differing regio- and enantiospecificities are achieved with the different enzymes, despite each apparently going through similar enone intermediates. Initially, the three-dimensional x-ray crystal structures of both PLR_Tp1 and PCBER_Pt1 were solved and refined to 2.5 and 2.2 A resolutions, respectively. Not only do they share high gene sequence similarity, but their structures are similar, having a continuous alpha/beta NADPH-binding domain and a smaller substrate-binding domain. IFR (whose crystal structure is not yet obtained) was also compared (modeled) with PLR and PCBER and was deduced to have the same overall basic structure. The basis for the distinct enantio-specific and regio-specific reactions of PCBER, PLR, and IFR, as well as the reaction mechanism and participating residues involved (as identified by site-directed mutagenesis), are discussed.

  16. Microevolution of Anthrax from a Young Ancestor (M.A.Y.A. Suggests a Soil-Borne Life Cycle of Bacillus anthracis.

    Directory of Open Access Journals (Sweden)

    Peter Braun

    Full Text Available During an anthrax outbreak at the Pollino National Park (Basilicata, Italy in 2004, diseased cattle were buried and from these anthrax-foci Bacillus anthracis endospores still diffuse to the surface resulting in local accumulations. Recent data suggest that B. anthracis multiplies in soil outside the animal-host body. This notion is supported by the frequent isolation of B. anthracis from soil lacking one or both virulence plasmids. Such strains represent an evolutionary dead end, as they are likely no longer able to successfully infect new hosts. This loss of virulence plasmids is explained most simply by postulating a soil-borne life cycle of the pathogen. To test this hypothesis we investigated possible microevolution at two natural anthrax foci from the 2004 outbreak. If valid, then genotypes of strains isolated from near the surface at these foci should be on a different evolutionary trajectory from those below residing in deeper-laying horizons close to the carcass. Thus, the genetic diversity of B. anthracis isolates was compared conducting Progressive Hierarchical Resolving Assays using Nucleic Acids (PHRANA and next generation Whole Genome Sequencing (WGS. PHRANA was not discriminatory enough to resolve the fine genetic relationships between the isolates. Conversely, WGS of nine isolates from near-surface and nine from near-carcass revealed five isolate specific SNPs, four of which were found only in different near-surface isolates. In support of our hypothesis, one surface-isolate lacked plasmid pXO1 and also harbored one of the unique SNPs. Taken together, our results suggest a limited soil-borne life cycle of B. anthracis.

  17. Antimicrobial effects of gold/copper sulphide (Gold/Copper monosulfide) core/shell nanoparticles on Bacillus anthracis spores and cells

    Science.gov (United States)

    Addae, Ebenezer

    Bacillus anthracis is a gram positive, rod shaped and spore forming bacteria. It causes anthrax, a deadly human and animal disease that can kill its victims in three days. The spores of B. anthracis can survive extreme environmental conditions for decades and germinate when exposed to proper conditions. Due to its potential as a bio-weapon, effective disinfectants that pose less harm to the environment and animals are urgently needed. Metal nanoparticles have the potential of killing microbial cells and spores. We present here the effect of Gold/Copper Sulphide core/shell (Au/CuS) nanoparticles on B. anthracis cells and spores. The results indicated that the continuous presence of 0.83 microM during the spore growth in nutrient medium completely inhibited spore outgrowth. Au/CuS nanoparticles at concentration of 4.15 μM completely inactivated B. anthracis cells (x 107) after 30 min of pre-treatment in any of the three buffers including water, PBS, and nutrient broth. However, the same and even higher concentrations of nanoparticles produce no significant spore (x 105) killing after 24 h of pre-treatment. SEM imaging, EDS analysis, and DNA extrusion experiments revealed that nanoparticles damaged the cell membrane causing DNA and cytosolic content efflux and eventually cell death. The study demonstrated the strong antimicrobial activity of Au/CuS nanoparticles to B. anthracis cells and revealed that Au/CuS NPs showed more effective inactivation effect against the cells than they did against the spores.

  18. Failure of Sterne- and Pasteur-like strains of Bacillus anthracis to replicate and survive in the urban bluebottle blow fly Calliphora vicina under laboratory conditions.

    Science.gov (United States)

    von Terzi, Britta; Turnbull, Peter C B; Bellan, Steve E; Beyer, Wolfgang

    2014-01-01

    This study aimed to elucidate the bacteriological events occurring within the gut of Calliphora vicina, selected as the European representative of blow flies held responsible for the spread of anthrax during epidemics in certain parts of the world. Green-fluorescent-protein-carrying derivatives of Bacillus anthracis were used. These lacked either one of the virulence plasmids pXO1 and pXO2 and were infected, or not infected, with a worm intestine phage (Wip4) known to influence the phenotype and survival of the pathogen. Blood meals were prepared for the flies by inoculation of sheep blood with germinated and, in case of pXO2+ strains, encapsulated cells of the four B. anthracis strains. After being fed for 4 h an initial 10 flies were externally disinfected with peracetic acid to ensure subsequent quantitation representing ingested B. anthracis only. Following neutralization, they were crushed in sterile saline. Over each of the ensuing 7 to 10 days, 10 flies were removed and processed the same way. In the absence of Wip4, strains showed steady declines to undetectable in the total B. anthracis counts, within 7-9 days. With the phage infected strains, the falls in viable counts were significantly more rapid than in their uninfected counterparts. Spores were detectable in flies for longer periods than vegetative bacteria. In line with the findings in both biting and non-biting flies of early workers our results indicate that B. anthracis does not multiply in the guts of blow flies and survival is limited to a matter of days. PMID:24392098

  19. Failure of Sterne- and Pasteur-like strains of Bacillus anthracis to replicate and survive in the urban bluebottle blow fly Calliphora vicina under laboratory conditions.

    Directory of Open Access Journals (Sweden)

    Britta von Terzi

    Full Text Available This study aimed to elucidate the bacteriological events occurring within the gut of Calliphora vicina, selected as the European representative of blow flies held responsible for the spread of anthrax during epidemics in certain parts of the world. Green-fluorescent-protein-carrying derivatives of Bacillus anthracis were used. These lacked either one of the virulence plasmids pXO1 and pXO2 and were infected, or not infected, with a worm intestine phage (Wip4 known to influence the phenotype and survival of the pathogen. Blood meals were prepared for the flies by inoculation of sheep blood with germinated and, in case of pXO2+ strains, encapsulated cells of the four B. anthracis strains. After being fed for 4 h an initial 10 flies were externally disinfected with peracetic acid to ensure subsequent quantitation representing ingested B. anthracis only. Following neutralization, they were crushed in sterile saline. Over each of the ensuing 7 to 10 days, 10 flies were removed and processed the same way. In the absence of Wip4, strains showed steady declines to undetectable in the total B. anthracis counts, within 7-9 days. With the phage infected strains, the falls in viable counts were significantly more rapid than in their uninfected counterparts. Spores were detectable in flies for longer periods than vegetative bacteria. In line with the findings in both biting and non-biting flies of early workers our results indicate that B. anthracis does not multiply in the guts of blow flies and survival is limited to a matter of days.

  20. Recominant Pinoresino-Lariciresinol Reductase, Recombinant Dirigent Protein And Methods Of Use

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, Norman G. (Pullman, WA); Davin, Laurence B. (Pullman, WA); Dinkova-Kostova, Albena T. (Baltimore, MD); Fujita, Masayuki (Kita-gun, JP), Gang; David R. (Ann Arbor, MI), Sarkanen; Simo (Minneapolis, MN), Ford; Joshua D. (Pullman, WA)

    2003-10-21

    Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided from source species Forsythia intermedia, Thuja plicata, Tsuga heterophylla, Eucommia ulmoides, Linum usitatissimum, and Schisandra chinensis, which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.

  1. Recombinant pinoresinol/lariciresinol reductase, recombinant dirigent protein, and methods of use

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, Norman G. (Pullman, WA); Davin, Laurence B. (Pullman, WA); Dinkova-Kostova, Albena T. (Baltimore, MD); Fujita, Masayuki (Kagawa, JP); Gang, David R. (Ann Arbor, MI); Sarkanen, Simo (S. Minneapolis, MN); Ford, Joshua D. (Pullman, WA)

    2001-04-03

    Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.

  2. CD4+ T cells targeting dominant and cryptic epitopes from Bacillus anthracis Lethal Factor

    Directory of Open Access Journals (Sweden)

    Stephanie eAscough

    2016-01-01

    Full Text Available Anthrax is an endemic infection in many countries, particularly in the developing world. The causative agent, Bacillus anthracis, mediates disease through the secretion of binary exotoxins. Until recently, research into adaptive immunity targeting this bacterial pathogen has largely focused on the humoral response to these toxins. There is, however, growing recognition that cellular immune responses involving IFNγ producing CD4+ T cells also contribute significantly to a protective memory response. An established concept in adaptive immunity to infection is that during infection of host cells, new microbial epitopes may be revealed, leading to immune recognition of so called ‘cryptic’ or ‘subdominant’ epitopes. We analysed the response to both cryptic and immunodominant T cell epitopes derived from the toxin component lethal factor and presented by a range of HLA-DR alleles. Using IFNγ-ELISPOT assays we characterised epitopes that elicited a response following immunisation with synthetic peptide and the whole protein and tested their capacities to bind purified HLA-DR molecules in vitro. We found that DR1 transgenics demonstrated T cell responses to a greater number of domain III cryptic epitopes than other HLA-DR transgenics, and that this pattern was repeated with the immunodominant epitopes, a greater proportion of these epitopes induced a T cell response when presented within the context of the whole protein. Immunodominant epitopes LF457-476 and LF467-487 were found to induce a T cell response to the peptide, as well as to the whole native LF protein in DR1 and DR15, but not in DR4 trangenics. The analysis of Domain I revealed the presence of several unique cryptic epitopes all of which showed a strong to moderate relative binding affinity to HLA-DR4 molecules. However, none of the cryptic epitopes from either domain III or I displayed notably high binding affinities across all HLA-DR alleles assayed. These responses were

  3. Identification of Novel Raft Marker Protein, FlotP in Bacillus anthracis.

    Science.gov (United States)

    Somani, Vikas K; Aggarwal, Somya; Singh, Damini; Prasad, Tulika; Bhatnagar, Rakesh

    2016-01-01

    Lipid rafts are dynamic, nanoscale assemblies of specific proteins and lipids, distributed heterogeneously on eukaryotic membrane. Flotillin-1, a conserved eukaryotic raft marker protein (RMP) harbor SPFH (Stomatin, Prohibitin, Flotillin, and HflK/C) and oligomerization domains to regulate various cellular processes through its interactions with other signaling or transport proteins. Rafts were thought to be absent in prokaryotes hitherto, but recent report of its presence and significance in physiology of Bacillus subtilis prompted us to investigate the same in pathogenic bacteria (PB) also. In prokaryotes, proteins of SPFH2a subfamily show highest identity to SPFH domain of Flotillin-1. Moreover, bacterial genome organization revealed that Flotillin homolog harboring SPFH2a domain exists in an operon with an upstream gene containing NFeD domain. Here, presence of RMP in PB was initially investigated in silico by analyzing the presence of SPFH2a, oligomerization domains in the concerned gene and NfeD domain in the adjacent upstream gene. After investigating 300 PB, four were found to harbor RMP. Among them, domains of Bas0525 (FlotP) of Bacillus anthracis (BA) showed highest identity with characteristic domains of RMP. Considering the global threat of BA as the bioterror agent, it was selected as a model for further in vitro characterization of rafts in PB. In silico and in vitro analysis showed significant similarity of FlotP with numerous attributes of Flotillin-1. Its punctate distribution on membrane with exclusive localization in detergent resistant membrane fraction; strongly favors presence of raft with RMP FlotP in BA. Furthermore, significant effect of Zaragozic acid (ZA), a raft associated lipid biosynthesis inhibitor, on several patho-physiological attributes of BA such as growth, morphology, membrane rigidity etc., were also observed. Specifically, a considerable decrease in membrane rigidity, strongly recommended presence of an unknown raft associated

  4. Identification of Novel Raft Marker Protein, FlotP in Bacillus anthracis.

    Science.gov (United States)

    Somani, Vikas K; Aggarwal, Somya; Singh, Damini; Prasad, Tulika; Bhatnagar, Rakesh

    2016-01-01

    Lipid rafts are dynamic, nanoscale assemblies of specific proteins and lipids, distributed heterogeneously on eukaryotic membrane. Flotillin-1, a conserved eukaryotic raft marker protein (RMP) harbor SPFH (Stomatin, Prohibitin, Flotillin, and HflK/C) and oligomerization domains to regulate various cellular processes through its interactions with other signaling or transport proteins. Rafts were thought to be absent in prokaryotes hitherto, but recent report of its presence and significance in physiology of Bacillus subtilis prompted us to investigate the same in pathogenic bacteria (PB) also. In prokaryotes, proteins of SPFH2a subfamily show highest identity to SPFH domain of Flotillin-1. Moreover, bacterial genome organization revealed that Flotillin homolog harboring SPFH2a domain exists in an operon with an upstream gene containing NFeD domain. Here, presence of RMP in PB was initially investigated in silico by analyzing the presence of SPFH2a, oligomerization domains in the concerned gene and NfeD domain in the adjacent upstream gene. After investigating 300 PB, four were found to harbor RMP. Among them, domains of Bas0525 (FlotP) of Bacillus anthracis (BA) showed highest identity with characteristic domains of RMP. Considering the global threat of BA as the bioterror agent, it was selected as a model for further in vitro characterization of rafts in PB. In silico and in vitro analysis showed significant similarity of FlotP with numerous attributes of Flotillin-1. Its punctate distribution on membrane with exclusive localization in detergent resistant membrane fraction; strongly favors presence of raft with RMP FlotP in BA. Furthermore, significant effect of Zaragozic acid (ZA), a raft associated lipid biosynthesis inhibitor, on several patho-physiological attributes of BA such as growth, morphology, membrane rigidity etc., were also observed. Specifically, a considerable decrease in membrane rigidity, strongly recommended presence of an unknown raft associated

  5. Naturally acquired antibodies to Bacillus anthracis protective antigen in vultures of southern Africa

    Directory of Open Access Journals (Sweden)

    P. C.B. Turnbull

    2008-08-01

    Full Text Available TURNBULLP, P.C.B. DIEKMANNM,M., KILIAN, J.W., VERSFELDW, W.,DE VOS, V., ARNTZENL, L.,WOLTER, K., BARTELS, P. & KOTZE, A. 2008.N aturally acquired antibodies to Bacillusa nthracisp rotective antigeni n vultureso f southern Africa. Onderstepoort Journal of Veterinary Research, T5:95-102 Sera from 19 wild caught vultures in northern Namibia and 15 (12 wild caught and three captive bred but with minimal histories in North West Province, South Africa, were examined by an enzyme-linked immunosorbenats say( ELISAf or antibodiesto the Bacillus anthracis toxin protective antigen (PA. As assessed from the baseline established with a control group of ten captive reared vultures with well-documented histories, elevated titres were found in 12 of the 19 (63% wild caught Namibian birds as compared with none of the 15 South African ones. There was a highly significant difference between the Namibian group as a hole and the other groups (P < 0.001 and no significant difference between the South African and control groups (P > 0.05. Numbers in the Namibian group were too small to determine any significances in species-, sex- or age-related differences within the raw data showing elevated titres in four out of six Cape Vultures, Gyps coprotheress, six out of ten Whitebacked Vultures, Gyps africanus, and one out of three Lappet-faced Vultures, Aegypiust racheliotus, or in five of six males versus three of seven females, and ten of 15 adults versus one of four juveniles. The results are in line with the available data on the incidence of anthrax in northern Namibia and South Africa and the likely contact of the vultures tested with anthrax carcasses. lt is not known whether elevated titre indicates infection per se in vultures or absorption of incompletely digested epitopes of the toxin or both. The results are discussed in relation to distances travelled by vultures as determined by new tracking techniques, how serology can reveal anthrax activity in an area and

  6. National validation study of a swab protocol for the recovery of Bacillus anthracis spores from surfaces.

    Science.gov (United States)

    Hodges, Lisa R; Rose, Laura J; O'Connell, Heather; Arduino, Matthew J

    2010-05-01

    Twelve Laboratory Response Network (LRN) affiliated laboratories participated in a validation study of a macrofoam swab protocol for the recovery, detection, and quantification of viable B. anthracis (BA) Sterne spores from steel surfaces. CDC personnel inoculated steel coupons (26cm(2)) with 1-4 log(10) BA spores and recovered them by sampling with pre-moistened macrofoam swabs. Phase 1 (P1) of the study evaluated swabs containing BA only, while dust and background organisms were added to swabs in Phase 2 (P2) to mimic environmental conditions. Laboratories processed swabs and enumerated spores by culturing eluted swab suspensions and counting colonies with morphology consistent with BA. Processed swabs were placed in enrichment broth, incubated 24h, and cultured by streaking for isolation. Real-time PCR was performed on selected colonies from P2 samples to confirm the identity of BA. Mean percent recovery (%R) of spores from the surface ranged from 15.8 to 31.0% (P1) and from 27.9 to 55.0% (P2). The highest mean percent recovery was 31.0% (sd 10.9%) for P1 (4 log(10) inoculum) and 55.0% (sd 27.6%) for P2 (1 log(10) inoculum). The overall %R was higher for P2 (44.6%) than P1 (24.1%), but the overall reproducibility (between-lab variability) was lower in P2 than in P1 (25.0 vs 16.5%CV, respectively). The overall precision (within-lab variability) was close to identical for P1 and P2 (44.0 and 44.1, respectively), but varied greatly between inoculum levels. The protocol demonstrated linearity in %R over the three inoculum levels and is able to detect between 26 and 5x10(6)spores/26cm(2). Sensitivity as determined by culture was >98.3% for both phases and all inocula, suggesting that the culture method maintains sensitivity in the presence of contaminants. The enrichment broth method alone was less sensitive for sampled swabs (66.4%) during P2, suggesting that the presence of background organisms inhibited growth or isolation of BA from the broth. The addition of

  7. Identification of novel raft marker protein, FlotP in Bacillus anthracis

    Directory of Open Access Journals (Sweden)

    Vikas Kumar Somani

    2016-02-01

    Full Text Available Lipid rafts are dynamic, nanoscale assemblies of specific proteins and lipids, distributed heterogeneously on eukaryotic membrane. Flotillin-1, a conserved eukaryotic raft marker protein (RMP harbor SPFH (Stomatin, Prohibitin, Flotillin, and HflK/C and oligomerization domains to regulate various cellular processes through its interactions with other signaling or transport proteins. Rafts were thought to be absent in prokaryotes hitherto, but recent report of its presence and significance in physiology of Bacillus subtilis prompted us to investigate the same in pathogenic bacteria (PB also. In prokaryotes, proteins of SPFH2a subfamily show highest identity to SPFH domain of Flotillin-1. Moreover, bacterial genome organization revealed that Flotillin homologue harbouring SPFH2a domain exists in an operon with an upstream gene containing NFeD domain. Here, presence of RMP in PB was initially investigated in silico by analyzing the presence of SPFH2a, oligomerization domains in the concerned gene and NfeD domain in the adjacent upstream gene. After investigating 300 PB, 4 were found to harbor RMP. Among them, domains of Bas0525 (FlotP of Bacillus anthracis (BA showed highest identity with characteristic domains of RMP. Considering the global threat of BA as the bioterror agent, it was selected as a model for further in vitro characterization of rafts in PB. In silico and in vitro analysis showed significant similarity of FlotP with numerous attributes of Flotillin-1. Its punctate distribution on membrane with exclusive localization in detergent resistant membrane fraction; strongly favors presence of raft with RMP FlotP in BA. Furthermore, significant effect of Zaragozic acid (ZA, a raft associated lipid biosynthesis inhibitor, on several patho-physiological attributes of BA such as growth, morphology, membrane rigidity etc., were also observed. Specifically, a considerable decrease in membrane rigidity, strongly recommended presence of an unknown

  8. A single-dose PLGA encapsulated protective antigen domain 4 nanoformulation protects mice against Bacillus anthracis spore challenge.

    Directory of Open Access Journals (Sweden)

    Manish Manish

    Full Text Available Bacillus anthracis, the etiological agent of anthrax, is a major bioterror agent. Vaccination is the most effective prophylactic measure available against anthrax. Currently available anthrax vaccines have issues of the multiple booster dose requirement, adjuvant-associated side effects and stability. Use of biocompatible and biodegradable nanoparticles to deliver the antigens to immune cells could solve the issues associated with anthrax vaccines. We hypothesized that the delivery of a stable immunogenic domain 4 of protective antigen (PAD4 of Bacillus anthracis encapsulated in a poly (lactide-co-glycolide (PLGA--an FDA approved biocompatible and biodegradable material, may alleviate the problems of booster dose, adjuvant toxicity and stability associated with anthrax vaccines. We made a PLGA based protective antigen domain 4 nanoparticle (PAD4-NP formulation using water/oil/water solvent evaporation method. Nanoparticles were characterized for antigen content, morphology, size, polydispersity and zeta potential. The immune correlates and protective efficacy of the nanoparticle formulation was evaluated in Swiss Webster outbred mice. Mice were immunized with single dose of PAD4-NP or recombinant PAD4. The PAD4-NP elicited a robust IgG response with mixed IgG1 and IgG2a subtypes, whereas the control PAD4 immunized mice elicited low IgG response with predominant IgG1 subtype. The PAD4-NP generated mixed Th1/Th2 response, whereas PAD4 elicited predominantly Th2 response. When we compared the efficacy of this single-dose vaccine nanoformulation PAD4-NP with that of the recombinant PAD4 in providing protective immunity against a lethal challenge with Bacillus anthracis spores, the median survival of PAD4-NP immunized mice was 6 days as compared to 1 day for PAD4 immunized mice (p<0.001. Thus, we demonstrate, for the first time, the possibility of the development of a single-dose and adjuvant-free protective antigen based anthrax vaccine in the form

  9. Possession, Use, and Transfer of Select Agents and Toxins-- Addition of Bacillus Cereus Biovar Anthracis to the HHS List of Select Agents and Toxins. Interim final rule and request for comments.

    Science.gov (United States)

    2016-09-14

    The Centers for Disease Control and Prevention (CDC) in the Department of Health and Human Services (HHS) is adding Bacillus cereus Biovar anthracis to the list of HHS select agents and toxins as a Tier 1 select agent. We are taking this action to regulate this agent that is similar to B. anthracis to prevent its misuse, which could cause a biological threat to public health and/or national security.

  10. The respiratory arsenate reductase from Bacillus selenitireducens strain MLS10

    Science.gov (United States)

    Afkar, E.; Lisak, J.; Saltikov, C.; Basu, P.; Oremland, R.S.; Stolz, J.F.

    2003-01-01

    The respiratory arsenate reductase from the Gram-positive, haloalkaliphile, Bacillus selenitireducens strain MLS10 was purified and characterized. It is a membrane bound heterodimer (150 kDa) composed of two subunits ArrA (110 kDa) and ArrB (34 kDa), with an apparent Km for arsenate of 34 ??M and Vmax of 2.5 ??mol min-1 mg-1. Optimal activity occurred at pH 9.5 and 150 g l-1 of NaCl. Metal analysis (inductively coupled plasma mass spectrometry) of the holoenzyme and sequence analysis of the catalytic subunit (ArrA; the gene for which was cloned and sequenced) indicate it is a member of the DMSO reductase family of molybdoproteins. ?? 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

  11. Methionine sulfoxide reductase A is a stereospecific methionine oxidase

    OpenAIRE

    Lim, Jung Chae; You, Zheng; Kim, Geumsoo; Levine, Rodney L.

    2011-01-01

    Methionine sulfoxide reductase A (MsrA) catalyzes the reduction of methionine sulfoxide to methionine and is specific for the S epimer of methionine sulfoxide. The enzyme participates in defense against oxidative stresses by reducing methionine sulfoxide residues in proteins back to methionine. Because oxidation of methionine residues is reversible, this covalent modification could also function as a mechanism for cellular regulation, provided there exists a stereospecific methionine oxidase....

  12. Determination of plasma gluthatione reductase enzyme activity in osteoporotic women

    OpenAIRE

    Sadeghi N; Oveisi M.R.; Jannat B.; Hajimahmoodi M; Jamshidi A.R; Sajadian Z.

    2008-01-01

    Background: Osteoporosis is a disease of high prevalence with increased bone loss. Free radicals have been proved to be involved in bone resorption. Glutathione reductase (GR) plays an essential role in cell defense against reactive oxygen metabolites by sustaining the reduced status of an important antioxidant, glutathione. In the present study GR activity of plasma as an antioxidant enzyme in relation to Bone Mineral Density (BMD) was investigated.Material and Method: GR activity was measur...

  13. Aldo-keto reductase (AKR) superfamily: Genomics and annotation

    OpenAIRE

    Mindnich Rebekka D; Penning Trevor M

    2009-01-01

    Abstract Aldo-keto reductases (AKRs) are phase I metabolising enzymes that catalyse the reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H)-dependent reduction of carbonyl groups to yield primary and secondary alcohols on a wide range of substrates, including aliphatic and aromatic aldehydes and ketones, ketoprostaglan-dins, ketosteroids and xenobiotics. In so doing they functionalise the carbonyl group for conjugation (phase II enzyme reactions). Although functionally diverse, AK...

  14. Internal electron transfer within mitochondrial succinate-cytochrome C reductase

    International Nuclear Information System (INIS)

    Internal electron transfer within succinate-cytochrome C reductase from pigeon breast muscle mitochondria was followed by the pulse radiolytic technique. The electron equivalent is transferred from an unknown donor to b type cytochrome(s), in a first order process with a rate constant of: 660 +- 150s-1. This process might be the rate determining step of electron transfer in mitochondria, since it is similar in rate to the turnover number of the mitochondrial respiratory chain

  15. A strain-variable bacteriocin in Bacillus anthracis and Bacillus cereus with repeated Cys-Xaa-Xaa motifs

    Directory of Open Access Journals (Sweden)

    Haft Daniel H

    2009-04-01

    Full Text Available Abstract Bacteriocins are peptide antibiotics from ribosomally translated precursors, produced by bacteria often through extensive post-translational modification. Minimal sequence conservation, short gene lengths, and low complexity sequence can hinder bacteriocin identification, even during gene calling, so they are often discovered by proximity to accessory genes encoding maturation, immunity, and export functions. This work reports a new subfamily of putative thiazole-containing heterocyclic bacteriocins. It appears universal in all strains of Bacillus anthracis and B. cereus, but has gone unrecognized because it is always encoded far from its maturation protein operon. Patterns of insertions and deletions among twenty-four variants suggest a repeating functional unit of Cys-Xaa-Xaa. Reviewers This article was reviewed by Andrei Osterman and Lakshminarayan Iyer.

  16. 炭疽芽胞杆菌的基因分型%The genotyping of Bacillus anthracis

    Institute of Scientific and Technical Information of China (English)

    周伟(综述); 郭学军(审校)

    2013-01-01

    In order to identify the genotype of the Bacillus anthracis,multi-locus variable number tandem repeat analysis (MLVA), single nucleotide polymorphism analysis (SNP) and single nucleotide repeat analysis (SNR) have been used, and become the main methods gradually .This paper reviewes the status of their application .%为了鉴别炭疽芽胞杆菌( Bacillus anthracis)的基因型,多位点可变数量串联重复序列分析( MLVA)、单核苷酸多态性分析( SNP )和单核苷酸重复序列分析( SNR )被广泛应用,并逐渐成为炭疽分型的基本方法,因而对其应用现状进行了综述。

  17. The cytotoxic activity of Bacillus anthracis lethal factor is inhibited by leukotriene A4 hydrolase and metallopeptidase inhibitors.

    Science.gov (United States)

    Menard, A; Papini, E; Mock, M; Montecucco, C

    1996-01-01

    The lethal factor of Bacillus anthracis is central to the pathogenesis of anthrax. Its mechanism of action is still unknown. Recently, on the basis of sequence similarities, we suggested that lethal factor might act similarly to leukotriene A4 hydrolase (LTA4), a bifunctional enzyme also endowed with a metallopeptidase activity. Here we show that some inhibitors of the LTA4 hydrolase and metallopeptidase activities of LTA4 hydrolase also affect the cytotoxicity of the anthrax lethal factor on macrophage cell lines, without interfering with the ability of the lethal factor to enter cells. These results support the proposal that anthrax lethal factor might display in the cytosol of intoxicated cells a peptidase activity similar to that of LTA4 hydrolase. PMID:8973585

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  19. Evaluation of New Dihydrophthalazine-Appended 2,4-Diaminopyrimidines against Bacillus anthracis: Improved Syntheses Using a New Pincer Complex

    Directory of Open Access Journals (Sweden)

    Nagendra Prasad Muddala

    2015-04-01

    Full Text Available The synthesis and evaluation of ten new dihydrophthalazine-appended 2,4-diaminopyrimidines as potential drugs to treat Bacillus anthracis is reported. An improved synthesis utilizing a new pincer catalyst, dichlorobis[1-(dicyclohexylphosphanyl-piperidine]palladium(II, allows the final Heck coupling to be performed at 90 °C using triethylamine as the base. These milder conditions have been used to achieve improved yields for new and previously reported substrates with functional groups that degrade or react at the normal 140 °C reaction temperature. An analytical protocol for separating the S and R enantiomers of two of the most active compounds is also disclosed. Finally, the X-ray structure for the most active enantiomer of the lead compound, (S-RAB1, is given.

  20. Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues.

    Science.gov (United States)

    Youngblut, Matthew D; Tsai, Chi-Lin; Clark, Iain C; Carlson, Hans K; Maglaqui, Adrian P; Gau-Pan, Phonchien S; Redford, Steven A; Wong, Alan; Tainer, John A; Coates, John D

    2016-04-22

    Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-Å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6 μm) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mm) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO3 (2-) bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth.

  1. Aldo-Keto Reductases 1B in Adrenal Cortex Physiology.

    Science.gov (United States)

    Pastel, Emilie; Pointud, Jean-Christophe; Martinez, Antoine; Lefrançois-Martinez, A Marie

    2016-01-01

    Aldose reductase (AKR1B) proteins are monomeric enzymes, belonging to the aldo-keto reductase (AKR) superfamily. They perform oxidoreduction of carbonyl groups from a wide variety of substrates, such as aliphatic and aromatic aldehydes or ketones. Due to the involvement of human aldose reductases in pathologies, such as diabetic complications and cancer, AKR1B subgroup enzymatic properties have been extensively characterized. However, the issue of AKR1B function in non-pathologic conditions remains poorly resolved. Adrenal activities generated large amount of harmful aldehydes from lipid peroxidation and steroidogenesis, including 4-hydroxynonenal (4-HNE) and isocaproaldehyde (4-methylpentanal), which can both be reduced by AKR1B proteins. More recently, some AKR1B isoforms have been shown to be endowed with prostaglandin F synthase (PGFS) activity, suggesting that, in addition to possible scavenger function, they could instigate paracrine signals. Interestingly, the adrenal gland is one of the major sites for human and murine AKR1B expression, suggesting that their detoxifying/signaling activity could be specifically required for the correct handling of adrenal function. Moreover, chronic effects of ACTH result in a coordinated regulation of genes encoding the steroidogenic enzymes and some AKR1B isoforms. This review presents the molecular mechanisms accounting for the adrenal-specific expression of some AKR1B genes. Using data from recent mouse genetic models, we will try to connect their enzymatic properties and regulation with adrenal functions.

  2. Phosphoglycerate kinase acts in tumour angiogenesis as a disulphide reductase

    Science.gov (United States)

    Lay, Angelina J.; Jiang, Xing-Mai; Kisker, Oliver; Flynn, Evelyn; Underwood, Anne; Condron, Rosemary; Hogg, Philip J.

    2000-12-01

    Disulphide bonds in secreted proteins are considered to be inert because of the oxidizing nature of the extracellular milieu. An exception to this rule is a reductase secreted by tumour cells that reduces disulphide bonds in the serine proteinase plasmin. Reduction of plasmin initiates proteolytic cleavage in the kringle 5 domain and release of the tumour blood vessel inhibitor angiostatin. New blood vessel formation or angiogenesis is critical for tumour expansion and metastasis. Here we show that the plasmin reductase isolated from conditioned medium of fibrosarcoma cells is the glycolytic enzyme phosphoglycerate kinase. Recombinant phosphoglycerate kinase had the same specific activity as the fibrosarcoma-derived protein. Plasma of mice bearing fibrosarcoma tumours contained several-fold more phosphoglycerate kinase, as compared with mice without tumours. Administration of phosphoglycerate kinase to tumour-bearing mice caused an increase in plasma levels of angiostatin, and a decrease in tumour vascularity and rate of tumour growth. Our findings indicate that phosphoglycerate kinase not only functions in glycolysis but is secreted by tumour cells and participates in the angiogenic process as a disulphide reductase.

  3. Cloning and sequence of the human adrenodoxin reductase gene

    International Nuclear Information System (INIS)

    Adrenodoxin reductase is a flavoprotein mediating electron transport to all mitochondrial forms of cytochrome P450. The authors cloned the human adrenodoxin reductase gene and characterized it by restriction endonuclease mapping and DNA sequencing. The entire gene is approximately 12 kilobases long and consists of 12 exons. The first exon encodes the first 26 of the 32 amino acids of the signal peptide, and the second exon encodes the remainder of signal peptide and the apparent FAD binding site. The remaining 10 exons are clustered in a region of only 4.3 kilobases, separated from the first two exons by a large intron of about 5.6 kilobases. Two forms of human adrenodoxin reductase mRNA, differing by the presence or absence of 18 bases in the middle of the sequence, arise from alternate splicing at the 5' end of exon 7. This alternately spliced region is directly adjacent to the NADPH binding site, which is entirely contained in exon 6. The immediate 5' flanking region lacks TATA and CAAT boxes; however, this region is rich in G+C and contains six copies of the sequence GGGCGGG, resembling promoter sequences of housekeeping genes. RNase protection experiments show that transcription is initiated from multiple sites in the 5' flanking region, located about 21-91 base pairs upstream from the AUG translational initiation codon

  4. 炭疽芽孢杆菌S-层蛋白功能研究进展%S-layer proteins of Bacillus anthracis: research progress

    Institute of Scientific and Technical Information of China (English)

    王雪芳; 刘先凯; 陈福生; 王恒樑

    2011-01-01

    Bacillus anthracis is the etiological agent of anthrax. Currently, plasmids pXOl and pX02 of B. anthracis, which were encoding the virulence genes and capsule synthesis genes respectively, have been studied thoroughly. However, the S-layer, a protein paracrystalline structure that exists between the capsule and the cell wall of B. anthracis, is less known. Sap( surface array protein ) and EA1( extracellular antigen 1 ) are the major proteins of S-layer as well as some other relative proteins in B. anthracis. Investigation of the interaction of these proteins and their immunologic mechanism is vital for understanding the pathogenesis of B. anthracis. This brief review will focus on recent researches on the components of S-layer proteins, their linkage with the cell wall and the regulation patterns of their encoding genes of S-layer proteins, their role in the pathogenesis of B. anthracis, and the relationship between S-layer proteins and host immune systems.%炭疽芽孢杆菌是人畜共患病炭疽的病原菌.目前,对炭疽杆菌2个毒力大质粒(编码主要毒力基因的pXO1与编码合成荚膜基因的pXO2)的研究比较深入;炭疽杆菌细胞壁与荚膜间还存在一种蛋白性质的类晶体(paracrystalline)结构:S-层(surface-layer,表层)结构.炭疽杆菌的S-层蛋白主要为表面排列蛋白(surface array protein,Sap)和胞外抗原1(extracellular antigen 1,EA1),此外,在炭疽杆菌中还存在其他一些与S-层相关的蛋白,了解这些蛋白的相互作用及免疫机制对深入认识炭疽杆菌的致病机制具有重要意义.本文将就近年来关于炭疽杆菌S-层蛋白成分、与细胞壁的连接、S-层基因的调控、致病性及其与宿主免疫机制的关系等方面的研究进展做一简要综述.

  5. Two-component system cross-regulation integrates Bacillus anthracis response to heme and cell envelope stress.

    Directory of Open Access Journals (Sweden)

    Laura A Mike

    2014-03-01

    Full Text Available Two-component signaling systems (TCSs are one of the mechanisms that bacteria employ to sense and adapt to changes in the environment. A prototypical TCS functions as a phosphorelay from a membrane-bound sensor histidine kinase (HK to a cytoplasmic response regulator (RR that controls target gene expression. Despite significant homology in the signaling domains of HKs and RRs, TCSs are thought to typically function as linear systems with little to no cross-talk between non-cognate HK-RR pairs. Here we have identified several cell envelope acting compounds that stimulate a previously uncharacterized Bacillus anthracis TCS. Furthermore, this TCS cross-signals with the heme sensing TCS HssRS; therefore, we have named it HssRS interfacing TCS (HitRS. HssRS reciprocates cross-talk to HitRS, suggesting a link between heme toxicity and cell envelope stress. The signaling between HssRS and HitRS occurs in the parental B. anthracis strain; therefore, we classify HssRS-HitRS interactions as cross-regulation. Cross-talk between HssRS and HitRS occurs at both HK-RR and post-RR signaling junctions. Finally, HitRS also regulates a previously unstudied ABC transporter implicating this transporter in the response to cell envelope stress. This chemical biology approach to probing TCS signaling provides a new model for understanding how bacterial signaling networks are integrated to enable adaptation to complex environments such as those encountered during colonization of the vertebrate host.

  6. The central nervous system as target of Bacillus anthracis toxin independent virulence in rabbits and guinea pigs.

    Directory of Open Access Journals (Sweden)

    Haim Levy

    Full Text Available Infection of the central nervous system is considered a complication of Anthrax and was reported in humans and non-human primates. Previously we have reported that Bacillus anthracis possesses a toxin-independent virulent trait that, like the toxins, is regulated by the major virulence regulator, AtxA, in the presence of pXO2. This toxin-independent lethal trait is exhibited in rabbits and Guinea pigs following significant bacteremia and organ dissemination. Various findings, including meningitis seen in humans and primates, suggested that the CNS is a possible target for this AtxA-mediated activity. In order to penetrate into the brain tissue, the bacteria have to overcome the barriers isolating the CNS from the blood stream. Taking a systematic genetic approach, we compared intracranial (IC inoculation and IV/SC inoculation for the outcome of the infection in rabbits/GP, respectively. The outstanding difference between the two models is exhibited by the encapsulated strain VollumΔpXO1, which is lethal when injected IC, but asymptomatic when inoculated IV/SC. The findings demonstrate that there is an apparent bottleneck in the ability of mutants to penetrate into the brain. Any mutant carrying either pXO1 or pXO2 will kill the host upon IC injection, but only those carrying AtxA either on pXO1 or in the chromosome in the background of pXO2 can penetrate into the brain following peripheral inoculation. The findings were corroborated by histological examination by H&E staining and immunofluorescence of rabbits' brains following IV and IC inoculations. These findings may have major implications on future research both on B. anthracis pathogenicity and on vaccine development.

  7. Comparison of Packed Beds and Qiagen Columns for Recovering Trace Amounts of B. anthracis DNA from Liquid Suspensions

    Energy Technology Data Exchange (ETDEWEB)

    Sorensen, K; Arroyo, E; Erler, A; Christian, A T; Camp, D; Wheeler, E K

    2006-06-23

    The goal of this work was to optimize and evaluate LLNL's in-bed amplification technology to improve the level of detection for suspensions containing trace amounts of anthracis DNA. The binding/cleaning performance of the packed bed is compared to the conventional commercial approach; Qiagen column cleanup and elution, followed by detection through an ex-situ amplification process. Five liquid suspensions were spiked with B.anthracis DNA in concentration series. These suspensions were: (1) water, (2) water with EDTA, (3) dirty water from carpet extraction, (4) dirty carpet extraction with phosphate buffered saline (PBS) plus 0.1% Tween 20 plus 0.1% gelatin, and (5) a subway aerosol collected in water. Each suspension matrix was spiked with DNA and injected (in replicate) into either Qiagen Microcolumns (using the kit processing instructions) or LLNL's packed bed (using the LLNL in-bed purification and amplification protocol). The process output was assayed by quantitative polymerase chain reaction (QPCR). Table ES-1 shows the level of DNA (pg per 100 uL of input suspension) that resulted in successful amplification for all reactions (X=Y), and the level for which at least one of the reactions was successful (X>0). For each suspension and DNA concentration, there were Y QPCR assays of which X showed successful amplification. LLNL's packed bed technology outperformed Qiagen Microcolumns for all five suspensions, typically by one order of magnitude in both the limit of assured detection (all reactions positive), and the lower limit of detection (some reactions positive).

  8. The central nervous system as target of Bacillus anthracis toxin independent virulence in rabbits and guinea pigs.

    Science.gov (United States)

    Levy, Haim; Glinert, Itai; Weiss, Shay; Bar-David, Elad; Sittner, Assa; Schlomovitz, Josef; Altboum, Zeev; Kobiler, David

    2014-01-01

    Infection of the central nervous system is considered a complication of Anthrax and was reported in humans and non-human primates. Previously we have reported that Bacillus anthracis possesses a toxin-independent virulent trait that, like the toxins, is regulated by the major virulence regulator, AtxA, in the presence of pXO2. This toxin-independent lethal trait is exhibited in rabbits and Guinea pigs following significant bacteremia and organ dissemination. Various findings, including meningitis seen in humans and primates, suggested that the CNS is a possible target for this AtxA-mediated activity. In order to penetrate into the brain tissue, the bacteria have to overcome the barriers isolating the CNS from the blood stream. Taking a systematic genetic approach, we compared intracranial (IC) inoculation and IV/SC inoculation for the outcome of the infection in rabbits/GP, respectively. The outstanding difference between the two models is exhibited by the encapsulated strain VollumΔpXO1, which is lethal when injected IC, but asymptomatic when inoculated IV/SC. The findings demonstrate that there is an apparent bottleneck in the ability of mutants to penetrate into the brain. Any mutant carrying either pXO1 or pXO2 will kill the host upon IC injection, but only those carrying AtxA either on pXO1 or in the chromosome in the background of pXO2 can penetrate into the brain following peripheral inoculation. The findings were corroborated by histological examination by H&E staining and immunofluorescence of rabbits' brains following IV and IC inoculations. These findings may have major implications on future research both on B. anthracis pathogenicity and on vaccine development.

  9. Baulamycins A and B, broad-spectrum antibiotics identified as inhibitors of siderophore biosynthesis in Staphylococcus aureus and Bacillus anthracis.

    Science.gov (United States)

    Tripathi, Ashootosh; Schofield, Michael M; Chlipala, George E; Schultz, Pamela J; Yim, Isaiah; Newmister, Sean A; Nusca, Tyler D; Scaglione, Jamie B; Hanna, Philip C; Tamayo-Castillo, Giselle; Sherman, David H

    2014-01-29

    Siderophores are high-affinity iron chelators produced by microorganisms and frequently contribute to the virulence of human pathogens. Targeted inhibition of the biosynthesis of siderophores staphyloferrin B of Staphylococcus aureus and petrobactin of Bacillus anthracis hold considerable potential as a single or combined treatment for methicillin-resistant S. aureus (MRSA) and anthrax infection, respectively. The biosynthetic pathways for both siderophores involve a nonribosomal peptide synthetase independent siderophore (NIS) synthetase, including SbnE in staphyloferrin B and AsbA in petrobactin. In this study, we developed a biochemical assay specific for NIS synthetases to screen for inhibitors of SbnE and AsbA against a library of marine microbial-derived natural product extracts (NPEs). Analysis of the NPE derived from Streptomyces tempisquensis led to the isolation of the novel antibiotics baulamycins A (BmcA, 6) and B (BmcB, 7). BmcA and BmcB displayed in vitro activity with IC50 values of 4.8 μM and 19 μM against SbnE and 180 μM and 200 μM against AsbA, respectively. Kinetic analysis showed that the compounds function as reversible competitive enzyme inhibitors. Liquid culture studies with S. aureus , B. anthracis , E. coli , and several other bacterial pathogens demonstrated the capacity of these natural products to penetrate bacterial barriers and inhibit growth of both Gram-positive and Gram-negative species. These studies provide proof-of-concept that natural product inhibitors targeting siderophore virulence factors can provide access to novel broad-spectrum antibiotics, which may serve as important leads for the development of potent anti-infective agents. PMID:24401083

  10. cis-Acting elements that control expression of the master virulence regulatory gene atxA in Bacillus anthracis.

    Science.gov (United States)

    Dale, Jennifer L; Raynor, Malik J; Dwivedi, Prabhat; Koehler, Theresa M

    2012-08-01

    Transcription of the Bacillus anthracis structural genes for the anthrax toxin proteins and biosynthetic operon for capsule is positively regulated by AtxA, a transcription regulator with unique properties. Consistent with the role of atxA in virulence factor expression, a B. anthracis atxA-null mutant is avirulent in a murine model for anthrax. In culture, multiple signals impact atxA transcript levels, and the timing and steady-state level of atxA expression are critical for optimal toxin and capsule synthesis. Despite the apparent complex control of atxA transcription, only one trans-acting protein, the transition state regulator AbrB, has been demonstrated to interact directly with the atxA promoter. Here we employ 5' and 3' deletion analysis and site-directed mutagenesis of the atxA control region to demonstrate that atxA transcription from the major start site P1 is dependent upon a consensus sequence for the housekeeping sigma factor SigA and an A+T-rich upstream element for RNA polymerase. We also show that an additional trans-acting protein(s) binds specifically to atxA promoter sequences located between -13 and +36 relative to P1 and negatively impacts transcription. Deletion of this region increases promoter activity up to 15-fold. Site-directed mutagenesis of a 9-bp palindromic sequence within the region prevents binding of the trans-acting protein(s), increasing promoter activity 7-fold and resulting in a corresponding increase in AtxA and anthrax toxin production. Notably, an atxA promoter mutant that produced elevated levels of AtxA and toxin proteins during culture was unaffected for virulence in a murine model for anthrax. PMID:22636778

  11. Immunological comparison of the NADH:nitrate reductase from different cucumber tissues

    Directory of Open Access Journals (Sweden)

    Jolanta Marciniak

    2014-02-01

    Full Text Available Soluble nitrate reductase from cucumber roots (Cucumis sativus L. was isolated and purified with blue-Sepharose 4B. Specific antibodies against the NR protein were raised by immunization of a goat. Using polyclonal antibodies anti-NR properties of the nitrate reductase from various cucumber tissues were examined. Experiments showed difference in immuno-logical properties of nitrate reductase (NR from cotyledon roots and leaves.

  12. Development of a killed but metabolically active anthracis vaccine candidate strain%一种KBMA炭疽疫苗候选株的研制

    Institute of Scientific and Technical Information of China (English)

    沈非; 刘纯杰; 袁盛凌; 展德文; 王艳春; 任敏; 陶好霞; 王芃; 王令春; 陈冬生

    2011-01-01

    Anthrax is a zoonosis caused by Bacillus anthracis, which seriously affects human health. In recent years, a special phenomenon is found that the metabolic active of a bacterium remains after it is killed. To development of a KBMA (killed but metabolically active) Bacillus anthracis vaccine candidate strain, a plasmid pMAD and a recombinase system Cre-loxP were used to knockout the uvrAB gene of B. anthracis AP422 which lacks both of two plasmids pXOl and pXO2. The results of PCR and RT-PCR shows that uvrAB genes were deleted from B. anthracis AP422 chromosome successfully. The constructed B. anthracis AP422ΔuvrAB was inactivated by photochemical treatment (PCT) including an exposure in a long-wave-length ultraviolet (UVA)light and a treatment of 8-Methoxypsoralen (8-MOP), then the metabolic activity were detected by the method of MTS. The results showed that the killed B. anthracis AP422ΔuvrAB maintained a highly metabolic activity for at least 4 hours, showing a state of KBMA. The KBMA strain of B. anthracis AP422ΔuvrAB provides the prospective vaccine candidate strain for anthrax.%炭疽病是由炭疽芽胞杆菌Bacillus anthracis 引起的一种人畜共患传染病,严重影响着人类的健康.近年来在细菌疫苗的研究中发现一种特殊的现象:细菌被杀死后,体内的代谢活性却仍然维持(Killed but metabolically active,KBMA).此发现为炭疽新型疫苗候选株的研制提供了新思路.先通过同源重组的方法,利用pMAD质粒和Cre-loxP重组酶系统完成对缺失两个毒性大质粒的炭疽芽胞杆菌减毒株AP422的uvrAB基因的敲除,得到AP422△uvrAB菌株,然后通过光化学处理(包括长波紫外光的照射和8-甲氧基补骨脂素处理),使炭疽芽胞杆菌AP422△uvrAB失去繁殖能力.利用四氮唑化合物MTS检测其代谢活性,表明光化学处理杀死后的炭疽芽胞杆菌AP422△uvrAB在至少4 h内维持一个很高的代谢活性水平,即具备

  13. Progress on the Molecular Genotyping Techniques of Bacillus anthracis%炭疽芽孢杆菌分子分型研究进展

    Institute of Scientific and Technical Information of China (English)

    吴松羽; 刘先凯; 宋丽; 魏华; 王恒樑

    2011-01-01

    随着分子生物学的发展,分子分型技术被广泛应用于鉴别炭疽芽孢杆菌菌株间的遗传相关性和流行病学特征.我们就近年来常用的炭疽芽孢杆菌分子分型方法的优缺点和研究进展进行综述.%With the development of molecular biology, genotyping techniques are widely used in identification of strains of Bacillus anthracis in the genetic correlation and epidemiological characteristics. In this paper, we reviewed the advantages and disadvantages of the commonly methods used in genotyping of B.anthracis in recent years.

  14. Histochemical Localization of Glutathione Dependent NBT-Reductase in Mouse Skin

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective Localization of the glutathione dependent Nitroblue tetrazolium (NBT) reductase in fresh frozen sections of mouse skin and possible dependence of NBT reductase on tissue thiol levels has been investigated. Methods The fresh frozen tissue sections (8m thickness) were prepared and incubated in medium containing NBT, reduced glutathione (GSH) and phosphate buffer. The staining for GSH was performed with mercury orange. Results  The activity of the NBT-reductase in mouse skin has been found to be localized in the areas rich in glutathione and actively proliferating area of the skin. Conclusion The activity of the NBT-reductase seems to be dependent on the glutathione contents.

  15. Two new sequence type isolates of Bacillus anthracis by multilocus sequence typing%两株炭疽芽胞杆菌MLST新序列型(ST)

    Institute of Scientific and Technical Information of China (English)

    左庭婷; 李岩伟; 韩雪莲; 何君; 端青

    2012-01-01

    [目的]2株炭疽芽胞杆菌(Bacillus anthracis)17003-14和17003-32的多位点序列分型(Multilocussequence typing,MLST)研究.[方法]选取B.anthracis基因组7个常见管家基因位点glpF、gmk、ilvD、pta、pur、pycA和tpi进行PCR扩增、测序,与MLST数据库中的等位基因序列进行比对,确定菌株的序列型( sequence type,ST).[结果]B.anthracis 17003-14和17003-32的等位基因编号分别为113、31、1、43、1、53、7和113、31、1、43、1、53、37,比对结果显示这2株细菌的等位基因编号组合未见报道.[结论]17003-14和17003-32为新ST菌株,已被MLST数据库确认,注册号(pubMLST id)分别为id-1053和id-1054.%[Objective] To define the sequence type (ST) isolates of Bacillus anthracis by multilocus sequence typing (MLST). [Methods] Fragments of seven housekeeping genes (glpF, gmk, ilvD, pta, pur, pycA , and tpi) were amplified by PCR using the standard primers as described on the website for MLST of Bacillus and the sequences were compared with existing allele sequences on the MLST website. [Results] Two novel allele combinations of the seven loci were found in two isolates 17003-14 and 17003-32. [Conclusion] Two novel ST isolates of B. anthracis were identified by this study and confirmed by the MLST website, and the pubMLST ids were id-1053 and id-1054.

  16. Cloning and Expression of Fusion Genes of Domain A-1 Protective Antigen of Bacillus Anthracis and Shigella Enterotoxin B Subunit (Stxb In E. Coil

    Directory of Open Access Journals (Sweden)

    AH ahmadi

    2015-02-01

    Conclusion: The findings of the current study revealed that this antigen can be raised as an anti-cancer and recombinant vaccine candidate against types of Shigella, Escherichia coli and Bacillus anthracis which can be due to such factors as identification of antigen(PA by antibody PA20, its apoptosis induction properties, property of immunogenicity, adjuvant and delivery of STxB protein and high expression levels of Gb3 in human cancer cells.

  17. Immunization with a Recombinant, Pseudomonas fluorescens-Expressed, Mutant Form of Bacillus anthracis-Derived Protective Antigen Protects Rabbits from Anthrax Infection

    OpenAIRE

    Reed, Matthew D.; Wilder, Julie A.; Mega, William M.; Hutt, Julie A.; Kuehl, Philip J.; Valderas, Michelle W.; Chew, Lawrence L.; Liang, Bertrand C.; Squires, Charles H.

    2015-01-01

    Protective antigen (PA), one of the components of the anthrax toxin, is the major component of human anthrax vaccine (Biothrax). Human anthrax vaccines approved in the United States and Europe consist of an alum-adsorbed or precipitated (respectively) supernatant material derived from cultures of toxigenic, non-encapsulated strains of Bacillus anthracis. Approved vaccination schedules in humans with either of these vaccines requires several booster shots and occasionally causes adverse inject...

  18. A Novel Immunogenic Spore Coat-Associated Protein in Bacillus Anthracis: Characterization via Proteomics Approaches and a Vector-Based Vaccine System

    OpenAIRE

    Liu, Yu-Tsueng; Lin, Shwu-Bin; Huang, Cheng-Po; Huang, Chun-Ming

    2007-01-01

    New generation anthrax vaccines have been actively explored with the aim of enhancing efficacies and decreasing undesirable side effects that could be caused by licensed vaccines. Targeting novel antigens and/or eliminating the requirements for multiple needle injections and adjuvants are major objectives in the development of new anthrax vaccines. Using proteomics approaches, we identified a spore coat-associated protein (SCAP) in Bacillus anthracis. An E. coli vector-based vaccine system wa...

  19. Immunological Correlates for Protection against Intranasal Challenge of Bacillus anthracis Spores Conferred by a Protective Antigen-Based Vaccine in Rabbits

    OpenAIRE

    Weiss, Shay; Kobiler, David; Levy, Haim; Marcus, Hadar; Pass, Avi; Rothschild, Nili; Altboum, Zeev

    2006-01-01

    Correlates between immunological parameters and protection against Bacillus anthracis infection in animals vaccinated with protective antigen (PA)-based vaccines could provide surrogate markers to evaluate the putative protective efficiency of immunization in humans. In previous studies we demonstrated that neutralizing antibody levels serve as correlates for protection in guinea pigs (S. Reuveny et al., Infect. Immun. 69:2888-2893, 2001; H. Marcus et al., Infect. Immun. 72:3471-3477, 2004). ...

  20. Bacillus anthracis spores germinate extracellularly at air–liquid interface in an in vitro lung model under serum‐free conditions

    OpenAIRE

    Powell, J D; Hutchison, J.R.; Hess, B.M.; Straub, T.M.

    2015-01-01

    Abstract Aims To better understand the parameters that govern spore dissemination after lung exposure using in vitro cell systems. Methods and Results We evaluated the kinetics of uptake, germination and proliferation of Bacillus anthracis Sterne spores in association with human primary lung epithelial cells, Calu‐3 and A549 cell lines. We also analysed the influence of various cell culture medium formulations related to spore germination. Conclusions We found negligible spore uptake by epith...

  1. Anthrax toxin targeting of myeloid cells through the CMG2 receptor is essential for establishment of Bacillus anthracis infections in mice

    OpenAIRE

    Liu, Shihui; Miller-Randolph, Sharmina; Crown, Devorah; Moayeri, Mahtab; Sastalla, Inka; Okugawa, Shu; Leppla, Stephen H.

    2010-01-01

    Bacillus anthracis kills through a combination of bacterial infection and toxemia. Anthrax toxin working via the CMG2 receptor mediates lethality late in infection, but its roles early in infection remain unclear. We generated myeloid-lineage specific CMG2-deficient mice to examine the roles of macrophages, neutrophils, and other myeloid cells in anthrax pathogenesis. Macrophages and neutrophils isolated from these mice were resistant to anthrax toxin. However, the myeloid-specific CMG2-defic...

  2. Matrix Assisted Laser Desorption Ionization Mass Spectrometric Analysis of Bacillus anthracis: From Fingerprint Analysis of the Bacterium to Quantification of its Toxins in Clinical Samples

    Science.gov (United States)

    Woolfitt, Adrian R.; Boyer, Anne E.; Quinn, Conrad P.; Hoffmaster, Alex R.; Kozel, Thomas R.; de, Barun K.; Gallegos, Maribel; Moura, Hercules; Pirkle, James L.; Barr, John R.

    A range of mass spectrometry-based techniques have been used to identify, characterize and differentiate Bacillus anthracis, both in culture for forensic applications and for diagnosis during infection. This range of techniques could usefully be considered to exist as a continuum, based on the degrees of specificity involved. We show two examples here, a whole-organism fingerprinting method and a high-specificity assay for one unique protein, anthrax lethal factor.

  3. Bacillus anthracis Capsule Activates Caspase-1 and Induces Interleukin-1β Release from Differentiated THP-1 and Human Monocyte-Derived Dendritic Cells▿ †

    OpenAIRE

    Cho, Min-Hee; Ahn, Hae-Jeong; Ha, Hyun-Joon; Park, Jungchan; Chun, Jeong-Hoon; Kim, Bong-Su; Oh, Hee-Bok; Rhie, Gi-eun

    2009-01-01

    The poly-γ-d-glutamic acid (PGA) capsule is one of the major virulence factors of Bacillus anthracis, which causes a highly lethal infection. The antiphagocytic PGA capsule disguises the bacilli from immune surveillance and allows unimpeded growth of bacilli in the host. Recently, efforts have been made to include PGA as a component of anthrax vaccine; however, the innate immune response of PGA itself has been poorly investigated. In this study, we characterized the innate immune response eli...

  4. Characterization of Bacillus anthracis-Like Bacteria Isolated from Wild Great Apes from Côte d'Ivoire and Cameroon

    OpenAIRE

    Klee, Silke R.; Özel, Muhsin; Appel, Bernd; Boesch, Christophe; Ellerbrok, Heinz; Jacob, Daniela; Holland, Gudrun; Leendertz, Fabian H; Pauli, Georg; Grunow, Roland; Nattermann, Herbert

    2006-01-01

    We present the microbiological and molecular characterization of bacteria isolated from four chimpanzees and one gorilla thought to have died of an anthrax-like disease in Côte d'Ivoire and Cameroon. These isolates differed significantly from classic Bacillus anthracis by the following criteria: motility, resistance to the gamma phage, and, for isolates from Cameroon, resistance to penicillin G. A capsule was expressed not only after induction by CO2 and bicarbonate but also under normal grow...

  5. Generierung und genotypische Untersuchung eines Ciprofloxacin-resistenten Bacillus cereus Stammes und Entwicklung von real-time-PCR-Schnelltests zum Nachweis von Resistenzen gegen Ciprofloxacin in Bacillus anthracis

    OpenAIRE

    Hübner, Anika

    2014-01-01

    Die gebräuchliche Therapie gegen Milzbrand besteht aus der Gabe von Antibiotika. Als Therapie der Wahl gilt hierbei das Fluorochinolon Ciprofloxacin. Resistenzen gegen dieses Antibiotikum wurden bei B. anthracis in vivo noch nicht, in vitro jedoch im Rahmen mehrerer Studien beschrieben. Es existieren herkömmliche Resistenztests, wie der Gradientendiffusions- oder der Mikrodilutionstest, welche bei einer Milzbranderkrankung genutzt werden können. Diese nehmen jedoch aufgrund der kulturellen An...

  6. A Field Investigation of Bacillus anthracis Contamination of U.S. Department of Agriculture and Other Washington, D.C., Buildings during the Anthrax Attack of October 2001

    OpenAIRE

    Higgins, James A.; Cooper, Mary; Schroeder-Tucker, Linda; Black, Scott; Miller, David; Karns, Jeffrey S.; Manthey, Erlynn; Breeze, Roger; Perdue, Michael L

    2003-01-01

    In response to a bioterrorism attack in the Washington, D.C., area in October 2001, a mobile laboratory (ML) was set up in the city to conduct rapid molecular tests on environmental samples for the presence of Bacillus anthracis spores and to route samples for further culture analysis. The ML contained class I laminar-flow hoods, a portable autoclave, two portable real-time PCR devices (Ruggedized Advanced Pathogen Identification Device [RAPID]), and miscellaneous supplies and equipment to pr...

  7. Immunoelectrophoretic analysis, toxicity, and kinetics of in vitro production of the protective antigen and lethal factor components of Bacillus anthracis toxin.

    OpenAIRE

    Ezzell, J W; Ivins, B E; Leppla, S H

    1984-01-01

    The kinetics of Bacillus anthracis toxin production in culture and its lethal activity in rats, mice, and guinea pigs were investigated. Lethal toxin activity was produced in vitro throughout exponential growth at essentially identical rates in both encapsulated virulent and nonencapsulated avirulent strains. The two toxin proteins which produce lethality when in combination, lethal factor (LF) and protective antigen (PA), could be quantitated directly from culture fluids by rocket immunoelec...

  8. Human Monoclonal Antibody AVP-21D9 to Protective Antigen Reduces Dissemination of the Bacillus anthracis Ames Strain from the Lungs in a Rabbit Model▿

    Science.gov (United States)

    Peterson, Johnny W.; Comer, Jason E.; Baze, Wallace B.; Noffsinger, David M.; Wenglikowski, Autumn; Walberg, Kristin G.; Hardcastle, Jason; Pawlik, Jennifer; Bush, Kathryn; Taormina, Joanna; Moen, Scott; Thomas, John; Chatuev, Bagram M.; Sower, Laurie; Chopra, Ashok K.; Stanberry, Lawrence R.; Sawada, Ritsuko; Scholz, Wolfgang W.; Sircar, Jagadish

    2007-01-01

    Dutch-belted and New Zealand White rabbits were passively immunized with AVP-21D9, a human monoclonal antibody to protective antigen (PA), at the time of Bacillus anthracis spore challenge using either nasal instillation or aerosol challenge techniques. AVP-21D9 (10 mg/kg) completely protected both rabbit strains against lethal infection with Bacillus anthracis Ames spores, regardless of the inoculation method. Further, all but one of the passively immunized animals (23/24) were completely resistant to rechallenge with spores by either respiratory challenge method at 5 weeks after primary challenge. Analysis of the sera at 5 weeks after primary challenge showed that residual human anti-PA levels decreased by 85 to 95%, but low titers of rabbit-specific anti-PA titers were also measured. Both sources of anti-PA could have contributed to protection from rechallenge. In a subsequent study, bacteriological and histopathology analyses revealed that B. anthracis disseminated to the bloodstream in some naïve animals as early as 24 h postchallenge and increased in frequency with time. AVP-21D9 significantly reduced the dissemination of the bacteria to the bloodstream and to various organs following infection. Examination of tissue sections from infected control animals, stained with hematoxylin-eosin and the Gram stain, showed edema and/or hemorrhage in the lungs and the presence of bacteria in mediastinal lymph nodes, with necrosis and inflammation. Tissue sections from infected rabbits dosed with AVP-21D9 appeared comparable to corresponding tissues from uninfected animals despite lethal challenge with B. anthracis Ames spores. Concomitant treatment with AVP-21D9 at the time of challenge conferred complete protection in the rabbit inhalation anthrax model. Early treatment increased the efficacy progressively and in a dose-dependent manner. Thus, AVP-21D9 could offer an adjunct or alternative clinical treatment regimen against inhalation anthrax. PMID:17452469

  9. Human monoclonal antibody AVP-21D9 to protective antigen reduces dissemination of the Bacillus anthracis Ames strain from the lungs in a rabbit model.

    Science.gov (United States)

    Peterson, Johnny W; Comer, Jason E; Baze, Wallace B; Noffsinger, David M; Wenglikowski, Autumn; Walberg, Kristin G; Hardcastle, Jason; Pawlik, Jennifer; Bush, Kathryn; Taormina, Joanna; Moen, Scott; Thomas, John; Chatuev, Bagram M; Sower, Laurie; Chopra, Ashok K; Stanberry, Lawrence R; Sawada, Ritsuko; Scholz, Wolfgang W; Sircar, Jagadish

    2007-07-01

    Dutch-belted and New Zealand White rabbits were passively immunized with AVP-21D9, a human monoclonal antibody to protective antigen (PA), at the time of Bacillus anthracis spore challenge using either nasal instillation or aerosol challenge techniques. AVP-21D9 (10 mg/kg) completely protected both rabbit strains against lethal infection with Bacillus anthracis Ames spores, regardless of the inoculation method. Further, all but one of the passively immunized animals (23/24) were completely resistant to rechallenge with spores by either respiratory challenge method at 5 weeks after primary challenge. Analysis of the sera at 5 weeks after primary challenge showed that residual human anti-PA levels decreased by 85 to 95%, but low titers of rabbit-specific anti-PA titers were also measured. Both sources of anti-PA could have contributed to protection from rechallenge. In a subsequent study, bacteriological and histopathology analyses revealed that B. anthracis disseminated to the bloodstream in some naïve animals as early as 24 h postchallenge and increased in frequency with time. AVP-21D9 significantly reduced the dissemination of the bacteria to the bloodstream and to various organs following infection. Examination of tissue sections from infected control animals, stained with hematoxylin-eosin and the Gram stain, showed edema and/or hemorrhage in the lungs and the presence of bacteria in mediastinal lymph nodes, with necrosis and inflammation. Tissue sections from infected rabbits dosed with AVP-21D9 appeared comparable to corresponding tissues from uninfected animals despite lethal challenge with B. anthracis Ames spores. Concomitant treatment with AVP-21D9 at the time of challenge conferred complete protection in the rabbit inhalation anthrax model. Early treatment increased the efficacy progressively and in a dose-dependent manner. Thus, AVP-21D9 could offer an adjunct or alternative clinical treatment regimen against inhalation anthrax. PMID:17452469

  10. Next-Generation Bacillus anthracis Live Attenuated Spore Vaccine Based on the htrA(-) (High Temperature Requirement A) Sterne Strain.

    Science.gov (United States)

    Chitlaru, Theodor; Israeli, Ma'ayan; Bar-Haim, Erez; Elia, Uri; Rotem, Shahar; Ehrlich, Sharon; Cohen, Ofer; Shafferman, Avigdor

    2016-01-01

    Anthrax is a lethal disease caused by the gram-positive spore-producing bacterium Bacillus anthracis. Live attenuated vaccines, such as the nonencapsulated Sterne strain, do not meet the safety standards mandated for human use in the Western world and are approved for veterinary purposes only. Here we demonstrate that disrupting the htrA gene, encoding the chaperone/protease HtrA (High Temperature Requirement A), in the virulent Bacillus anthracis Vollum strain results in significant virulence attenuation in guinea pigs, rabbits and mice, underlying the universality of the attenuated phenotype associated with htrA knockout. Accordingly, htrA disruption was implemented for the development of a Sterne-derived safe live vaccine compatible with human use. The novel B. anthracis SterneΔhtrA strain secretes functional anthrax toxins but is 10-10(4)-fold less virulent than the Sterne vaccine strain depending on animal model (mice, guinea pigs, or rabbits). In spite of this attenuation, double or even single immunization with SterneΔhtrA spores elicits immune responses which target toxaemia and bacteremia resulting in protection from subcutaneous or respiratory lethal challenge with a virulent strain in guinea pigs and rabbits. The efficacy of the immune-protective response in guinea pigs was maintained for at least 50 weeks after a single immunization. PMID:26732659

  11. Comprehensive Laboratory Evaluation of a Highly Specific Lateral Flow Assay for the Presumptive Identification of Bacillus anthracis Spores in Suspicious White Powders and Environmental Samples

    Science.gov (United States)

    Ramage, Jason G.; Prentice, Kristin W.; DePalma, Lindsay; Venkateswaran, Kodumudi S.; Chivukula, Sruti; Chapman, Carol; Bell, Melissa; Datta, Shomik; Singh, Ajay; Hoffmaster, Alex; Sarwar, Jawad; Parameswaran, Nishanth; Joshi, Mrinmayi; Thirunavkkarasu, Nagarajan; Krishnan, Viswanathan; Morse, Stephen; Avila, Julie R.; Sharma, Shashi; Estacio, Peter L.; Stanker, Larry; Hodge, David R.

    2016-01-01

    We conducted a comprehensive, multiphase laboratory evaluation of the Anthrax BioThreat Alert® test strip, a lateral flow immunoassay (LFA) for the rapid detection of Bacillus anthracis spores. The study, conducted at 2 sites, evaluated this assay for the detection of spores from the Ames and Sterne strains of B. anthracis, as well as those from an additional 22 strains. Phylogenetic near neighbors, environmental background organisms, white powders, and environmental samples were also tested. The Anthrax LFA demonstrated a limit of detection of about 106 spores/mL (ca. 1.5 × 105 spores/assay). In this study, overall sensitivity of the LFA was 99.3%, and the specificity was 98.6%. The results indicated that the specificity, sensitivity, limit of detection, dynamic range, and repeatability of the assay support its use in the field for the purpose of qualitatively evaluating suspicious white powders and environmental samples for the presumptive presence of B. anthracis spores. PMID:27661796

  12. Characterization of Genetic Diversity of Bacillus anthracis in France by Using High-Resolution Melting Assays and Multilocus Variable-Number Tandem-Repeat Analysis ▿ †

    Science.gov (United States)

    Derzelle, S.; Laroche, S.; Le Flèche, P.; Hauck, Y.; Thierry, S.; Vergnaud, G.; Madani, N.

    2011-01-01

    Using high-resolution melting (HRM) analysis, we developed a cost-effective method to genotype a set of 13 phylogenetically informative single-nucleotide polymorphisms (SNPs) within the genome of Bacillus anthracis. SNP discrimination assays were performed in monoplex or duplex and applied to 100 B. anthracis isolates collected in France from 1953 to 2009 and a few reference strains. HRM provided a reliable and cheap alternative to subtype B. anthracis into one of the 12 major sublineages or subgroups. All strains could be correctly positioned on the canonical SNP (canSNP) phylogenetic tree, except the divergent Pasteur vaccine strain ATCC 4229. We detected the cooccurrence of three canSNP subgroups in France. The dominant B.Br.CNEVA sublineage was found to be prevalent in the Alps, the Pyrenees, the Auvergne region, and the Saône-et-Loire department. Strains affiliated with the A.Br.008/009 subgroup were observed throughout most of the country. The minor A.Br.001/002 subgroup was restricted to northeastern France. Multiple-locus variable-number tandem-repeat analysis using 24 markers further resolved French strains into 60 unique profiles and identified some regional patterns. Diversity found within the A.Br.008/009 and B.Br.CNEVA subgroups suggests that these represent old, ecologically established clades in France. Phylogenetic relationships with strains from other parts of the world are discussed. PMID:21998431

  13. Discrimination of Bacillus anthracis Spores by Direct in-situ Analysis of Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Youngsu; Lee, Jonghee; Kim, Seongsoo [Agency for Defense Development, Daejeon (Korea, Republic of)

    2013-09-15

    The rapid and accurate identification of biological agents is a critical step in the case of bio-terror and biological warfare attacks. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has been widely used for the identification of microorganisms. In this study, we describe a method for the rapid and accurate discrimination of Bacillus anthracis spores using MALDI-TOF MS. Our direct in-situ analysis of MALDI-TOF MS does not involve subsequent high-resolution mass analyses and sample preparation steps. This method allowed the detection of species-specific biomarkers from each Bacillus spores. Especially, B. anthracis spores had specific biomarker peaks at 2503, 3089, 3376, 6684, 6698, 6753, and 6840 m/z. Cluster and PCA analyses of the mass spectra of Bacillus spores revealed distinctively separated clusters and within-groups similarity. Therefore, we believe that this method is effective in the real-time identification of biological warfare agents such as B. anthracis as well as other microorganisms in the field.

  14. Comprehensive Laboratory Evaluation of a Highly Specific Lateral Flow Assay for the Presumptive Identification of Bacillus anthracis Spores in Suspicious White Powders and Environmental Samples.

    Science.gov (United States)

    Ramage, Jason G; Prentice, Kristin W; DePalma, Lindsay; Venkateswaran, Kodumudi S; Chivukula, Sruti; Chapman, Carol; Bell, Melissa; Datta, Shomik; Singh, Ajay; Hoffmaster, Alex; Sarwar, Jawad; Parameswaran, Nishanth; Joshi, Mrinmayi; Thirunavkkarasu, Nagarajan; Krishnan, Viswanathan; Morse, Stephen; Avila, Julie R; Sharma, Shashi; Estacio, Peter L; Stanker, Larry; Hodge, David R; Pillai, Segaran P

    2016-01-01

    We conducted a comprehensive, multiphase laboratory evaluation of the Anthrax BioThreat Alert(®) test strip, a lateral flow immunoassay (LFA) for the rapid detection of Bacillus anthracis spores. The study, conducted at 2 sites, evaluated this assay for the detection of spores from the Ames and Sterne strains of B. anthracis, as well as those from an additional 22 strains. Phylogenetic near neighbors, environmental background organisms, white powders, and environmental samples were also tested. The Anthrax LFA demonstrated a limit of detection of about 10(6) spores/mL (ca. 1.5 × 10(5) spores/assay). In this study, overall sensitivity of the LFA was 99.3%, and the specificity was 98.6%. The results indicated that the specificity, sensitivity, limit of detection, dynamic range, and repeatability of the assay support its use in the field for the purpose of qualitatively evaluating suspicious white powders and environmental samples for the presumptive presence of B. anthracis spores. PMID:27661796

  15. Structures of an alanine racemase from Bacillus anthracis (BA0252) in the presence and absence of (R)-1-aminoethylphosphonic acid (l-Ala-P)

    International Nuclear Information System (INIS)

    Structures of BA0252, an alanine racemase from B. anthracis, in the presence and absence of the inhibitor (R)-1-aminoethylphosphonic acid (l-Ala-P) and determined by X-ray crystallography to resolutions of 2.1 and 1.47 Å, respectively, are described. Bacillus anthracis, the causative agent of anthrax, has been targeted by the Oxford Protein Production Facility to validate high-throughput protocols within the Structural Proteomics in Europe project. As part of this work, the structures of an alanine racemase (BA0252) in the presence and absence of the inhibitor (R)-1-aminoethylphosphonic acid (l-Ala-P) have determined by X-ray crystallo@@graphy to resolutions of 2.1 and 1.47 Å, respectively. Difficulties in crystallizing this protein were overcome by the use of reductive methylation. Alanine racemase has attracted much interest as a possible target for anti-anthrax drugs: not only is d-alanine a vital component of the bacterial cell wall, but recent studies also indicate that alanine racemase, which is accessible in the exosporium, plays a key role in inhibition of germination in B. anthracis. These structures confirm the binding mode of l-Ala-P but suggest an unexpected mechanism of inhibition of alanine racemase by this compound and could provide a basis for the design of improved alanine racemase inhibitors with potential as anti-anthrax therapies

  16. Discrimination of Bacillus anthracis Spores by Direct in-situ Analysis of Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry

    International Nuclear Information System (INIS)

    The rapid and accurate identification of biological agents is a critical step in the case of bio-terror and biological warfare attacks. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has been widely used for the identification of microorganisms. In this study, we describe a method for the rapid and accurate discrimination of Bacillus anthracis spores using MALDI-TOF MS. Our direct in-situ analysis of MALDI-TOF MS does not involve subsequent high-resolution mass analyses and sample preparation steps. This method allowed the detection of species-specific biomarkers from each Bacillus spores. Especially, B. anthracis spores had specific biomarker peaks at 2503, 3089, 3376, 6684, 6698, 6753, and 6840 m/z. Cluster and PCA analyses of the mass spectra of Bacillus spores revealed distinctively separated clusters and within-groups similarity. Therefore, we believe that this method is effective in the real-time identification of biological warfare agents such as B. anthracis as well as other microorganisms in the field

  17. The effect of copper and gallium compounds on ribonucleotide reductase

    Energy Technology Data Exchange (ETDEWEB)

    Narasimhan, J.

    1992-01-01

    The mode of action of copper complexes (CuL and CuKTS) and gallium compounds (gallium nitrate and citrate) in cytotoxicity was studied. The effects of these agents on the enzyme ribonucleotide reductase was investigated by monitoring the tyrosyl free radical present in the active site of the enzyme through electron spin resonance (ESR) spectroscopy. Ribonucleotide reductase, a key enzyme in cellular proliferation, consists of two subunits. M1, a dimer of molecular weight 170,000 contains the substrate and effector binding sites. M2, a dimer of molecular weight 88,000, contains non-heme iron and tyrosyl free radical essential for the activity of the enzyme. In studies using copper complexes, the cellular oxidative chemistry was examined by ESR studies on adduct formation with membranes, and oxidation of thiols. Membrane thiols were oxidized through the reduction of the ESR signal of the thiol adduct and the analysis of sulfhydryl content. Using the radiolabel [sup 59]Fe, the inhibitory action of copper thiosemicarbazones on cellular iron uptake was shown. The inhibitory action of CuL on ribonucleotide reductase was shown by the quenching of the tyrosyl free radical on the M2 subunit. The hypothesis that gallium directly interacts with the M2 subunit of the enzyme and displaces the iron from it was proven. The tyrosyl free radical signal from cell lysates was inhibited by the direct addition of gallium compounds. Gallium content in the cells was measured by a fluorimetric method, to ensure the presence of sufficient amounts of gallium to compete with the iron in the M2 subunit. The enzyme activity, measured by the conversion of [sup 14]C-CDP to the labeled deoxy CDP, was inhibited by the addition of gallium nitrate in a cell free assay system. The immunoprecipitation studies of the [sup 59]Fe labeled M2 protein using the monoclonal antibody directed against this subunit suggested that gallium releases iron from the M2 subunit.

  18. Aldo-keto reductases 1B in adrenal cortex physiology

    Directory of Open Access Journals (Sweden)

    Emilie PASTEL

    2016-07-01

    Full Text Available Aldose reductase proteins are cytosolic monomeric enzymes, belonging to the aldo-keto reductase (AKR superfamily. They perform oxidoreduction of carbonyl groups from a wide variety of substrates such as aliphatic and aromatic aldehydes or ketones. The Aldose reductase subgroup (AKR1B is one of the most characterized because of its involvement in human diseases such as diabetic complications resulting from the ability of its human archetype AKR1B1 to reduce glucose into sorbitol. However the issue of AKR1B function in non pathologic condition remains poorly resolved. Adrenal steroidogenesis is strongly associated with high production of endogenous harmful lipid aldehyde by-products including isocaproaldehyde (4-methylpentanal derived from cholesterol side chain cleavage (the first step of steroid synthesis and 4-hydroxynonenal (4- HNE that can both be reduced by AKR1B proteins. More recently, some AKR1B isoforms have been shown to be endowed with prostaglandin F synthase activity, suggesting that in addition to possible scavenger function, they could instigate paracrine signals. Interestingly, previous studies have established that the adrenal gland is one of the major site for human and murine AKR1B expression suggesting that their detoxifying/signaling activity could be specifically required for the correct handling of adrenal function. Moreover chronic effects of ACTH result in a coordinated regulation of genes encoding the steroidogenic enzymes and some AKR1B isoforms.This review presents the molecular mechanisms accounting for the adrenal specific expression of some AKR1B genes. Using data from recent mouse genetic models, we will try to connect their enzymatic properties and regulation with adrenal functions.

  19. Methylenetetrahydrofolate reductase (MTHFR) deficiency presenting as a rash.

    LENUS (Irish Health Repository)

    Crushell, Ellen

    2012-09-01

    We report on the case of a 2-year-old girl recently diagnosed with Methylenetetrahydrofolate reductase (MTHFR) deficiency who originally presented in the neonatal period with a distinctive rash. At 11 weeks of age she developed seizures, she had acquired microcephaly and developmental delay. The rash deteriorated dramatically following commencement of phenobarbitone; both rash and seizures abated following empiric introduction of pyridoxine and folinic acid as treatment of possible vitamin responsive seizures. We postulate that phenobarbitone in combination with MTHFR deficiency may have caused her rash to deteriorate and subsequent folinic acid was helpful in treating the rash and preventing further acute neurological decline as commonly associated with this condition.

  20. Vibrio harveyi Nitroreductase Is Also a Chromate Reductase

    OpenAIRE

    Kwak, Young Hak; Lee, Dong Seok; Kim, Han Bok

    2003-01-01

    The chromate reductase purified from Pseudomonas ambigua was found to be homologous with several nitroreductases. Escherichia coli DH5α and Vibrio harveyi KCTC 2720 nitroreductases were chosen for the present study, and their chromate-reducing activities were determined. A fusion between glutathione S-transferase (GST) and E. coli DH5α NfsA (GST-EcNfsA), a fusion between GST and E. coli DH5α NfsB (GST-EcNfsB), and a fusion between GST and V. harveyi KCTC 2720 NfsA (GST-VhNfsA) were prepared f...

  1. Mediated electrochemistry of dimethyl sulfoxide reductase promoted by carbon nanotubes

    Institute of Scientific and Technical Information of China (English)

    BERNHARDT; Paul; V

    2010-01-01

    Mediated electrochemistry of dimethyl sulfoxide reductase from Rhodobacter capsulatus (DMSOR) which is immobilized on a bare glassy carbon (GC) electrode and a carbon nanotube (CNT)-modified GC electrode was studied using the Co complex (trans-6,13-dimethyl-1,4,8,11-tetraazacyclotetradecane-6,13-diamine)cobalt(III) ([Co(trans-diammac)] +) as a mediator.The cyclic voltammograms of different electrodes were carried out at different substrate (DMSO) concentrations.The results demonstrated that the catalytic current was increased by employing CNT as a promoter.

  2. Applications of Carboxylic Acid Reductases in Oleaginous Microbes

    Energy Technology Data Exchange (ETDEWEB)

    Resch, Michael G.; Linger, Jeffrey; McGeehan, John; Tyo, Keith; Beckham, Gregg

    2016-04-24

    Carboxylic acid reductases (CARs) are recently emerging reductive enzymes for the direct production of aldehydes from biologically-produced carboxylic acids. Recent work has demonstrated that these powerful enzymes are able to reduce a very broad range of volatile- to long-chain fatty acids as well as aromatic acids. Here, we express four CAR enzymes from different fungal origins to test their activity against fatty acids commonly produced in oleaginous microbes. These in vitro results will inform metabolic engineering strategies to conduct mild biological reduction of carboxylic acids in situ, which is conventionally done via hydrotreating catalysis at high temperatures and hydrogen pressures.

  3. Functions of Flavin Reductase and Quinone Reductase in 2,4,6-Trichlorophenol Degradation by Cupriavidus necator JMP134▿

    OpenAIRE

    Belchik, Sara Mae; Xun, Luying

    2007-01-01

    The tcpRXABCYD operon of Cupriavidus necator JMP134 is involved in the degradation of 2,4,6-trichlorophenol (2,4,6-TCP), a toxic pollutant. TcpA is a reduced flavin adenine dinucleotide (FADH2)-dependent monooxygenase that converts 2,4,6-TCP to 6-chlorohydroxyquinone. It has been implied via genetic analysis that TcpX acts as an FAD reductase to supply TcpA with FADH2, whereas the function of TcpB in 2,4,6-TCP degradation is still unclear. In order to provide direct biochemical evidence for t...

  4. MEK2 regulates ribonucleotide reductase activity through functional interaction with ribonucleotide reductase small subunit p53R2

    OpenAIRE

    Piao, Chunmei; Youn, Cha-Kyung; Jin, Min; Yoon, Sang Pil; Chang, In-Youb; Lee, Jung Hee; You, Ho Jin

    2012-01-01

    The p53R2 protein, a newly identified member of the ribonucleotide reductase family that provides nucleotides for DNA damage repair, is directly regulated by p53. We show that p53R2 is also regulated by a MEK2 (ERK kinase 2/MAP kinase kinase 2)-dependent pathway. Increased MEK1/2 phosphorylation by serum stimulation coincided with an increase in the RNR activity in U2OS and H1299 cells. The inhibition of MEK2 activity, either by treatment with a MEK inhibitor or by transfection with MEK2 siRN...

  5. Evaluation of 5α-reductase inhibitory activity of certain herbs useful as antiandrogens.

    Science.gov (United States)

    Nahata, A; Dixit, V K

    2014-08-01

    This study demonstrates 5α-reductase inhibitory activity of certain herbs useful in the management of androgenic disorders. Ganoderma lucidum (Curtis) P. Karst (GL), Urtica dioica Linn. (UD), Caesalpinia bonducella Fleming. (CB), Tribulus terrestris Linn. (TT), Pedalium murex Linn. (PM), Sphaeranthus indicus Linn. (SI), Cuscuta reflexa Roxb. (CR), Citrullus colocynthis Schrad. (CC), Benincasa hispida Cogn. (BH), Phyllanthus niruri Linn. (PN) and Echinops echinatus Linn. (EE) were included in the study. Petroleum ether, ethanol and aqueous extracts of these herbs were tested for their 5α-reductase inhibitory activity against the standard 5α-reductase inhibitor, finasteride. A biochemical method to determine the activity of 5α-reductase was used to evaluate the inhibition of different extracts to the enzyme. The optical density (OD) value of each sample was measured continuously with ultraviolet spectrophotometer for the reason that the substrate NADPH has a specific absorbance at 340 nm. As the enzyme 5α-reductase uses NADPH as a substrate, so in the presence of 5α-reductase inhibitor, the NADPH concentration will increase with the function of time. This method thus implicates the activity of 5α-reductase. The method proved to be extremely useful to screen the herbs for their 5α-reductase inhibitory potential. GL, UD, BH, SI and CR came out to be promising candidates for further exploring their antiandrogenic properties. PMID:23710567

  6. THE EFFECTS OF AN ALDOSE REDUCTASE INHIBITOR ON THE PROGRESSION OF DIABETIC-RETINOPATHY

    NARCIS (Netherlands)

    TROMP, A; HOOYMANS, JMM; BARENDSEN, BC; VONDOORMAAL, JJ

    1991-01-01

    The polyol pathway has long been associated with diabetic retinopathy. Glucose is converted to sorbitol with the aid of the enzyme aldose reductase. Aldose reductase inhibitors can prevent changes induced by diabetes. A total of 30 patients with minimal background retinopathy were randomly divided i

  7. Sucrose mimics the light induction of Arabidopsis nitrate reductase gene transcription

    DEFF Research Database (Denmark)

    Cheng, Chi-Lien; Acedo, Gregoria N; Kristensen, Michael;

    1992-01-01

    Nitrate reductase, the first enzyme in nitrate assimilation, is located at the crossroad of two energy-consuming pathways: nitrate assimilation and carbon fixation. Light, which regulates the expression of many higher-plant carbon fixation genes, also regulates nitrate reductase gene expression. ...

  8. Determination of the specific activities of methionine sulfoxide reductase A and B by capillary electrophoresis

    Science.gov (United States)

    A capillary electrophoresis (CE) method for the determination of methionine sulfoxide reductase A and methionine sulfoxide reductase B activities in mouse liver is described. The method is based on detection of the 4-(dimethylamino)azobenzene-4’-sulfonyl derivative of L-methionine (dabsyl Met), the ...

  9. Stereospecificity of (+)-pinoresinol and (+)-lariciresinol reductases from Forsythia intermedia.

    Science.gov (United States)

    Chu, A; Dinkova, A; Davin, L B; Bedgar, D L; Lewis, N G

    1993-12-25

    Pinoresinol/lariciresinol reductase catalyzes the first known example of a highly unusual benzylic ether reduction in plants; its mechanism of hydride transfer is described. The enzyme was found in Forsythia intermedia and catalyzes the presumed regulatory branch-points in the pathway leading to benzylaryltetrahydrofuran, dibenzylbutane, dibenzylbutyrolactone, and aryltetrahydronaphthalene lignans. Using [7,7'-2H2]-pinoresinol and [7,7'-2H3]lariciresinol as substrates, the hydride transfers of the highly unusual reductase were demonstrated to be completely stereospecific (> 99%). The incoming hydrides were found to take up the pro-R position at C-7' (and/or C-7) in lariciresinol and secoisolariciresinol, thereby eliminating the possibility of random hydride delivery to a planar quinone methide intermediate. As might be expected, the mode of hydride abstraction from NADPH was also stereospecific: using [4R-3H] and [4S-3H]NADPH, it was found that only the 4 pro-R hydrogen was abstracted for enzymatic hydride transfer.

  10. Optimisation of nitrate reductase enzyme activity to synthesise silver nanoparticles.

    Science.gov (United States)

    Khodashenas, Bahareh; Ghorbani, Hamid Reza

    2016-06-01

    Today, the synthesis of silver nanoparticles (Ag NPs) is very common since it has many applications in different areas. The synthesis of these nanoparticles is done by means of physical, chemical, or biological methods. However, due to its inexpensive and environmentally friendly features, the biological method is more preferable. In the present study, using nitrate reductase enzyme available in the Escherichia coli (E. coli) bacterium, the biosynthesis of Ag NPs was investigated. In addition, the activity of the nitrate reductase enzyme was optimised by changing its cultural conditions, and the effects of silver nitrate (AgNO3) concentration and enzyme amount on nanoparticles synthesis were studied. Finally, the produced nanoparticles were studied using ultraviolet -visible (UV-Vis) spectrophotometer, dynamic light scattering technique, and transmission electron microscopy. UV-Visible spectrophotometric study showed the characteristic peak for Ag NPs at wavelength 405-420 nm for 1 mM metal precursor solution (AgNO3) with 1, 5, 10, and 20 cc supernatant and 435 nm for 0.01M AgNO3 with 20 cc supernatant. In this study, it was found that there is a direct relationship between the AgNO3 concentration and the size of produced Ag NPs. PMID:27256897

  11. Azotobacter vinelandii NADPH:ferredoxin reductase cloning, sequencing, and overexpression.

    Science.gov (United States)

    Isas, J M; Yannone, S M; Burgess, B K

    1995-09-01

    Azotobacter vinelandii ferredoxin I (AvFdI) controls the expression of another protein that was originally designated Protein X. Recently we reported that Protein X is a NADPH-specific flavoprotein that binds specifically to FdI (Isas, J.M., and Burgess, B.K. (1994) J. Biol. Chem. 269, 19404-19409). The gene encoding this protein has now been cloned and sequenced. Protein X is 33% identical and has an overall 53% similarity with the fpr gene product from Escherichia coli that encodes NADPH:ferredoxin reductase. On the basis of this similarity and the similarity of the physical properties of the two proteins, we now designate Protein X as A. vinelandii NADPH:ferredoxin reductase and its gene as the fpr gene. The protein has been overexpressed in its native background in A. vinelandii by using the broad host range multicopy plasmid, pKT230. In addition to being regulated by FdI, the fpr gene product is overexpressed when A. vinelandii is grown under N2-fixing conditions even though the fpr gene is not preceded by a nif specific promoter. By analogy to what is known about fpr expression in E. coli, we propose that FdI may exert its regulatory effect on fpr by interacting with the SoxRS regulon. PMID:7673160

  12. Nitrate metabolism in tobacco leaves overexpressing Arabidopsis nitrite reductase.

    Science.gov (United States)

    Davenport, Susie; Le Lay, Pascaline; Sanchez-Tamburrrino, Juan Pablo

    2015-12-01

    Primary nitrogen assimilation in plants includes the reduction of nitrite to ammonium in the chloroplasts by the enzyme nitrite reductase (NiR EC:1.7.7.1) or in the plastids of non-photosynthetic organs. Here we report on a study overexpressing the Arabidopsis thaliana NiR (AtNiR) gene in tobacco plants under the control of a constitutive promoter (CERV - Carnation Etched Ring Virus). The aim was to overexpress AtNiR in an attempt to alter the level of residual nitrite in the leaf which can act as precursor to the formation of nitrosamines. The impact of increasing the activity of AtNiR produced an increase in leaf protein and a stay-green phenotype in the primary transformed AtNiR population. Investigation of the T1 homozygous population demonstrated elevated nitrate reductase (NR) activity, reductions in leaf nitrite and nitrate and the amino acids proline, glutamine and glutamate. Chlorophyl content of the transgenic lines was increased, as evidenced by the stay-green phenotype. This reveals the importance of NiR in primary nitrogen assimilation and how modification of this key enzyme affects both the nitrogen and carbon metabolism of tobacco plants. PMID:26447683

  13. The effect of ionic and non-ionic surfactants on the growth, nitrate reductase and nitrite reductase activities of Spirodela polyrrhiza (L. Schleiden

    Directory of Open Access Journals (Sweden)

    Józef Buczek

    2014-02-01

    Full Text Available Inclusion into the medium of 5 mg•dm-3 of non-ionic (ENF or ionic (DBST surfactant caused 50-60% inhibition of nitrite reductase MR activity in S. polyrrhiza. At the same time, increased accumulation of NO2- in the plant tissues and lowering of the total and soluble protein contents were found. DBST also lowered the nitrate reductase (NR activity and the dry mass of the plants.

  14. 炭疽芽胞杆菌(Bacillus anthracis)检测质粒的构建及其应用%Construction of Plasmid pBIB2006 for Detecting Bacillus anthracis and Its Application

    Institute of Scientific and Technical Information of China (English)

    吕和平; 闵勇; 杨东明; 郑应华; 李旭; 武轶

    2007-01-01

    根据炭疽芽胞杆菌(Bacillus anthracis)毒性质粒pX01和pX02上的2个毒力相关基因cya和capA的序列特点,以pIJ2925为出发载体,采用一步重叠延伸PCR技术(One-step Overlap Extension PCR,简称OOE-PCR)构建了包含cya基因和capA基因保守区DNA片段的炭疽检测质粒pBIB2006.采用复合PCR对模拟炭疽危险品进行分析,结果表明pBIB2006可以为炭疽芽胞杆菌的检测提供准确、安全和方便的阳性参照品,从而为检测炭疽芽胞杆菌和炭疽芽胞杆菌灭活疫苗提供了便利.

  15. Pinpointing a Mechanistic Switch Between Ketoreduction and "Ene" Reduction in Short-Chain Dehydrogenases/Reductases.

    Science.gov (United States)

    Lygidakis, Antonios; Karuppiah, Vijaykumar; Hoeven, Robin; Ní Cheallaigh, Aisling; Leys, David; Gardiner, John M; Toogood, Helen S; Scrutton, Nigel S

    2016-08-01

    Three enzymes of the Mentha essential oil biosynthetic pathway are highly homologous, namely the ketoreductases (-)-menthone:(-)-menthol reductase and (-)-menthone:(+)-neomenthol reductase, and the "ene" reductase isopiperitenone reductase. We identified a rare catalytic residue substitution in the last two, and performed comparative crystal structure analyses and residue-swapping mutagenesis to investigate whether this determines the reaction outcome. The result was a complete loss of native activity and a switch between ene reduction and ketoreduction. This suggests the importance of a catalytic glutamate vs. tyrosine residue in determining the outcome of the reduction of α,β-unsaturated alkenes, due to the substrate occupying different binding conformations, and possibly also to the relative acidities of the two residues. This simple switch in mechanism by a single amino acid substitution could potentially generate a large number of de novo ene reductases. PMID:27411040

  16. Aminoadipate reductase gene: a new fungal-specific gene for comparative evolutionary analyses

    Directory of Open Access Journals (Sweden)

    Miura Yoshiharu

    2002-04-01

    Full Text Available Abstract Background In fungi, aminoadipate reductase converts 2-aminoadipate to 2-aminoadipate 6-semialdehyde. However, other organisms have no homologue to the aminoadipate reductase gene and this pathway appears to be restricted to fungi. In this study, we designed degenerate primers for polymerase chain reaction (PCR amplification of a large fragment of the aminoadipate reductase gene for divergent fungi. Results Using these primers, we amplified DNA fragments from the archiascomycetous yeast Saitoella complicata and the black-koji mold Aspergillus awamori. Based on an alignment of the deduced amino acid sequences, we constructed phylogenetic trees. These trees are consistent with current ascomycete systematics and demonstrate the potential utility of the aminoadipete reductase gene for phylogenetic analyses of fungi. Conclusions We believe that the comparison of aminoadipate reductase among species will be useful for molecular ecological and evolutionary studies of fungi, because this enzyme-encoding gene is a fungal-specific gene and generally appears to be single copy.

  17. Trypanothione Reductase: A Viable Chemotherapeutic Target for Antitrypanosomal and Antileishmanial Drug Design

    Directory of Open Access Journals (Sweden)

    M. Omar F. Khan

    2007-01-01

    Full Text Available Trypanosomiasis and leishmaniasis are two debilitating disease groups caused by parasites of Trypanosoma and Leishmania spp. and affecting millions of people worldwide. A brief outline of the potential targets for rational drug design against these diseases are presented, with an emphasis placed on the enzyme trypanothione reductase. Trypanothione reductase was identified as unique to parasites and proposed to be an effective target against trypanosomiasis and leishmaniasis. The biochemical basis of selecting this enzyme as a target, with reference to the simile and contrast to human analogous enzyme glutathione reductase, and the structural aspects of its active site are presented. The process of designing selective inhibitors for the enzyme trypanothione reductase has been discussed. An overview of the different chemical classes of inhibitors of trypanothione reductase with their inhibitory activities against the parasites and their prospects as future chemotherapeutic agents are briefl y revealed.

  18. The flavin inhibitor diphenyleneiodonium renders Trichomonas vaginalis resistant to metronidazole, inhibits thioredoxin reductase and flavin reductase, and shuts off hydrogenosomal enzymatic pathways.

    Science.gov (United States)

    Leitsch, David; Kolarich, Daniel; Duchêne, Michael

    2010-05-01

    Infections with the microaerophilic protozoan parasite Trichomonas vaginalis are commonly treated with metronidazole, a 5-nitroimidazole drug. Metronidazole is selectively toxic to microaerophiles and anaerobes because reduction at the drug's nitro group, which is a precondition for toxicity, occurs only quantitatively in these organisms. In our previous work we identified the flavin enzyme thioredoxin reductase as an electron donor to 5-nitroimidazole drugs in T. vaginalis and observed that highly metronidazole-resistant cell lines lack thioredoxin reductase and flavin reductase activities. In this study we added the flavin inhibitor diphenyleneiodonium (DPI) to T. vaginalis cultures in order to test our hypothesis that metronidazole reduction is catalyzed by flavin enzymes, e.g. thioredoxin reductase, and intracellular free flavins. Indeed, within hours, DPI rendered T. vaginalis insensitive to metronidazole concentrations as high as 1mM and prevented the formation of metronidazole adducts with proteins. Thioredoxin reductase activity was absent from DPI-treated cells and flavin reductase activity was sharply decreased. In addition, DPI-treated cells also upregulated the expression of antioxidant enzymes, i.e. thioredoxin peroxidases and superoxide dismutases, and displayed a fundamentally altered metabolism caused by inactivation of pyruvate:ferredoxin oxidoreductase (PFOR) and concomitant upregulation of lactate dehydrogenase (LDH) activity. Thus, the disruption of the cellular flavin metabolism by DPI mediated metabolic steps which are similar to that of cells with metronidazole resistance induced in vitro. Finally, we present direct evidence that the increased expression of antioxidant enzymes is dispensable for acquiring resistance to metronidazole. PMID:20093143

  19. Naegleria fowleri: a free-living highly pathogenic amoeba contains trypanothione/trypanothione reductase and glutathione/glutathione reductase systems.

    Science.gov (United States)

    Ondarza, Raúl N; Hurtado, Gerardo; Tamayo, Elsa; Iturbe, Angélica; Hernández, Eva

    2006-11-01

    This paper presents definitive data showing that the thiol-bimane compound isolated and purified by HPLC from Naegleria fowleri trophozoites unequivocally corresponds by matrix assisted laser-desorption ionization-time-of-flight MS, to the characteristic monoprotonated ion of trypanothione-(bimane)(2) [M(+)H(+)] of m/z 1104.57 and to the trypanothione-(bimane) of m/z 914.46. The trypanothione disulfide T(S)(2) was also found to have a molecular ion of m/z 723.37. Additionally HPLC demonstrated that thiol-bimane compounds corresponding to cysteine and glutathione were present in Naegleria. The ion patterns of the thiol-bimane compounds prepared from commercial trypanothione standard, Entamoeba histolytica and Crithidia luciliae are identical to the Naegleria thiol-bimane compound. Partially purified extracts from N. fowleri showed the coexistence of glutathione and trypanothione reductases activities. There is not doubt that the thiol compound trypanothione, which was previously thought to occur only in Kinetoplastida, is also present in the human pathogens E. histolytica and N. fowleri, as well as in the non-pathogenic euglenozoan E. gracilis. The presence of the trypanothione/trypanothione reductase system in N. fowleri creates the possibility of using this enzyme as a new "drug target" for rationally designed drugs to eliminate the parasite, without affecting the human host.

  20. Peach MYB7 activates transcription of the proanthocyanidin pathway gene encoding leucoanthocyanidin reductase, but not anthocyanidin reductase

    Directory of Open Access Journals (Sweden)

    Hui eZhou

    2015-10-01

    Full Text Available Proanthocyanidins (PAs are a group of natural phenolic compounds that have a great effect on both flavour and nutritious value of fruit. It has been shown that PA synthesis is regulated by R2R3-MYB transcription factors (TFs via activation of PA-specific pathway genes encoding leucoanthocyanidin reductase (LAR and anthocyanidin reductase (ANR. Here, we report the isolation and characterization of a MYB gene designated PpMYB7 in peach. The peach PpMYB7 represents a new group of R2R3-MYB genes regulating PA synthesis in plants. It is able to activate transcription of PpLAR1 but not PpANR, and has a broader selection of potential bHLH partners compared with PpMYBPA1. Transcription of PpMYB7 can be activated by the peach basic leucine-zipper 5 TF (PpbZIP5 via response to ABA. Our study suggests a transcriptional network regulating PA synthesis in peach, with the results aiding the understanding of the functional divergence between R2R3-MYB TFs in plants.