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Bacillus Anthracis (bacillus + anthraci)
Terms modified by Bacillus Anthracis Selected AbstractsDistribution of S-layers on the surface of Bacillus cereus strains: phylogenetic origin and ecological pressureENVIRONMENTAL MICROBIOLOGY, Issue 8 2001Tâm Mignot Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis have been described as members of the Bacillus cereus group but are, in fact, one species. B. anthracis is a mammal pathogen, B. thuringiensis an entomopathogen and B. cereus a ubiquitous soil bacterium and an occasional human pathogen. In two clinical isolates of B. cereus, in some B. thuringiensis strains and in B. anthracis, an S-layer has been described. We investigated how the S-layer is distributed in B. cereus, and whether phylogeny or ecology could explain its presence on the surface of some but not all strains. We first developed a simple biochemical assay to test for the presence of the S-layer. We then used the assay with 51 strains of known genetic relationship: 26 genetically diverse B. cereus and 25 non- B. anthracis of the B. anthracis cluster. When present, the genetic organization of the S-layer locus was analysed further. It was identical in B. cereus and B. anthracis. Nineteen strains harboured an S-layer, 16 of which belonged to the B. anthracis cluster. All 19 were B. cereus clinical isolates or B. thuringiensis, except for one soil and one dairy strain. These findings suggest a common phylogenetic origin for the S-layer at the surface of B. cereus strains and, presumably, ecological pressure on its maintenance. [source] Synthesis and in vitro Efficacy Studies of Silver Carbene Complexes on Biosafety Level 3 BacteriaEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 13 2009Matthew J. Panzner Abstract A series of N-heterocyclic carbene silver complexes have been synthesized and tested against the select group of biosafety level 3 bacteria Burkholderia pseudomallei, Burkholderia mallei, Bacillus anthracis, methicillin-resistant Staphylococcus aureus and Yersinia pestis. Minimal inhibitory concentrations, minimal bactericidal and killing assays demonstrated the exceptional efficacy of the complexes against these potentially weaponizable pathogens. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009) [source] Mycobacterium tuberculosis possesses a functional enzyme for the synthesis of vitamin C, L -gulono-1,4-lactone dehydrogenaseFEBS JOURNAL, Issue 19 2006Beata A. Wolucka The last step of the biosynthesis of l -ascorbic acid (vitamin C) in plants and animals is catalyzed by l -gulono-1,4-lactone oxidoreductases, which use both l -gulono-1,4-lactone and l -galactono-1,4-lactone as substrates. l -Gulono-1,4-lactone oxidase is missing in scurvy-prone, vitamin C-deficient animals, such as humans and guinea pigs, which are also highly susceptible to tuberculosis. A blast search using the rat l -gulono-1,4-lactone oxidase sequence revealed the presence of closely related orthologs in a limited number of bacterial species, including several pathogens of human lungs, such as Mycobacterium tuberculosis, Pseudomonas aeruginosa, Burkholderia cepacia and Bacillus anthracis. The genome of M. tuberculosis, the etiologic agent of tuberculosis, encodes a protein (Rv1771) that shows 32% identity with the rat l -gulono-1,4-lactone oxidase protein. The Rv1771 gene was cloned and expressed in Escherichia coli, and the corresponding protein was affinity-purified and characterized. The FAD-binding motif-containing Rv1771 protein is a metalloenzyme that oxidizes l -gulono-1,4-lactone (Km 5.5 mm) but not l -galactono-1,4-lactone. The enzyme has a dehydrogenase activity and can use both cytochrome c (Km 4.7 µm) and phenazine methosulfate as exogenous electron acceptors. Molecular oxygen does not serve as a substrate for the Rv1771 protein. Dehydrogenase activity was measured in cellular extracts of a Mycobacterium bovis BCG strain. In conclusion, M. tuberculosis produces a novel, highly specific l -gulono-1,4-lactone dehydrogenase (Rv1771) and has the capacity to synthesize vitamin C. [source] From soil to gut: Bacillus cereus and its food poisoning toxinsFEMS MICROBIOLOGY REVIEWS, Issue 4 2008Lotte P. Stenfors Arnesen Abstract Bacillus cereus is widespread in nature and frequently isolated from soil and growing plants, but it is also well adapted for growth in the intestinal tract of insects and mammals. From these habitats it is easily spread to foods, where it may cause an emetic or a diarrhoeal type of food-associated illness that is becoming increasingly important in the industrialized world. The emetic disease is a food intoxication caused by cereulide, a small ring-formed dodecadepsipeptide. Similar to the virulence determinants that distinguish Bacillus thuringiensis and Bacillus anthracis from B. cereus, the genetic determinants of cereulide are plasmid-borne. The diarrhoeal syndrome of B. cereus is an infection caused by vegetative cells, ingested as viable cells or spores, thought to produce protein enterotoxins in the small intestine. Three pore-forming cytotoxins have been associated with diarrhoeal disease: haemolysin BL (Hbl), nonhaemolytic enterotoxin (Nhe) and cytotoxin K. Hbl and Nhe are homologous three-component toxins, which appear to be related to the monooligomeric toxin cytolysin A found in Escherichia coli. This review will focus on the toxins associated with foodborne diseases frequently caused by B. cereus. The disease characteristics are described, and recent findings regarding the associated toxins are discussed, as well as the present knowledge on virulence regulation. [source] Difference between the spore sizes of Bacillus anthracis and other Bacillus speciesJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2007M. Carrera Abstract Aims:, To determine the size distribution of the spores of Bacillus anthracis, and compare its size with other Bacillus species grown and sporulated under similar conditions. Methods and Results:, Spores from several Bacillus species, including seven strains of B. anthracis and six close neighbours, were prepared and studied using identical media, protocols and instruments. Here, we report the spore length and diameter distributions, as determined by transmission electron microscopy (TEM). We calculated the aspect ratio and volume of each spore. All the studied strains of B. anthracis had similar diameter (mean range between 0·81 ± 0·08 ,m and 0·86 ± 0·08 ,m). The mean lengths of the spores from different B. anthracis strains fell into two significantly different groups: one with mean spore lengths 1·26 ± 0·13 ,m or shorter, and another group of strains with mean spore lengths between 1·49 and 1·67 ,m. The strains of B. anthracis that were significantly shorter also sporulated with higher yield at relatively lower temperature. The grouping of B. anthracis strains by size and sporulation temperature did not correlate with their respective virulence. Conclusions:, The spores of Bacillus subtilis and Bacillus atrophaeus (previously named Bacillus globigii), two commonly used simulants of B. anthracis, were considerably smaller in length, diameter and volume than all the B. anthracis spores studied. Although rarely used as simulants, the spores of Bacillus cereus and Bacillus thuringiensis had dimensions similar to those of B. anthracis. Significance and Impact of the Study:, Spores of nonvirulent Bacillus species are often used as simulants in the development and testing of countermeasures for biodefence against B. anthracis. The data presented here should help in the selection of simulants that better resemble the properties of B. anthracis, and thus, more accurately represent the performance of collectors, detectors and other countermeasures against this threat agent. [source] Virulent spores of Bacillus anthracis and other Bacillus species deposited on solid surfaces have similar sensitivity to chemical decontaminantsJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2007J-L. Sagripanti Abstract Aims:, To compare the relative sensitivity of Bacillus anthracis and spores of other Bacillus spp. deposited on different solid surfaces to inactivation by liquid chemical disinfecting agents. Methods and Results:, We prepared under similar conditions spores from five different virulent and three attenuated strains of B. anthracis, as well as spores of Bacillus subtilis, Bacillus atrophaeus (previously known as Bacillus globigii), Bacillus cereus, Bacillus thuringiensis and Bacillus megaterium. As spore-surface interactions may bias inactivation experiments, we evaluated the relative binding of different spores to carrier materials. The survival of spores deposited on glass, metallic or polymeric surfaces were quantitatively measured by ASTM standard method E-2414-05 which recovers spores from surfaces by increasing stringency. The number of spores inactivated by each decontaminant was similar and generally within 1 log among the 12 different Bacillus strains tested. This similarity among Bacillus strains and species was observed through a range of sporicidal efficacy on spores deposited on painted metal, polymeric rubber or glass. Conclusions:, The data obtained indicate that the sensitivity of common simulants (B. atrophaeus and B. subtilis), as well as spores of B. cereus, B. thuringiensis, and B. megaterium, to inactivation by products that contain either: peroxide, chlorine or oxidants is similar to that shown by spores from all eight B. anthracis strains studied. Significance and Impact of the Study:, The comparative results of the present study suggest that decontamination and sterilization data obtained with simulants can be safely extrapolated to virulent spores of B. anthracis. Thus, valid conclusions on sporicidal efficacy could be drawn from safer and less costly experiments employing non-pathogenic spore simulants. [source] Differential identification of Bacillus anthracis from environmental Bacillus species using microarray analysisJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2006J.E. Burton Abstract Aims:, To determine whether microarray analysis could be employed for the differential identification of a range of environmental Bacillus sp. from four strains of Bacillus anthracis. Methods and Results:, Oligonucleotide probes were designed that were specific to virulence factor genes of B. anthracis (pag, lef and cap), the variable number tandem repeat region of the B. anthracis vrrA gene and to the 16S-23S rRNA intergenic transcribed spacer region (ITS) and pleiotropic regulator (plcR) regions of the Bacillus cereus subgroup species. Generic probes were also designed to hybridize with conserved regions of the 16S rRNA genes of Bacillus (as a positive control), Neisseria sp., Pseudomonas sp., Streptococcus sp., Mycobacterium sp. and to all members of the Enterobacteriaceae to allow simultaneous detection of these bacteria. Identification of B. anthracis was found to rely entirely on hybridization of DNA specific to regions of the pag, lef and cap genes. Cross-reaction was observed between B. anthracis and other Bacillus species with all the other Bacillus probes tested. Results obtained using microarray hybridizations were confirmed using conventional microbiological techniques and found to have very high comparability. Conclusions:, Microarray-based assays are an effective method for the identification of B. anthracis from mixed-culture environmental samples without problems of false-positivity that have been observed with conventional PCR assays. Significance and Impact of the Study:, Identification of environmental Bacillus sp. by conventional PCR is prone to potential for reporting false-positives. This study provides a method for the exclusion of such isolates. [source] Anthrax: the challenges for decontaminationJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 10 2006Richard J Sharp Abstract Anthrax remains endemic in many parts of the world with regular infections of livestock presenting a consequent risk to public health. In the United Kingdom anthrax has diminished as a significant threat to human health with only sporadic outbreaks in farm animals derived from ingestion of spores from soil at sites associated with previous outbreaks and the burial of carcasses. Occupationally-derived anthrax, associated with industries involved in the processing of animal products, has historically had an impact on the occurrence of outbreaks of infection. The introduction, in 1965, of vaccination for workers in high-risk occupations contributed significantly to the eradication of the disease from the UK. During 2001 the deliberate release of anthrax spores in the USA, disseminated through the postal system, resulted in the infection of 22 people, five of which resulted in death through inhalational anthrax. At that time anthrax was unheard of in many clinical practices and there was a lack of training and preparedness to handle such incidents; the emergency resulted in medical and public health personnel across the world having a significantly raised awareness of both the organism and the clinical symptoms of infection, and the new threat posed by bioterrorism. In the USA, the immediate public health emergency was followed by the legacy of contaminated buildings and facilities. There had been little previous systematic study of the issues surrounding sampling and decontamination of areas contaminated with Bacillus anthracis. The decontamination of large complex buildings and the equipment they contained required the urgent development and validation of new procedures for both sampling and decontamination. Copyright © 2006 Society of Chemical Industry [source] Real-time monitoring of the membrane-binding and insertion properties of the cholesterol-dependent cytolysin anthrolysin O from Bacillus anthracis,JOURNAL OF MOLECULAR RECOGNITION, Issue 4 2006Simon Cocklin Abstract Bacillus anthracis has recently been shown to secrete a potently hemolytic/cytolytic protein that has been designated anthrolysin O (ALO). In this work, we initiated a study of this potential anthrax virulence factor in an effort to understand the membrane,binding properties of this protein. Recombinant anthrolysin O (rALO35,512) and two N-terminally truncated versions of ALO (rALO390,512 and rALO403,512) from B. anthracis were overproduced in Escherichia coli and purified to homogeneity. The role of cholesterol in the cytolytic activity of ALO was probed in cellular cholesterol depletion assays using mouse and human macrophage-like lines, and also Drosophila Schneider 2 cells. Challenging the macrophage cells with rALO35,512, but not rALO390,512 or rALO403,512, resulted in cell death by lysis, with this cytolysis being abolished by depletion of the membrane cholesterol. Drosophila cells, which contain ergosterol as their major membrane sterol, were resistant to rALO-mediated cytolysis. In order to determine the molecular mechanism of this resistance, the interaction of rALO with model membranes comprised of POPC alone, or with a variety of structurally similar sterols including ergosterol, was probed using Biacore. Both rALO35,512 and rALO403,512 demonstrated robust binding to model membranes composed of POPC and cholesterol, with amount of protein bound proportional to the cholesterol content. Ergosterol supported greatly reduced binding of both rALO35,512 and rALO403,512, whereas other sterols tested did not support binding. The rALO403,512,membrane interaction demonstrated an equilibrium dissociation constant (KD) in the low nanomolar range, whereas rALO35,512 exhibited complex kinetics likely due to the multiple events involved in pore formation. These results establish the pivotal role of cholesterol in the action of rALO. The biosensor method developed to measure ALO recognition of cholesterol in a membrane environment could be extended to provide a platform for the screening of inhibitors of other membrane-binding proteins and peptides. Copyright© 2006 John Wiley & Sons, Ltd. [source] Anthrax vaccine powder formulations for nasal mucosal deliveryJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 1 2006Ge Jiang Abstract Anthrax remains a serious threat worldwide as a bioterror agent. A second-generation anthrax vaccine currently under clinical evaluation consists of a recombinant Protective Antigen (rPA) of Bacillus anthracis. We have previously demonstrated that complete protection against inhalational anthrax can be achieved in a rabbit model, by intranasal delivery of a powder rPA formulation. Here we describe the preformulation and formulation development of such powder formulations. The physical stability of rPA was studied in solution as a function of pH and temperature using circular dichroism (CD), and UV-visible absorption and fluorescence spectroscopies. Extensive aggregation of rPA was observed at physiological temperatures. An empirical phase diagram, constructed using a combination of CD and fluorescence data, suggests that rPA is most thermally stable within the pH range of 6,8. To identify potential stabilizers, a library of GRAS excipients was screened using an aggregation sensitive turbidity assay, CD, and fluorescence. Based on these stability profiles, spray freeze-dried (SFD) formulations were prepared at pH 7,8 using trehalose as stabilizer and a CpG-containing oligonucleotide adjuvant. SFD formulations displayed substantial improvement in storage stability over liquid formulations. In combination with noninvasive intranasal delivery, such powder formulations may offer an attractive approach for mass biodefense immunization. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 95:80,96, 2006 [source] Evaluation of the immune response induced by a nasal anthrax vaccine based on the protective antigen protein in anaesthetized and non-anaesthetized miceJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 4 2006Brian R. Sloat To better protect against inhalational anthrax infection, a nasal anthrax vaccine based on the protective antigen (PA) protein of Bacillus anthracis could be an attractive alternative to the current Anthrax-Vaccine-Adsorbed (AVA), which was licensed for cutaneous anthrax prevention. Previously, we have demonstrated that an anti-PA immune response comparable with that in mice subcutaneously immunized with PA protein adjuvanted with aluminium hydroxide was induced in both the systemic compartment and the mucosal secretions of the nose and lung of anaesthetized mice when they were nasally immunized with PA protein incorporated into previously reported LPD (Liposome,Protamine,DNA) particles. In this study, we evaluated the anti-PA immune response induced by the nasal PA/LPD particles in non-anaesthetized mice and compared it with that in anaesthetized mice. Our data showed that the anti-PA antibody response and the anthrax lethal toxin-neutralization activity induced by the nasal PA/LPD in non-anaesthetized mice was relatively weaker than that in anaesthetized mice. However, the splenocytes isolated from the nasally immunized mice, anaesthetized and non-anaesthetized, proliferated comparably after in-vitro re-stimulation. By evaluating the uptake of fluorescence-labelled LPD particles by phagocytes in the nasal and broncho-alveolar lavages of mice after the nasal administration, we concluded that the relatively weaker anti-PA immune response in the non-anaesthetized mice might be partially attributed to the reduced retention of the PA/LPD particles in the nasal cavity of the non-anaesthetized mice. Data collected in this study are expected to be useful for future anthrax nasal vaccine studies when mice are used as a model. [source] Bacillus anthracis, a story of nature subverted by manLETTERS IN APPLIED MICROBIOLOGY, Issue 3 2005L.W.J. Baillie Summary Bacillus anthracis is a pathogen of animals which rarely infects humans. Its use as a bioweapon has stimulated efforts to develop genetic typing methods and therapeutics to respond to an attack. Of particular concern is the transfer of virulence genes from B. anthracis to other closely related strains of bacillus. [source] Two small c -type cytochromes affect virulence gene expression in Bacillus anthracisMOLECULAR MICROBIOLOGY, Issue 1 2009Adam C. Wilson Summary Regulated expression of the genes for anthrax toxin proteins is essential for the virulence of the pathogenic bacterium Bacillus anthracis. Induction of toxin gene expression depends on several factors, including temperature, bicarbonate levels, and metabolic state of the cell. To identify factors that regulate toxin expression, transposon mutagenesis was performed under non-inducing conditions and mutants were isolated that untimely expressed high levels of toxin. A number of these mutations clustered in the haem biosynthetic and cytochrome c maturation pathways. Genetic analysis revealed that two haem-dependent, small c -type cytochromes, CccA and CccB, located on the extracellular surface of the cytoplasmic membrane, regulate toxin gene expression by affecting the expression of the master virulence regulator AtxA. Deregulated AtxA expression in early exponential phase resulted in increased expression of toxin genes in response to loss of the CccA-CccB signalling pathway. This is the first function identified for these two small c -type cytochromes of Bacillus species. Extension of the transposon screen identified a previously uncharacterized protein, BAS3568, highly conserved across many bacterial and archeal species, as involved in cytochrome c activity and virulence regulation. These findings are significant not only to virulence regulation in B. anthracis, but also to analysis of virulence regulation in many pathogenic bacteria and to the study of cytochrome c activity in Gram-positive bacteria. [source] Non-uniform assembly of the Bacillus anthracis exosporium and a bottle cap model for spore germination and outgrowthMOLECULAR MICROBIOLOGY, Issue 2 2007Christopher T. Steichen Summary Spores of Bacillus anthracis are enclosed by an exosporium composed of a basal layer and an external hair-like nap. The nap is formed by a collagen-like glycoprotein called BclA, while the basal layer contains many different proteins, one of which is a spore-specific alanine racemase (Alr). In this study, we employed fluorescence microscopy and a fluorescently labelled anti-Alr monoclonal antibody (mAb) to examine the distribution of Alr within the exosporium. Binding of the mAb occurred over approximately three-quarters of the exosporium but not in a cap-like region at one end of the spore, indicating the absence or inaccessibility of Alr in this region. We also determined that the cap-like region, or cap, corresponds to the first part of the exosporium assembled within the mother cell during sporulation and the only part of the exosporium assembled in a ,exsY mutant strain of B. anthracis. Our results provide the first direct evidence that exosporium assembly is a non-uniform process and suggest that exosporium formation is discontinuous. Finally, we demonstrated that during spore germination and outgrowth, the outgrowing cell always escapes from its exosporium shell by popping through the cap, suggesting that the cap is designed to facilitate the emergence of the outgrowing cell. [source] Characterization of CidR-mediated regulation in Bacillus anthracis reveals a previously undetected role of S-layer proteins as murein hydrolasesMOLECULAR MICROBIOLOGY, Issue 4 2006Jong-Sam Ahn Summary Recent studies have shown that the Staphylococcus aureus cidABC and lrgAB operons are involved in the regulation of cell death and lysis. The transcription of cidABC and lrgAB was shown to be induced by acetic acid and was dependent on the cidR gene encoding a new member of the LysR-type transcription regulator (LTTR) family of proteins. In the study presented here, we examined the phenotypic and regulatory effects of disrupting a cidR homologue in Bacillus anthracis. As in S. aureus, the cidR mutation affected expression of the B. anthracis cid and lrg homologues, murein hydrolase activity and cell viability in stationary phase. Interestingly, the predominant murein hydrolase affected was an 85 kDa protein that was identified as Sap, a primary constituent of the S-layer in B. anthracis. The ability of Sap, as well as its counterpart EA1, to exhibit murein hydrolase activity was confirmed by cloning their respective genes in Escherichia coli and showing that the overexpressed proteins contained this activity. Northern blot analyses revealed that the cidR mutation caused reduced transcription of the genes encoding Sap and EA1, as well as CsaB involved in the attachment of the S-layer proteins to the cell wall. The results of these studies not only establish the existence of the cid and lrg murein hydrolase regulatory network in B. anthracis, but also help to define the function and regulation of the S-layer proteins. [source] Bacillus anthracis requires siderophore biosynthesis for growth in macrophages and mouse virulenceMOLECULAR MICROBIOLOGY, Issue 2 2004Stephen Cendrowski Summary Systemic anthrax infections can be characterized as proceeding in stages, beginning with an early intracellular establishment stage within phagocytes that is followed by extracelluar stages involving massive bacteraemia, sepsis and death. Because most bacteria require iron, and the host limits iron availability through homeostatic mechanisms, we hypothesized that B. anthracis requires a high-affinity mechanism of iron acquisition during its growth stages. Two putative types of siderophore synthesis operons, named Bacillus anthracis catechol, bac (anthrabactin), and anthrax siderophore biosynthesis, asb (anthrachelin), were identified. Directed gene deletions in both anthrabactin and anthrachelin pathways were generated in a B. anthracis (Sterne) 34F2 background resulting in mutations in asbA and bacCEBF. A decrease in siderophore production was observed during iron-depleted growth in both the ,asbA and ,bacCEBF strains, but only the ,asbA strain was attenuated for growth under these conditions. In addition, the ,asbA strain was severely attenuated both for growth in macrophages (M,) and for virulence in mice. In contrast, the ,bacCEBF strain did not differ phenotypically from the parental strain. These findings support a requirement for anthrachelin but not anthrabactin in iron assimilation during the intracellular stage of anthrax. [source] Evolution of signalling in the sporulation phosphorelayMOLECULAR MICROBIOLOGY, Issue 2 2002Keith Stephenson Summary Two-component and phosphorelay signal transduction systems are believed to function as environ-mental sensors that programme gene expression to the composition of the ecological niche in which a microbe normally resides. The question of how evolutionarily related bacteria that occupy different environments change their signal transduction pathways to adapt to such environments was asked of the sporulation phosphorelay of Bacillus subtilis, Bacillus halodurans, Bacillus anthracis and Bacillus stearothermophilus. Comparison of the primary amino acid sequence of phosphorelay proteins with the known structural and interactive properties of the B. subtilis proteins revealed that the amino acid residues of interaction surfaces between phosphorelay proteins and between a phosphorelay protein and DNA resist evolutionary change. The absolute conservation of interaction surfaces allowed the identification of sporulation sensor kinases in B. halodurans, B. anthracis and B. stearothermophilus. In these sensor kinases, the signal-sensing domains are vastly different in size and subdomain composition, with little apparent conservation between species, whereas the catalytic domains of these sensor kinases retain the high level of homology observed for the other phosphorelay proteins. Adaptation to new environments appears to result in rapid evolution of signalling domains to maximize environmental impact while maintaining identical protein,protein and protein,DNA contacts in the entire phosphorelay. In Clostridial genomes, only the Spo0A protein was found, suggesting that the anaerobic relatives of the Bacilli do not use a phosphorelay and phosphorylate Spo0A directly with sensor kinases. [source] Increased US prescription trends associated with the CDC Bacillus anthracis antimicrobial postexposure prophylaxis campaign,,PHARMACOEPIDEMIOLOGY AND DRUG SAFETY, Issue 3 2003Douglas Shaffer MD Abstract Purpose We evaluated national outpatient antimicrobial prescription trends in relation to the first United States case of inhalational anthrax due to the intentional delivery of Bacillus anthracis (B. anthracis) spores. Methods We queried IMS HEALTH's National Prescription Audit Plus7Ô database for two 6-month periods (July,December) in 2001 and 2000 to describe outpatient prescription trends of antimicrobials recommended during the Centers for Disease Control and Prevention's (CDC) postexposure prophylaxis campaign. Results Overall, antimicrobial utilization for the referent 6-month time frame was greater in 2000 compared to 2001. In contrast, ciprofloxacin utilization was greater in 2001 during October, the month following the index case, increasing by more than 40% over utilization in October 2000. Similarly, doxycycline utilization increased by 30% during October/November. This corresponded to relative increases in US utilization for ciprofloxacin of approximately 160,000 prescriptions for the month of October and for doxycycline of approximately 96,000 prescriptions during October and 120,000 prescriptions for November. Conclusions We conclude more widespread prescribing of ciprofloxacin and doxycycline occurred in response to the first US bioterrorist-associated anthrax attacks than was warranted based upon confirmed or suspected B. anthracis exposure alone. Published in 2003 by John Wiley & Sons, Ltd. [source] Insights into the anthrax lethal factor,substrate interaction and selectivity using docking and molecular dynamics simulationsPROTEIN SCIENCE, Issue 8 2009Georgios A. Dalkas Abstract The anthrax toxin of the bacterium Bacillus anthracis consists of three distinct proteins, one of which is the anthrax lethal factor (LF). LF is a gluzincin Zn-dependent, highly specific metalloprotease with a molecular mass of ,90 kDa that cleaves most isoforms of the family of mitogen-activated protein kinase kinases (MEKs/MKKs) close to their amino termini, resulting in the inhibition of one or more signaling pathways. Previous studies on the crystal structures of uncomplexed LF and LF complexed with the substrate MEK2 or a MKK-based synthetic peptide provided structure-activity correlations and the basis for the rational design of efficient inhibitors. However, in the crystallographic structures, the substrate peptide was not properly oriented in the active site because of the absence of the catalytic zinc atom. In the current study, docking and molecular dynamics calculations were employed to examine the LF-MEK/MKK interaction along the catalytic channel up to a distance of 20 Å from the zinc atom. This residue-specific view of the enzyme-substrate interaction provides valuable information about: (i) the substrate selectivity of LF and its inactivation of MEKs/MKKs (an issue highly important not only to anthrax infection but also to the pathogenesis of cancer), and (ii) the discovery of new, previously unexploited, hot-spots of the LF catalytic channel that are important in the enzyme/substrate binding and interaction. [source] Differential analysis of Bacillus anthracis after pX01 plasmid curing and comprehensive data on Bacillus anthracis infection in macrophages and glial cellsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 20 2007Sung-Ha Park Abstract Bacillus anthracis is a gram-positive bacterial organism responsible for anthrax. This organism has two pathogenic plasmids: pX01 and pX02. The genetic function of pX01, which comprises about 198,kb, is not known, except for a region called the pathogenic island, which contains three genes,pag, lef, and cya,that code for three toxic proteins. A 2-D difference gel electrophoresis (2-D DIGE) system was used to verify the existence of proteins controlled by the pX01 plasmid, and protein regulation data were obtained using DeCyder software. A total of 1728 proteins were identified in the wild-type strain of this organism and 1684 in the pX01 plasmid. Twenty-seven of these proteins disappeared and eight appeared when the pX01 plasmid was removed. An additional 52 proteins were downregulated and 15 were upregulated when this plasmid was removed. A total of 102 proteins have been identified using the MALDI-TOF method of analysis, including 49 whose functions are unknown. Among these, 31 participate in metabolic processes, two in cellular processes, 15 in the processing of genetic information, and five in the processing of extracellular information. Another seven proteins participate in bacterial virulence and pathogenesis. We investigated the functions of these proteins in other bacteria, particularly the B. anthracis derivative H9041. Bacterial growth differed between pX01+/pX02+ B. anthracis and its pX01,/pX02+ derivative as did the cytotoxicity of macrophages infected by pX01+/pX02+ B. anthracis and the pX01,pX02+ derivative. We also found that S100B protein levels increased in the host infected with pX01+/pX02+ B. anthracis or its pX01,/pX02+ derivative. These data suggest that the pX01 plasmid plays a key role in the regulation of protein functions in B. anthracis. [source] Proteomic analysis of the response of the human neutrophil-like cell line NB-4 after exposure to anthrax lethal toxinPROTEOMICS - CLINICAL APPLICATIONS, Issue 10 2007Jun X. Wheeler Abstract We used 2-D DIGE to analyze the early response of NB-4 cells, a human promyelotic leukemia cell line, exposed to lethal toxin from Bacillus anthracis at the proteome level. After a 2,h exposure, cells were still viable and 43% of spots (n,=,1042) showed a significant change in protein level. We identified 59 spots whose expression had changed significantly, and these reflected cytoskeleton damage, mitochondrial lysis and endoplasmic reticulum stress. Actin filament assembly was disrupted as evidenced by an increase in both actin subunits and phosphorylated cofilin, whilst levels of tropomyosin, tropomodulin and actin related protein 2/3 complex subunit decreased. Lower levels of ATP synthase subunits and mitochondrial inner membrane protein were identified as markers of mitochondrial lysis. Levels of various stress response proteins rose and, uniquely, levels of Ca2+ binding proteins such as translationally controlled tumor protein rose and hippocalcin-like protein 1 decreased. This response may have mitigated effects brought about by mitochondrial lysis and endoplasmic reticulum stress, and delayed or prevented apoptosis in NB-4 cells. These results resemble findings of similar proteomics studies in murine macrophages, although quantitative differences were observed. [source] Structural study and thermodynamic characterization of inhibitor binding to lumazine synthase from Bacillus anthracisACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2010Ekaterina Morgunova The crystal structure of lumazine synthase from Bacillus anthracis was solved by molecular replacement and refined to Rcryst = 23.7% (Rfree = 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. [source] Crystallization and initial crystallographic analysis of phosphoglucosamine mutase from Bacillus anthracisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2009Ritcha Mehra-Chaudhary The enzyme phosphoglucosamine mutase catalyzes the conversion of glucosamine 6-phosphate to glucosamine 1-phosphate, an early step in the formation of the nucleotide sugar UDP- N -acetylglucosamine, which is involved in peptidoglycan biosynthesis. These enzymes are part of the large ,- d -phosphohexomutase enzyme superfamily, but no proteins from the phosphoglucosamine mutase subgroup have been structurally characterized to date. Here, the crystallization of phosphoglucosamine mutase from Bacillus anthracis in space group P3221 by hanging-drop vapor diffusion is reported. The crystals diffracted to 2.7,Å resolution under cryocooling conditions. Structure determination by molecular replacement was successful and refinement is under way. The crystal structure of B. anthracis phosphoglucosamine mutase should shed light on the substrate-specificity of these enzymes and will also serve as a template for inhibitor design. [source] Expression, purification, crystallization and preliminary X-ray diffraction analysis of dihydrodipicolinate synthase from Bacillus anthracis in the presence of pyruvateACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2009Jarrod E. Voss Dihydrodipicolinate synthase (DHDPS) catalyses the first committed step in the lysine-biosynthesis pathway in bacteria, plants and some fungi. In this study, the expression of DHDPS from Bacillus anthracis (Ba -DHDPS) and the purification of the recombinant enzyme in the absence and presence of the substrate pyruvate are described. It is shown that DHDPS from B. anthracis purified in the presence of pyruvate yields greater amounts of recombinant enzyme with more than 20-fold greater specific activity compared with the enzyme purified in the absence of substrate. It was therefore sought to crystallize Ba -DHDPS in the presence of the substrate. Pyruvate was soaked into crystals of Ba -DHDPS prepared in 0.2,M sodium fluoride, 20%(w/v) PEG 3350 and 0.1,M bis-tris propane pH 8.0. Preliminary X-ray diffraction data of the recombinant enzyme soaked with pyruvate at a resolution of 2.15,Å are presented. The pending crystal structure of the pyruvate-bound form of Ba -DHDPS will provide insight into the function and stability of this essential bacterial enzyme. [source] The structure of Rph, an exoribonuclease from Bacillus anthracis, at 1.7,Å resolutionACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2009Andrea E. Rawlings Maturation of tRNA precursors into functional tRNA molecules requires trimming of the primary transcript at both the 5, and 3, ends. Cleavage of nucleotides from the 3, stem of tRNA precursors, releasing nucleotide diphosphates, is accomplished in Bacillus by a phosphate-dependent exoribonuclease, Rph. The crystal structure of this enzyme from B. anthracis has been solved by molecular replacement to a resolution of 1.7,Å and refined to an R factor of 19.3%. There is one molecule in the asymmetric unit; the crystal packing reveals the assembly of the protein into a hexamer arranged as a trimer of dimers. The structure shows two sulfate ions bound in the active-site pocket, probably mimicking the phosphate substrate and the phosphate of the 3,-terminal nucleotide of the tRNA precursor. Three other bound sulfate ions point to likely RNA-binding sites. [source] Structures of an alanine racemase from Bacillus anthracis (BA0252) in the presence and absence of (R)-1-aminoethylphosphonic acid (l -Ala-P)ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2008Kinfai Au 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 crystallography 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. [source] Purification, crystallization and preliminary structural analysis of nucleoside diphosphate kinase from Bacillus anthracisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2007Gauri Misra 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,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 P6322. The crystals contain a monomer in the asymmetric unit, which corresponds to a Matthews coefficient (VM) of 2.1,Å3,Da,1 and a solvent content of about 36.9%. [source] Structures of two superoxide dismutases from Bacillus anthracis reveal a novel active centreACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2005Ian W. Boucher 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. [source] Structure of purine nucleoside phosphorylase (DeoD) from Bacillus anthracisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2005Rosa Grenha 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. [source] Anthrax lethal toxin promotes dephosphorylation of TTP and formation of processing bodiesCELLULAR MICROBIOLOGY, Issue 4 2010Edith M. C. Chow Summary Anthrax lethal toxin (LeTx) is composed of protective antigen (PA) and lethal factor (LF) , PA is the receptor-binding moiety and LF is a protease that cleaves mitogen-activated protein kinase kinases (MAPKKs). LeTx subverts the immune response to Bacillus anthracis in several ways, such as downregulating interleukin-8 (IL-8) by increasing the rate of IL-8 mRNA degradation. Many transcripts are regulated through cis -acting elements that bind proteins that either impede or promote degradation. Some of these RNA-binding proteins are regulated by MAPKs and previous work has demonstrated that interfering with MAPK signalling decreases the half-life of IL-8 mRNA. Here, we have localized a segment within the IL-8 3, untranslated region responsible for LeTx-induced transcript destabilization and show that this is caused by inhibition of the p38, ERK and JNK pathways. TTP, an RNA-binding protein involved in IL-8 mRNA decay, became hypophosphorylated in LeTx-treated cells and knock-down of TTP prevented LeTx from destabilizing the IL-8 transcript. Cells that were treated with LeTx exhibited increased localization of TTP to Processing bodies, which are structures that accumulate transcripts targeted for degradation. We furthermore observed that LeTx promoted the formation of Processing bodies, revealing a link between the toxin and a major mRNA decay pathway. [source] | |||||||||||||||||||||||||||||||