B. anthracis

Cell Mol Life Sci, 2004 Nov, 61(22), 2859 - 65
Anthrax lethal toxin: a weapon of multisystem destruction; Agrawal A et al.; Lethal toxin (LT) is a major virulence factor secreted by anthrax bacteria . It is composed of two proteins, PA (protective antigen) and LF (lethal factor) . PA transports the LF inside the cell, where LF, a zinc-dependent metalloprotease cleaves the mitogen activated protein kinase kinase (MAPKK) enzymes of the mitogen activated protein kinase (MAPK) signaling pathway, thereby impairing their function . This disruption of the MAPK pathway, which serves essential functions such as proliferation, survival and inflammation in all cell types, results in multisystem dysfunction in the host . The inactivation of the MAPK pathway in both macrophages and dendritic cells leads to inhibition of proinflammatory cytokine secretion, downregulation of costimulatory molecules such as CD80 and CD86, and ineffective T cell priming . The net result is an impaired innate and adaptive immune response . Endothelial cells of the vascular system undergo apoptosis upon LT exposure, also likely due to inactivation of the MAPK pathway . The activity of various hormone receptors such as glucocorticoids, progesterone and estrogen is also blocked, due to inhibition of p38 MAPK phosphorylation, thus affecting the body's response to stress . The present review summarizes the various disarming effects of Bacillus anthracis through the use of a single weapon, the lethal toxin.

Infect Immun, 2004 Dec, 72(12), 7096 - 106
Adaptation of the endogenous Salmonella enterica serovar Typhi clyA-encoded hemolysin for antigen export enhances the immunogenicity of anthrax protective antigen domain 4 expressed by the attenuated live-vector vaccine strain CVD 908-htrA; Galen JE et al.; Bacterial live-vector vaccines aim to deliver foreign antigens to the immune system and induce protective immune responses, and surface-expressed or secreted antigens are generally more immunogenic than cytoplasmic constructs . We hypothesize that an optimum expression system will use an endogenous export system to avoid the need for large amounts of heterologous DNA encoding additional proteins . Here we describe the cryptic chromosomally encoded 34-kDa cytolysin A hemolysin of Salmonella enterica serovar Typhi (ClyA) as a novel export system for the expression of heterologous antigens in the supernatant of attenuated Salmonella serovar Typhi live-vector vaccine strains . We constructed a genetic fusion of ClyA to the reporter green fluorescent protein and showed that in Salmonella serovar Typhi CVD 908-htrA, the fusion protein retains biological activity in both domains and is exported into the supernatant of an exponentially growing live vector in the absence of detectable bacterial lysis . The utility of ClyA for enhancing the immunogenicity of an otherwise problematic antigen was demonstrated by engineering ClyA fused to the domain 4 (D4) moiety of Bacillus anthracis protective antigen (PA) . A total of 11 of 15 mice immunized intranasally with Salmonella serovar Typhi exporting the protein fusion manifested fourfold or greater rises in serum anti-PA immunoglobulin G, compared with only 1 of 16 mice immunized with the live vector expressing cytoplasmic D4 (P = 0.0002) . In addition, the induction of PA-specific gamma interferon and interleukin 5 responses was observed in splenocytes . This technology offers exceptional versatility for enhancing the immunogenicity of bacterial live-vector vaccines.

Biochem Biophys Res Commun, 2004 Dec 24, 325(4), 1236 - 9
Rapid profiling of the infection of Bacillus anthracis on human macrophages using SELDI-TOF mass spectroscopy; Seo GM et al.; Anthrax is an infectious disease caused by toxigenic strains of the Gram-positive bacterium Bacillus anthracis, which is mainly present in the environment in the form of highly resistant spores . In order to elucidate a surface enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectroscopy analysis to profile different expressed proteins when B . anthracis spores are infected in human macrophages, we analyzed human macrophage cytosolic fractions for the infection of B . anthracis spores . Eleven different protein peaks were obtained . The 8217.8 kDa was increased specifically in inactivated-Sterne spores at 90 min . At 120 min, the peak of 8552.1 kDa in the inactivate-Sterne spores increased more than fourfold compared to live-Sterne spores . The protein peak at 8552.1kDa suggests that inactivated-Sterne spores could cause the phagolysosome formation of macrophages . And the protein peaks that increased in live-Sterne spores suggest that it could escape from the phagolysosome of the macrophage . These SELDI-TOF profiles assume an important role in human macrophage for the survival and escape of the infected B . anthracis spores.

Rev Physiol Biochem Pharmacol, 2004, 152, 135 - 64 Epub 2004.
Anthrax toxins; Mourez M; Bacillus anthracis, the etiological agent of anthrax, secretes three polypeptides that assemble into toxic complexes on the cell surfaces of the host it infects . One of these polypeptides, protective antigen (PA), binds to the integrin-like domains of ubiquitously expressed membrane proteins of mammalian cells . PA is then cleaved by membrane endoproteases of the furin family . Cleaved PA molecules assemble into heptamers, which can then associate with the two other secreted polypeptides: edema factor (EF) and/or lethal factor (LF) . The heptamers of PA are relocalized to lipid rafts where they are quickly endocytosed and routed to an acidic compartment . The low pH triggers a conformational change in the heptamers, resulting in the formation of cation-specific channels and the translocation of EF/LF . EF is a calcium- and calmodulin-dependent adenylate cyclase that dramatically raises the intracellular concentration of cyclic adenosine monophosphate (cAMP) . LF is a zinc-dependent endoprotease that cleaves the amino terminus of mitogen-activated protein kinase kinases (Meks) . Cleaved Meks cannot bind to their substrates and have reduced kinase activity, resulting in alterations of the signaling pathways they govern . The structures of PA, PA heptamer, EF, and LF have been solved and much is now known about the molecular details of the intoxication mechanism . The in vivo action of the toxins, on the other hand, is still poorly understood and hotly debated . A better understanding of the toxins will help in the design of much-needed anti-toxin drugs and the development of new toxin-based medical applications.

J Bacteriol, 2004 Dec, 186(23), 7959 - 70
Population structure and evolution of the Bacillus cereus group; Priest FG et al.; Representative strains of the Bacillus cereus group of bacteria, including Bacillus anthracis (11 isolates), B . cereus (38 isolates), Bacillus mycoides (1 isolate), Bacillus thuringiensis (53 isolates from 17 serovars), and Bacillus weihenstephanensis (2 isolates) were assigned to 59 sequence types (STs) derived from the nucleotide sequences of seven alleles, glpF, gmk, ilvD, pta, pur, pycA, and tpi . Comparisons of the maximum likelihood (ML) tree of the concatenated sequences with individual gene trees showed more congruence than expected by chance, indicating a generally clonal structure to the population . The STs followed two major lines of descent . Clade 1 comprised B . anthracis strains, numerous B . cereus strains, and rare B . thuringiensis strains, while clade 2 included the majority of the B . thuringiensis strains together with some B . cereus strains . Other species were allocated to a third, heterogeneous clade . The ML trees and split decomposition analysis were used to assign STs to eight lineages within clades 1 and 2 . These lineages were defined by bootstrap analysis and by a preponderance of fixed differences over shared polymorphisms among the STs . Lineages were named with reference to existing designations: Anthracis, Cereus I, Cereus II, Cereus III, Kurstaki, Sotto, Thuringiensis, and Tolworthi . Strains from some B . thuringiensis serovars were wholly or largely assigned to a single ST, for example, serovar aizawai isolates were assigned to ST-15, serovar kenyae isolates were assigned to ST-13, and serovar tolworthi isolates were assigned to ST-23, while other serovars, such as serovar canadensis, were genetically heterogeneous . We suggest a revision of the nomenclature in which the lineage and clone are recognized through name and ST designations in accordance with the clonal structure of the population.

Curr Opin Mol Ther, 2004 Oct, 6(5), 506 - 12
Development of anthrax DNA vaccines; Ferrari ME et al.; Over 120 years ago, Pasteur and Greenfield developed an in vitro procedure for producing a live-attenuated Bacillus anthracis bacterial culture capable of protecting livestock from anthrax disease . Since then, anthrax has become one of the best characterized bacterial pathogens with regard to mechanism of toxicity and vaccine development . Most developments have used live-attenuated strains, bacterial supernatants or protein subunit approaches . Recently, novel plasmid DNA (pDNA) approaches to a safe and effective anthrax vaccine have been proposed . This review summarizes the history of anthrax, the need for new vaccines and recent developments in pDNA-based vaccines, leading to the initiation of a human phase I clinical trial in a significantly shorter timeframe than in traditional vaccine development.

Biologicals, 2004 Sep, 32(3), 157 - 63
Validation of an anti-PA-ELISA for the potency testing of anthrax vaccine in mice; Pombo M et al.; The potency test for the anthrax vaccine currently licensed for human use in the United States (Anthrax Vaccine Adsorbed) involves the protection of actively immunized guinea pigs from a lethal challenge with a virulent strain of Bacillus anthracis . Lethal challenge tests entail the use of specialized containment facilities for the safe and secure handling of the challenge strain . This potential difficulty, plus humane considerations, have prompted us to investigate non-lethal, alternative immunogenicity assays that could be considered as potency tests not only for the current vaccine, but also for vaccines under development . Immunogenicity tests will require suitable measurement of an antibody response to relevant antigens, by methods such as enzyme linked immunosorbent assay (ELISA) or a toxin neutralization assay . Any assay chosen for this purpose should be adequately validated and reproducible by other laboratories . Validation of an analytical procedure requires the demonstration that the assay is suitable for its intended purpose . The objective of this work was to study the performance of an anti-PA-ELISA designed to assess the antibody response to anthrax vaccines in mice . Validation studies were performed according to the guidelines of the International Conference of Harmonization (ICH), and we have established the working range of the assay (37-1159 EU/mL) on the bases of the following parameters: linearity (20-1159 EU/mL; r2=0.99; p-value=0.21), accuracy (91-118% recovery), precision (< or =20%CV, repeatability; < or =9 and < or =21%CV, intermediate precision per day and per analyst, respectively), detection limit (5 EU/mL), and quantification limit (37 EU/mL) . We believe that assay specificity and the above characteristics are adequate to allow this ELISA to be considered for use in a mouse immunogenicity (potency) test of anthrax vaccines, and for the standardization of reagents.

Structure (Camb), 2004 Nov, 12(11), 2059 - 66
Large-scale structural changes accompany binding of lethal factor to anthrax protective antigen: a cryo-electron microscopic study; Ren G et al.; Anthrax toxin (AT), secreted by Bacillus anthracis, is a three-protein cocktail of lethal factor (LF, 90 kDa), edema factor (EF, 89 kDa), and the protective antigen (PA, 83 kDa) . Steps in anthrax toxicity involve (1) binding of ligand (EF/LF) to a heptamer of PA63 (PA63h) generated after N-terminal proteolytic cleavage of PA and, (2) following endocytosis of the complex, translocation of the ligand into the cytosol by an as yet unknown mechanism . The PA63h.LF complex was directly visualized from analysis of images of specimens suspended in vitrified buffer by cryo-electron microscopy, which revealed that the LF molecule, localized to the nonmembrane-interacting face of the oligomer, interacts with four successive PA63 monomers and partially unravels the heptamer, thereby widening the central lumen . The observed structural reorganization in PA63h likely facilitates the passage of the large 90 kDa LF molecule through the lumen en route to its eventual delivery across the membrane bilayer.

Biosens Bioelectron, 2004 Nov 1, 20(4), 807 - 13
Identification and characterization of Bacillus anthracis by multiplex PCR on DNA chip; Wang SH et al.; Bacillus anthracis can be identified by detecting virulence factor genes located on two plasmids, pXO1 and pXO2 . Combining multiplex PCR with arrayed anchored primer PCR and biotin-avidin alkaline phosphatase indicator system, we developed a qualitative DNA chip method for characterization of B . anthracis, and simultaneous confirmation of the species identity independent of plasmid contents . The assay amplifies pag gene (in pXO1), cap gene (in pXO2) and Ba813 gene (a B . anthracis specific chromosomal marker), and the results were indicated by an easy-to-read profile based on the color reaction of alkaline phosphatase . About 1 pg of specific DNA fragments on the chip wells could be detected after PCR . With the proposed method, the avirulent (pXO1+/2-, pXO1-/2+ and pXO1-/2-) strains of B . anthracis and distinguished 'anthrax-like' strains from other B . cereus group bacteria were unambiguously identified, while the genera other than Bacillus gave no positive signal.

Biosens Bioelectron, 2004 Nov 1, 20(4), 719 - 27
Modeling of SEB-induced host gene expression to correlate in vitro to in vivo responses; Hammamieh R et al.; Detection of exposure to biological threat agents has relied on ever more sensitive methods for pathogen identification, but that usually requires pathogen proliferation to dangerous, near untreatable levels . Recent events have demonstrated that assessing exposure to a biological threat agent well in advance of onset of illness or at various stages post-exposure is invaluable among the diagnostic options . There is an urgent need for better diagnostic tools that will be sensitive, rapid, and unambiguous . Since human clinical cases of illness induced by biothreat agents are, fortunately, rare, use of animal models that closely mimic the human illness is the only in vivo option . Such studies can be very difficult and expensive; therefore, maximizing the information obtained from in vitro exposures to peripheral blood mononuclear cells (PBMCs) provide an opportunity to investigate dose/time variability in host responses . In our quest to study staphylococcal enterotoxin B (SEB) induced host gene expression patterns, we addressed two core issues using microarray analysis and predictive modeling . Our first objective was to determine gene expression patterns in human PBMCs exposed to SEB in vitro . Second, we compared the in vitro data with host responses gene expression patterns in vivo using PBMCs from an animal model of SEB intoxication that closely replicates the progression of illness in humans . We used cDNA microarrays to study global gene expression patterns in piglets intoxicated with SEB . We applied a supervised learning method for class prediction based on the k-nearest neighbor algorithm for the data obtained in piglets exposed to SEB in vivo against a training data set . This data set included gene expression profiles derived from in vitro exposures to eight different pathogens (Bacillus anthracis, Yersinia pestis, Brucella melitensis, SEB, cholera toxin, Clostridium botulinum toxin A, Venezuelan equine encephalitis, and Dengue-2) in PBMCs . We found that despite differences in gene expression profiles between in vitro and in vivo systems, there exists a subset of genes that show correlations between in vitro and in vivo exposures, which can be used as a predictor of exposure to SEB in vivo.

Biosens Bioelectron, 2004 Nov 1, 20(4), 706 - 18
Virulence signatures: microarray-based approaches to discovery and analysis; Pannucci J et al.; Rapid, accurate, and sensitive detection of biothreat agents requires a broad-spectrum assay capable of discriminating between closely related microbial or viral pathogens . Moreover, in cases where a biological agent release has been identified, forensic analysis demands detailed genetic signature data for accurate strain identification and attribution . To date, nucleic acid sequences have provided the most robust and phylogentically illuminating signature information . Nucleic acid signature sequences are not often linked to genomic or extrachromosomal determinants of virulence, a link that would further facilitate discrimination between pathogens and closely related species . Inextricably coupling genetic determinants of virulence with highly informative nucleic acid signatures would provide a robust means of identifying human, livestock, and agricultural pathogens . By means of example, we present here an overview of two general applications of microarray-based methods for: (1) the identification of candidate virulence factors; and (2) the analysis of genetic polymorphisms that are coupled to Bacillus anthracis virulence factors using an accurate, low cost solid-phase mini-sequencing assay . We show that microarray-based analysis of gene expression can identify potential virulence associated genes for use as candidate signature targets, and, further, that microarray-based single nucleotide polymorphism assays provide a robust platform for the detection and identification of signature sequences in a manner independent of the genetic background in which the signature is embedded . We discuss the strategy as a general approach or pipeline for the discovery of virulence-linked nucleic acid signatures for biothreat agents.

FEMS Microbiol Lett, 2004 Nov 15, 240(2), 215 - 23
Oligonucleotide microarray for identification of Bacillus anthracis based on intergenic transcribed spacers in ribosomal DNA; Nubel U et al.; We developed a DNA microarray for identification of Bacillus anthracis and other phylogenetic groupings within the "Bacillus cereus group" . Nucleotide sequences of 16S-23S ribosomal DNA internal transcribed spacers containing genes for tRNA(Ile) from 52 B . anthracis strains were found to be identical to sequences from seven strains published previously and different from all other bacteria . When 42 oligonucleotide probes targeting polymorphic sites were immobilized on glass slides and hybridized to fluorescently labeled PCR amplification products, one or more mismatches could be discriminated in all but one cases . Hence, hybridization events were highly specific and identification of B . anthracis was straightforward.

Vaccine, 2004 Nov 15, 23(1), 43 - 7
Anthrax capsule vaccine protects against experimental infection; Chabot DJ et al.; Efficacy of a poly-gamma-D-glutamic acid anthrax capsule vaccine was assessed in a mouse model of infection . Capsule by itself was protective against lethal challenge with a toxin(-), capsule(+) Bacillus anthracis strain . Conjugation of capsule to bovine serum albumin resulted in enhanced IgG anti-capsule antibodies measured by ELISA, but completely abrogated the protection . The protective unconjugated capsule vaccine elicited significantly higher IgM titers and opsonic activity than did the non-protective capsule conjugate . When tested against a fully virulent toxin(+), capsule(+) B . anthracis strain, neither capsule nor protective antigen alone was protective . However, the combination of the two protected against a lethal challenge . These results suggest that capsule may enhance the protection afforded by protective antigen vaccines against anthrax if opsonizing antibodies are produced . Surprisingly, some protection was also observed when protective antigen was conjugated to itself.

Biotechniques, 2004 Oct, 37(4), 654 - 8, 660
Organism identification using a genome sequence-independent universal microarray probe set; Belosludtsev YY et al.; There has been increasing interest and efforts devoted to developing biosensor technologies for identifying pathogens, particularly in the biothreat area . In this study, a universal set of short 12- and 13-mer oligonucleotide probes was derived independently of a priori genomic sequence information and used to generate unique species-dependent genomic hybridization signatures . The probe set sequences were algorithmically generated to be maximally distant in sequence space and not dependent on the sequence of any particular genome . The probe set is universally applicable because it is unbiased and independent of hybridization predictions based upon simplified assumptions regarding probe-target duplex formation from linear sequence analysis . Tests were conducted on microarrays containing 14,283 unique probes synthesized using an in situ light-directed synthesis methodology . The genomic DNA hybridization intensity patterns reproducibly differentiated various organisms (Bacillus subtilis, Yersinia pestis, Streptococcus pneumonia, Bacillus anthracis, and Homo sapiens), including the correct identification of a blinded "unknown" sample . Applications of this method include not only pathological and forensic genome identification in medicine and basic science, but also potentially a novel method for the discovery of unknown targets and associations inherent in dynamic nucleic acid populations such as represented by differential gene expression.

Biotechniques, 2004 Oct, 37(4), 642 - 4, 646, 648 passim
Mass spectrometry provides accurate characterization of two genetic marker types in Bacillus anthracis; Van Ert MN et al.; Epidemiological and forensic analyses of bioterrorism events involving Bacillus anthracis could be improved if both variable number tandem repeats (VNTRs) and single nucleotide polymorphisms (SNPs) could be combined on a single analysis platform . Here we present the use of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) to characterize 24 alleles from 6 VNTR loci and 11 alleles from 7 SNP loci in B . anthracis . The results obtained with ESI-FTICR-MS were consistent with independent results obtained from traditional approaches using electrophoretic detection of fluorescent products . However, ESI-FTICR-MS improves on the traditional approaches because it does not require fluorescent labeling of PCR products, minimizes post-PCR processing, obviates electrophoresis, and provides unambiguous base composition of both SNP and VNTR PCR products . In addition, ESI-FTICR-MS allows both marker types to be examined simultaneously and at a rate of approximately 1 sample per min . This technology represents a significant advance in our ability to rapidly characterize B . anthracis isolates using VNTR and SNP loci.

Przegl Epidemiol, 2004, 58(2), 335 - 42
{Anthrax--continuous threat to humans and animals}; Mizak L; Gram-positive, spore-forming, aerobic bacterium Bacillus anthracis is an etiological agent of anthrax a disease very dangerous to humans and all warm-blooded animals . The spore forms are markedly resistant to unfavourable environmental extremes of heat, cold, desiccation, chemicals, irradiation etc . The vegetative forms characterised virulence factors: the antiphagocytic poly-gamma-D-polipeptide capsule and three proteins, edema factor (EF), lethal factor (LF) and protective antigen (PA) . Anthrax is mainly transmitted from animals to man through food of animal origin, animal products and contamination of the environment with B . anthracis and its spores . There are three types of this disease: cutaneous, intestinal and inhalation anthrax . Research on anthrax as a biological weapon began more then 80 years ago . Depending on the target chosen and the scale of the attack the anthrax spores may by used to contaminate of foodstuffs or liquids and water . The aerosolised release of anthrax spore can cause illness with a high fatality rate.

Anal Chem, 2004 Nov 1, 76(21), 6492 - 9
Correlation of mass spectrometry identified bacterial biomarkers from a fielded pyrolysis-gas chromatography-ion mobility spectrometry biodetector with the microbiological gram stain classification scheme; Snyder AP et al.; A pyrolysis-gas chromatography-ion mobility spectrometry (Py-GC-IMS) briefcase system has been shown to detect and classify deliberately released bioaerosols in outdoor field scenarios . The bioaerosols included Gram-positive and Gram-negative bacteria, MS-2 coliphage virus, and ovalbumin protein species . However, the origin and structural identities of the pyrolysate peaks in the GC-IMS data space, their microbiological information content, and taxonomic importance with respect to biodetection have not been determined . The present work interrogates the identities of the peaks by inserting a time-of-flight mass spectrometry system in parallel with the IMS detector through a Tee connection in the GC module . Biological substances producing ion mobility peaks from the pyrolysis of microorganisms were identified by their GC retention time, matching of their electron ionization mass spectra with authentic standards, and the National Institutes for Standards and Technology mass spectral database . Strong signals from 2-pyridinecarboxamide were identified in Bacillus samples including Bacillus anthracis, and its origin was traced to the cell wall peptidoglycan macromolecule . 3-Hydroxymyristic acid is a component of lipopolysaccharides in the cell walls of Gram-negative organisms . The Gram-negative Escherichia coli organism showed significant amounts of 3-hydroxymyristic acid derivatives and degradation products in Py-GC-MS analyses . Some of the fatty acid derivatives were observed in very low abundance in the ion mobility spectra, and the higher boiling lipid species were absent . Evidence is presented that the Py-GC-ambient temperature and pressure-IMS system generates and detects bacterial biochemical information that can serve as components of a biological classification scheme directly correlated to the Gram stain reaction in microorganism taxonomy.

J Public Health Manag Pract, 2003 Sep-Oct, 9(5), 352 - 6
Public health response to bioterrorism with Bacillus anthracis: coordinating public health laboratory, communication, and law enforcement; Nolan PA et al.; In October 2001, public health departments across the United States were part of an intensive response to a bioterrorism event using anthrax spores delivered by mail . It is useful to examine this experience as an unscripted exercise of bioterrorism response capacity, more realistic than scenarios of planned exercises . The event particularly challenged public health laboratory and communications capacity, but it also tested surveillance and training capacity . The bioterrorism response demonstrated the importance of strong partnerships between the public health laboratory and emergency response agencies as well as medical providers and the usefulness of open, flexible communication strategies.

Infect Immun, 2004 Nov, 72(11), 6382 - 9
Cytokine response to infection with Bacillus anthracis spores; Pickering AK et al.; 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 . Therefore, the ability of B . anthracis to transit through the host macrophage or dendritic cell appears to be an early and critical step in B . anthracis pathogenesis . In this work, we examined the cytokine responses to spore infection in mouse primary peritoneal macrophages, in primary human dendritic cells, and during a spore aerosol infection model utilizing the susceptible A/J mouse strain . We demonstrated that both mouse peritoneal macrophages and human dendritic cells exhibited significant intracellular bactericidal activity during the first hours following uptake, providing the necessary time to mount a cytokine response prior to cell lysis . Strong tumor necrosis factor (TNF-alpha) and interleukin-6 (IL-6) responses were seen in mouse peritoneal macrophages . In addition to TNF-alpha and IL-6, human dendritic cells produced the cytokines IL-1beta, IL-8, and IL-12 . A mixture of Th1 and Th2 cytokines were detected in sera obtained from infected animals . In this study, we provide further evidence of an acute cytokine response when cells in culture and mice are infected with B . anthracis spores.

Infect Immun, 2004 Nov, 72(11), 6313 - 7
Functional analysis of Bacillus anthracis protective antigen by using neutralizing monoclonal antibodies; Brossier F et al.; Protective antigen (PA) is central to the action of the lethal and edema toxins produced by Bacillus anthracis . It is the common cell-binding component, mediating the translocation of the enzymatic moieties (lethal factor {LF} and edema factor) into the cytoplasm of the host cell . Monoclonal antibodies (MAbs) against PA, able to neutralize the activities of the toxins in vitro and in vivo, were screened . Two such MAbs, named 7.5 and 48.3, were purified and further characterized . MAb 7.5 binds to domain 4 of PA and prevents the binding of PA to its cell receptor . MAb 48.3 binds to domain 2 and blocks the cleavage of PA into PA63, a step necessary for the subsequent interaction with the enzymatic moieties . The epitope recognized by this antibody is in a region involved in the oligomerization of PA63; thus, MAb 48.3 does not recognize the oligomer form . MAbs 7.5 and 48.3 neutralize the activities of anthrax toxins produced by B . anthracis in mice . Also, there is an additive effect between the two MAbs against PA and a MAb against LF, in protecting mice against a lethal challenge by the Sterne strain . This work contributes to the functional analysis of PA and offers immunotherapeutic perspectives for the treatment of anthrax disease.

Crit Rev Microbiol, 2004, 30(3), 187 - 96
The adenylate cyclase toxins; Ahuja N et al.; Cyclic AMP is a ubiquitous messenger that integrates many processes of the cell . Diverse families of adenylate cyclases and phosphodiesterases stringently regulate the intracellular concentration of cAMP . Any alteration in the cytosolic concentration of cAMP has a profound effect on the various processes of the cell . Disruption of these cellular processes in vivo is often the most critical event in the pathogenesis of infectious diseases for animals and humans . Many pathogenic bacteria secrete toxins to alter the intracellular concentration of cAMP . These toxins either disrupt the normal regulation of the host cell's adenylate cyclases/phosphodiesterases or they themselves catalyze the synthesis of cAMP in the host cell . The latter are known as the adenylate cyclase toxins . Four such toxins have been identified: the invasive adenylate cyclase of Bordetella pertussis, the edema factor of Bacillus anthracis, ExoY of Pseudomonas aeruginosa, and the adenylate cyclase of Yersinia pestis . These adenylate cyclase toxins enter the eukaryotic host cells and get activated by eukaryotic cofactors, like calmodulin, to trigger the synthesis of cAMP in these cells . By accumulating cAMP in the target cells, these toxins either modulate the cellular function or completely deactivate the cell for further function . The immune effector cells appear to be the primary target of these adenylate cyclase toxins . By accumulating cAMP in the immune effector cells, these adenylate cyclase toxins poison the immune system and thus facilitate the survival of the bacteria in the host.

J Med Chem, 2004 Oct 21, 47(22), 5347 - 55
The anthrax protective antigen (PA63) bound conformation of a peptide inhibitor of the binding of lethal factor to PA63: as determined by trNOESY NMR and molecular modeling; Hicks RP et al.; Anthrax protective antigen (PA) is one of the three proteins produced by the gram positive bacteria Bacillus anthracis collectively known as the "anthrax toxin" (Ascenzi, P.; Visca, P.; Ippolito, G.; Spallarossa, A.; Bolognesi, M.; et al . Anthrax toxin: a tripartite lethal combination . FEBS Lett . 2002, 531, 384-388) . The role played by PA in anthrax intoxication is to transport the two enzymes lethal factor (LF) and edema factor (EF) into the cell . Collier and co-workers (Mourez, M.; Kane, R . S.; Mogridge, J.; Metallo, S.; Deschatelets, P.; et al . Designing a polyvalent inhibitor of anthrax toxin . Nat . Biotechnol . 2001, 958) . reported the isolation of two peptides via phage display that bind to the PA63 heptamer and inhibit its interaction with LF and EF, and thereby prevent the transport of LF and EF into the cell . One of these peptides, His-Thr-Ser-Thr-Try-Trp-Trp-Leu-Asp-Gly-Ala-Pro (P1), was selected for structural investigation on the basis of its ability to prevent the binding of LF to the PA63 heptamer bundle . Two-dimensional trNOESY experiments coupled with NOE restrained simulated annealing calculations were used to determine the PA63-bound conformation of P1 . On binding to PA63, P1 adopts a helical conformation involving residues 3-9 while the C- and N-terminal residues exhibit dynamic fraying.

Zh Mikrobiol Epidemiol Immunobiol, 2004 Jul-Aug, (4), 61 - 3
{Changes in fine morphological structures of Escherichia coli, Staphylococcus aureus and spores of Bacillus anthracis vaccine strain STI under the action of the disinfectant "Veltolen"}; Chloroquine enhances survival in Bacillus anthracis intoxication; Center for Biodefense and Emerging Pathogens, Division of Infectious Diseases, Memorial Hospital of Rhode Island, Pawtucket 02860, USA . artenstein@brown.eduThe intentional release of anthrax in the United States in 2001 resulted in 11 cases of inhalational disease, with an attendant mortality rate of 45% . Current therapeutic options for anthrax are limited; antimicrobials target only replicating organisms, thus allowing bacterial toxins to cause unchecked, devastating physiological derangements in the host . Novel approaches that target the cytotoxic effects of anthrax exotoxins are needed . Chloroquine (CQ), a commonly used antimalarial agent, endows anthrax-intoxicated murine peritoneal macrophages with a 50% and 35% marginal survival advantage at 2 and 4 h, respectively, over that of untreated control cells . The cell rescue is dose dependent and, at lower concentrations, results in delayed cell death . We subsequently studied the effect of CQ in BALB/c mice challenged with anthrax lethal toxin . CQ-treated mice demonstrated reduced tissue injury, as assessed by histopathological examination of the spleen and by peripheral blood differential cell count ratios . CQ significantly enhanced survival and may augment current treatment and prophylaxis options for this otherwise lethal infection.

Protein Expr Purif, 2004 Nov, 38(1), 145 - 52
Production and purification of Bacillus anthracis protective antigen from Escherichia coli; Laird MW et al.; 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.

FEMS Microbiol Lett, 2004 Oct 15, 239(2), 235 - 40
Intriguing diversity of Bacillus anthracis in eastern Poland--the molecular echoes of the past outbreaks; Gierczynski R et al.; The multiple locus VNTRs analysis (MLVA) revealed the presence of five genotypes in a group of 10 Bacillus anthracis isolates from epidemiologically unrelated cases of bovine-anthrax in eastern Poland . Eight tested isolates possessed the pagA and capB genes indicating the presence of both virulence plasmids, while two isolates revealed only pagA and lacked pXO2 . The MLVA and DNA sequence analysis indicated that seven tested isolates represent four novel genotypes . Five tested strains revealed a unique 144 bp vrrB2 variant as well as 220 bp variant of vrrB1, implying the relatedness to the lineage B2 . Consequently, we propose establishing of novel B2 strains sub-lineage . Multiple anthrax outbreaks, which took place in Poland several decades ago were proposed as a cause of intriguing diversity of B . anthracis observed in this study.

Vaccine, 2004 Oct 22, 22(31-32), 4374 - 84
Expression of Bacillus anthracis protective antigen in transgenic chloroplasts of tobacco, a non-food/feed crop; Watson J et al.; The Centers for Disease Control (CDC) lists Bacillus anthracis as a category A agent and estimates the cost of an anthrax attack to exceed US$ 26 billion per 100,000 exposed individuals . Concerns regarding anthrax vaccine purity, a requirement for multiple injections, and a limited supply of the protective antigen (PA), underscore the urgent need for an improved vaccine . Therefore, the 83 kDa immunogenic Bacillus anthracis protective antigen was expressed in transgenic tobacco chloroplasts . The PA gene (pag) was cloned into a chloroplast vector along with the psbA regulatory signals to enhance translation . Chloroplast integration of the transgenes was confirmed by PCR and Southern blot analyses . Crude plant extracts contained up to 2.5 mg full length PA/g of fresh leaf tissue and this showed exceptional stability for several months in stored leaves or crude extracts . Maximum levels of expression were observed in mature leaves under continuous illumination . Co-expression of the ORF2 chaperonin from Bacillus thuringiensis did not increase PA accumulation or induce folding into cuboidal crystals in transgenic chloroplasts . Trypsin, chymotrypsin and furin proteolytic cleavage sites present in PA were protected in transgenic chloroplasts because only full length PA 83 was observed without any degradation products . Both CHAPS and SDS detergents extracted PA with equal efficiency and PA was observed in the soluble fraction . Chloroplast-derived PA was functionally active in lysing mouse macrophages when combined with lethal factor (LF) . Crude leaf extracts contained up to 25 microg functional PA/ml . With an average yield of 172 mg of PA per plant using an experimental transgenic cultivar grown in a greenhouse, 400 million doses of vaccine (free of contaminants) could be produced per acre, a yield that could be further enhanced 18-fold using a commercial cultivar in the field.

Vaccine, 2004 Oct 22, 22(31-32), 4245 - 51
Characterisation of adsorbed anthrax vaccine by two-dimensional gel electrophoresis; Whiting GC et al.; The current UK anthrax vaccine is an alum precipitate prepared from static culture filtrate of the avirulent, unencapsulated Sterne strain of Bacillus anthracis . Protective antigen (PA) is regarded as the major immunogen in the vaccine and production conditions are intended to maximize the PA content . However, the precise composition of the vaccine is unknown and there are concerns that the observed side effects of vaccination may be caused by residual enzymatically active toxin components . Two-dimensional gel electrophoresis (2DGE) was used to define the protein components of the current UK anthrax vaccine . Consistency of composition was assessed by examining batches spanning 14 years of vaccine production . The reproducibility of the 2DGE technique was assessed by repeated analysis of selected vaccine batches . For two recently produced batches, between 86.7 and 88.8% of the spots could be matched . However, for one older batch, reproducibility of the spot pattern was considerably less, with a mean similarity of 53.4% . This difference may be explained by a change in production or because of decay during storage . Variation between the recently produced batches ranged from 72.9 to 84.3%, whereas the similarity between these and old batches was comparatively low at between 30 and 59% . Our results demonstrate that, as expected, the major antigen present in the vaccine is PA . The 83 and 63 kDa species are dominant but there are numerous lower molecular weight fragments resulting from proteolytic cleavage . In addition, we have established the presence of the toxin components, oedema factor and lethal factor, and S-layer proteins, EA1 and SAP . Mass spectrometry has also enabled us to identify several bacterial cell-derived proteins present in the vaccine, including PA, enolase, fructose-bisphosphate aldolase, nucleoside diphosphate kinase and a 60 kDa heat shock protein . The use of proteomics can provide useful information on the antigenic make up of this vaccine and the consistency of vaccine production.

J Clin Microbiol, 2004 Oct, 42(10), 4859 - 62
Comparative analysis of the Schleicher and Schuell IsoCode Stix DNA isolation device and the Qiagen QIAamp DNA Mini Kit; Coyne SR et al.; Efficient, rapid, and reproducible procedures for isolating high-quality DNA before PCR gene amplification are essential for the diagnostic and molecular identification of pathogenic bacteria . This study evaluated the Qiagen QIAamp DNA Mini Kit and the Schleicher and Schuell IsoCode Stix DNA isolation device for isolating nucleic acid . Buffer, serum, and whole-blood samples were spiked with Bacillus anthracis Sterne vegetative cells and Yersinia pestis, while water was spiked with B . anthracis Sterne spores . Although minimal variations in limit of detection occurred among matrices, both the IsoCode Stix extraction method and the Qiagen procedure have comparable detection limits.

Appl Environ Microbiol, 2004 Oct, 70(10), 6173 - 80
Quorum quenching: enzymatic disruption of N-acylhomoserine lactone-mediated bacterial communication in Burkholderia thailandensis; Ulrich RL; Many species of gram-negative bacteria communicate by synthesizing, secreting, and responding to N-acylhomoserine lactones (AHLs), a mechanism termed quorum sensing . Several investigations have characterized numerous AHL-degrading enzymes (AiiA lactonases) encoded by environmental isolates of Bacillus spp . The Burkholderia thailandensis quorum system is comprised of at least three AHL synthases (AHSs) and five transcriptional regulators belonging to the LuxIR class of proteins . Expression of the Bacillus anthracis (Ames strain) AiiA lactonase in B . thailandensis completely abolished the accumulation of N-decanoylhomoserine lactone (C(10)-HSL) and N-octanoylhomoserine lactone (C(8)-HSL), reduced N-hexanoylhomoserine lactone (C(6)-HSL) levels, altered both swarming and twitching motility, caused a significant increase in generation time, and affected carbon metabolism . In contrast, heterologous expression of the Bacillus cereus strain A24 AiiA lactonase in B . thailandensis reduced the concentrations of C(6)-HSL, C(8)-HSL, and C(10)-HSL to nondetectable levels; altered both swarming and twitching motility; and caused fluctuations in carbon utilization . Individual disruption of the B . thailandensis AHSs, specifically disruption of the btaI1 and btaI3 genes, which encode the proteins that direct the synthesis of C(8)-HSL and C(6)-HSL, respectively, caused the hyper-beta-hemolysis of sheep erythrocytes on blood agar plates . In contrast, AHL cleavage in B . thailandensis by the Bacillus AiiA lactonases failed to enhance beta-hemolytic activity . The results of this study demonstrate that heterologous expression of Bacillus sp . AiiA lactonases in B . thailandensis reduced AHL accumulation, affected both swarming and twitching motility, increased generation time, altered substrate utilization, and prevented the beta-hemolysis of sheep erythrocytes.

Microb Pathog, 2004 Oct, 37(4), 169 - 75
The use of a model of in vivo macrophage depletion to study the role of macrophages during infection with Bacillus anthracis spores; Cote CK et al.; The pathogenesis of infection by Bacillus anthracis has been the subject of many investigations, but remains incompletely understood . It has been shown that B . anthracis spores germinate in macrophages and perhaps require this intracellular niche to germinate in vivo before outgrowth of the vegetative organism . However, it has also been reported that macrophages are sporicidal in vitro . In our in vivo model, macrophages were depleted from mice by either silica treatment or treatment with liposome-encapsulated dichloromethylene disphosphonate (Cl(2)MDP), and the animals were infected parenterally with virulent ungerminated B . anthracis (Ames strain) spores . The mice in which macrophages had been depleted were killed more rapidly than untreated mice . In addition, augmenting peritoneal populations of macrophages with cultured RAW264.7 cells partially protected mice from disease, increasing the survival rate in a dose dependent relationship . Alveolar macrophages were depleted by intranasal instillation of liposome-encapsulated Cl(2)MDP . The animals with normal alveolar macrophage numbers had significantly greater survival rates after inhaling B . anthracis spores than the macrophage-depleted mice . These findings do not preclude the observations that macrophages provide a site permissive for spore germination, however, these data suggest that macrophages do play an important role in limiting and/or clearing a B . anthracis infection.

J Biol Chem, 2004 Dec 10, 279(50), 51760 - 8 Epub 2004 Dec 10.
Identification and biochemical characterization of two novel collagen binding MSCRAMMs of Bacillus anthracis; Xu Y et al.; Cell wall-anchored proteins play critical roles in the pathogenesis of infections caused by Gram-positive bacteria . Through the analysis of the genome of Bacillus anthracis Ames strain, we identified two novel putative cell wall-anchored proteins, BA0871 and BA5258, which have sequence homology to CNA, a cell wall-anchored collagen adhesin of Staphylococcus aureus . The two proteins have similar domain organization to that of CNA, with typical signal peptide sequences, a non-repetitive A region followed by repeats, and a characteristic cell wall-anchoring region . They are expressed on the surface of B . anthracis . The A regions of the two proteins were predicted to adopt similar structural folds as CNA . Circular dichroism analysis of the recombinant A regions of the two proteins (rBA0871A and rBA5258A) indicate that their secondary structure compositions are similar to those of the A regions of CNA and other cell wall-anchored adhesins . We demonstrate through solid phase binding assays and surface plasmon resonance analyses that rBA0871A and rBA5258A specifically bound type I collagen in a dose-dependent and saturable manner . Their dissociation constants (KD) for collagen are 1.6-3.2 microm for rBA0871A and 0.6-0.9 microm for rBA5258A, respectively . We further demonstrate that BA0871 and BA5258 can mediate cell attachment to collagen when expressed on the surface of a heterologous host bacterium . To our knowledge these are the first two adhesins of B . anthracis described, which may have important implications for our understanding of the pathogenic mechanisms explored by this organism.

Am J Infect Control, 2004 Oct, 32(6), 355 - 7
Prevalence of presumptive Bacillus anthracis in the human population examined by nasal swabs; Godyn JJ et al.; Routine cultures may reveal presumptive Bacillus anthracis microorganisms in human samples . Our study of 1336 individuals showed the probability of encountering presumptively positive cultures using routine microbiologic examination was approximately 0.4% (5 individuals) when nasal swabs were examined for the following characteristics indicative of Bacillus anthracis : nonhemolytic ground-glass colonies retaining their shape when manipulated, nonmotile in testing media, and microscopically revealing spore-forming gram-positive rods . Confirmatory tests revealed that those cultures were of Bacillus nonanthracis microorganisms . The probability appears significantly high to suggest a need for the confirmatory tests to be available at the community laboratories . In addition, our study confirmed the Gram's staining method does not kill all the spores of the spore-forming gram-positive rods, necessitating special safety requirements.

Infect Genet Evol, 2004 Sep, 4(3), 205 - 13
Anthrax molecular epidemiology and forensics: using the appropriate marker for different evolutionary scales; Keim P et al.; Precise identification of Bacillus anthracis isolates has aided forensic and epidemiological analyses of natural anthrax cases, bioterrorism acts and industrial scale accidents by state-sponsored bioweapons programs . Because there is little molecular variation among B . anthracis isolates, identifying and using rare variation is crucial for precise strain identification . We think that mutation is the primary diversifying force in a clonal, recently emerged pathogen, such as B . anthracis, since mutation rate is correlated with diversity on a per locus basis . While single nucleotide polymorphisms (SNPs) are rare, their detection is facilitated by whole genome discovery approaches . As highly stable phylogenetic markers, SNPs are useful for identifying long branches or key phylogenetic positions . Selection of single, diagnostic "Canonical SNPs" (canSNPs) for these phylogenetic positions allows for efficient and defining assays . We have taken a nested hierarchal strategy for subtyping B . anthracis, which is consistent with traditional diagnostics and applicable to a wide range of pathogens . Progressive hierarchical resolving assays using nucleic acids (PHRANA) uses a progression of diagnostic genomic loci that are initially highly stable but with low resolution and, ultimately, very unstable but with high resolution . This approach mitigates the need for data weighting and provides both a deeply rooted phylogenetic hypothesis and high resolution discrimination among closely related isolates.

Infect Immun, 2004 Oct, 72(10), 5814 - 23
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; Pomerantsev AP et al.; 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 spontaneous plcR-papR intergenic region deletion . Transformation of the resulting plasmid pFP12, encoding a fused PlcR-PapR protein, into the nonhemolytic B . anthracis parental strain produced strong activation of B . anthracis hemolysins, including phosphatidylcholine-specific phospholipase C and sphingomyelinase . The fused PlcR-PapR protein present in a lysate of B . anthracis containing pFP12 bound strongly and specifically to the double-stranded palindrome 5'-TATGCATTATTTCATA-3' that matches the consensus PlcR-binding site . In contrast, native PlcR protein in a lysate from a B . anthracis strain expressing large amounts of this protein did not demonstrate binding with the palindrome . The results suggest that the activation of PlcR by binding of a PapR pentapeptide as normally occurs in Bacillus thuringiensis and B . cereus can be mimicked by tethering the peptide to PlcR in a translational fusion, thereby obviating the need for PapR secretion, extracellular processing, retrieval into the bacterium, and binding with PlcR.

Clin Diagn Lab Immunol, 2004 Sep, 11(5), 919 - 23
Mass value assignment of total and subclass immunoglobulin G in a human standard anthrax reference serum; Semenova VA et al.; An anti-Anthrax Vaccine Adsorbed (anti-AVA) standard human reference serum pool, AVR414, has been prepared, and the total and protective antigen (PA)-specific immunoglobulin G (IgG) were quantified . AVR414 was prepared by plasmapheresis of healthy adults who had received a minimum of four subcutaneous injections of AVA . Mass values (in milligrams per milliliter) for total IgG and IgG subclasses 1 to 4 were determined by radial immunodiffusion . Anti-PA-specific IgG assignment (in micrograms per milliliter) was done by consensus of two complementary approaches: homologous enzyme-linked immunosorbent assay (ELISA) with affinity-purified anti-PA IgG as a calibrator and summation of mean PA-specific IgG subclass concentrations determined by IgG subclass-specific ELISA using the United States National Reference Preparation for Human Serum Proteins as a standard . The total IgG concentration assigned to AVR414 reference serum was 8.33 mg/ml . IgG subclass concentrations were the following: for IgG1, 4.48 mg/ml; for IgG2, 3.35 mg/ml; for IgG3, 0.37 mg/ml; and for IgG4, 0.30 mg/ml . The assigned mass value for total anti-PA-specific IgG was 141.2 microg/ml . Anti-PA-specific IgG subclass concentrations were the following: for IgG1, 79.6 microg/ml; for IgG2, 35.3 microg/ml; for IgG3, 3.2 microg/ml; and for IgG4, 25.3 microg/ml . Human reference serum pool AVR414 will have direct application in the standardization of anthrax serological assays, in reagent qualification, and as a standard for quantification of PA-specific IgG in humans who have been vaccinated with or otherwise exposed to Bacillus anthracis PA.

Biochem Biophys Res Commun, 2004 Aug 27, 321(3), 601 - 5
Cross-inhibition between furin and lethal factor inhibitors; Peinado JR et al.; Bacillus anthracis synthesizes two toxins composed of the three proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF) . The cleavage of PA on the cell surface by the convertase furin leads to the translocation of LF and EF into the cytosol . We have investigated the cross-inhibitory activities of the furin inhibitors hexa-d-arginine amide (D6R) and nona-d-arginine amide (D9R), which block the proteolytic activation of PA; and of the LF inhibitor In-2-LF, a peptide hydroxamate . D6R and D9R inhibit LF with IC(50s) of 300 and 10microM, respectively; conversely, In-2-LF also inhibits furin (IC(50) 2microM) . In-2-LF was efficiently cleaved by furin with the concomitant loss of inhibitory activity on both LF and furin . Incubation of In-2-LF with LF however generated a product that retained partial inhibitory activity against LF . Combined treatment of cells with D6R and In-2-LF enhanced protection against anthrax lethal toxin, indicating that combined administration of inhibitors could represent an effective therapeutic approach.

Proteomics, 2004 Sep, 4(9), 2653 - 61
Identification of Bacillus anthracis proteins associated with germination and early outgrowth by proteomic profiling of anthrax spores; Huang CM et al.; The use of anthrax spores as a bioweapon has spurred efforts aimed at identifying key proteins expressed in Bacillus anthracis . Because spore germination and outgrowth occur prior to and are required for disease manifestations, blocking germination and early outgrowth with novel vaccines or inhibitors targeting critical B . anthracis germination and outgrowth-associated factors is a promising strategy in mitigating bioterror . By screening 587 paired protein spots that were isolated from dormant and germinating anthrax spores, respectively, we identified 10 spore proteins with statistically significant germination-associated increases and decreases . It is likely that proteins whose levels change during germination may play key roles in the germination and outgrowth processes, and they should be listed as priority targets for development of prophylactic and therapeutic agents against anthrax . The 31 new proteins identified in this study also complement an emerging proteomic database of B . anthracis.

Microb Pathog, 2004 Sep, 37(3), 149 - 54
The NheA component of the non-hemolytic enterotoxin of Bacillus cereus is produced by Bacillus anthracis but is not required for virulence; Mendelson I et al.; A non-hemolytic enterotoxin (NHE) is one of the two enterotoxins thought to cause diarrhea produced by Bacillus cereus . We identified genes in Bacillus anthracis homologous to the B . cereus nheAB genes encoding proteins of the NHE complex . The NheA component was detected immunologically in culture supernatants from B . anthracis but not from a NheA(-) mutant, suggesting that B . anthracis produces and secretes the NheA subunit of NHE . A NheA deletion mutant was not attenuated in the guinea pig suggesting that NheA is not absolutely required for virulence.

Proc Natl Acad Sci U S A, 2004 Sep 14, 101(37), 13536 - 41 Epub 2004 Sep 03.
Phylogenetic discovery bias in Bacillus anthracis using single-nucleotide polymorphisms from whole-genome sequencing; Pearson T et al.; Phylogenetic reconstruction using molecular data is often subject to homoplasy, leading to inaccurate conclusions about phylogenetic relationships among operational taxonomic units . Compared with other molecular markers, single-nucleotide polymorphisms (SNPs) exhibit extremely low mutation rates, making them rare in recently emerged pathogens, but they are less prone to homoplasy and thus extremely valuable for phylogenetic analyses . Despite their phylogenetic potential, ascertainment bias occurs when SNP characters are discovered through biased taxonomic sampling; by using whole-genome comparisons of five diverse strains of Bacillus anthracis to facilitate SNP discovery, we show that only polymorphisms lying along the evolutionary pathway between reference strains will be observed . We illustrate this in theoretical and simulated data sets in which complex phylogenetic topologies are reduced to linear evolutionary models . Using a set of 990 SNP markers, we also show how divergent branches in our topologies collapse to single points but provide accurate information on internodal distances and points of origin for ancestral clades . These data allowed us to determine the ancestral root of B . anthracis, showing that it lies closer to a newly described "C" branch than to either of two previously described "A" or "B" branches . In addition, subclade rooting of the C branch revealed unequal evolutionary rates that seem to be correlated with ecological parameters and strain attributes . Our use of nonhomoplastic whole-genome SNP characters allows branch points and clade membership to be estimated with great precision, providing greater insight into epidemiological, ecological, and forensic questions.

Zh Mikrobiol Epidemiol Immunobiol, 2004 May-Jun, (3), 81 - 3
{Heterogeneity of Bacillus anthracis strains in terms of their adhesive capacity}; Kurilova AA et al.; The homogeneity of colonies of two B . anthracis vaccine strains in R- and RS- forms (100 colonies of each strain) in terms of their adhesive capacity was studied . B . anthracis strain 228/8 showed more heterogeneous composition than B . anthracis strain 71/12, moderately and highly adhesive colonies prevailing in both forms and nonadhesive colonies being absent . The prevalence of highly adhesive clones was established in the RS- form of B . anthracis strain 72/12 in comparison with R- form . By the average value of the adhesion index the RS- form colonies of this strain were classified as highly adhesive, while the colonies in the R- form were characterized as moderately adhesive.

J Infect Dis, 2004 Oct 1, 190(7), 1228 - 36 Epub 2004 Aug 30.
Immune responses to Bacillus anthracis protective antigen in patients with bioterrorism-related cutaneous or inhalation anthrax; Quinn CP et al.; Anti-protective antigen (PA) immunoglobulin (Ig) G, toxin neutralization, and PA-specific IgG memory B cell responses were studied in patients with bioterrorism-related cutaneous or inhalation anthrax and in a patient with laboratory-acquired cutaneous anthrax . Responses were determined for >1 year after the onset of symptoms . Eleven days after the onset of symptoms (15 days after likely exposure), anti-PA IgG was detected in 16 of 17 patients with confirmed or suspected clinical anthrax who were tested . Anti-PA IgG remained detectable 8-16 months after the onset of symptoms in all 6 survivors of inhalation anthrax and in 7 of 11 survivors of cutaneous anthrax who were tested . Anti-PA IgG levels and serum toxin neutralizing activity were strongly associated (R2=0.83) . PA-specific IgG memory B cells were detectable in all 6 survivors of inhalation anthrax but in only 2 of 7 patients with cutaneous anthrax who were tested . Anti-PA IgG is an important diagnostic marker of anthrax, a predictor of serum anti-toxin activity, and a marker of immunological memory against anthrax.

Structure (Camb), 2004 Sep, 12(9), 1705 - 17
Crystal structure of 7,8-dihydropteroate synthase from Bacillus anthracis: mechanism and novel inhibitor design; Babaoglu K et al.; Dihydropterate synthase (DHPS) is the target for the sulfonamide class of antibiotics, but increasing resistance has encouraged the development of new therapeutic agents against this enzyme . One approach is to identify molecules that occupy the pterin binding pocket which is distinct from the pABA binding pocket that binds sulfonamides . Toward this goal, we present five crystal structures of DHPS from Bacillus anthracis, a well-documented bioterrorism agent . Three DHPS structures are already known, but our B . anthracis structures provide new insights into the enzyme mechanism . We show how an arginine side chain mimics the pterin ring in binding within the pterin binding pocket . The structures of two substrate analog complexes and the first structure of a DHPS-product complex offer new insights into the catalytic mechanism and the architecture of the pABA binding pocket . Finally, as an initial step in the development of pterin-based inhibitors, we present the structure of DHPS complexed with 5-nitro-6-methylamino-isocytosine.

Biochem Biophys Res Commun, 2004 Sep 24, 322(3), 1029 - 37
Thermal inactivation of protective antigen of Bacillus anthracis and its prevention by polyol osmolytes; Singh S et al.; Protective antigen (PA) of Bacillus anthracis is the main immunogen of all anthrax vaccines . It is a highly thermolabile molecule and loses its activity rapidly when exposed to higher temperatures . Earlier some cosolvents had been used to stabilize PA with variable success but no study has been done to find out the primary cause of PA thermal inactivation . This study aims at elucidating the predominant cause of thermal inactivation of PA in order to develop more effective strategies for its thermostabilization . The prime cause for the loss of biological activity of PA at high temperature was its aggregation and an inverse correlation between PA activity and its aggregation on heating was observed . Inactivation of the protein by autolysis did not occur . This paper reports the use of a series of polyol osmolytes to stabilize PA . Different polyols stabilized PA to a different extent against thermal inactivation in a concentration dependent manner, with glycerol stabilizing to the maximum extent . Addition of NaCl to glycerol solution further enhanced the thermal stability of PA . An increase in the T(1/2) value, the temperature at which 50% of the activity is retained during short-term incubation, of more than 20 degrees C was observed . The half-life (t(1/2)) of PA thermal inactivation at 40 degrees C increased by more than 6 times in the presence of the mixture of glycerol and NaCl as compared to control . This study demonstrates for the first time that aggregation of the PA molecule is the predominant cause of its thermal inactivation, and can be very effectively prevented by the use of glycerol and other polyols to increase the shelf life of the recombinant vaccine against anthrax.

Biochem Biophys Res Commun, 2004 Sep 24, 322(3), 854 - 9
Targeting of Bacillus anthracis interaction factors for human macrophages using two-dimensional gel electrophoresis; Seo GM et al.; Bacillus anthracis, a gram-positive, endospore-forming, aerobic rod-shaped bacterium, interacts with macrophages at various stages of the disease . Spore germination and the outgrowth of vegetative bacilli are crucial steps enabling the bacteria to proliferate actively and to synthesize the virulence factors leading to a massive septicemia . In this study, we performed a proteomic analysis and MALDI-TOF/MS were carried out to identify proteins using human macrophages infected with the spores of B . anthracis live-Sterne or inactivated-Sterne . We identified 21 proteins which are related to the infection of B . anthracis spores on human macrophages at the early stage events . These proteins function in processes such as cytoskeleton regulation, apoptosis, cell division, and protein degradation . Proteins such as PAK 2 revealed a relationship to apoptosis in human macrophages . These proteins play an important role in the macrophage survival and death on human macrophages with infected B . anthracis spores.

J Food Prot, 2004 Aug, 67(8), 1702 - 8
Lethality of chlorine, chlorine dioxide, and a commercial fruit and vegetable sanitizer to vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis; Beuchat LR et al.; Chlorine, ClO2, and a commercial raw fruit and vegetable sanitizer were evaluated for their effectiveness in killing vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis . The ultimate goal was to use one or both species as a potential surrogate(s) for Bacillus anthracis in studies that focus on determining the efficacy of sanitizers in killing the pathogen on food contact surfaces and foods . Treatment with alkaline (pH 10.5 to 11.0) ClO2 (200 microg/ml) produced by electrochemical technologies reduced populations of a five-strain mixture of vegetative cells and a five-strain mixture of spores of B . cereus by more than 5.4 and more than 6.4 log CFU/ml respectively, within 5 min . This finding compares with respective reductions of 4.5 and 1.8 log CFU/ml resulting from treatment with 200 microg/ml of chlorine . Treatment with a 1.5% acidified (pH 3.0) solution of Fit powder product was less effective, causing 2.5- and 0.4-log CFU/ml reductions in the number of B . cereus cells and spores, respectively . Treatment with alkaline ClO2 (85 microg/ml), acidified (pH 3.4) ClO2 (85 microg/ml), and a mixture of ClO2 (85 microg/ml) and Fit powder product (0.5%) (pH 3.5) caused reductions in vegetative cell/spore populations of more than 5.3/5.6, 5.3/5.7, and 5.3/6.0 log CFU/ml, respectively . Treatment of B . cereus and B . thuringiensis spores in a medium (3.4 mg/ml of organic and inorganic solids) in which cells had grown and produced spores with an equal volume of alkaline (pH 12.1) ClO2 (400 microg/ml) for 30 min reduced populations by 4.6 and 5.2 log CFU/ml, respectively, indicating high lethality in the presence of materials other than spores that would potentially react with and neutralize the sporicidal activity of ClO2.

J Microbiol Methods, 2004 Oct, 59(1), 127 - 30
Rapid detection of Bacillus anthracis spores directly from powders with an evanescent wave fiber-optic biosensor; Tims TB et al.; There currently are no rapid, sensitive tests to directly and reliably detect Bacillus anthracis spores in common powders . Traditional culture is time consuming and molecular techniques cannot directly process powders . This study describes a biosensor assay that detects B . anthracis at concentrations of 3.2 x 10(5) spores/mg or higher in spiked powders in less than 1 h with minimal sample preparation.

Anesthesiol Clin North America, 2004 Sep, 22(3), 533 - 40, vii
Anthrax; Kalamas AG; Anthrax is an often fatal bacterial infection that occurs when Bacillus anthracis endospores enter the body through one of three major routes: inhalational, cutaneous, or gastrointestinal . Before the anthrax terrorist attacks in the United States in 2001, there was very little interest in anthrax as a serious human pathogen; anthrax was viewed mainly as a veterinarian problem of minor importance, with most cases attributed to occupational exposure . However, this cavalier attitude toward anthrax changed following the 2001 terrorist attacks . Although the number of cases was relatively small, the attacks have heightened concern about the feasibility of large-scale aerosol bioweapons attacks by terrorist groups . Many, if not most patients, would require some degree of critical care in the form of ventilator or hemodynamic support . It is for this reason that anesthesiologists and other critical care physicians have specific knowledge of the diagnosis, treatment, and prevention of anthrax.

Chem Biol, 2004 Aug, 11(8), 1139 - 46
Discovery of a small molecule that inhibits the interaction of anthrax edema factor with its cellular activator, calmodulin; Lee YS et al.; The catalytic efficiency of adenylyl cyclase activity of edema factor (EF) from Bacillus anthracis is enhanced by approximately 1000-fold upon its binding to mammalian protein calmodulin (CaM) . A tandem cell-based and protein binding-based screen of a 10,000 member library identified a molecule that inhibits the EF-CaM interaction and therefore the adenylyl cyclase activity . A combination of fluorescence spectroscopy and photolabeling studies showed that the molecule targets the CaM binding region of EF . A series of related compounds were synthesized and evaluated to identify one compound, 4-{4-(4-nitrophenyl)-thiazolylamino}-benzenesulfonamide, that maintained activity against EF but showed minimal toxicity to two cultured cell lines . This compound represents an important reagent to study the role of EF in anthrax pathology and may represent a drug lead against anthrax infection.

Infect Immun, 2004 Sep, 72(9), 5460 - 3
The capsule of Bacillus anthracis behaves as a thymus-independent type 2 antigen; Wang TT et al.; Bacillus anthracis elaborates a homopolymeric capsule composed of gamma-D-glutamic acid residues . Mice were immunized with formalin-fixed encapsulated B . anthracis bacilli, and the serum antibody response to a gamma-D-glutamyl capsular epitope was measured . Antiglutamyl antibodies were elicited in athymic BALB/c Nu/Nu, BALB/c Nu/+, and CBA/J mice but not in CBA/N xid mice . These response patterns define the capsule of B . anthracis as a thymus-independent type 2 antigen.

J Med Microbiol, 2004 Sep, 53(Pt 9), 833 - 40
Common oligosaccharide moieties inhibit the adherence of typical and atypical respiratory pathogens; Thomas R et al.; Intervention in bacterial adhesion to host cells is a novel method of overcoming current problems associated with antibiotic resistance . Antibiotic-resistant strains of bacteria that cause respiratory tract infections are a problem in hospitals and could be used in bioterrorist attacks . A range of bacterial species was demonstrated to attach to an alveolar epithelial (A549) cell line . In all cases, cell surface oligosaccharides were important in attachment, demonstrated by reduced adhesion when A549 cells were pre-treated with tunicamycin . Bacillus anthracis and Yersinia pestis displayed a restricted tropism for oligosaccharides compared to the environmental, opportunistic pathogens, Pseudomonas aeruginosa, Burkholderia cenocepacia, Burkholderia pseudomallei and Legionella pneumophila . The compound with the greatest anti-adhesion activity was p-nitrophenol . Other generic attachment inhibitors included the polymeric saccharides (dextran and heparin), GalNAcbeta1-4Gal, GalNAcbeta1-3Gal, Galbeta1-4GlcNAc and Galbeta1-3GlcNAc . Burkholderia pseudomallei attachment was particularly susceptible to oligosaccharide inhibition . Combinations of such compounds may serve as a novel generic therapeutics for respiratory tract infections.

Clin Chem, 2004 Oct, 50(10), 1899 - 906 Epub 2004 Aug 12.
Diagnostic probes for Bacillus anthracis spores selected from a landscape phage library; Brigati J et al.; BACKGROUND: Recent use of Bacillus anthracis spores as a bioweapon has highlighted the need for a continuous monitoring system . Current monitoring systems rely on antibody-derived probes, which are not hardy enough to withstand long-term use under extreme conditions . We describe new, phage-derived probes that can be used as robust substitutes for antibodies . METHODS: From a landscape phage library with random octapeptides displayed on all copies of the major phage coat protein of the phage fd-tet, we selected clones that bound to spores of B . anthracis (Sterne strain) . ELISA, micropanning, and coprecipitation assays were used to evaluate the specificity and selectivity with which these phage bound to B . anthracis spores . RESULTS: Peptides on the selected clones directed binding of the phage to B . anthracis spores . Most clones exhibited little or no binding to spores of distantly related Bacillus species, but some binding was observed with spores of closely related species . Our most specific spore-binding phage displayed a peptide EPRLSPHS (several thousand peptides per phage) and bound 3.5- to 70-fold better to spores of B . anthracis Sterne than to spores of other Bacillus species . CONCLUSIONS: The selected phage probes bound preferentially to B . anthracis Sterne spores compared with other Bacillus species . These phage could possibly be further developed into highly specific and robust probes suitable for long-term use in continuous monitoring devices and biosorbents.

Curr Microbiol, 2004 Aug, 49(2), 89 - 94
Temperature control of a 3,4-dihydroxybenzoate (protocatechuate)-based siderophore in Bacillus anthracis; Garner BL et al.; Bacillus anthracis Sterne produced a catecholate siderophore named anthrachelin that was based on 3,4-dihydroxybenzoic acid (3,4-DHB, or protocatechuic acid), a catechol moiety previously unreported as a siderophore component . During iron restriction, both anthrachelin and free 3,4-DHB were excreted . Growth at 37 degrees C (as compared with 23 degrees C) decreased excretion of anthrachelin but not its precursor 3,4-DHB, suggesting that anthrachelin assembly is temperature regulated . A plasmidless strain also produced anthrachelin in an iron- and temperature-regulated fashion, indicating that anthrachelin genes are chromosomal . In addition to anthrachelin-mediated iron delivery, B . anthracis also used heme, hemoproteins, iron-transferrin, and certain heterologous siderophores (xenosiderophores) produced by other microorganisms as iron sources . Downregulation of anthrachelin production at the temperature of the mammalian host (which triggers toxin production in this pathogen) may focus the B . anthracis iron acquisition systems to exploit the iron sources prevailing in the infected host.

J Clin Microbiol, 2004 Aug, 42(8), 3711 - 30
Use of 16S rRNA, 23S rRNA, and gyrB gene sequence analysis to determine phylogenetic relationships of Bacillus cereus group microorganisms; Bavykin SG et al.; In order to determine if variations in rRNA sequence could be used for discrimination of the members of the Bacillus cereus group, we analyzed 183 16S rRNA and 74 23S rRNA sequences for all species in the B . cereus group . We also analyzed 30 gyrB sequences for B . cereus group strains with published 16S rRNA sequences . Our findings indicated that the three most common species of the B . cereus group, B . cereus, Bacillus thuringiensis, and Bacillus mycoides, were each heterogeneous in all three gene sequences, while all analyzed strains of Bacillus anthracis were found to be homogeneous . Based on analysis of 16S and 23S rRNA sequence variations, the microorganisms within the B . cereus group were divided into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, and these seven subgroups were further organized into two distinct clusters . This classification of the B . cereus group conflicts with current taxonomic groupings, which are based on phenotypic traits . The presence of B . cereus strains in six of the seven subgroups and the presence of B . thuringiensis strains in three of the subgroups do not support the proposed unification of B . cereus and B . thuringiensis into one species . Analysis of the available phenotypic data for the strains included in this study revealed phenotypic traits that may be characteristic of several of the subgroups . Finally, our results demonstrated that rRNA and gyrB sequences may be used for discriminating B . anthracis from other microorganisms in the B . cereus group.

J Clin Microbiol, 2004 Aug, 42(8), 3626 - 34
MICs of selected antibiotics for Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides from a range of clinical and environmental sources as determined by the Etest; Turnbull PC et al.; This paper presents Etest determinations of MICs of selected antimicrobial agents for 76 isolates of Bacillus anthracis chosen for their diverse histories and 67, 12, and 4 cultures, respectively, of its close relatives B . cereus, B . thuringiensis, and B . mycoides derived from a range of clinical and environmental sources . NCCLS breakpoints are now available for B . anthracis and ciprofloxacin, penicillin, and tetracycline; based on these breakpoints, the B . anthracis isolates were all fully susceptible to ciprofloxacin and tetracycline, and all except four cultures, three of which had a known history of penicillin resistance and were thought to originate from the same original parent, were susceptible to penicillin . Based on NCCLS interpretive standards for gram-positive and/or aerobic bacteria, all cultures were susceptible to amoxicillin-clavulanic acid and gentamicin and 99% (one with intermediate sensitivity) of cultures were susceptible to vancomycin . No group trends were apparent among the different categories of B . cereus (isolates from food poisoning incidents and nongastrointestinal infections and food and environmental specimens not associated with illness) . Differences between B . anthracis and the other species were as expected for amoxicillin and penicillin, with all B . anthracis cultures, apart from the four referred to above, being susceptible versus high proportions of resistant isolates for the other three species . Four of the B . cereus and one of the B . thuringiensis cultures were resistant to tetracycline and a further six B . cereus and one B . thuringiensis cultures fell into the intermediate category . There was a slightly higher resistance to azithromycin among the B . anthracis strains than for the other species . The proportion of B . anthracis strains fully susceptible to erythromycin was also substantially lower than for the other species, although just a single B . cereus strain was fully resistant . The Etest compared favorably with agar dilution in a subsidiary test set up to test the readings, and it compared with other published studies utilizing a variety of test methods.

Appl Environ Microbiol, 2004 Aug, 70(8), 4740 - 7
Determination of the efficacy of two building decontamination strategies by surface sampling with culture and quantitative PCR analysis; Buttner MP et al.; The efficacy of currently available decontamination strategies for the treatment of indoor furnishings contaminated with bioterrorism agents is poorly understood . Efficacy testing of decontamination products in a controlled environment is needed to ensure that effective methods are used to decontaminate domestic and workplace settings . An experimental room supplied with materials used in office furnishings (i.e., wood laminate, painted metal, and vinyl tile) was used with controlled dry aerosol releases of endospores of Bacillus atrophaeus ("Bacillus subtilis subsp . niger," also referred to as BG), a Bacillus anthracis surrogate . Studies were performed using two test products, a foam decontaminant and chlorine dioxide gas . Surface samples were collected pre- and posttreatment with three sampling methods and analyzed by culture and quantitative PCR (QPCR) . Additional aerosol releases with environmental background present on the surface materials were also conducted to determine if there was any interference with decontamination or sample analysis . Culture results indicated that 10(5) to 10(6) CFU per sample were present on surfaces before decontamination . After decontamination with the foam, no culturable B . atrophaeus spores were detected . After decontamination with chlorine dioxide gas, no culturable B . atrophaeus was detected in 24 of 27 samples (89%) . However, QPCR analysis showed that B . atrophaeus DNA was still present after decontamination with both methods . Environmental background material had no apparent effect on decontamination, but inhibition of the QPCR assay was observed . These results demonstrate the effectiveness of two decontamination methods and illustrate the utility of surface sampling and QPCR analysis for the evaluation of decontamination strategies.

Int J Med Microbiol, 2004 Jul, 294(1), 35 - 44
Protection of mice against challenge with Bacillus anthracis STI spores after DNA vaccination; Hahn UK et al.; Immune responses against the protective antigen (PA) of Bacillus anthracis are known to confer immunity against anthrax . We evaluated the efficacy of genetic vaccination with plasmid vectors encoding PA, in protecting mice from a lethal challenge with B . anthracis STI spores . BALB/c and A/J mice were immunized via gene gun inoculation, using eukaryotic expression vectors with different cellular targeting signals for the encoded antigen . The vector pSecTag PA83, encoding the full-length PA protein, has a signal sequence for secretion of the expressed protein . The plasmids pCMV/ER PA83 and pCMV/ER PA63, encoding the full-length and the physiologically active form of PA, respectively, target and retain the expressed antigen in the endoplasmic reticulum of transfected cells . All three plasmids induced PA-specific humoral immune responses, predominantly IgG1 antibodies, in mice . Spleen cells collected from plasmid-vaccinated BALB/c mice produced PA-specific interleukin-4, interleukin-5, and interferon-gamma in vitro . Vaccination with either pSecTag PA83 or pCMV/ER PA83 showed significant protection of A/J mice against infection with B . anthracis STI spores.

J Biol Chem, 2004 Oct 8, 279(41), 42860 - 6 Epub 2004 Jul 28.
Dissecting the protein-protein interface between beta-lactamase inhibitory protein and class A beta-lactamases; Zhang Z et al.; beta-Lactamase inhibitory protein (BLIP) binds and inhibits a diverse collection of class A beta-lactamases at a wide range of affinities . Alanine-scanning mutagenesis was previously performed to identify the amino acid sequence requirements of BLIP for inhibiting TEM-1 beta-lactamase and SME-1 beta-lactamase . Two hotspots of binding energy, one from each domain of BLIP, were identified (Zhang, Z., and Palzkill, T . (2003) J . Biol . Chem . 278, 45706-45712) . This study has been extended to examine the amino acid sequence requirements of BLIP for binding to the SHV-1 beta-lactamase, which is a poor binding substrate (Ki= 1.1 microm), and the Bacillus anthracis Bla1 enzyme (Ki= 2.5 nm) . The two hotspots previously identified as important for binding TEM-1 and SME-1 beta-lactamase were also found to be important for binding Bla1 . The hotspot from the second domain of BLIP, however, does not make substantial contributions to SHV-1 binding . This may explain why BLIP binds to SHV-1 beta-lactamase with much weaker affinity than to the other three enzymes . Three regions, including two loops that insert into the active pocket of TEM-1 beta-lactamase and the Glu-73-Lys-74 buried charge motif, exhibit strikingly different effects on the binding affinity of BLIP toward the various enzymes when mutated and, therefore, act as specificity determinants . Analysis of double mutants of BLIP that combine specificity-determining residues suggests that these residues contribute to the poor affinity between the second domain of BLIP and SHV-1 beta-lactamase.

Rev Physiol Biochem Pharmacol . 2004 Jul 27; {Epub ahead of print}
Anthrax toxins; Mourez M; Bacillus anthracis, the etiological agent of anthrax, secretes three polypeptides that assemble into toxic complexes on the cell surfaces of the host it infects . One of these polypeptides, protective antigen (PA), binds to the integrin-like domains of ubiquitously expressed membrane proteins of mammalian cells . PA is then cleaved by membrane endoproteases of the furin family . Cleaved PA molecules assemble into heptamers, which can then associate with the two other secreted polypeptides: edema factor (EF) and/or lethal factor (LF) . The heptamers of PA are relocalized to lipid rafts where they are quickly endocytosed and routed to an acidic compartment . The low pH triggers a conformational change in the heptamers, resulting in the formation of cation-specific channels and the translocation of EF/LF . EF is a calcium- and calmodulin-dependent adenylate cyclase that dramatically raises the intracellular concentration of cyclic adenosine monophosphate (cAMP) . LF is a zinc-dependent endoprotease that cleaves the amino terminus of mitogen-activated protein kinase kinases (Meks) . Cleaved Meks cannot bind to their substrates and have reduced kinase activity, resulting in alterations of the signaling pathways they govern . The structures of PA, PA heptamer, EF, and LF have been solved and much is now known about the molecular details of the intoxication mechanism . The in vivo action of the toxins, on the other hand, is still poorly understood and hotly debated . A better understanding of the toxins will help in the design of much-needed anti-toxin drugs and the development of new toxin-based medical applications.

Antimicrob Agents Chemother, 2004 Aug, 48(8), 3024 - 7
Activities of different fluoroquinolones against Bacillus anthracis mutants selected in vitro and harboring topoisomerase mutations; Grohs P et al.; Three sets of mutants of Bacillus anthracis resistant to fluoroquinolones were selected on ciprofloxacin and moxifloxacin in a stepwise manner from a nalidixic acid-resistant but fluoroquinolone-susceptible plasmidless strain harboring a Ser85Leu GyrA mutation . A high level of resistance to fluoroquinolones could be obtained in four or five selection steps . In each case, ParC was the secondary target . However, in addition to the GyrA mutation, expression of high-level resistance required (i) in the first set of mutants, active drug efflux associated with a mutation in the QRDR of ParC; (ii) in the second set, two mutations in the QRDR of ParC associated with a mutation in GyrB; and (iii) in the third set, two QRDR mutations, one in ParC and one in GyrA . Interestingly, several selection steps occurred without obvious mutations in the QRDR of any topoisomerase, thereby implying the existence of other resistance mechanisms . Among the fluoroquinolones tested, garenoxacin showed the best activity.

J Infect Dis, 2004 Aug 15, 190(4), 774 - 82 Epub 2004 Jul 13.
Induction of protective immunity against lethal anthrax challenge with a patch; Kenney RT et al.; BACKGROUND: Transcutaneous immunization (TCI) is a needle-free technique that delivers antigens and adjuvants to potent epidermal immune cells . To address critical unmet needs in biodefense against anthrax, we have designed a novel vaccine delivery system using a dry adhesive patch that simplifies administration and improves tolerability of a subunit anthrax vaccine . METHODS: Mice and rabbits were vaccinated with recombinant protective antigen of Bacillus anthracis and the heat-labile toxin of Escherichia coli . Serologic changes, levels of toxin-neutralizing antibodies (TNAs), and pulmonary and nodal responses were monitored in the mice . A lethal aerosolized B . anthracis challenge model was used in A/J mice, to demonstrate efficacy . RESULTS: The level of systemic immunity and protection induced by TCI was comparable to that induced by intramuscular vaccination, and peak immunity could be achieved with only 2 doses . The addition of adjuvant in the patch induced superior TNA levels, compared with injected vaccination . CONCLUSIONS: Anthrax vaccine patches stimulated robust and functional immune responses that protected against lethal challenge . Demonstration of responses in the lung suggests that a mechanism exists for protection against challenge with aerosolized anthrax spores . A formulated, pressure-sensitive, dry adhesive patch, which is stable and can be manufactured in large scale, elicited comparable immunoglobulin G and TNA responses, suggesting that an anthrax vaccine patch is feasible and should advance into clinical evaluation.

Infect Immun, 2004 Aug, 72(8), 4801 - 9
Murine model of pulmonary anthrax: kinetics of dissemination, histopathology, and mouse strain susceptibility; Lyons CR et al.; Bioweapons are most often designed for delivery to the lung, although this route is not the usual portal of entry for many of the pathogens in the natural environment . Vaccines and therapeutics that are efficacious for natural routes of infection may not be effective against the pulmonary route . Pulmonary models are needed to investigate the importance of specific bacterial genes in virulence, to identify components of the host immune system that are important in providing innate and acquired protection, and for testing diagnostic and therapeutic strategies . This report describes the characteristics of host and Bacillus anthracis interactions in a murine pulmonary-infection model . The infective dose varied depending on the route and method of inoculation . The germination process in the lung began within 1 h of inoculation into the lung, although growth within the lung was limited . B . anthracis was found in the lung-associated lymph nodes approximately 5 h after infection . Minimal pneumonitis was associated with the lung infection, but significant systemic pathology was noted after dissemination . Infected mice typically succumbed to infection approximately 3 to 4 days after inoculation . The 50% lethal doses differed among inbred strains of mice, but within a given mouse strain, neither the age nor the sex of the mice influenced susceptibility to B . anthracis.

Infect Immun, 2004 Aug, 72(8), 4439 - 47
Mouse susceptibility to anthrax lethal toxin is influenced by genetic factors in addition to those controlling macrophage sensitivity; Moayeri M et al.; Bacillus anthracis lethal toxin (LT) produces symptoms of anthrax in mice and induces rapid lysis of macrophages (M phi) derived from certain inbred strains . We used nine inbred strains and two inducible nitric oxide synthase (iNOS) knockout C57BL/6J strains polymorphic for the LT M phi sensitivity Kif1C locus to analyze the role of M phi sensitivity (to lysis) in LT-mediated cytokine responses and lethality . LT-mediated induction of cytokines KC, MCP-1/JE, MIP-2, eotaxin, and interleukin-1 beta occurred only in mice having LT-sensitive M phi . However, while iNOS knockout C57BL/6J mice having LT-sensitive M phi were much more susceptible to LT than the knockout mice with LT-resistant M phi, a comparison of susceptibilities to LT in the larger set of inbred mouse strains showed a lack of correlation between M phi sensitivity and animal susceptibility to toxin . For example, C3H/HeJ mice, harboring LT-sensitive M phi and having the associated LT-mediated cytokine response, were more resistant than mice with LT-resistant M phi and no cytokine burst . Toll-like receptor 4 (Tlr4)-deficient, lipopolysaccharide-nonresponsive mice were not more resistant to LT . We also found that CAST/Ei mice are uniquely sensitive to LT and may provide an economical bioassay for toxin-directed therapeutics . The data indicate that while the cytokine response to LT in mice requires M phi lysis and while M phi sensitivity in the C57BL/6J background is sufficient for BALB/cJ-like mortality of that strain, the contribution of M phi sensitivity and cytokine response to animal susceptibility to LT differs among other inbred strains . Thus, LT-mediated lethality in mice is influenced by genetic factors in addition to those controlling M phi lysis and cytokine response and is independent of Tlr4 function.

Nature, 2004 Jul 22, 430(6998), 451 - 2
Anthrax kills wild chimpanzees in a tropical rainforest; Leendertz FH et al.; Infectious disease has joined habitat loss and hunting as threats to the survival of the remaining wild populations of great apes . Nevertheless, relatively little is known about the causative agents . We investigated an unusually high number of sudden deaths observed over nine months in three communities of wild chimpanzees (Pan troglodytes verus) in the Tai National Park, Ivory Coast . Here we report combined pathological, cytological and molecular investigations that identified Bacillus anthracis as the cause of death for at least six individuals . We show that anthrax can be found in wild non-human primates living in a tropical rainforest, a habitat not previously known to harbour B . anthracis . Anthrax is an acute disease that infects ruminants, but other mammals, including humans, can be infected through contacting or inhaling high doses of spores or by consuming meat from infected animals . Respiratory and gastrointestinal anthrax are characterized by rapid onset, fever, septicaemia and a high fatality rate without early antibiotic treatment . Our results suggest that epidemic diseases represent substantial threats to wild ape populations, and through bushmeat consumption also pose a hazard to human health.

Gac Med Mex, 2004 May-Jun, 140(3), 321 - 7
{Historical perspective of smallpox in Mexico: emergence, elimination, and risk of reemergence due to bioterrorism}; Franco-Paredes C et al.; Smallpox has been considered a disease of historical interest . However, given the 2001 terrorist events in the U.S . with intentional release of spores of Bacillus anthracis, and the current political worldwide agenda, the risk of bioterrorism has become a global public health concern . The risk of an intentional release of Variola virus as a biological weapon mandates a critical review of the historical impact of the disease in our country and the possible risk of its intentional reemergence . Smallpox was introduced into susceptible Indian populations in the Americas in the 16th century, contributing to the collapse of the Aztec Empire . Francisco Xavier Balmis start a vaccination campaign in the New World, and his efforts are considered the first eradication campaign of vaccine preventable diseases . Due to his efforts, smallpox was eliminated in Mexico in 1951 . In the posteradication era, there is small but finite risk of intentional release of Variola virus . In response to this risk, Mexico has developed a comprehensive National preparedness plan . The impact of a new epidemic of smallpox will be considered a catastrophic event from both a historical and public health perspectives.

Occup Environ Med, 2004 Aug, 61(8), 703 - 8
Determination of serum IgG antibodies to Bacillus anthracis protective antigen in environmental sampling workers using a fluorescent covalent microsphere immunoassay; Biagini RE et al.; AIMS: To evaluate potential exposure to Bacillis anthracis (Ba) spores in sampling/decontamination workers in the aftermath of an anthrax terror attack . METHODS: Fifty six serum samples were obtained from workers involved in environmental sampling for Ba spores at the American Media, Inc . (AMI) building in Boca Raton, FL after the anthrax attack there in October 2001 . Nineteen sera were drawn from individuals both pre-entry and several weeks after entrance into the building . Nine sera each were drawn from unique individuals at the pre-entry and follow up blood draws . Thirteen donor control sera were also evaluated . Individuals were surveyed for Ba exposure by measurement of serum Ba anti-protective antigen (PA) specific IgG antibodies using a newly developed fluorescent covalent microsphere immunoassay (FCMIA) . RESULTS: Four sera gave positive anti-PA IgG results (defined as anti-PA IgG concentrations > or = the mean microg/ml anti-PA IgG from donor control sera (n = 13 plus 2 SD which were also inhibited > or = 85% when the serum was pre-adsorbed with PA) . The positive sera were the pre-entry and follow up samples of two workers who had received their last dose of anthrax vaccine in 2000 . CONCLUSION: It appears that the sampling/decontamination workers of the present study either had insufficient exposure to Ba spores to cause the production of anti-PA IgG antibodies or they were exposed to anthrax spores without producing antibody . The FCMIA appears to be a fast, sensitive, accurate, and precise method for the measurement of anti-PA IgG antibodies.

Microb Drug Resist, 2004 Summer, 10(2), 77 - 82
The structure of the cell wall peptidoglycan of Bacillus cereus RSVF1, a strain closely related to Bacillus anthracis; Severin A et al.; The peptidoglycan of Bacillus cereus RSVF1, a close relative of Bacillus anthracis, has several distinguishing features: the overwhelming majority of cross-linked muropeptides are dimers, higher oligomers are only present in minute quantities; and virtually all muropeptides lack the N-acetyl group from glucosamine residues, thus explaining resistance of the cell walls to lysozyme.

Indian J Exp Biol, 2003 Feb, 41(2), 177 - 80
Detection of spores of Bacillus anthracis from environment using polymerase chain reaction; Alam SI et al.; A sensitive PCR based detection of Bacillus anthracis spores from environnment was standardized . Specific 1247bp amplicon could be detected with template concentration as low as 13 pg . Sensitivity was enhanced to 10 fold by nesting with second set of primers, forming 208bp amplicon . Extraction of DNA from spores purified from soil samples by aqueous polymer two-phase system followed by partial germination and freeze-thaw treatment yielded best results . Soil sample spiked with spores (8x10(2)/g of sample) could be detected with this method.

Indian J Exp Biol, 2003 Feb, 41(2), 123 - 8
Generation and characterization of monoclonal antibodies to protective antigen of Bacillus anthracis; Sastry KS et al.; Monoclonal antibodies (MoAbs) were generated following immunization of BALB/c mice with protective antigen (PA) of B . anthracis . Five clones reactive to this protein were stabilized and preserved . These MoAbs could detect nanogram levels of PA when tested in ELISA . In Western blotting, they reacted with all PA preparations tested and no cross-reactivity was observed with lethal factor, edema factor of B . anthracis and with other organisms . These MoAbs could detect PA from 22 confirmed clinical isolates of B . anthracis on Western blotting and hold promise for direct detection of PA in clinical samples for diagnosing anthrax.

Vaccine, 2004 Jul 29, 22(21-22), 2843 - 52
Development of an in vitro-based potency assay for anthrax vaccine; Little SF et al.; The potency assay currently used to evaluate consistency of manufacture for the anthrax vaccine is contingent upon meeting specified parameters after statistical analysis of the percent survival and time to death of vaccinated guinea pigs after challenge with spores of a virulent strain of Bacillus anthracis . During the development of a new anthrax vaccine based upon recombinant protective antigen (rPA) adsorbed to aluminum hydroxide gel (Alhydrogel), we found that the serological response of female A/J mice, as measured by a quantitative anti-rPA IgG ELISA, may be an effective method to monitor a manufacturer's consistency for rPA-based vaccines . An advantage of the proposed in vitro-based potency assay is that it will not need stringent biosafety containment measures as required by the current guinea pig potency assay.

Diagn Microbiol Infect Dis, 2004 Jul, 49(3), 163 - 71
Identification of Bacillus anthracis by multiprobe microarray hybridization; Volokhov D et al.; We have developed a rapid assay based on microarray analysis of amplified genetic markers for reliable identification of Bacillus anthracis and its discrimination from other closely related bacterial species of the Bacillus cereus group . By combining polymerase chain reaction (PCR) amplification of six B . anthracis-specific genes (plasmid-associated genes encoding virulence factors (cyaA, pagA, lef, and capA, capB, capC) and one chromosomal marker BA-5449) with analysis of amplicons by microarray hybridization, we were able to unambiguously identify and discriminate B . anthracis among other closely related species . Bacillus identification relied on hybridization with multiple individual microarray oligonucleotide probes (oligoprobes) specific to each target B . anthracis gene . Evaluation of the assay was conducted using several B . anthracis strains (with or without pXO1 and pXO2 plasmids) as well as over 50 other species phylogenetically related to B . anthracis, including B . cereus, B . thuringiensis, B . mycoides, and B . subtilis . The developed microarray analysis of amplified genetic markers protocol provides an efficient method for (i) unambiguous identification and discrimination of B . anthracis from other Bacillus species and (ii) distinguishing between plasmid-containing and plasmid-free Bacillus anthracis strains.

Structure (Camb), 2004 Jul, 12(7), 1147 - 56
Structures of sortase B from Staphylococcus aureus and Bacillus anthracis reveal catalytic amino acid triad in the active site; Zhang R et al.; Surface proteins attached by sortases to the cell wall envelope of bacterial pathogens play important roles during infection . Sorting and attachment of these proteins is directed by C-terminal signals . Sortase B of S . aureus recognizes a motif NPQTN, cleaves the polypeptide after the Thr residue, and attaches the protein to pentaglycine cross-bridges . Sortase B of B . anthracis is thought to recognize the NPKTG motif, and attaches surface proteins to m-diaminopimelic acid cross-bridges . We have determined crystal structure of sortase B from B . anthracis and S . aureus at 1.6 and 2.0 A resolutions, respectively . These structures show a beta-barrel fold with alpha-helical elements on its outside, a structure thus far exclusive to the sortase family . A putative active site located on the edge of the beta-barrel is comprised of a Cys-His-Asp catalytic triad and presumably faces the bacterial cell surface . A putative binding site for the sorting signal is located nearby.

Anal Chem, 2004 Jul 1, 76(13), 3492 - 7
Development of an automated sample preparation module for environmental monitoring of biowarfare agents; Hindson BJ et al.; An automated sample preparation module, based upon sequential injection analysis (SIA), has been developed for use within an autonomous pathogen detection system . The SIA system interfaced aerosol sampling with multiplexed microsphere immunoassay-flow cytometric detection . Metering and sequestering of microspheres using SIA was found to be reproducible and reliable, over 24-h periods of autonomous operation . Four inbuilt immunoassay controls showed excellent immunoassay and system stability over five days of unattended continuous operation . Titration curves for two biological warfare agents, Bacillus anthracis and Yersinia pestis, obtained using the automated SIA procedure were shown to be similar to those generated using a manual microtiter plate procedure.

J Exp Med, 2004 Jul 5, 200(1), 35 - 46 Epub 2004 Jun 28.
A new family of potent AB(5) cytotoxins produced by Shiga toxigenic Escherichia coli; Paton AW et al.; The Shiga toxigenic Escherichia coli (STEC) O113:H21 strain 98NK2, which was responsible for an outbreak of hemolytic uremic syndrome, secretes a highly potent and lethal subtilase cytotoxin that is unrelated to any bacterial toxin described to date . It is the prototype of a new family of AB(5) toxins, comprising a single 35-kilodalton (kD) A subunit and a pentamer of 13-kD B subunits . The A subunit is a subtilase-like serine protease distantly related to the BA_2875 gene product of Bacillus anthracis . The B subunit is related to a putative exported protein from Yersinia pestis, and binds to a mimic of the ganglioside GM2 . Subtilase cytotoxin is encoded by two closely linked, cotranscribed genes (subA and subB), which, in strain 98NK2, are located on a large, conjugative virulence plasmid . Homologues of the genes are present in 32 out of 68 other STEC strains tested . Intraperitoneal injection of purified subtilase cytotoxin was fatal for mice and resulted in extensive microvascular thrombosis, as well as necrosis in the brain, kidneys, and liver . Oral challenge of mice with E . coli K-12-expressing cloned subA and subB resulted in dramatic weight loss . These findings suggest that the toxin may contribute to the pathogenesis of human disease.

J Immunol, 2004 Jul 1, 173(1), 521 - 30
High bactericidal efficiency of type iia phospholipase A2 against Bacillus anthracis and inhibition of its secretion by the lethal toxin; Gimenez AP et al.; There is a considerable body of evidence supporting the role of secretory type II-A phospholipase A(2) (sPLA(2)-IIA) as an effector of the innate immune response . This enzyme also exhibits bactericidal activity especially toward Gram-positive bacteria . In this study we examined the ability of sPLA(2)-IIA to kill Bacillus anthracis, the etiological agent of anthrax . Our results show that both germinated B . anthracis spores and encapsulated bacilli were sensitive to the bactericidal activity of recombinant sPLA(2)-IIA in vitro . In contrast, nongerminated spores were resistant . This bactericidal effect was correlated to the ability of sPLA(2)-IIA to hydrolyze bacterial membrane phospholipids . Guinea pig alveolar macrophages, the major source of sPLA(2)-IIA in an experimental model of acute lung injury, released enough sPLA(2)-IIA to kill extracellular B . anthracis . The production of sPLA(2)-IIA was significantly inhibited by B . anthracis lethal toxin . Human bronchoalveolar lavage fluids from acute respiratory distress syndrome patients are known to contain sPLA(2)-IIA; bactericidal activity against B . anthracis was detected in a high percentage of these samples . This anthracidal activity was correlated to the levels of sPLA(2)-IIA and was abolished by an sPLA(2)-IIA inhibitor . These results suggest that sPLA(2)-IIA may play a role in innate host defense against B . anthracis infection and that lethal toxin may help the bacteria to escape from the bactericidal action of sPLA(2)-IIA by inhibiting the production of this enzyme.

Emerg Infect Dis, 2004 Jun, 10(6), 1023 - 9
Swab materials and Bacillus anthracis spore recovery from nonporous surfaces; Rose L et al.; Four swab materials were evaluated for their efficiency in recovery of Bacillus anthracis spores from steel coupons . Cotton, macrofoam, polyester, and rayon swabs were used to sample coupons inoculated with a spore suspension of known concentration . Three methods of processing for the removal of spores from the swabs (vortexing, sonication, or minimal agitation) and two swab preparations (premoistened and dry) were evaluated . Results indicated that premoistened swabs were more efficient at recovering spores than dry swabs (14.3% vs . 4.4%) . Vortexing swabs for 2 min during processing resulted in superior extraction of spores when compared to sonicating them for 12 min or subjecting them to minimal agitation . Premoistened macrofoam and cotton swabs that were vortexed during processing recovered the greatest proportions of spores with a mean recovery of 43.6% (standard deviation {SD} 11.1%) and 41.7% (SD 14.6%), respectively . Premoistened and vortexed polyester and rayon swabs were less efficient, at 9.9% (SD 3.8%) and 11.5% (SD 7.9%), respectively.

Emerg Infect Dis, 2004 Jun, 10(6), 996 - 1002
Airborne infection with Bacillus anthracis--from mills to mail; Fennelly KP et al.; The lack of identified exposures in 2 of the 11 cases of bioterrorism-related inhalation anthrax in 2001 raised uncertainty about the infectious dose and transmission of Bacillus anthracis . We used the Wells-Riley mathematical model of airborne infection to estimate 1) the exposure concentrations in postal facilities where cases of inhalation anthrax occurred and 2) the risk for infection in various hypothetical scenarios of exposure to B . anthracis aerosolized from contaminated mail in residential settings . These models suggest that a small number of cases of inhalation anthrax can be expected when large numbers of persons are exposed to low concentrations of B . anthracis . The risk for inhalation anthrax is determined not only by bacillary virulence factors but also by infectious aerosol production and removal rates and by host factors.

J Antimicrob Chemother, 2004 Aug, 54(2), 424 - 8 Epub 2004 Jun 17.
Selection of Bacillus anthracis isolates resistant to antibiotics; Athamna A et al.; OBJECTIVE: Long-term therapy for anthrax might induce antimicrobial resistance in Bacillus anthracis . The aim of the present study was to investigate the potential of 18 different antibiotics to select resistant isolates of B . anthracis, (ST-1 and Sterne strains) . METHODS: Resistant isolates were selected by serial passages on brain heart infusion agar containing increasing concentrations of antibiotics (from the MIC upwards) . RESULTS: The MICs of ciprofloxacin, ofloxacin and levofloxacin increased from 0.125-0.25 to 8 mg/L, that of moxifloxacin increased from 0.03-0.06 to 8 mg/L, in both strains, and the MIC of garenoxacin increased from 0.015 to 0.5 mg/L for the ST-1 strain and from 0.03 to 8 mg/L for the Sterne strain . The MICs of tetracycline and minocycline increased from 0.125 to 2-8 mg/L and 0.06 to 1 mg/L, respectively . The MIC of vancomycin increased from 2.5 to 20 mg/L for the ST-1 strain and from 5 to 20 mg/L for the Sterne strain . Linezolid exhibited an MIC increase from 2 to 4 mg/L for both strains . The MIC of quinupristin/dalfopristin increased from 0.125 to 64-128 mg/L . Erythromycin demonstrated an MIC increase from 1 to 128 mg/L, that of clarithromycin increased from 0.125 to 8-64 mg/L and that of telithromycin increased from 0.06-0.125 to 1-4 mg/L . The clindamycin MIC increased from 0.125-0.25 to 8 mg/L . Penicillin G and amoxicillin MICs increased from <1 mg/L to 128-512 mg/L . Isolates made resistant to one fluoroquinolone exhibited cross-resistance to the other quinolones except the ST-1 mutant strain which remained susceptible to garenoxacin . Cross-resistance to fluoroquinolones did not correlate with resistance to other antibiotics . CONCLUSION: The ease with which B . anthracis can be made resistant in vitro suggests that close monitoring of patients treated for anthrax is mandatory.

J Occup Environ Hyg, 2004 Mar, 1(3), 127 - 38
Assessment of electrical charge on airborne microorganisms by a new bioaerosol sampling method; Lee SA et al.; Bioaerosol sampling is necessary to monitor and control human exposure to harmful airborne microorganisms . An important parameter affecting the collection of airborne microorganisms is the electrical charge on the microorganisms . Using a new design of an electrostatic precipitator (ESP) for bioaerosol sampling, the polarity and relative strength of the electrical charges on airborne microorganisms were determined in several laboratory and field environments by measuring the overall physical collection efficiency and the biological collection efficiency at specific precipitation voltages and polarities . First, bacteria, fungal spores, and dust dispersed from soiled carpets were sampled in a walk-in test chamber . Second, a simulant of anthrax-causing Bacillus anthracis spores was dispersed and sampled in the same chamber . Third, bacteria were sampled in a small office while four adults were engaged in lively discussions . Fourth, bacteria and fungal spores released from hay and horse manure were sampled in a horse barn during cleanup operations . Fifth, bacteria in metalworking fluid droplets were sampled in a metalworking simulator . It was found that the new ESP differentiates between positively and negatively charged microorganisms, and that in most of the tested environments the airborne microorganisms had a net negative charge . This adds a signature to the sampled microorganisms that may assist in their identification or differentiation, for example, in an anti-bioterrorism network.

Emerg Infect Dis, 2004 Apr, 10(4), 732 - 5
Ruling out Bacillus anthracis; Papaparaskevas J et al.; Optimization of methods for ruling out Bacillus anthracis leads to increased yields, faster turnaround times, and a lighter workload . We used 72 environmental non-B . anthracis bacilli to validate methods for ruling out B . anthracis . Most effective were the use of horse blood agar, motility testing after isolates had a 2-h incubation in trypticase soy broth, and screening isolates with a B . anthracis-selective agar.

Proc Natl Acad Sci U S A, 2004 Jun 22, 101(25), 9193 - 8 Epub 2004 Jun 14.
Anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from Escherichia coli-expressed libraries; Harvey BR et al.; Anchored periplasmic expression (APEx) is a technology for the isolation of ligand-binding proteins from combinatorial libraries anchored on the periplasmic face of the inner membrane of Escherichia coli . After disruption of the outer membrane by Tris-EDTA-lysozyme, the inner-membrane-anchored proteins readily bind fluorescently labeled ligands as large as 240 kDa . Fluorescently labeled cells are isolated by flow cytometry, and the DNA of isolated clones is rescued by PCR . By using two rounds of APEx, the affinity of a neutralizing antibody to the Bacillus anthracis protective antigen was improved >200-fold, exhibiting a final K(D) of 21 pM . This approach has several technical advantages compared with previous library screening technologies, including the unique ability to screen for ligand-binding proteins that bind endogenously expressed ligands fused to a short-lived GFP . Further, APEx is able to display proteins either as an N-terminal fusion to a six-residue sequence derived from the native E . coli lipoprotein NlpA, or as a C-terminal fusion to the phage gene three minor coat protein of M13 . The latter fusions allow hybrid phage display/APEx strategies without the need for further subcloning.

J Antimicrob Chemother, 2004 Jul, 54(1), 90 - 4 Epub 2004 Jun 09.
Type II topoisomerase mutations in Bacillus anthracis associated with high-level fluoroquinolone resistance; Bast DJ et al.; OBJECTIVES: To identify and characterize the mechanisms of high-level fluoroquinolone resistance in two strains of Bacillus anthracis following serial passage in increasing concentrations of fluoroquinolones . METHODS: Fluoroquinolone-resistant isolates of the Sterne and Russian Anthrax Vaccine STi strains were obtained following serial passage in the presence of increasing concentrations of four different fluoroquinolones . The quinolone-resistance-determining regions of the type II topoisomerase genes from the resistant strains were amplified by PCR and characterized by DNA sequence analysis . The MICs in the presence and absence of reserpine were determined using broth microdilution as a means of detecting active efflux . RESULTS: Single and double amino acid substitutions in the GyrA (Ser-85-Leu; Glu-89-Arg/Gly/Lys) and GrlA (Ser-81-Tyr; Val-96-Ala; Asn-70-Lys) were most common . A single amino acid substitution in GyrB (Asp-430-Asn) was also identified . Efflux only applied to isolates selected for by either levofloxacin or ofloxacin . CONCLUSIONS: Specific amino acid substitutions in the type II topoisomerase enzymes significantly contributed to the development of high-level fluoroquinolone resistance in B . anthracis . However, notable differences between the strains and the drugs tested were identified including the role of efflux and the numbers and types of mutations identified.

Zh Mikrobiol Epidemiol Immunobiol, 2004 Mar-Apr, (2), 87 - 8
{Immunobiological properties of 100 years old Bacillus anthracis vaccine strains Lange}; Sheremet'eva IuV et al.; The results of the study of B . anthracis vaccine strains Lange 1 and 2 in the form of spores after prolonged storage in 30% glycerine solution at room temperature are presented . The study revealed that spores stored for 100 years germinated and bacterial cells proved to be viable when cultivated in artificial nutrient media and in vivo . They exhibited typical cultural, morphological, biochemical, virulent, antigenic and immunogenic properties.

Biochem Biophys Res Commun, 2004 Jul 2, 319(3), 854 - 8
Esterase activity as a novel parameter of spore germination in Bacillus anthracis; Ferencko L et al.; Spores of Bacillus anthracis were shown to produce esterase activity about 4 min after exposure to conventional germinants such as combinations of amino acids and purine ribosides . Neither amino acids nor ribosides alone induce germination and esterase activity . Expression of esterase activity was chloramphenicol resistant, and correlated with loss of spore refractivity, a traditional parameter of early germination . Based on these observations, we hypothesized that esterase activity could be used as a novel parameter for quantifying early events during spore germination . To test this hypothesis, we measured expression of esterase activity under a variety of germinating conditions . Using diacetyl fluorescein as fluorogenic substrate of esterases, we demonstrated that esterase activity was invariably induced whenever spores were triggered by known germinants . Moreover, D-alanine, an inhibitor of L-alanine-mediated germination, was found to significantly inhibit expression of esterase activity . In terms of molecular mechanisms, esterase expression could represent activation of proteases at the onset of spore germination.

Curr Pharm Biotechnol, 2004 Jun, 5(3), 279 - 84
Applications of single-molecule detection to the analysis of pathogenic DNA; Marina O et al.; We have devised a new technique based on single fluorescent molecule detection for the analysis of specific sequences of DNA . The method consists of synthesizing a fluorescent reporter molecule using a polymerase extension reaction and labeled nucleotides . The fluorescent reporter products are analyzed in a laser-based single-molecule detection system . We have applied this method to the detection of pUC19 and Bacillus anthracis DNA targets . We expect that this method will have applications in rapid detection and identification of DNA from pathogens as well as other sources, and that it will be used for processing of large number of samples in a short period of time.

MMWR Recomm Rep, 2004 Jun 4, 53(RR-7), 1 - 12
Responding to detection of aerosolized Bacillus anthracis by autonomous detection systems in the workplace; Meehan PJ et al.; Autonomous detection systems (ADSs) are under development to detect agents of biologic and chemical terror in the environment . These systems will eventually be able to detect biologic and chemical hazards reliably and provide approximate real-time alerts that an agent is present . One type of ADS that tests specifically for Bacillus anthracis is being deployed in hundreds of postal distribution centers across the United States . Identification of aerosolized B . anthracis spores in an air sample can facilitate prompt on-site decontamination of workers and subsequent administration of postexposure prophylaxis to prevent inhalational anthrax . Every employer who deploys an ADS should develop detailed plans for responding to a positive signal . Responding to ADS detection of B . anthracis involves coordinating responses with community partners and should include drills and exercises with these partners . This report provides guidelines in the following six areas: 1) response and consequence management planning, including the minimum components of a facility response plan; 2) immediate response and evacuation; 3) decontamination of potentially exposed workers to remove spores from clothing and skin and prevent introduction of B . anthracis into the worker's home and conveyances; 4) laboratory confirmation of an ADS signal; 5) steps for evaluating potentially contaminated environments; and 6) postexposure prophylaxis and follow-up.

J Microbiol Methods, 2004 Jul, 58(1), 23 - 30
Ruthenium red staining for ultrastructural visualization of a glycoprotein layer surrounding the spore of Bacillus anthracis and Bacillus subtilis; Waller LN et al.; Ruthenium red is a polycationic stain used to visualize acid polysaccharides on the outer surface of cells . Ruthenium red staining followed by electron microscopic analysis was used to demonstrate the presence of an external glycoprotein layer surrounding the spore of both Bacillus anthracis and Bacillus subtilis . This layer is less apparent with traditional staining methods used for electron microscopy . Renografin gradients were used to purify B . subtilis spores . These purified spores displayed greatly enhanced staining with ruthenium red, indicating nonspecific binding of renografin, which has a major carbohydrate constituent, methylglucamine . For B . anthracis, staining with ruthenium red was sufficiently intense that it was not significantly enhanced by renografin purification . In addition to demonstrating a previously undiscovered layer surrounding the spores of B . subtilis, the results help explain a long-standing controversy as to ultrastructural differences among these genetically closely related organisms . Ruthenium red staining provides an important addition to the identification of surface glycoproteins in studies to define similarities and differences in the exosporium layers of Bacillus species .

Jpn J Ophthalmol, 2004 May-Jun, 48(3), 268 - 71
Cutaneous anthrax on eyelids; Caca I et al.; BACKGROUND: Ophthalmologists should be aware of the signs and symptoms of anthrax, although it is a rare disease in humans . We report our successful treatment of three patients with cutaneous lesions in the periorbital area . CASES: In this study, we report on the treatment of three female patients who were initially diagnosed as having preseptal cellulitis . OBSERVATIONS: Gram-positive robs were revealed in the microscopic examination of scrapings taken from the lesions . Bacillus anthracis was found in only two of the three scraping-material cultures . Intravenous penicillin G was administered in all cases . Black and necrotic eschar, which is characteristic of anthrax, developed on the eyelids of all three patients during treatment . At the final examinations of the patients after the completion of treatment, we recognized the development of cicatrisation, lagophthalmos, and slight ectropion in the upper eyelid of the first patient, and, in the second patient, restriction of upper eyelid movement and development of a corneal scar from exposure keratopathy and ectropion . The cutaneous lesions healed without any eyelid pathology in the third patient . CONCLUSION: Although it is a rare disease in humans, anthrax should be considered in the differential diagnosis of preseptal and orbital cellulitis.

J Biol Inorg Chem, 2004 Jul, 9(5), 609 - 15 Epub 2004 May 26.
Geobacillus stearothermophilus V ubiE gene product is involved in the evolution of dimethyl telluride in Escherichia coli K-12 cultures amended with potassium tellurate but not with potassium tellurite; Araya MA et al.; A 3.8-kb fragment of chromosomal DNA of Geobacillus stearothermophilus V cloned in pSP72 (p1VH) confers resistance to potassium tellurite (K(2)TeO(3)) and to potassium tellurate (K(2)TeO(4)) when the encoded genes are expressed in Escherichia coli K-12 . The nt sequence of the cloned fragment predicts three ORFs of 780, 399, and 600 bp, whose encoded protein products exhibit about 80% similarity with the SUMT methyltransferase and the BtuR protein of Bacillus megaterium, and with the UbiE methyltransferase of Bacillus anthracis A2012, respectively . In addition, E . coli/p1VH cells evolved dimethyl telluride, which was released into the headspace gas above liquid cultures when amended with K(2)TeO(3) or with K(2)TeO(4) . After 48 h of growth in the presence of these compounds, a protein of about 25 kDa was found at a significantly higher level when crude extracts were analyzed by SDS-PAGE . The N-terminal amino acid (aa) sequence of this protein, obtained by Edman degradation, matched the deduced aa sequence predicted by the G . stearothermophilus V ubiE gene . This gene was amplified by PCR, subcloned in pET21b, and transformed into E . coli JM109(DE3) . Interestingly, DMTe evolution occurred when these modified cells were grown in K(2)TeO(4) - but not in K(2)TeO(3) - amended media . These results may be indicative that the two Te oxyanions could be detoxified in the cell by different metabolic pathways.

J Antimicrob Chemother, 2004 Jul, 54(1), 95 - 9 Epub 2004 May 26.
Post-exposure prophylaxis of systemic anthrax in mice and treatment with fluoroquinolones; Steward J et al.; OBJECTIVES: To compare the fluoroquinolones gatifloxacin and moxifloxacin with ciprofloxacin for post-exposure prophylaxis of systemic anthrax in a BALB/c mouse model . METHODS: Treated mice and controls were inoculated subcutaneously with 5 x 10(4) spores/mouse of Bacillus anthracis Ames strain and observed for 37 days after challenge . Treated mice were given 100 mg/kg of antibiotic orally twice daily for 14 days, starting at various times post-challenge . RESULTS: Treatment starting 6 h post-challenge resulted in survival rates of 90%, 15% and 40% for gatifloxacin, moxifloxacin and ciprofloxacin, respectively . Treatment commencing 24 h post-challenge resulted in survival rates of 65%, 10% and 5%, respectively . Treatment starting more than 24 h after exposure had little effect on survival . CONCLUSIONS: Gatifloxacin appeared to be more effective than moxifloxacin or ciprofloxacin, at similar doses, for early post-exposure treatment of murine systemic anthrax . However, these results might be due to differences in potency or pharmacokinetic properties.

J Infect Chemother, 2004 Apr, 10(2), 97 - 100
Pharmacokinetic-pharmacodynamic analysis of fluoroquinolones against Bacillus anthracis; Kihira T et al.; Based on the pharmacokinetic-pharmacodynamic (PK-PD) parameters of ciprofloxacin in rhesus monkeys, the efficacies of levofloxacin, sparfloxacin, norfloxacin, and tosufloxacin against anthrax in humans were examined . The optimal PK-PD parameter for the prophylaxis or treatment of infection with Bacillus anthracis is not clearly defined . To evaluate the efficacy of fluoroquinolones against anthrax, PK-PD parameters and the protein-binding effect of fluoroquinolones are used . B . anthracis is very susceptible to fluoroquinolones in vitro, and levofloxacin, sparfloxacin, and tosufloxacin may be as effective against anthrax as ciprofloxacin by PK-PD analysis . However, additional studies of the in vivo model are necessary to define more clearly efficacy against anthrax and the pharmacodynamic relationship of fluoroquinolones.

Hum Antibodies, 2003, 12(4), 129 - 35
Neutralizing monoclonal antibody against anthrax lethal factor inhibits intoxication in a mouse model; Zhao P et al.; Anthrax toxin is the dominant virulence factor of Bacillus anthracis; drugs blocking its action could therefore have therapeutic benefit . We report here the production of a neutralizing monoclonal antibody (mAb) against anthrax lethal factor (LF) and the inhibition by the antibody of anthrax lethal toxin (LeTx) formation . The anti-LF monoclonal antibody LF8 neutralized the LeTx challenge both in vitro with macrophage J774A.1 cells and in vivo in nude mice . Our data suggested that LF8 binds LF at or near the PA binding domain . A set of dodecameric peptides was selected from a phage-displayed peptide library through their specific binding to anti-LF neutralizing mAb LF8 . These small peptides compete with LF to bind LF8 . Further investigation is undergoing to test the potential application of these peptides to the clinical treatment of anthrax infection by blocking LeTx formation.

Proc Natl Acad Sci U S A, 2004 Jun 1, 101(22), 8449 - 54 Epub 2004 May 21.
Identification of anthrax toxin genes in a Bacillus cereus associated with an illness resembling inhalation anthrax; Hoffmaster AR et al.; Bacillus anthracis is the etiologic agent of anthrax, an acute fatal disease among mammals . It was thought to differ from Bacillus cereus, an opportunistic pathogen and cause of food poisoning, by the presence of plasmids pXO1 and pXO2, which encode the lethal toxin complex and the poly-gamma-d-glutamic acid capsule, respectively . This work describes a non-B . anthracis isolate that possesses the anthrax toxin genes and is capable of causing a severe inhalation anthrax-like illness . Although initial phenotypic and 16S rRNA analysis identified this isolate as B . cereus, the rapid generation and analysis of a high-coverage draft genome sequence revealed the presence of a circular plasmid, named pBCXO1, with 99.6% similarity with the B . anthracis toxin-encoding plasmid, pXO1 . Although homologues of the pXO2 encoded capsule genes were not found, a polysaccharide capsule cluster is encoded on a second, previously unidentified plasmid, pBC218 . A/J mice challenged with B . cereus G9241 confirmed the virulence of this strain . These findings represent an example of how genomics could rapidly assist public health experts responding not only to clearly identified select agents but also to novel agents with similar pathogenic potentials . In this study, we combined a public health approach with genome analysis to provide insight into the correlation of phenotypic characteristics and their genetic basis.

Infect Immun, 2004 Jun, 72(6), 3471 - 7
Contribution of immunological memory to protective immunity conferred by a Bacillus anthracis protective antigen-based vaccine; Marcus H et al.; Protective antigen (PA)-based vaccination is an effective countermeasure to anthrax infection . While neutralizing anti-PA antibody titers elicited by this vaccine serve as good correlates for protection against anthrax (S . Reuveny, M . D . White, Y . Y . Adar, Y . Kafri, Z . Altboum, Y . Gozes, D . Kobiler, A . Shafferman, and B . Velan, Infect . Immun . 69:2888-2893, 2001), no data are available on the contribution of the immunological memory for PA itself to protection . We therefore developed a guinea pig model in which a primary immunization with threshold levels of PA can induce a long-term T-cell immunological memory response without inducing detectable anti-PA antibodies . A revaccination of primed animals with the same threshold PA levels was effective for memory activation, yielding a robust and rapid secondary response . A challenge with a lethal dose (40 50% lethal doses; 2,000 spores) of spores after the booster vaccinations indicated that animals were not protected at days 2, 4, and 6 postboosting . Protection was achieved only from the 8th day postboosting, concomitant with the detection of protective levels of neutralizing antibody titers in the circulation . The practical implications from the studies reported herein are that, as expected, the protective capacity of memory depends on the PA dose used for the primary immunization and that the effectiveness of booster immunizations for the postexposure treatment of anthrax may be very limited when no detectable antibodies are present in primed animals prior to Bacillus anthracis spore exposure . Therefore, to allow for the establishment of memory-dependent protection prior to the expected onset of disease, booster immunizations should not be used without concomitant antimicrobial treatment in postexposure scenarios.

Infect Immun, 2004 Jun, 72(6), 3276 - 83
Enhancement of anthrax lethal toxin cytotoxicity: a subset of monoclonal antibodies against protective antigen increases lethal toxin-mediated killing of murine macrophages; Mohamed N et al.; We investigated the ability of using monoclonal antibodies (MAbs) against anthrax protective antigen (PA), an anthrax exotoxin component, to modulate exotoxin cytotoxic activity on target macrophage cell lines . Anthrax PA plays a critical role in the pathogenesis of Bacillus anthracis infection . PA is the cell-binding component of the two anthrax exotoxins: lethal toxin (LeTx) and edema toxin . Several MAbs that bind the PA component of LeTx are known to neutralize LeTx-mediated killing of target macrophages . Here we describe for the first time an overlooked population of anti-PA MAbs that, in contrast, function to increase the potency of LeTx against murine macrophage cell lines . The results support a possible mechanism of enhancement: binding of MAb to PA on the macrophage cell surface stabilizes the PA by interaction of MAb with macrophage Fcgamma receptors . This results in an increase in the amount of PA bound to the cell surface, which in turn leads to an enhancement in cell killing, most likely due to increased internalization of LF . Blocking of PA-receptor binding eliminates enhancement by MAb, demonstrating the importance of this step for the observed enhancement . The additional significance of these results is that, at least in mice, immunization with PA appears to elicit a poly-clonal response that has a significant prevalence of MAbs that enhance LeTx-mediated killing in macrophages.

J Biol Chem, 2004 Jul 23, 279(30), 30945 - 53 Epub 2004 May 19.
Novel oligosaccharide side chains of the collagen-like region of BclA, the major glycoprotein of the Bacillus anthracis exosporium; Daubenspeck JM et al.; Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by a prominent loose fitting layer called the exosporium . The exosporium consists of a basal layer and an external hairlike nap . The filaments of the nap are composed of a highly immunogenic glycoprotein called BclA, which has a long, central collagen-like region with multiple XXG repeats . Most of the triplet repeats are PTG, and nearly all of the triplet repeats contain a threonine residue, providing multiple potential sites for O-glycosylation . In this study, we demonstrated that two O-linked oligosaccharides, a 715-Da tetrasaccharide and a 324-Da disaccharide, are released from spore- and exosporium-associated BclA by hydrazinolysis . Each oligosaccharide is probably attached to BclA through a GalNAc linker, which was lost during oligosaccharide release . We found that multiple copies of the tetrasaccharide are linked to the collagen-like region of BclA, whereas the disaccharide may be attached outside of this region . Using NMR, mass spectrometry, and other analytical techniques, we determined that the structure of the tetrasaccharide is 2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-beta-d-glucopyranosyl-(1-->3)-alpha-l-rhamnopyranosyl-(1-->3)-alpha-l-rhamnopyranosyl-(1-->2)-l-rhamnopyranose . The previously undescribed nonreducing terminal sugar (i.e . 2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-d-glucose) was given the trivial name anthrose . Anthrose was not found in spores of either Bacillus cereus or Bacillus thuringiensis, two species that are the most phylogenetically similar to B . anthracis . Thus, anthrose may be useful for species-specific detection of B . anthracis spores or as a new target for therapeutic intervention.

J Bacteriol, 2004 Jun, 186(11), 3531 - 8
Distinct mutations in PlcR explain why some strains of the Bacillus cereus group are nonhemolytic; Slamti L et al.; Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis are closely related species belonging to the Bacillus cereus group . B . thuringiensis and B . cereus generally produce extracellular proteins, including phospholipases and hemolysins . Transcription of the genes encoding these factors is controlled by the pleiotropic regulator PlcR . Disruption of plcR in B . cereus and B . thuringiensis drastically reduces the hemolytic, lecithinase, and cytotoxic properties of these organisms . B . anthracis does not produce these proteins due to a nonsense mutation in the plcR gene . We screened 400 B . thuringiensis and B . cereus strains for their hemolytic and lecithinase properties . Eight Hly- Lec- strains were selected and analyzed to determine whether this unusual phenotype was due to a mutation similar to that found in B . anthracis . Sequence analysis of the DNA region including the plcR and papR genes of these strains and genetic complementation of the strains with functional copies of plcR and papR indicated that different types of mutations were responsible for these phenotypes . We also found that the plcR genes of three B . anthracis strains belonging to different phylogenetic groups contained the same nonsense mutation, suggesting that this mutation is a distinctive trait of this species.

Aust Vet J, 2004 Apr, 82(4), 220 - 2
Specificity of an immunochromatographic test for anthrax; Muller JD et al.; OBJECTIVE: To evaluate the specificity of an immunochromatographic test (ICT) for anthrax in cattle . DESIGN: A comparison of an ICT with blood smear and culture in uninfected cattle . PROCEDURE: Two hundred and forty blood samples were collected from dead cattle at two knackeries within Victoria and tested on-site with an ICT for the detection of protective antigen (PA) of Bacillus anthracis . Blood smears were prepared on-site and blood samples transported to the laboratory for aerobic and anaerobic culture . The results of the ICT were compared with blood smear and culture . Animals were regarded as not infected with B anthracis if the organism was not detected in a stained blood smear or on culture . Ten healthy yearling cattle were vaccinated with live spore anthrax vaccine and blood samples collected on days 0 to 7 and day 15 were tested in the ICT for the presence of PA . RESULTS: All blood samples from the 240 knackery cattle were ICT, smear and culture negative . All blood samples from the 10 vaccinated cattle were ICT negative . CONCLUSION: The ICT is a test with high specificity in cattle (98.5 to 100%; 95% CI) and recent vaccination of cattle does not give rise to positive reactions.

Int J Med Microbiol, 2004 Apr, 293(7-8), 619 - 24
In vivo Bacillus anthracis gene expression requires PagR as an intermediate effector of the AtxA signalling cascade; Mignot T et al.; Transcription of the major Bacillus anthracis virulence genes is triggered by CO2, a signal mimicking the host environment . A 182-kb plasmid, pXO1, carries the anthrax toxin genes and the genes responsible for their regulation of transcription, namely atxA and, pagR, the second gene of the pag operon . AtxA has major effects on the physiology of B . anthracis . It coordinates the transcription activation of the toxin genes with that of the capsule biosynthetic enzyme operon, located on the second virulence plasmid, pXO2 . In rich medium, B . anthracis synthesises alternatively two S-layer proteins (Sap and EA1) . An exponential phase "Sap-layer" is subsequently replaced by a stationary phase "EA1-layer" . S-layer gene transcription is controlled by alternative sigma factors and by Sap acting as a transcriptional repressor of eag . Furthermore, in vitro in presence of CO2 and in vivo, AtxA is part of the sap and eag regulatory network . Only eag is significantly expressed in these conditions and this is due to AtxA activating eag and repressing sap transcription . PagR, and not AtxA itself, is the direct effector of this regulation by binding to sap and eag promoter regions . Therefore, PagR mediates the effect of AtxA on eag and sap and is the most downstream element of a signalling cascade initiated by AtxA . Taken together, these results indicate that the B . anthracis transcriptional regulator AtxA is controlling the synthesis of the three toxin components and of the surface elements (capsule and S-layer) . Thus, AtxA is a master regulator that coordinates the response to host signals by orchestrating positive and negative controls over genes located on all genetic elements.

Prehospital Disaster Med, 2003 Jul-Sep, 18(3), 179 - 83
Aum Shinrikyo and the Japanese law on bioterrorism; Sugishima M; Before the sarin incidents in Tokyo and Matsumoto, the Aum Shinrikyo (now Aleph) had tried to conduct bioterrorism with botulinum toxin and Bacillus anthracis . Followers of the Aum could not overcome technical difficulties inherent in developing biological weapons, and the perpetrators had not been prosecuted for their failed attempts of bioterrorism . But the Aum's biological attack revealed several shortcomings in the Japanese law that regulated biological weapons . Since the missile experiment of North Korea conducted in 1998, the Japanese government has come to consider the threat posed by biological weapons more seriously . In 2001, after the 11 September 2001 terrorist attacks and the series of anthrax letter scares in the United States of America, the Japanese government established its Five Basic Principles for Chemical and Biological Weapons Terrorism and several measures were taken at the central and local levels . Activities of the Aum have been monitored by the Public Security Investigation Agency and the National Police Agency under the Anti-Aum Law since 2000.

Biosens Bioelectron, 2004 Mar 15, 19(8), 849 - 59
Low-level detection of a bacillus anthracis simulant using Love-wave biosensors on 36 degrees YX LiTaO3; Branch DW et al.; We present an acoustic Love-wave biosensor for detection of the Bacillus anthracis simulant, Bacillus thuringiensis at or below inhalational infectious levels . The present work is an experimental study of 36 degrees YX cut LiTaO3 based Love-wave devices for detection of pathogenic spores in aqueous conditions . Given that the detection limit (D1) of Love-wave-based sensors is a strong function of the overlying waveguide, two waveguide materials have been investigated, which are polyimide and polystyrene . To determine the mass sensitivity of Love-wave sensor, bovine serum albumin (BSA) protein was injected into the Love-wave test cell while recording the magnitude and phase shift across each sensor . Polyimide had the lowest mass detection limit with an estimated value of 1.0-2.0 ng/cm2, as compared to polystyrene where D1 = 2.0 ng/cm2 . Suitable chemistries were used to orient antibodies on the Love-wave sensor using protein G . The thickness of each biofilm was measured using ellipsometry from which the surface concentrations were calculated . The monoclonal antibody BD8 with a high degree of selectivity for anthrax spores was used to capture the non-pathogenic simulant B . thuringiensis B8 spores . Bacillus subtilis spores were used as a negative control to determine whether significant non-specific binding would occur . Spore aliquots were prepared using an optical counting method, which permitted removal of background particles for consistent sample preparation . This work demonstrates that Love-wave biosensors are promising for low-level detection for whole-cell biological pathogens.

J Bacteriol, 2004 May, 186(10), 3108 - 16
An extracytoplasmic-function sigma factor is involved in a pathway controlling beta-exotoxin I production in Bacillus thuringiensis subsp . thuringiensis strain 407-1; Espinasse S et al.; Beta-exotoxin I is an insecticidal nucleotide analogue secreted by various Bacillus thuringiensis strains . In this report, we describe the characterization and transcriptional analysis of a gene cluster, designated sigW-ecfX-ecfY, that is essential for beta-exotoxin I production in B . thuringiensis subsp . thuringiensis strain 407-1 . In this strain, the disruption of the sigW cluster resulted in nontoxic culture supernatants . sigW encodes a protein of 177 residues that is 97 and 94% identical to two putative RNA polymerase extracytoplasmic-function-type sigma factors from Bacillus anthracis strain Ames and Bacillus cereus strain ATCC 14579, respectively . It is also 50, 30, and 26% identical to SigW from Clostridium perfringens and SigW and SigX from Bacillus subtilis, respectively . EcfX, encoded by the gene following sigW, significantly repressed the expression of sigW when both genes were overtranscribed, suggesting that it could be the anti-sigma factor of SigW . Following the loss of its curable cry plasmid, strain 407 became unable to synthesize crystal toxins, in contrast to the mutant strain 407-1(Cry-)(Pig+), which overproduced this molecule in the absence of this plasmid . Transcriptional analysis of sigW indicated that this gene was expressed during the stationary phase and only in the 407-1(Cry-)(Pig+) mutant . This suggests that in the wild type-407(Cry+) strain, beta-exotoxin I was produced from determinants located on a cry gene-bearing plasmid and that sigW is able to induce beta-exotoxin I production in B . thuringiensis in the absence of cry gene-bearing plasmids . Although the signal responsible for this activation is unknown, these results indicate that beta-exotoxin I production in B . thuringiensis can be restored or induced via an alternative pathway that requires sigW expression.

JAMA, 2004 Apr 28, 291(16), 1994 - 8
One-year health assessment of adult survivors of Bacillus anthracis infection; Reissman DB et al.; CONTEXT: Little is known about potential long-term health effects of bioterrorism-related Bacillus anthracis infection . OBJECTIVE: To describe the relationship between anthrax infection and persistent somatic symptoms among adults surviving bioterrorism-related anthrax disease approximately 1 year after illness onset in 2001 . DESIGN, SETTING, AND PARTICIPANTS: Cross-sectional study of 15 of 16 adult survivors from September through December 2002 using a clinical interview, a medical review-of-system questionnaire, 2 standardized self-administered questionnaires, and a review of available medical records . MAIN OUTCOME MEASURES: Health complaints summarized by the body system affected and by symptom categories; psychological distress measured by the Revised 90-Item Symptom Checklist; and health-related quality-of-life indices by the Medical Outcomes Study 36-Item Short-Form Health Survey (version 2) . RESULTS: The anthrax survivors reported symptoms affecting multiple body systems, significantly greater overall psychological distress (P<.001), and significantly reduced health-related quality-of-life indices compared with US referent populations . Eight survivors (53%) had not returned to work since their infection . Comparing disease manifestations, inhalational survivors reported significantly lower overall physical health than cutaneous survivors (mean scores, 30 vs 41; P =.02) . Available medical records could not explain the persisting health complaints . CONCLUSION: The anthrax survivors continued to report significant health problems and poor life adjustment 1 year after onset of bioterrorism-related anthrax disease.

Clin Microbiol Infect, 2004 May, 10(5), 421 - 4
Evaluation of Bacillus anthracis extractable antigen for testing anthrax immunity; Shlyakhov E et al.; Three extractable Bacillus anthracis cell-wall-associated antigens were evaluated for potential use as skin testing agents, and as possible candidates for in-vitro diagnosis of anthrax immunity . Anthraxin and a partially purified extractable antigen (EAP) were produced from avirulent B . anthracis strain 34F2 (Sterne) . The thermoextractable antigen used for the Ascoli reaction was obtained commercially . Guinea-pigs were immunised and boosted several times subcutaneously with the Sterne live veterinary anthrax vaccine . Four weeks after the last booster dose, animals were skin-tested with the three antigens . Serum antibody levels were also determined by ELISA, and the in-vitro T-cell response was evaluated by {3H}-thymidine incorporation . EAP was the most active antigen in both the serological and cellular reactions . EAP also elicited a distinct positive skin reaction in animals immunised with B . anthracis . The data obtained in this preliminary study indicated that extractable cell-wall antigens obtained from the vegetative form of B . anthracis may be used for skin tests and in-vitro testing of specific humoral and cell-mediated anthrax immunity.

Med Dosw Mikrobiol, 2003, 55(4), 315 - 23
{Gamma radiation resistance of Bacillus anthracis spores}; Mizak L et al.; The aim of the presented study was determined the effectiveness of action the gamma radiation on water suspension B . anthracis spores . The irradiation was performed using a Cobalt 60 (Co 60) source, by using single and fractionary irradiation doses . In the investigations was used B . anthracis stain "Sterne" 34F2 . The obtained results show, that gamma radiation effectively inactivates B . anthracis spores . On the efficiency of sterilization process influence the irradiation's method and the number of spores in 1 ml suspension . In the suspension 1.5 x 10(9) spore in 1 ml, sporicidal doses gamma radiation amount to 25.0 kGy (single dose) or 41.5 kGy (fractionary dose) . The volume suspension about definite inoculum of spores, subjected working the gamma rays has not influence on sporicidal effectiveness of radiation sterilization.

Infect Immun, 2004 May, 72(5), 3069 - 72
Macrophages release tumor necrosis factor alpha and interleukin-12 in response to intracellular Bacillus anthracis spores; Pickering AK et al.; Herein we report that infection of a murine macrophage cell line with Bacillus anthracis results in the production of tumor necrosis factor alpha and interleukin-12 (IL-12) . When infected with B . anthracis spores in combination with lipopolysaccharide, macrophages release increased amounts of IL-12 . We found no evidence of inhibition of cytokine responses in macrophages infected with B . anthracis spores.

RNA, 2004 May, 10(5), 854 - 62
Principles of 3' splice site selection and alternative splicing for an unusual group II intron from Bacillus anthracis; Robart AR et al.; We investigated the self-splicing properties of two introns from the bacterium Bacillus anthracis . One intron (B.a.I1) splices poorly in vitro despite having typical structural motifs, while the second (B.a.I2) splices well while having apparently degenerated features . The spliced exons of B.a.I2 were sequenced, and splicing was found to occur at a 3' site shifted one nucleotide from the expected position, thus restoring missing gamma-gamma' and IBS3-EBS3 pairings, but leaving the two conserved exonic ORFs out of frame . Because of the unexpected splice site, the principles for 3' intron definition were examined, which showed that the 3' splice site is flexible but contingent on gamma-gamma' and IBS3-EBS3 pairings, and can be as far away as four nucleotides from the wild-type site . Surprisingly, alternative splicing occurs at position +4 for wild-type B.a.I2 intron, both in vitro and in vivo, and the alternative event fuses the two conserved exon ORFs, presumably leading to translation of the downstream ORF . The finding suggests that the structural irregularities of B.a.I2 may be an adaptation to facilitate gene expression in vivo.

J Bacteriol, 2004 May, 186(9), 2717 - 23
Isolation of a minireplicon of the virulence plasmid pXO2 of Bacillus anthracis and characterization of the plasmid-encoded RepS replication protein; Tinsley E et al.; A minireplicon of plasmid pXO2 of Bacillus anthracis was isolated by molecular cloning in Escherichia coli and shown to replicate in B . anthracis, Bacillus cereus, and Bacillus subtilis . The pXO2 replicon included (i) an open reading frame encoding the putative RepS replication initiation protein and (ii) the putative origin of replication . The RepS protein was expressed as a fusion with the maltose binding protein (MBP) at its amino-terminal end and purified by affinity chromatography . Electrophoretic mobility shift assays showed that the purified MBP-RepS protein bound specifically to a 60-bp region corresponding to the putative origin of replication of pXO2 located immediately downstream of the RepS open reading frame . Competition DNA binding experiments showed that the 5' and central regions of the putative origin were important for RepS binding . MBP-RepS also bound nonspecifically to single-stranded DNA with a lower affinity.

Proc Natl Acad Sci U S A, 2004 Apr 27, 101(17), 6367 - 72 Epub 2004 Apr 12.
Crystal structure of the von Willebrand factor A domain of human capillary morphogenesis protein 2: an anthrax toxin receptor; Lacy DB et al.; Anthrax toxin is released from Bacillus anthracis as three monomeric proteins, which assemble into toxic complexes at the surface of receptor-bearing host cells . One of the proteins, protective antigen (PA), binds to receptors and orchestrates the delivery of the other two (the lethal and edema factors) into the cytosol . PA has been shown to bind to two cellular receptors: anthrax toxin receptor/tumor endothelial marker 8 and capillary morphogenesis protein 2 (CMG2) . Both are type 1 membrane proteins that include an approximately 200-aa extracellular von Willebrand factor A (VWA) domain with a metal ion-dependent adhesion site (MIDAS) motif . The anthrax toxin receptor/tumor endothelial marker 8 and CMG2 VWA domains share approximately 60% amino acid identity and bind PA directly in a metal-dependent manner . Here, we report the crystal structure of the CMG2 VWA domain, with and without its intramolecular disulfide bond, to 1.5 and 1.8 A, respectively . Both structures contain a carboxylate ligand-mimetic bound at the MIDAS and appear as open conformations when compared with the VWA domains from alpha-integrins . The CMG2 structures provide a template to begin probing the high-affinity CMG2-PA interaction (200 pM) and may facilitate understanding of toxin assembly/internalization and the development of new anthrax treatments . The structural data also allow molecular interpretation of known CMG2 VWA domain mutations linked to the genetic disorders, juvenile hyaline fibromatosis, and infantile systemic hyalinosis.

J Appl Microbiol, 2004, 96(5), 1048 - 56
Bacillus anthracis contamination and inhalational anthrax in a mail processing and distribution center; Sanderson WT et al.; AIMS: Four inhalational anthrax cases occurred in a large mail processing and distribution center in Washington, DC, after envelopes containing Bacillus anthracis spores were processed . This report describes the results of sampling for B . anthracis spores during investigations conducted in October and December 2001 . METHODS AND RESULTS: Wet swabs, wet wipes, vacuum sock, and air-filter samples were collected throughout the facility to characterize the extent of building contamination . The results showed widespread contamination of B . anthracis spores, particularly associated with one delivery bar code sorter (DBCS) machine that had sorted the spore-containing envelopes and an area where the envelopes were handled by postal workers . Spore concentrations decreased as distance from the DBCS machine increased, but spores were widely dispersed into surrounding areas . CONCLUSION: The spatial distribution of culture positive samples was closely related to the work areas of the inhalational anthrax cases and supported epidemiological evidence that the workers became ill from exposure to B . anthracis spores in areas where the contaminated envelopes had travelled . SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this investigation were used to guide decontamination efforts and provided baseline spore concentrations for follow-up measurements after the building had been cleaned . Implementing methods to reduce aerosolization and dispersion of dust within the facility would reduce postal workers' potential exposures to bioterrorism agents.

Vaccine, 2004 Apr 16, 22(13-14), 1604 - 8
Genetic immunization against anthrax; Galloway D et al.; The objective of this study was to determine whether a DNA prime-protein boost immunization against the Bacillus anthracis protective antigen (PA) and lethal factor (LF) antigens could induce a protective immune response against significant aerosol challenge in the rabbit model . Rabbits were vaccinated with different regimens of DNA vaccines (Table 1) and aerosol challenged with B . anthracis spores, Ames strain, with an average dose of 50 LD(50s) with a range from 18 to 169 LD(50s.) Of the five vaccinated rabbits that survived, two were immunized intramuscularly (i.m.) with DNA followed with a protein boost and three were immunized subcutaneous (s.q.) with recombinant protein . A major factor predicting survival was the ability of the animal to mount a lasting antibody response to PA . Rabbit sera were collected prior to and following aerosol challenge and titrated for PA antibodies by indirect ELISA . The results of this study indicate that DNA-based immunization against PA and LF induces significant protective immunity against aerosol challenge in the rabbit model and compares favorably with protein-based immunization.

Biochem Biophys Res Commun, 2004 Apr 30, 317(2), 309 - 14
Structure of anthrax edema factor-calmodulin-adenosine 5'-(alpha,beta-methylene)-triphosphate complex reveals an alternative mode of ATP binding to the catalytic site; Shen Y et al.; Anthrax edema factor (EF) is a key virulence factor secreted by Bacillus anthracis . Here, we report a structure, at 3.0 A resolution, of the catalytic domain of EF (EF3) in complex with calmodulin (CaM) and adenosine 5'-(alpha,beta-methylene)-triphosphate (AMPCPP) . Although the binding of the triphosphate of AMPCPP to EF3 can be superimposed on that of previously determined 3'deoxy-ATP (3'dATP) and 2'deoxy 3' anthraniloyl-ATP (2'd3' ANT-ATP) in EF3-CaM, the ribose and the adenine rings of AMPCPP are rotated approximately 105 and 180 degrees, respectively, relative to those of 3'dATP and 2'd3'ANT-ATP . Based on this model, K382 and F586 should play key roles in the recognition of adenine . However, mutations of these residues to alanine either separately or together cause only modest changes in Michaelis-Menten constants and IC50 values of AMPCPP and cAMP . Therefore, this alternate binding mode of the adenosine of AMPCPP binds to EF likely playing only a minor role in ATP binding and in catalysis.

J Biol Chem, 2004 Jun 4, 279(23), 24861 - 5 Epub 2004 Apr 01.
The 26 Nudix hydrolases of Bacillus cereus, a close relative of Bacillus anthracis; Xu W et al.; The genome of Bacillus cereus contains 26 Nudix hydrolase genes, second only to its closest relative, Bacillus anthracis which has 30 . All 26 genes have been cloned, 25 have been expressed, and 21 produced soluble proteins suitable for analysis . Substrates for 16 of the enzymes were identified; these included ADP-ribose, diadenosine polyphosphates, sugar nucleotides, and deoxynucleoside triphosphates . One of the enzymes was a CDP-choline pyrophosphatase, the first Nudix hydrolase active on this substrate . Furthermore, as a result of this and previous work we have identified a new sub-family of the Nudix hydrolase superfamily recognizable by a specific amino acid motif outside of the Nudix box.

J Bacteriol, 2004 Apr, 186(8), 2413 - 7
Characterization of a major Bacillus anthracis spore coat protein and its role in spore inactivation; Kim HS et al.; A major Bacillus anthracis spore coat protein of 13.4 kDa, designated Cot alpha, was found only in the Bacillus cereus group . A stable ca . 30-kDa dimer of this protein was also present in spore coat extracts . Cot alpha, which is encoded by a monocistronic gene, was first detected late in sporulation, consistent with a sigma(K)-regulated gene . On the basis of immunogold labeling, the protein is in the outer spore coat and absent from the exosporium . In addition, disruption of the gene encoding Cot alpha resulted in spores lacking a dark-staining outer spore coat in thin-section electron micrographs . The mutant spores were stable upon heating or storage, germinated at the same rate as the wild type, and were resistant to lysozyme . They were, however, more sensitive than the wild type to phenol, chloroform, and hypochlorite but more resistant to diethylpyrocarbonate . In all cases, resistance or sensitivity to these reagents was restored by introducing a clone of the cot alpha gene into the mutant . Since Cot alpha is an abundant outer spore coat protein of the B . cereus group with a prominent role in spore resistance and sensitivity, it is a promising target for the inactivation of B . anthracis spores.

Trends Biochem Sci, 2004 Mar, 29(3), 106 - 10
NERD: a DNA processing-related domain present in the anthrax virulence plasmid, pXO1; Grynberg M et al.; We have identified a new domain in a broad range of bacterial, as well as single archaeal and plant proteins . Its presence in the virulence-related pXO1 plasmid of Bacillus anthracis as well as in several other pathogens makes it a possible drug target . We term the new domain nuclease-related domain (NERD) because of its distant similarity to endonucleases.

Proc Natl Acad Sci U S A, 2004 Apr 6, 101(14), 5042 - 7 Epub 2004 Mar 29.
mAbs to Bacillus anthracis capsular antigen for immunoprotection in anthrax and detection of antigenemia; Kozel TR et al.; Bacillus anthracis is surrounded by an antiphagocytic polypeptide capsule composed of poly gamma-D-glutamic acid (gammaDPGA) . gammaDPGA has been identified recently as a potential target for vaccine development . Studies of the role of gammaDPGA in disease have been hampered by the poor Ab response to this antigen and the lack of immunochemical reagents . As a consequence, neither the extent of gammaDPGA production during anthrax nor the protective activity of gammaDPGA Abs in inhalation anthrax are known . Here we report production of IgG Abs to gammaDPGA in mice following an immunization regimen using gammaDPGA in combination with agonist mAbs to CD40 . mAbs were produced that are specific for gammaDPGA . Passive immunization with gammaDPGA mAbs protected >90% of mice in a pulmonary model of anthrax that was lethal in control mice (P < 0.0001) . Use of gammaDPGA mAb in an antigen detection immunoassay found that the appearance of gammaDPGA in serum coincided with the emergence of bacteremia . These studies identify CD40 stimulation as a means for production of Ab and generation of mAbs against a weakly immunogenic antigen and demonstrate that the capsule is an effective target for immunoprotection and for antigen detection in the diagnosis of anthrax.

Biochem Biophys Res Commun, 2004 Apr 23, 317(1), 294 - 300
Western blot analysis of the exotoxin components from Bacillus anthracis separated by isoelectric focusing gel electrophoresis; Little SF; The components of the Bacillus anthracis exotoxins, protective antigen (PA), lethal factor (LF), and edema factor (EF), from 24 isolates were separated by isoelectric focusing gel electrophoresis and detected by Western blot with monoclonal antibodies . Only two isoforms each were observed for PA and EF . Four isoforms were identified for LF . The biological activities of both lethal toxin and edema toxin were measured by using in vitro cell-based assays . This study provides another method of characterizing various isolates of B . anthracis by determining the isoelectric points of the exotoxin components and may be useful in the development of protective vaccines against B . anthracis infection.

J Natl Med Assoc, 2004 Mar, 96(3), 344 - 50
Anthrax in America 2001-2003; Joshi SG et al.; Anthrax caused by Bacillus anthracis in humans is rare . Two recent outbreaks that were intentionally caused occurred among postal employees, politicians, and journalists in the United States . This has caused tremendous fear, and our experience with these "anthrax incidents" has changed our views on the natural history of this disease in people . In this paper, we review the lifecycle and biology of this micro-organism . Anthrax that occurs from a weaponized form of this micro-organism has a specific clinical presentation that requires a suspicion of anthrax exposure to be diagnosed . New methods of testing for anthrax have been developed and may simplify diagnosis in the future . The range of illness caused by B . anthracis from the molecular level to the clinical symptoms is discussed . We also review the diagnostic criteria and differential diagnosis as well as treatment of this condition.

Biosecur Bioterror, 2003, 1(3), 161 - 8
Biotechnology: impact on biological warfare and biodefense; Petro JB et al.; Advances in biological research likely will permit development of a new class of advanced biological warfare (ABW) agents engineered to elicit novel effects . In addition, biotechnology will have applications supporting ABW weaponization, dissemination, and delivery . Such new agents and delivery systems would provide a variety of new use options, expanding the BW paradigm . Although ABW agents will not replace threats posed by traditional biological agents such as Bacillus anthracis (anthrax) and Variola (smallpox), they will necessitate novel approaches to counterproliferation, detection, medical countermeasures, and attribution.

FEMS Immunol Med Microbiol, 2004 Apr 9, 40(3), 231 - 7
Induction of opsonic antibodies to the gamma-D-glutamic acid capsule of Bacillus anthracis by immunization with a synthetic peptide-carrier protein conjugate; Wang TT et al.; The capsule of Bacillus anthracis, a polymer of gamma-D-glutamic acid, functions as a virulence determinant and is a poor immunogen . In this study we show that antibodies reactive with the B . anthracis capsule can be elicited in mice by immunization with a conjugate consisting of a synthetic gamma-D-glutamic acid nonamer peptide (gamma-D-glu9) covalently coupled to keyhole limpet hemocyanin . The serum response to gamma-D-glu9 was comprised primarily of IgG antibodies that recognized an epitope requiring a minimum of four gamma-linked D-glutamic acid residues . Antibodies to (gamma-D-glu9) bound to the surface of encapsulated B . anthracis cells and mediated opsonophagoctosis . These findings suggest that anti-capsular antibodies could mediate the clearance of vegetative B . anthracis cells in vivo . Thus, inclusion of an immunogenic capsular component as well as protective antigen in new anthrax vaccines would generate immune responses targeting both the bacteremic and toxigenic aspects of anthrax infection and thus may increase protective efficacy.

Curr Opin Microbiol, 2004 Feb, 7(1), 19 - 24
The roles of anthrax toxin in pathogenesis; Moayeri M et al.; Anthrax lethal toxin is a multi-functional virulence factor that has evolved to target multiple host functions to allow for optimal establishment of Bacillus anthracis infection . The toxin appears to play a role in all stages of infection, from germination to the induction of vascular collapse leading to host death . Early in infection, at sublethal doses, it acts to suppress immune cell and cytokine responses, thereby promoting bacterial outgrowth . Later in the disease, lethal levels of toxin induce the cytokine-independent shock-like death associated with anthrax . The understanding of the molecular events induced by anthrax toxin in different target cells at each stage of infection will aid in deciphering the pathogenesis of this bacterium and developing therapies.

J Infect Dis, 2004 Apr 1, 189(7), 1313 - 6 Epub 2004 Mar 19.
Time-lapse confocal imaging of development of Bacillus anthracis in macrophages; Ruthel G et al.; Macrophages attempt to battle infection with Bacillus anthracis spores by phagocytosis of the spores . However, it is believed that B . anthracis spores may survive phagocytosis and may actually use the macrophages that ingest them as a means of transport to lymph nodes . Thus far, the events that occur after spores undergo phagocytosis have remained unclear . To elucidate the fate of spores internalized by macrophages, we have used time-lapse confocal microscopy to follow individual fluorescent spores over time . By use of this method, we have determined that some phagocytized spores survive beyond germination, to become bacilli that then replicate within the macrophages.

Nature, 2004 Mar 18, 428(6980), 341 - 5
The protein kinase PKR is required for macrophage apoptosis after activation of Toll-like receptor 4; Hsu LC et al.; Macrophages are pivotal constituents of the innate immune system, vital for recognition and elimination of microbial pathogens . Macrophages use Toll-like receptors (TLRs) to detect pathogen-associated molecular patterns--including bacterial cell wall components, such as lipopolysaccharide or lipoteichoic acid, and viral nucleic acids, such as double-stranded (ds)RNA--and in turn activate effector functions, including anti-apoptotic signalling pathways . Certain pathogens, however, such as Salmonella spp., Shigellae spp . and Yersiniae spp., use specialized virulence factors to overcome these protective responses and induce macrophage apoptosis . We found that the anthrax bacterium, Bacillus anthracis, selectively induces apoptosis of activated macrophages through its lethal toxin, which prevents activation of the anti-apoptotic p38 mitogen-activated protein kinase . We now demonstrate that macrophage apoptosis by three different bacterial pathogens depends on activation of TLR4 . Dissection of anti- and pro-apoptotic signalling events triggered by TLR4 identified the dsRNA responsive protein kinase PKR as a critical mediator of pathogen-induced macrophage apoptosis . The pro-apoptotic actions of PKR are mediated both through inhibition of protein synthesis and activation of interferon response factor 3.

J Bacteriol, 2004 Apr, 186(7), 2195 - 9
Bacillus anthracis and Bacillus cereus PcrA helicases can support DNA unwinding and in vitro rolling-circle replication of plasmid pT181 of Staphylococcus aureus; Anand SP et al.; Replication of rolling-circle replicating (RCR) plasmids in gram-positive bacteria requires the unwinding of initiator protein-nicked plasmid DNA by the PcrA helicase . In this report, we demonstrate that heterologous PcrA helicases from Bacillus anthracis and Bacillus cereus are capable of unwinding Staphylococcus aureus plasmid pT181 from the initiator-generated nick and promoting in vitro replication of the plasmid . These helicases also physically interact with the RepC initiator protein of pT181 . The ability of PcrA helicases to unwind noncognate RCR plasmids may contribute to the broad-host-range replication and dissemination of RCR plasmids in gram-positive bacteria.

BMC Genomics . 2004 Feb 12;5(1):15.
Identification of Bacillus anthracis specific chromosomal sequences by suppressive subtractive hybridization; Dwyer KG et al.; 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.

Biologicals, 2004 Mar, 32(1), 17 - 27
Validation of the anthrax lethal toxin neutralization assay; Hering D et al.; A validation of the performance characteristics of a toxin neutralization assay is presented . This in vitro assay measures the functional ability of antisera, containing antibodies to anthrax lethal toxin, to specifically protect J774A.1 cells against Bacillus anthracis lethal toxin cytotoxicity . This colormetric assay is based upon the reduction of MTT by living cells . Human and rabbit antisera produced against anthrax vaccine absorbed (AVA) were used to validate the assay . Results showed a high level of repeatability and reproducibility, particularly for a bio-assay . Inter-assay variability in absorbance values was the most prominent negative finding however, an acceptable level was demonstrated with a ratio {neutralization ratio (NR)} of the test serum 50% effective dose (ED(50)) to the reference standard ED(50) . Accuracy was maintained, even in samples with minimal neutralizing capacity, and linearity was noted when sample dilutions resulted in accurate prediction of the Y(max)and Y(min) . Specificity tests demonstrated that normal sera did not have an observable effect on the ability of the reference standard to neutralize toxin . The assay remained stable against time, temperature, and freeze/thaw effects on the reference standards, but not on the toxin . The assay also remained stable against media and solution storage effects . Cell passage number and cell plating density were two critical parameters identified during the robustness studies that may be responsible for inter-assay variability in absorbance values . The work was performed in accordance with the FDA's Bioanalytical Method Validation Guidance for Industry and the FDA's Good Laboratory Practice for Nonclinical Laboratory Studies (21 CFR Part 58).

Infez Med, 2003 Jun, 11(2), 108 - 13
{Anthrax and carbuncle: two sides of the same coin}; Mancini R et al.; The disease caused by Bacillus anthracis is one of the most critical concerns to the general public and public health authorities due both to the anthrax cases caused by the intentional release of the germ in the USA at the close of 2001 when letters and packages were contaminated with anthrax spores, and the current threat of biological warfare . After a brief excursus on the history of the terms Anthrax and Carbuncle, we survey the main evidence of anthrax found in the ancient literature, and deal with the identification of the pathogenic agent responsible for the disease and the subsequent discovery of the first anthrax vaccine and its use in order to control the spread of the disease in the cattle . Finally, we examine some of the most important episodes of occupational exposure to the Bacillus anthracis that occurred in the past two centuries and the preventive measures applied both to employees and the workplace.

Biochem Biophys Res Commun, 2004 Apr 2, 316(2), 559 - 64
The osmoprotectants glycine and its methyl derivatives prevent the thermal inactivation of protective antigen of Bacillus anthracis; Singh S et al.; Protective antigen (PA) is the main immunogenic constituent of all vaccines against anthrax . It is known to lose its biological activity even at 37 degrees C . Its thermolabile nature has, thus, remained a cause of concern as even transient exposure of the vaccine to higher temperature could compromise its efficacy . Various types of cosolvent excipients have been used to stabilize a number of proteins with variable success . However, no comprehensive and systematic study to stabilize anthrax PA molecule using this approach has ever been undertaken . We have carried out a systematic study on the effect of osmoprotectants like glycine and its methyl derivatives, sarcosine, dimethylglycine, and betaine, on the thermostability of PA . The thermal stability of PA was found to be highly sensitive to pH with maxima at pH 7.9 . All the cosolvent additives used were able to enhance the thermal stability of PA as inferred from an increase in T(1/2) values, the temperature at which 50% activity was retained during short-term incubation . Glycine was found to be the best stabilizer, while the ability of its methyl derivatives to stabilize PA decreased with an increase in the number of substituted methyl groups suggesting perturbation of hydrophobic interactions . On extended incubation at 40 degrees C the half-life of PA thermal inactivation increased more than four times in the presence of glycine . Thus, glycine could be used as an effective stabilizer to enhance the shelf life of recombinant vaccine against anthrax.

J Biol Chem, 2004 May 14, 279(20), 20563 - 6 Epub 2004 Mar 09.
Anthrax lethal toxin rapidly activates caspase-1/ICE and induces extracellular release of interleukin (IL)-1beta and IL-18; Cordoba-Rodriguez R et al.; Anthrax lethal toxin (LT), a critical virulence factor for Bacillus anthracis, has been demonstrated to cleave and to inactivate mitogen-activated protein kinase kinases (MAPKKs) that propagate prosurvival signals in macrophages (1-5) . Whether this action of anthrax LT leads to the production of proinflammatory cytokines by macrophages has been more controversial (6, 7) . We now report that anthrax LT treatment leads to the specific extracellular release of interleukin (IL)-1beta and IL-18 by the murine macrophage cell lines, RAW264.7 and J774A.1 . Studies of the processing of IL-1beta reveal that the levels of activated/cleaved IL-1beta in RAW264.7 and J774.A1 cells are increased following treatment with anthrax LT . Enhanced processing of IL-1beta directly correlates with increased levels in the activation of its upstream regulator, IL-1beta-converting enzyme/Caspase-1 (ICE) . The extracellular release of IL-1beta and IL-18 in response to anthrax LT is ICE-dependent, as an ICE-specific inhibitor blocks this process . These data indicate that ICE, IL-1beta, and IL-18 are downstream effectors of anthrax LT in macrophages, providing the basis for new bioassays for anthrax LT activity and representing potential therapeutic targets.

J Antimicrob Chemother, 2004 Apr, 53(4), 609 - 15 Epub 2004 Mar 03.
In vitro post-antibiotic effect of fluoroquinolones, macrolides, beta-lactams, tetracyclines, vancomycin, clindamycin, linezolid, chloramphenicol, quinupristin/dalfopristin and rifampicin on Bacillus anthracis; Athamna A et al.; OBJECTIVES: The aim of this study was to investigate in vitro the post-antibiotic effect (PAE) of 19 antibacterial agents against two strains of Bacillus anthracis (ST-1 and Sterne strains) . METHODS: PAE was determined by calculating the time required for the viable counts of antibiotic-exposed bacteria (at concentrations of 10x MIC and exposure for 2 h) at 37 degrees C to increase by 1 log10 above the counts observed immediately after antibiotic removal compared with the corresponding time for controls not exposed to antibiotics . RESULTS: The PAEs of the fluoroquinolones (ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin and garenoxacin) were 2-5 h . The macrolide (erythromycin, clarithromycin and telithromycin) PAEs were 1-4 h, and that of clindamycin was 2 h . The PAEs induced by tetracycline and minocycline were 1-3 h . The PAEs induced by the beta-lactams (penicillin G, amoxicillin and ceftriaxone), vancomycin, linezolid and chloramphenicol were 1-2 h . The PAE induced by rifampicin was 4-5 h . Quinupristin/dalfopristin had the longest PAE, lasting for 7-8 h . CONCLUSIONS: Our results indicate that the PAE is unrelated to the MIC but may be related to the rapidity of bacterial kill . These observations may bear importance on treatment regimens of human anthrax.

Lancet Infect Dis, 2004 Mar, 4(3), 166 - 70
Immune system paralysis by anthrax lethal toxin: the roles of innate and adaptive immunity; Fukao T; Since the deliberate use of anthrax as a bioweapon in the USA in 2001, an enormous amount of attention has been focused on the biology of Bacillus anthracis, the causative bacterium of anthrax . Fatal systemic anthrax involves massive bacteraemia and toxaemia with non-descript early symptoms until the onset of shock and sudden death . The outbreak of fatal symptoms after the incubation period of B anthracis suggests an impairment of the host immune system against this pathogen . Thus, it is likely that B anthracis will possess certain strategies to escape from the host immune system . However, the mechanisms of such immune-evasion strategies are not fully characterised yet . Given the critical role of B anthracis toxins in anthrax pathogenesis, much effort has been made to understand the pathological nature of the toxins . Recent studies have shown the pleiotropic actions of anthrax lethal toxin on host innate immune cells, and that several effects of anthrax lethal toxin may directly account for the mechanism of immune intervention by B anthracis.

Proteomics, 2004 Mar, 4(3), 677 - 91
Identification of chromosomally encoded membranal polypeptides of Bacillus anthracis by a proteomic analysis: prevalence of proteins containing S-layer homology domains; Chitlaru T et al.; Bacillus anthracis is the causative agent of anthrax disease . Improvement of existing anthrax vaccines, which are currently based on the administration of Protective Antigen (the highly immunogenic nontoxic subunit of the bacterial toxin) may entail other bacterial immunogenic elements, part of which are predicted to reside on the surface of bacterial cells . In the present study, membranal proteins extracted from a stationary-phase culture of a nonvirulent B . anthracis strain, devoid of the native virulence plasmids pXO1 and pXO2, were separated by two-dimensional electrophoresis (2-DE) and a characteristic protein map was defined . The proteomic analysis allowed matrix-assisted laser desorption/ionization-time of flight mass spectrometry-assisted identification of 86 protein spots which represent the product of 30 individual open reading frames (ORF) . Among these, a prevalent class of proteins was the S-layer proteins (which were found to represent more than 75% of the B . anthracis membranal fraction) and proteins containing S-layer homology (SLH)-membranal localization domains . Five novel SLH proteins, previously inferred only from bioinformatic ORF analysis (draft genome sequence), were identified and one was shown to be a highly abundant membranal protein . Western blots of the 2-DE gels were probed with sera from convalescent rabbits and guinea pigs infected with virulent B . anthracis (Vollum strain) . This analysis revealed that B . anthracis immune animals exhibit antibodies against at least 14 distinct membranal proteins present in the 2-DE map, establishing that these proteins are expressed in vivo and are able to elicit an immune response . The identification of the protein components of the B . anthracis membranal fraction, as well as the establishment of their potential immunogenicity, underscore the strength of the proteomic approach for identifying molecules which may serve for further analysis of immune and protective abilities.

Jpn J Infect Dis, 2004 Feb, 57(1), 29 - 32
Preparation of a positive control DNA for molecular diagnosis of Bacillus anthracis; Inoue S et al.; Bacillus anthracis is considered to be one of the most potent biological weapons because of its highly pathogenic nature and efficiency of transmission . Routinely, a presumptive diagnosis of anthrax is achieved if the bands with predicted sizes are detected after the PCR targeted to the pag and cap genes residing on pXO1 and pXO2 plasmids, respectively . A positive control DNA prepared from the standard strains of B . anthracis (PAI and PAII) is usually included in the PCR tests . The handling of living B . anthracis, however, requires physical containment . The inclusion of DNA from B . anthracis as a positive control in the PCR test also has a potential risk of cross contamination that may confuse the results . In order to circumvent such problems, we attempted to construct a recombinant plasmid harboring the fragments of the pag and cap genes that could be distinguished from authentic sequences by the presence the restriction-enzyme site, the EcoRV site for the pag gene and the BamHI site for the cap gene, respectively, which were newly introduced . The strategy reported here provides a safe and reproducible positive-control DNA template . It also allows the detection of possible cross contamination, indicating that this strategy would be useful and convenient for the molecular identification of not only B . anthracis but also other highly pathogenic microbes.

Antimicrob Agents Chemother, 2004 Mar, 48(3), 909 - 17
Novel chromosomally encoded multidrug efflux transporter MdeA in Staphylococcus aureus; Huang J et al.; Antibiotic efflux is an important mechanism of resistance in pathogenic bacteria . Here we describe the identification and characterization of a novel chromosomally encoded multidrug resistance efflux protein in Staphylococcus aureus, MdeA (multidrug efflux A) . MdeA was identified from screening an S . aureus open reading frame expression library for resistance to antibiotic compounds . When overexpressed, MdeA confers resistance on S . aureus to a range of quaternary ammonium compounds and antibiotics, but not fluoroquinolones . MdeA is a 52-kDa protein with 14 predicted transmembrane segments . It belongs to the major facilitator superfamily and is most closely related, among known efflux proteins, to LmrB of Bacillus subtilis and EmrB of Escherichia coli . Overexpression of mdeA in S . aureus reduced ethidium bromide uptake and enhanced its efflux, which could be inhibited by reserpine and abolished by an uncoupler . The mdeA promoter was identified by primer extension . Spontaneous mutants selected for increased resistance to an MdeA substrate had undergone mutations in the promoter for mdeA, and their mdeA transcription levels were increased by as much as 15-fold . The mdeA gene was present in the genomes of all six strains of S . aureus examined . Uncharacterized homologs of MdeA were present elsewhere in the S . aureus genome, but their overexpression did not mediate resistance to the antibacterials tested . However, MdeA homologs were identified in other bacteria, including Bacillus anthracis, some of which were shown to be functional orthologs of MdeA.

J Biol Chem, 2004 May 7, 279(19), 19955 - 69 Epub 2004 Feb 23.
Differential inhibition of adenylyl cyclase isoforms and soluble guanylyl cyclase by purine and pyrimidine nucleotides; Gille A et al.; Mammals express nine membranous adenylyl cyclase isoforms (ACs 1-9), a structurally related soluble guanylyl cyclase (sGC) and a soluble AC (sAC) . Moreover, Bacillus anthracis and Bacillus pertussis produce the AC toxins, edema factor (EF), and adenylyl cyclase toxin (ACT), respectively . 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-{gamma-thio}triphosphate is a potent competitive inhibitor of AC in S49 lymphoma cell membranes . These data prompted us to study systematically the effects of 24 nucleotides on AC in S49 and Sf9 insect cell membranes, ACs 1, 2, 5, and 6, expressed in Sf9 membranes and purified catalytic subunits of membranous ACs (C1 of AC5 and C2 of AC2), sAC, sGC, EF, and ACT in the presence of MnCl(2) . N-Methylanthraniloyl (MANT)-GTP inhibited C1.C2 with a K(i) of 4.2 nm . Phe-889 and Ile-940 of C2 mediate hydrophobic interactions with the MANT group . MANT-inosine 5'-{gamma-thio}triphosphate potently inhibited C1.C2 and ACs 1, 5, and 6 but exhibited only low affinity for sGC, EF, ACT, and G-proteins . Inosine 5'-{gamma-thio}triphosphate and uridine 5'-{gamma-thio}triphosphate were mixed G-protein activators and AC inhibitors . AC5 was up to 15-fold more sensitive to inhibitors than AC2 . EF and ACT exhibited unique inhibitor profiles . At sAC, 2',5'-dideoxyadenosine 3'-triphosphate was the most potent compound (IC(50), 690 nm) . Several MANT-adenine and MANT-guanine nucleotides inhibited sGC with K(i) values in the 200-400 nm range . UTP and ATP exhibited similar affinities for sGC as GTP and were mixed sGC substrates and inhibitors . The exchange of MnCl(2) against MgCl(2) reduced inhibitor potencies at ACs and sGC 1.5-250-fold, depending on the nucleotide and cyclase studied . The omission of the NTP-regenerating system from cyclase reactions strongly reduced the potencies of MANT-ADP, indicative for phosphorylation to MANT-ATP by pyruvate kinase . Collectively, AC isoforms and sGC are differentially inhibited by purine and pyrimidine nucleotides.

Infect Immun, 2004 Mar, 72(3), 1291 - 7
Exogenous gamma and alpha/beta interferon rescues human macrophages from cell death induced by Bacillus anthracis; Gold JA et al.; During the recent bioterrorism-related outbreaks, inhalational anthrax had a 45% mortality in spite of appropriate antimicrobial therapy, underscoring the need for better adjuvant therapies . The variable latency between exposure and development of disease suggests an important role for the host's innate immune response . Alveolar macrophages are likely the first immune cells exposed to inhalational anthrax, and the interferon (IFN) response of these cells comprises an important arm of the host innate immune response to intracellular infection with Bacillus anthracis . Furthermore, IFNs have been used as immunoadjuvants for treatment of another intracellular pathogen, Mycobacterium tuberculosis . We established a model of B . anthracis infection with the Sterne strain (34F(2)) which contains lethal toxin (LeTx) . 34F(2) was lethal to murine and human macrophages . Treatment with IFNs significantly improved cell viability and reduced the number of germinated intracellular spores . Infection with 34F(2) failed to induce the latent transcription factors signal transducer and activators of transcription 1 (STAT1) and ISGF-3, which are central to the IFN response . Furthermore, 34F(2) reduced STAT1 activation in response to exogenous alpha/beta IFN, suggesting direct inhibition of IFN signaling . Even though 34F(2) has LeTx, there was no mitogen-activated protein kinase kinase 3 cleavage and p38 was normally induced, suggesting that these early effects of B . anthracis infection in macrophages are independent of LeTx . These data suggest an important role for both IFNs in the control of B . anthracis and the potential benefit of using exogenous IFN as an immunoadjuvant therapy.

J Microbiol Methods, 2004 Mar, 56(3), 383 - 94
gyrB as a phylogenetic discriminator for members of the Bacillus anthracis-cereus-thuringiensis group; La Duc MT et al.; Bacillus anthracis, the causative agent of the human disease anthrax, Bacillus cereus, a food-borne pathogen capable of causing human illness, and Bacillus thuringiensis, a well-characterized insecticidal toxin producer, all cluster together within a very tight clade (B . cereus group) phylogenetically and are indistinguishable from one another via 16S rDNA sequence analysis . As new pathogens are continually emerging, it is imperative to devise a system capable of rapidly and accurately differentiating closely related, yet phenotypically distinct species . Although the gyrB gene has proven useful in discriminating closely related species, its sequence analysis has not yet been validated by DNA:DNA hybridization, the taxonomically accepted "gold standard" . We phylogenetically characterized the gyrB sequences of various species and serotypes encompassed in the "B . cereus group," including lab strains and environmental isolates . Results were compared to those obtained from analyses of phenotypic characteristics, 16S rDNA sequence, DNA:DNA hybridization, and virulence factors . The gyrB gene proved more highly differential than 16S, while, at the same time, as analytical as costly and laborious DNA:DNA hybridization techniques in differentiating species within the B . cereus group.

Anal Chem, 2004 Feb 15, 76(4), 888 - 94
A universal nucleic acid sequence biosensor with nanomolar detection limits; Baeumner AJ et al.; A quantitative universal biosensor was developed on the basis of olignucleotide sandwich hybridization for the rapid (30 min total assay time) and highly sensitive (1 nM) detection of specific nucleic acid sequences . The biosensor consists of a universal membrane and a universal dye-entrapping liposomal nanovesicle . Two oligonucleotides, a reporter and a capture probe that can hybridize specifically with the target nucleic acid sequence, can be coupled to the universal biosensor components within a 10-min incubation period, thus converting it into a specific assay . The liposomal nanovesicles bear a generic oligonucleotide sequence on their outer surface . The reporter probes consist of two parts: the 3' end is complementary to the generic liposomal oligonucleotide, and the 5' end is complementary to the target sequence . Streptavidin is immobilized in the detection zone of the universal membranes . The capture probes are biotinylated at the 5' end and are complementary to another segment in the target sequence . Thus, by incubating the liposomal nanovesicles with the reporter probes, the target sequence, and the capture probes in a hybridization buffer for 20 min, a sandwich complex is formed . The mixture is applied to the membrane, migrates along the strip, and is captured in the detection zone via streptavidin-biotin binding . The biosensor assay was optimized with respect to hybridization conditions, concentrations of all components, and length of the generic probe . It was tested using synthetic DNA sequences and authentic RNA sequences isolated and amplified using nucleic acid sequence-based amplification (NASBA) from Escherichia coli, Bacillus anthracis, and Cryptosporidium parvum . Dose-response curves were carried out using a portable reflectometer for the instantaneous quantification of liposomal nanovesicles in the detection zone . Limits of detection of 1 fmol per assay (1 nM) and dynamic ranges between 1 fmol and at least 750 fmol (1-750 nM) were obtained . The universal biosensors were compared to specific RNA biosensors developed earlier and were found to match or exceed their performance characteristics . In addition, no changes to hybridization conditions were required when switching to the detection of a new target sequence or when using actual nucleic acid sequence-based amplified RNA sequences . Therefore, the universal biosensor described is an excellent tool for use in laboratories or at test sites for rapidly investigating and quantifying any nucleic acid sequence of interest.

Nucleic Acids Res, 2004 Feb 11, 32(3), 977 - 88 Print 2004.
The genome sequence of Bacillus cereus ATCC 10987 reveals metabolic adaptations and a large plasmid related to Bacillus anthracis pXO1; Rasko DA et al.; We sequenced the complete genome of Bacillus cereus ATCC 10987, a non-lethal dairy isolate in the same genetic subgroup as Bacillus anthracis . Comparison of the chromosomes demonstrated that B.cereus ATCC 10987 was more similar to B.anthracis Ames than B.cereus ATCC 14579, while containing a number of unique metabolic capabilities such as urease and xylose utilization and lacking the ability to utilize nitrate and nitrite . Additionally, genetic mechanisms for variation of capsule carbohydrate and flagella surface structures were identified . Bacillus cereus ATCC 10987 contains a single large plasmid (pBc10987), of approximately 208 kb, that is similar in gene content and organization to B.anthracis pXO1 but is lacking the pathogenicity-associated island containing the anthrax lethal and edema toxin complex genes . The chromosomal similarity of B.cereus ATCC 10987 to B.anthracis Ames, as well as the fact that it contains a large pXO1-like plasmid, may make it a possible model for studying B.anthracis plasmid biology and regulatory cross-talk.

Microbiology, 2004 Feb, 150(Pt 2), 355 - 63
Identification of proteins in the exosporium of Bacillus anthracis; Redmond C et al.; Spores of Bacillus anthracis, the causative agent of anthrax, possess an exosporium . As the outer surface layer of these mature spores, the exosporium represents the primary contact surface between the spore and environment/host and is a site of spore antigens . The exosporium was isolated from the endospores of the B . anthracis wild-type Ames strain, from a derivative of the Ames strain cured of plasmid pXO2(-), and from a previously isolated pXO1(-), pXO2(-) doubly cured strain, B . anthracis UM23Cl2 . The protein profiles of SDS-PAGE-separated exosporium extracts were similar for all three . This suggests that avirulent variants lacking either or both plasmids are realistic models for studying the exosporium from spores of B . anthracis . A number of loosely adsorbed proteins were identified from amino acid sequences determined by either nanospray-MS/MS or N-terminal sequencing . Salt and detergent washing of the exosporium fragments removed these and revealed proteins that are likely to represent structural/integral exosporium proteins . Seven proteins were identified in washed exosporium: alanine racemase, inosine hydrolase, ExsF, CotY, ExsY, CotB and a novel protein, named ExsK . CotY, ExsY and CotB are homologues of Bacillus subtilis outer spore coat proteins, but ExsF and ExsK are specific to B . anthracis and other members of the Bacillus cereus group.

Appl Environ Microbiol, 2004 Feb, 70(2), 1068 - 80
Fluorescent amplified fragment length polymorphism analysis of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis isolates; Hill KK et al.; 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 . Department of Agriculture collection . Twenty-four diverse B . anthracis isolates were also included . Phylogenetic analysis of AFLP data revealed extensive diversity within B . thuringiensis and B . cereus compared to the monomorphic nature of B . anthracis . All of the B . anthracis strains were more closely related to each other than to any other Bacillus isolate, while B . cereus and B . thuringiensis strains populated the entire tree . Ten distinct branches were defined, with many branches containing both B . cereus and B . thuringiensis isolates . A single branch contained all the B . anthracis isolates plus an unusual B . thuringiensis isolate that is pathogenic in mice . In contrast, B . thuringiensis subsp . kurstaki (ATCC 33679) and other isolates used to prepare insecticides mapped distal to the B . anthracis isolates . The interspersion of B . cereus and B . thuringiensis isolates within the phylogenetic tree suggests that phenotypic traits used to distinguish between these two species do not reflect the genomic content of the different isolates and that horizontal gene transfer plays an important role in establishing the phenotype of each of these microbes . B . thuringiensis isolates of a particular subspecies tended to cluster together.

Cell Microbiol, 2004 Mar, 6(3), 225 - 33
Systemic cytokine response in murine anthrax; Popov SG et al.; Systemic pro-inflammatory cytokine release has been previously implicated as a major death-causing factor in anthrax, however, direct data have been absent . We determined the levels of IL-1 beta, IL-6 and TNF-alpha in serum of mice challenged with virulent (Ames) or attenuated (Sterne) strains of Bacillus anthracis . More than 10-fold increase in the IL-1beta levels was detected in Ames-challenged Balb/c mice, in contrast to more susceptible C57BL/6 mice, which showed no IL-1beta response . Balb/c mice have also responded with higher levels of IL-6 . The A/J mice demonstrated IL-1beta and IL-6 systemic response to either Ames or Sterne strain of B . anthracis, whereas no increase in TNF-alpha was detected in any murine strain . We used RT-PCR for gene expression analyses in the liver which often is a major source of cytokines and one of the main targets in infectious diseases . A/J mice challenged with B . anthracis (Sterne) showed increased gene expression for Fas, FasL, Bax, IL-1 beta, TNF-alpha, TGF-beta, MIP-1alpha, KC and RANTES . These data favour the hypothesis that apoptotic cell death during anthrax infection causes chemokine-induced transmigration of inflammatory cells to vitally important organs such as liver . Administration of caspase inhibitors z-VAD-fmk and ac-YVAD-cmk improved survival in Sterne-challenged mice indicating a pathogenic role of apoptosis in anthrax.

Mol Microbiol, 2004 Jan, 51(2), 407 - 17
Bacillus anthracis requires siderophore biosynthesis for growth in macrophages and mouse virulence; Cendrowski S et al.; 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 DeltaasbA and DeltabacCEBF strains, but only the DeltaasbA strain was attenuated for growth under these conditions . In addition, the DeltaasbA strain was severely attenuated both for growth in macrophages (MPhi) and for virulence in mice . In contrast, the DeltabacCEBF 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.

J Microbiol Methods, 2004 Feb, 56(2), 253 - 65
A microtiter fluorometric assay to detect the germination of Bacillus anthracis spores and the germination inhibitory effects of antibodies; Welkos SL et al.; Bacillus anthracis spore germination is usually detected in vitro by alterations in spore refractility, heat resistance, and stainability . We developed a more quantitative, sensitive, and semi-automated procedure for detecting germination by using a microtiter kinetic reader for fluorescence spectrophotometry . The procedure was based on the increase in fluorescence of spores with time during their incubation in germination medium containing a fluorescent nucleic acid-binding dye which stained germinated B . anthracis but not ungerminated (UG) spores . Spore germination in the presence of several germinants was characterized . Although L-alanine and inosine alone stimulated rapid germination in this assay, a medium containing optimal concentrations of L-alanine, adenosine, and casamino acids gave low background fluorescence, stimulated germination completely, and at a reasonable rate . Suspensions of heat-activated, UG spores of B . anthracis strain Ames were preincubated with antibodies (Abs) against whole spores to assess their effect on germination . Analyses of the germination data obtained revealed significant differences between spores pretreated with these Abs and those treated with non-immune sera or IgG . Germination inhibitory activity (GIA) was detected for several polyclonal rabbit anti-spore Ab preparations . These included anti-Ames strain spore antisera, IgG purified from the latter, and spore affinity-purified Abs from antisera elicited against four strains of B . anthracis . Abs elicited against UG as well as completely germinated Ames spores inhibited germination . Abs were ranked according to their GIA, and those specific for UG spores usually exhibited greater GIA . Direct binding to spores of these Abs was detected by an ELISA with whole un-germinated Ames spores . Although specific binding to spores by the anti-spore Abs was shown, their titers did not correlate with their GIA levels . Current efforts are focused on identifying the spore antigens recognized by the anti-spore Abs, characterizing the role of these targeted antigens in disease pathogenesis, and evaluating the ability of specific anti-spore Abs to protect against infection with B . anthracis.

FEMS Immunol Med Microbiol, 2004 Jan 15, 40(1), 71 - 4
Treatment of anthrax infection with combination of ciprofloxacin and antibodies to protective antigen of Bacillus anthracis; Karginov VA et al.; Currently there is no effective treatment for inhalational anthrax beyond administration of antibiotics shortly after exposure . There is need for new, safe and effective treatments to supplement traditional antibiotic therapy . Our study was based on the premise that simultaneous inhibition of lethal toxin action with antibodies and blocking of bacterial growth by antibiotics will be beneficial for the treatment of anthrax . In this study, we tested the effects of a combination treatment using purified rabbit or sheep anti-protective antigen (PA) antibodies and the antibiotic ciprofloxacin in a rodent anthrax model . In mice infected with a dose of Bacillus anthracis Sterne strain corresponding to 10 LD(50), antibiotic treatment with ciprofloxacin alone only cured 50% of infected animals . Administration of anti-PA IgG in combination with ciprofloxacin produced 90-100% survival . These data indicate that a combination of antibiotic/immunoglobulin therapy is more effective than antibiotic treatment alone in a rodent anthrax model.

Med Hypotheses, 2004, 62(1), 155 - 7
Anthrax and the etiology of the English sweating sickness; McSweegan E; In 2001, spores of Bacillus anthracis were deliberately sent through the United States postal system, resulting in five deaths from inhalational anthrax . Rarely observed clinical symptoms associated with these cases led to a hypothesis about the etiology of the English Sweating Sickness . The disease appeared sporadically in England between 1485 and 1551 . Numerous viruses have been proposed as possible causes of the "English Sweat" . Anthrax has not previously been considered because, documented cases of inhalational anthrax have been rare and pronounced sweating was not a noted symptom of the more common cutaneous and gastrointestinal forms of anthrax . Victims of the English Sweating Sickness have recently been identified in undisturbed tombs . It may be possible to examine those bodies and coffins for the presence of resilient anthrax spores and DNA using modern genomic tools.

Nat Struct Mol Biol, 2004 Jan, 11(1), 67 - 72 Epub 2003 Dec 29.
Identification of small molecule inhibitors of anthrax lethal factor; Panchal RG et al.; The virulent spore-forming bacterium Bacillus anthracis secretes anthrax toxin composed of protective antigen (PA), lethal factor (LF) and edema factor (EF) . LF is a Zn-dependent metalloprotease that inactivates key signaling molecules, such as mitogen-activated protein kinase kinases (MAPKK), to ultimately cause cell death . We report here the identification of small molecule (nonpeptidic) inhibitors of LF . Using a two-stage screening assay, we determined the LF inhibitory properties of 19 compounds . Here, we describe six inhibitors on the basis of a pharmacophoric relationship determined using X-ray crystallographic data, molecular docking studies and three-dimensional (3D) database mining from the US National Cancer Institute (NCI) chemical repository . Three of these compounds have K(i) values in the 0.5-5 microM range and show competitive inhibition . These molecular scaffolds may be used to develop therapeutically viable inhibitors of LF.

Zh Mikrobiol Epidemiol Immunobiol, 2003 Nov-Dec, (6), 104 - 7
{Bacteriological provision of anti-epidemic measures in the zones of a natural disaster in Southern Russia in 2002}; Evchenko IuM et al.; The bacteriological provision of the prophylactic and anti-epidemic measures, taken under the conditions of the unstable sanitary and epidemiological situation which arose as the result of the emergency situation due to the high flood in the Southern Federal District, was carried out by the efforts and means of microbiological laboratories forming a part of the territorial system of observation and laboratory control . On the whole, more than 20,000 samples of water supplied to the population for drinking and household use, more than 10,000 samples of foodstuffs and cooking raw materials were examined in the Southern Federal District during the period of the liquidation of the medico-sanitary consequences of the emergency situation (June-August) . To study the epidemic potential of the natural foci of quarantine infections and the probability of the spread of Bacillus anthracis from burial grounds for sick animals, the laboratory capacities of plague-control institutions were used.

Zh Mikrobiol Epidemiol Immunobiol, 2003 Nov-Dec, (6), 51 - 6
{Gene typing of Bacillus anthracis strains isolated on the territory of the countries of the Confederation of Independent States}; Tsygankova OI et al.; Thirty eight B . anthracis strains isolated on the territory of the former USSR from different sources at different periods were studied by the method of the multilocus analysis of 6 chromosomal and 2 plasmid regions of B . anthracis genome with a variable number of tandem repeats . The strains belonged to 18 different genotypes; of these, 14 genotypes were described for the first time . The analysis of the genetic relationship of the strains gave grounds to suggest that on this territory both closely related strains and strains whose genotypes were remote from those peculiar to the greater part of other strains could occur . The strains belonging to subgroup A1a of molecular variability were "endemic" for the European part of the former USSR . A modification of the method of gene typing was proposed, which permitted it to be made without the use of an automatic sequencer; this made it possible to greatly widen the circle of laboratories where this method of research could be used.

Zh Mikrobiol Epidemiol Immunobiol, 2003 Nov-Dec, (6), 47 - 51
{Improvement of the method for the indication of the causative agent of anthrax}; Abgarian AG et al.; A combined method for the indication of the causative agent of anthrax (Bacillus anthracis), including the preparation of the material to be tested, the exposure of the magneto-imunosorbent in the sample, cultivation in selective medium, DNA extraction with subsequent testing in the polymerase chain reaction with primers to genes cap, pag and chromosomal sequence Ba813, the registration and interpretation of results, has been developed . All determinations, including the preparation of samples, last not more than 6 hours . The indication of B . anthracis by the proposed method makes it possible not only to confirm its presence in the sample Under test, but also to evaluate its epidemic potential.

J Clin Microbiol, 2004 Jan, 42(1), 179 - 85
Detection of the Bacillus anthracis gyrA gene by using a minor groove binder probe; Hurtle W et al.; Identification of chromosomal markers for rapid detection of Bacillus anthracis is difficult because significant chromosomal homology exists among B . anthracis, Bacillus cereus, and Bacillus thuringiensis . We evaluated the bacterial gyrA gene as a potential chromosomal marker for B . anthracis . A real-time PCR assay was developed for the detection of B . anthracis . After analysis of the unique nucleotide sequence of the B . anthracis gyrA gene, a fluorescent 3' minor groove binding probe was tested with 171 organisms from 29 genera of bacteria, including 102 Bacillus strains . The assay was found to be specific for all 43 strains of B . anthracis tested . In addition, a test panel of 105 samples was analyzed to evaluate the potential diagnostic capability of the assay . The assay showed 100% specificity, demonstrating the usefulness of the gyrA gene as a specific chromosomal marker for B . anthracis.

Clin Diagn Lab Immunol, 2004 Jan, 11(1), 50 - 5
Comparison of a multiplexed fluorescent covalent microsphere immunoassay and an enzyme-linked immunosorbent assay for measurement of human immunoglobulin G antibodies to anthrax toxins; Biagini RE et al.; Recently, the Centers for Disease Control and Prevention reported an accurate, sensitive, specific, reproducible, and quantitative enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum (C . P . Quinn, V . A . Semenova, C . M . Elie et al., Emerg . Infect . Dis . 8:1103-1110, 2002) . The ELISA had a minimum detectable concentration (MDC) of 0.06 microgram/ml, which, when dilution adjusted, yielded a whole-serum MDC of 3.0 micro g of anti-PA IgG per ml . The reliable detection limit (RDL) was 0.09 microgram/ml, while the dynamic range was 0.06 to 1.7 microgram/ml . The diagnostic sensitivity of the assay was 97.6% and the diagnostic specificity was 94.2% for clinically verified cases of anthrax . A competitive inhibition anti-PA IgG ELISA was also developed to enhance the diagnostic specificity to 100% . We report a newly developed fluorescence covalent microbead immunosorbent assay (FCMIA) for B . anthracis PA which was Luminex xMap technology . The FCMIA MDC was 0.006 microgram of anti-PA IgG per ml, the RDL was 0.016 microgram/ml, and the whole-serum equivalent MDC was 1.5 micrograms/ml . The dynamic range was 0.006 to 6.8 microgram/ml . Using this system, we analyzed 20 serum samples for anti-PA IgG and compared our results to those measured by ELISA in a double-masked analysis . The two methods had a high positive correlation (r2 = 0.852; P < 0.001) . The FCMIA appears to have benefits over the ELISA for the measurement of anti-PA IgG, including greater sensitivity and speed, enhanced dynamic range and reagent stability, the use of smaller sample volumes, and the ability to be multiplexed (measurement of more than one analyte simultaneously), as evidenced by the multiplexed measurement in the present report of anti-PA and anti-lethal factor IgG in serum from a confirmed clinical anthrax infection.

Am J Physiol Regul Integr Comp Physiol, 2004 Apr, 286(4), R699 - 709 Epub 2004 Jan 08.
Lethality during continuous anthrax lethal toxin infusion is associated with circulatory shock but not inflammatory cytokine or nitric oxide release in rats; Cui X et al.; Although circulatory shock related to lethal toxin (LeTx) may play a primary role in lethality due to Bacillus anthracis infection, its mechanisms are unclear . We investigated whether LeTx-induced shock is associated with inflammatory cytokine and nitric oxide (NO) release . Sprague-Dawley rats with central venous and arterial catheters received 24-h infusions of LeTx (lethal factor 100 microg/kg; protective antigen 200 microg/kg) that produced death beginning at 9 h and a 7-day mortality rate of 53% . By 9 h, mean arterial blood pressure, heart rate, pH, and base excess were decreased and lactate and hemoglobin levels were increased in LeTx nonsurvivors compared with LeTx survivors and controls (diluent only) (P < or = 0.05 for each comparing the 3 groups) . Despite these changes, arterial oxygen and circulating leukocytes and platelets were not decreased and TNF-alpha, IL-beta, IL-6, and IL-10 levels were not increased comparing either LeTx nonsurvivors or survivors to controls . Nitrate/nitrite levels and tissue histology also did not differ comparing LeTx animals and controls . In additional experiments, although 24-h infusions of LeTx and Escherichia coli LPS produced similar mortality rates (54 and 56%, respectively) and times to death (13.2 +/- 0.8 vs . 11.0 +/- 1.7 h, respectively) compared with controls, only LPS reduced circulating leukocytes, platelets, and IL-2 levels and increased TNF-alpha, IL-1 alpha and -1 beta, IL-6, IL-10, interferon-gamma, granulocyte macrophage-colony stimulating factor, RANTES, migratory inhibitory protein-1 alpha, -2, and -3, and monocyte chemotactic protein-1, as well as nitrate/nitrite levels (all P < or = 0.05 for the effects of LPS) . Thus, in contrast to LPS, excessive inflammatory cytokine and NO release does not appear to contribute to the circulatory shock and lethality occurring with LeTx in this at model . Although therapies to modulate these host mediators may be applicable fo shock caused by LPS or other bacterial toxins, they may not with LeTx.

Appl Environ Microbiol, 2004 Jan, 70(1), 635 - 7
Combined effects of high hydrostatic pressure and temperature for inactivation of Bacillus anthracis spores; Clery-Barraud C et al.; Spores of Bacillus anthracis are known to be extremely resistant to heat treatment, irradiation, desiccation, and disinfectants . To determine inactivation kinetics of spores by high pressure, B . anthracis spores of a Sterne strain-derived mutant deficient in the production of the toxin components (strain RP42) were exposed to pressures ranging from 280 to 500 MPa for 10 min to 6 h, combined with temperatures ranging from 20 to 75 degrees C . The combination of heat and pressure resulted in complete destruction of B . anthracis spores, with a D value (exposure time for 90% inactivation of the spore population) of approximately 4 min after pressurization at 500 MPa and 75 degrees C, compared to 160 min at 500 MPa and 20 degrees C and 348 min at atmospheric pressure (0.1 MPa) and 75 degrees C . The use of high pressure for spore inactivation represents a considerable improvement over other available methods of spore inactivation and could be of interest for antigenic spore preparation.

Anal Chem, 2003 Oct 15, 75(20), 5293 - 9
Autonomous detection of aerosolized Bacillus anthracis and Yersinia pestis; McBride MT et al.; We have developed and tested a fully autonomous pathogen detection system (APDS) capable of continuously monitoring the environment for airborne biological threat agents . The system is designed to provide early warning to civilians in the event of a terrorist attack . The final APDS will be completely automated, offering aerosol sampling, in-line sample preparation fluidics, multiplexed detection and identification immunoassays, and orthogonal, multiplexed PCR (nucleic acid) amplification and detection . The system performance (current capabilities include aerosol collection, multiplexed immunoassays, sample archiving, data reporting, and alarming) was evaluated in a field test conducted in a Biosafety Level 3 facility, where the system was challenged with, and detected, a series of aerosolized releases containing two live, virulent biological threat agents (Bacillus anthracis and Yersinia pestis) . Results presented here represent the first autonomous, simultaneous measurement of these agents.

J Immunol, 2004 Jan 15, 172(2), 747 - 51
Cutting edge: anthrax lethal toxin inhibits activation of IFN-regulatory factor 3 by lipopolysaccharide; Dang O et al.; IFN-regulatory factor 3 (IRF3) is known to participate in the transcriptional induction of chemokines and cytokines, including IFNs, as a result of viral or bacterial infection . In this study, we demonstrate that the LPS-mediated activation of IRF3 and subsequent induction of chemokine genes or IRF3-responsive reporter constructs are inhibited after exposure of human or murine macrophages to the Bacillus anthracis toxin lethal factor . The inhibitory effect is caused by interference with the activation of the stress-activated protein kinase, p38, due to a proteolytic cleavage of mitogen-activated protein kinase kinase 6, and can be overcome by the ectopic expression of a cleavage-resistant mutant of mitogen-activated protein kinase kinase 6 or a constitutively active IRF3 . The lethal factor-mediated inhibition of IRF3 activation and subsequent cytokine production through bacterial membrane components offers Bacillus anthracis an efficient mechanism to evade the innate immune response.

J Bacteriol, 2004 Jan, 186(2), 566 - 9
Surface layer protein EA1 is not a component of Bacillus anthracis spores but is a persistent contaminant in spore preparations; Williams DD et al.; EA1 is an abundant, highly antigenic, surface layer protein of Bacillus anthracis vegetative cells . Recent studies indicate that EA1 is also a component of B . anthracis spores and a potential marker for spore detection . We show here that EA1 is not a spore component but a persistent contaminant in spore preparations.

J Bacteriol, 2004 Jan, 186(2), 307 - 15
atxA controls Bacillus anthracis capsule synthesis via acpA and a newly discovered regulator, acpB; Drysdale M et al.; Two regulatory genes, acpA and atxA, have been reported to control expression of the Bacillus anthracis capsule biosynthesis operon capBCAD . The atxA gene is located on the virulence plasmid pXO1, while pXO2 carries acpA and the cap genes . acpA has been viewed as the major regulator of the cap operon because it is essential for capsule gene expression in a pXO1(-) pXO2(+) strain . atxA is essential for toxin gene transcription but has also been implicated in control of the cap genes . The molecular functions of the regulatory proteins are unknown . We examined cap gene expression in a genetically complete pXO1(+) pXO2(+) strain . Our results indicate that another pXO2 gene, acpB (previously called pXO2-53; accession no . NC002146.1:49418-50866), has a role in cap expression . The predicted amino acid sequence of AcpB is 62% similar to that of AcpA and 50% similar to that of AtxA . Assessment of cap gene transcription revealed that cap expression was not affected in a pXO1(+) pXO2(+) acpB-null mutant and was slightly reduced in an isogenic acpA mutant . However, cap gene expression was abolished in an acpA acpB double mutant . Microscopic examination of capsule synthesis by the mutants corroborated these findings . acpA and acpB expression is controlled by atxA; capsule synthesis and transcription of acpA and acpB were markedly reduced in an atxA mutant . The data suggest that, in a strain containing both virulence plasmids, atxA is the major regulator of capsule synthesis and controls capBCAD expression indirectly, via positive regulation of acpA and acpB.

Biochem Biophys Res Commun, 2004 Jan 16, 313(3), 541 - 5
Evaluation of penicillin-based inhibitors of the class A and B beta-lactamases from Bacillus anthracis; Beharry Z et al.; Bacillus anthracis contains a class A (Bla1) and class B (Bla2) beta-lactamase, which confer resistance to beta-lactam antibiotics when expressed in Escherichia coli . In an effort to find new beta-lactamase inhibitors, several penicillin derivatives have been evaluated including experimental compounds incorporating a 6-mercaptomethyl group or a 6-pyridylmethylidene group, along with clavulanate and tazobactam, as inhibitors against Bla1 and Bla2 . The 6-mercaptomethyl-substituted penicillins showed much greater activity against the zinc-containing Bla2 than Bla1 . The compound that incorporated a 6-pyridylmethylidene substituent and a catecholic substituent at the 2' position was the most effective inhibitor of Bla1 with Ki=0.057 microM . Inhibitors containing iron-chelating functional groups have previously been shown to work in combination with antibiotics to inhibit growth of antibiotic-resistant bacteria expressing beta-lactamase . The development of similar compounds, incorporating these types of substituents, may help overcome resistance to currently used antibiotics.

Biochem Biophys Res Commun, 2004 Jan 16, 313(3), 496 - 502
Tyrosine-728 and glutamic acid-735 are essential for the metalloproteolytic activity of the lethal factor of Bacillus anthracis; Tonello F et al.; The lethal factor (LF) of Bacillus anthracis is a Zn2+-endopeptidase specific for the MAPK-kinase family of proteins . The catalytic zinc atom is coordinated by a first shell of residues including the two histidines and the glutamate of the zinc-binding motif HExxH and by Glu-735 . A characteristic feature of LF is the presence, within the second shell of residues, of a tyrosine (Tyr-728) in close proximity (3.3 A) to the zinc atom . To investigate the role of Tyr-728 and Glu-735, LF mutants with one or both of these two residues replaced by Ala were cloned, expressed, and purified from Escherichia coli . A fourth mutant was obtained by replacing Tyr-728 with Phe . Spectroscopic analysis of these mutants indicates that they fold in the same way as the parental molecule and that zinc stabilizes the structure of LF . These mutants have neither proteolytic activity nor in vivo toxicity . The possible role of Tyr-728 in catalysis is discussed.

Infect Immun, 2004 Jan, 72(1), 430 - 9
Anthrax lethal toxin induces human endothelial cell apoptosis; Kirby JE; Because of its ease of dispersal and high lethality, Bacillus anthracis is one of the most feared biowarfare agents . A better understanding of anthrax pathogenesis is urgently needed to develop new therapies for systemic disease that is relatively unresponsive to antibiotics . Although experimental evidence has implicated a role for macrophages in anthrax pathogenesis, clinical and pathological observations suggest that a direct insult to the host vasculature may also be important . Two bacterial toxins, lethal toxin and edema toxin, are believed to mediate the clinical sequelae of anthrax . Here, I examined whether these toxins are directly toxic to endothelial cells, the cell type that lines the interior of blood vessels . I show for the first time that lethal toxin but not edema toxin reduces the viability of cultured human endothelial cells and induces caspase-dependent endothelial apoptosis . In addition, this toxicity affects both microvascular and large vessel endothelial cells as well as endothelial cells that have differentiated into tubules within a type I collagen extracellular matrix . Finally, lethal toxin induces cleavage of mitogen-activated protein kinase kinases in endothelial cells and inhibits phosphorylation of ERK, p38, and JNK p46 . Based on the contributions of these pathways to endothelial survival, I propose that lethal toxin-mediated cytotoxicity/apoptosis results primarily through inhibition of the ERK pathway . I also hypothesize that the observed endothelial toxicity contributes to vascular pathology and hemorrhage during systemic anthrax.

J Antimicrob Chemother, 2004 Feb, 53(2), 247 - 51 Epub 2003 Dec 19.
In vitro susceptibility of Bacillus anthracis to various antibacterial agents and their time-kill activity; Athamna A et al.; OBJECTIVES: To investigate the in vitro acquisition of resistance to antibiotics by Bacillus anthracis . METHODS: The in vitro activities of 18 antibacterial agents against two strains of B . anthracis, the Sterne strain and the Russian anthrax vaccine strain ST-1, were tested by determining the MICs and by measuring the rates of antibiotic kill at 5x and 10x MIC . RESULTS: The fluoroquinolones ciprofloxacin, ofloxacin, levofloxacin and moxifloxacin, the beta-lactams penicillin G and amoxicillin, the macrolide clarithromycin, the ketolide telithromycin, as well as clindamycin, rifampicin and quinupristin/dalfopristin had MICs in the range of 0.03-0.25 mg/L . Minocycline had an MIC of 0.03 mg/L, as did penicillin, against the ST-1 strain . Ciprofloxacin had an MIC of 0.03 mg/L against both strains . Erythromycin, vancomycin and the oxazolidinone linezolid were less active (MIC 0.5-2.5 mg/L) . Ceftriaxone was the least active, having an MIC of 8.0 mg/L . Chloramphenicol was inactive (MIC > 256 mg/L) . Quinupristin/dalfopristin, rifampicin and moxifloxacin showed the most rapid bacterial killing, achieving a complete eradication of detectable organisms (2 log(10) reduction within 0.5-3 h and 4 log(10) reduction within 0.5-4 h for both strains at concentrations of 5x and 10x the MIC) . The beta-lactams and vancomycin demonstrated a 2-4 log(10) reduction within 5-15 h . Ceftriaxone had a similar effect to penicillin and amoxicillin against the ST-1 strain, but a slower effect than these two beta-lactams against the Sterne strain . The macrolides, tetracyclines and linezolid demonstrated a lower kill rate, while chloramphenicol did not kill at all . CONCLUSIONS: These data expand on the spectrum of agents recommended for the treatment of anthrax (ciprofloxacin, penicillin G and tetracyclines) and add new options, such as other fluoroquinolones, amoxicillin, rifampicin and quinupristin/dalfopristin, as potential therapeutic agents.

J Environ Health, 2003 Dec, 66(5), 19 - 26, 29
Minimizing pathogenic bacteria, including spores, in indoor air; Utrup LJ et al.; Five experiments were conducted to assess whether aerosolized bacteria, including spores, respond like particulate contaminants to the primary (electrical) forces that control the distribution of small particulate contaminants in indoor air . Such response would suggest an approach to minimizing infection in offices, hospitals, nursing homes, and other facilities . It also would have implications for protection against intentionally introduced pathogenic bacteria, including spores . The experiments used two different genera and five different strains of bacteria, including spores . Micrococcus luteus was used as a surrogate for Gram-positive cocci, because M . luteus is similar in size, shape, and cell wall composition to staphylococci, streptococci, and enterococci . Similarly, spore-forming and vegetative Bacillus subtilis were used as surrogates for Gram-positive bacilli such as Bacillus anthracis . The experiments were conducted in a dedicated aerosol physics test facility with culture-based measurements made at timed intervals . The results indicate that the organisms do respond like particulate contaminants to typical electrical forces in a room.

Acta Med Okayama, 2003 Oct, 57(5), 235 - 40
Cutaneous manifestations of anthrax in Eastern Anatolia: a review of 39 cases; Irmak H et al.; Anthrax is essentially a disease of grazing herbivorous animals . The most common form of the disease is cutaneous anthrax, which accounts for 95% of all cases . We report here 39 cutaneous anthrax cases in humans that were seen in Eastern Anatolia over a six-year period . The clinical presentation was malignant edema in 16 of the cases (41%) and malignant pustule in 23 (59%) . A secondary bacterial infection was present in 13 patients (33.3%) in the vicinity of the lesions . The agent was observed using Gram-stained smears in 25 patients (64%), and Bacillus anthracis was isolated from 15 patients (38.5%) . All of the patients were treated with penicillin G or penicillin procaine, except one patient who had a penicillin allergy . One patient with cervical edema (2.5%) died as a result of laryngeal edema and sepsis syndrome . In conclusion, we found that the appearance of the skin lesion of cutaneous anthrax may vary, and this fact, combined with the rarity of this disease, which contributes to a general lack of experience among medical personnel, may make diagnosis difficult in nonagricultural settings

J Bacteriol, 2004 Jan, 186(1), 164 - 78
Formation and composition of the Bacillus anthracis endospore; Liu H et al.; The endospores of Bacillus anthracis are the infectious particles of anthrax . Spores are dormant bacterial morphotypes able to withstand harsh environments for decades, which contributes to their ability to be formulated and dispersed as a biological weapon . We monitored gene expression in B . anthracis during growth and sporulation using full genome DNA microarrays and matched the results against a comprehensive analysis of the mature anthrax spore proteome . A large portion (approximately 36%) of the B . anthracis genome is regulated in a growth phase-dependent manner, and this regulation is marked by five distinct waves of gene expression as cells proceed from exponential growth through sporulation . The identities of more than 750 proteins present in the spore were determined by multidimensional chromatography and tandem mass spectrometry . Comparison of data sets revealed that while the genes responsible for assembly and maturation of the spore are tightly regulated in discrete stages, many of the components ultimately found in the spore are expressed throughout and even before sporulation, suggesting that gene expression during sporulation may be mainly related to the physical construction of the spore, rather than synthesis of eventual spore content . The spore also contains an assortment of specialized, but not obviously related, metabolic and protective proteins . These findings contribute to our understanding of spore formation and function and will be useful in the detection, prevention, and early treatment of anthrax . This study also highlights the complementary nature of genomic and proteomic analyses and the benefits of combining these approaches in a single study.

J Food Prot, 2003 Dec, 66(12), 2349 - 54
Detection and fate of Bacillus anthracis (Sterne) vegetative cells and spores added to bulk tank milk; Perdue ML et al.; A preparation of Bacillus anthracis (Sterne strain) spores was used to evaluate commercially available reagents and portable equipment for detecting anthrax contamination by using real-time PCR and was used to assess the fate of spores added directly to bulk tank milk . The Ruggedized Advanced Pathogen Identification Device (RAPID) was employed to detect spores in raw milk down to a concentration of 2,500 spores per ml . Commercially available primers and probes developed to detect either the protective antigen gene or the lethal factor gene both provided easily read positive signals with the RAPID following extraction from milk with a commercially available DNA extraction kit . Nucleotide sequence analysis of the vrrA gene with the use of DNA extracted from spiked milk provided molecular data that readily identified the spores as B . anthracis with a 100% BLAST match to the Sterne and Ames strains and easily distinguished them from B . cereus . Physical-fate and thermal-stability studies demonstrated that spores and vegetative cells have a strong affinity for the cream fraction of whole milk . A single treatment at standard pasteurization temperatures, while 100% lethal to vegetative cells, had no effect on spore viability even 14 days after the treatment . Twenty-four hours after the first treatment, a second treatment at 72 degrees C for 15 s reduced the viability of the population by ca . 99% but still did not kill all of the spores . From these studies, we conclude that standard pasteurization techniques for milk would have little effect on the viability of B . anthracis spores and that raw or pasteurized milk poses no obstacles to the rapid detection of the spores by molecular techniques.

Vaccine, 2004 Jan 2, 22(3-4), 422 - 30
Defining a serological correlate of protection in rabbits for a recombinant anthrax vaccine; Little SF et al.; In these studies, a serological correlate of protection against anthrax was identified in New Zealand white (NZW) rabbits that had been given one or two injections of various amounts of recombinant protective antigen (rPA) combined with aluminum hydroxide adjuvant (Alhydrogel) . Rabbits were subsequently challenged by the aerosol route with spores of the Ames isolate of Bacillus anthracis . Results suggested that the antibody response, as determined by the quantitative anti-rPA IgG ELISA and toxin neutralizing antibody (TNA) assay, were significant predictors (P<0.0015) of protection against a B . anthracis aerosol spore challenge in rabbits.

J Immunol Methods, 2003 Dec, 283(1-2), 77 - 89
A new epitope tag from hepatitis B virus preS1 for immunodetection, localization and affinity purification of recombinant proteins; Oh MS et al.; Previously, a murine monoclonal antibody (mAb) KR127 (IgG2a/kappa) that binds specifically to the preS1 of hepatitis B virus (HBV) was generated and the fine epitope was mapped to amino acids (aa) 37-45 (NSNNPDWDF) . In this current study, the epitope in combination with KR127 was tested for protein tagging . Initially, to evaluate the importance of each residue of the KR127 epitope in antibody binding, alanine substitution mutants of the epitope were constructed and characterized for KR127 binding by immunoblot analysis and competition ELISA . The results showed that substitution of Ser(38) by alanine (S38A) increased the affinity to KR127 . The mutated epitope (NANNPDWDF), designated S1 tag, was fused to the amino (N)- or carboxyl (C)-terminus of three human recombinant proteins, soluble B lymphocyte stimulator (sBLyS), the N-terminal domain of thrombopoietin (nTPO), and a mitochondrial ribosomal protein (CGI-113) for expression in mammalian cells, while it was fused to the N- or C-terminus of two proteins, a single-chain antibody fragment (ScFv) and the carboxyl-terminal domain (PAc) of the protective antigen of Bacillus anthracis for expression in Escherichia coli . The immunodetection, immunoprecipitation, and affinity purification of the expressed S1-tagged proteins by KR127 were successfully demonstrated . In addition, a KR127 mutant (AP2) with higher affinity, K(d) (0.9 nM), for the S1 tag compared to that (20 nM) of KR127 was obtained by mutational analysis of the heavy chain CDR3 (HCDR3) of KR127 . The AP2 antibody was 4-fold more sensitive in detecting the S1-tagged protein than KR127 . The S1 tag-KR127 or AP2 combination could be universally used for monitoring protein expression, localizing proteins, and protein purification, as well as studying protein interactions.

WMJ, 2003, 102(6), 60 - 4
The State Laboratory of Hygiene's role in terrorism preparedness and response; Hintzman PL; In the fall of 2001, the national public health system found itself responding to acts of terrorism . The intentional release of Bacillus anthracis spores on the East Coast tested the capacity of all state public health laboratories to respond . The impact on the public health system extended to the Wisconsin State Laboratory of Hygiene (WSLH) . Fortunately, participation in the National Laboratory Response Network helped the WSLH meet the challenge of 24 hour/7 days a week coverage, and subsequent federal funding increases have enabled the WSLH to expand its technical capabilities and provide training and outreach to other Wisconsin laboratories to prepare them for their roles in man-made or naturally-occurring public health emergencies.

Risk Anal, 2003 Dec, 23(6), 1135 - 45
Simulation modeling of anthrax spore dispersion in a bioterrorism incident; Reshetin VP et al.; Recent events have increased awareness of the risk posed by terrorist attacks . Bacillus anthracis has resurfaced in the 21st century as a deadly agent of bioterrorism because of its potential for causing massive civilian casualties . This analysis presents the results of a computer simulation of the dispersion of anthrax spores in a typical 50-story, high-rise building after an intentional release during a bioterrorist incident . The model simulates aerosol dispersion in the case of intensive, small-scale convection, which equalizes the concentration of anthrax spores over the building volume . The model can be used to predict the time interval required for spore dispersion throughout a building after a terrorist attack in a high-rise building . The analysis reveals that an aerosol release of even a relatively small volume of anthrax spores during a terrorist incident has the potential to quickly distribute concentrations that are infectious throughout the building.

Hum Gene Ther, 2003 Nov 20, 14(17), 1673 - 82
Protective immunity evoked against anthrax lethal toxin after a single intramuscular administration of an adenovirus-based vaccine encoding humanized protective antigen; Tan Y et al.; Because of the need to develop a vaccine to rapidly protect the civilian population in response to a bioterrorism attack with Bacillus anthracis, we designed AdsechPA, a replication-deficient human serotype 5 adenovirus encoding B . anthracis protective antigen (PA) with codons optimized for expression in mammalian cells . With a single intramuscular administration to mice of 10(9) particle units of AdsechPA, a dose that can be scaled to human use, anti-PA antibodies were evoked more rapidly and at a higher level than with a single administration of the new U.S . military recombinant PA/Alhydrogel vaccine . Importantly, AdsechPA afforded approximately 2.7-fold more protection than the recombinant PA vaccine against B . anthracis lethal toxin challenge 4 weeks after a single vaccination . Even at 11 days postvaccination, AdsechPA provided some survival benefit, whereas the rPA/Alhydrogel vaccine provided none . In the context that equivalent human doses of Ad vectors have already been demonstrated to be safe in humans, a single administration of AdsechPA may provide the means to rapidly protect the civilian population against B . anthracis in response to a bioterrorism attack.

Protein Eng, 2003 Nov, 16(11), 853 - 60
A broad-spectrum peptide inhibitor of beta-lactamase identified using phage display and peptide arrays; Huang W et al.; Hydrolysis of beta-lactam antibiotics by beta-lactamase enzymes is the most common mechanism of bacterial resistance to these agents . Several small-molecule, mechanism-based inhibitors of beta-lactamases such as clavulanic acid are clinically available although resistance to these inhibitors has been increasing in bacterial populations . In addition, these inhibitors act only on class A beta-lactamases . Here we utilized phage display to identify peptides that bind to the class A beta-lactamase, TEM-1 . The binding affinity of one of these peptides was further optimized by the synthesis of peptide arrays using SPOT synthesis technology . After two rounds of optimization, a linear 6-mer peptide with the sequence RRGHYY was obtained . A soluble version of this peptide was synthesized and found to inhibit TEM-1 beta-lactamase with a K(i) of 136 micro M . Surprisingly, the peptide inhibits the class A Bacillus anthracis Bla1 beta-lactamase with a K(i) of 42 micro M and the class C beta-lactamase, P99, with a K(i) of 140 micro M, despite the fact that it was not optimized to bind these enzymes . This peptide may be a useful starting point for the design of non-beta-lactam, broad-spectrum peptidomimetic inhibitors of beta-lactamases.

Nucleic Acids Res, 2003 Dec 1, 31(23), 6891 - 903
Computational identification of the Spo0A-phosphate regulon that is essential for the cellular differentiation and development in Gram-positive spore-forming bacteria; Liu J et al.; Spo0A-phosphate is essential for the initiation of cellular differentiation and developmental processes in Gram-positive spore-forming bacteria . Here we combined comparative genomics with analyses of microarray expression profiles to identify the Spo0A-phosphate regulon in Bacillus subtilis . The consensus Spo0A-phosphate DNA-binding motif identified from the training set based on different computational algorithms is an 8 bp sequence, TTGTCGAA . The same motif was identified by aligning the upstream regulatory sequences of spo0A-dependent genes obtained from the expression profile of Sad67 (a constitutively active form of Spo0A) and their orthologs . After the transcription units (TUs) having putative Spo0A-phosphate binding sites were obtained, conservation of regulons among the genomes of B.subtilis, Bacillus halodurans and Bacillus anthracis, and expression profiles were employed to identify the most confident predictions . Besides genes already known to be directly under the control of Spo0A-phosphate, 276 novel members (organized in 109 TUs) of the Spo0A-phosphate regulon in B.subtilis are predicted in this study . The sensitivity and specificity of our predictions are estimated based on known sites and combinations of different types of evidence . Further characterization of the novel candidates will provide information towards understanding the role of Spo0A-phosphate in the sporulation process, as well as the entire genetic network governing cellular differentiation and developmental processes in B.subtilis.

Clin Microbiol Infect, 2003 Oct, 9(10), 1051 - 6
Application of Bacillus anthracis PCR to simulated clinical samples; Rantakokko-Jalava K et al.; We evaluated PCR for the detection of Bacillus anthracis DNA from simulated clinical specimens relevant for the microbiological diagnosis of anthrax or exposure to B . anthracis spores . In simulated blood specimens, the lowest limit of detection was 400 CFU per mL of blood, which may be sufficient for samples from patients with septic anthrax . Screening nasal swabs by PCR may not be sensitive enough to rule out dangerous exposure to anthrax spores, as a minimum of 2000 spores per sample was required for detectable amplification . As spores survived some standard DNA purification methods, special attention should be paid to laboratory safety when preparing samples possibly containing live spores.

Clin Microbiol Infect, 2003 Sep, 9(9), 984 - 6
Antibiotic susceptibility of isolates of Bacillus anthracis, a bacterial pathogen with the potential to be used in biowarfare; Jones ME et al.; Bacillus anthracis is a bacterial species that could be used in a bioterrorist attack . We tested a collection of isolates with a range of relevant antimicrobial compounds . All isolates tested were susceptible to ciprofloxacin and doxycycline . Penicillin and amoxicillin, with or without clavulanate, showed in vitro activity against all B . anthracis isolates . Ceftriaxone demonstrated lower-level in vitro activity compared to penicillin-related compounds against B . anthracis . In vitro data from this study are in keeping with available guidelines.

Arch Intern Med, 2003 Nov 10, 163(20), 2527 - 31
Gastrointestinal anthrax: review of the literature; Beatty ME et al.; Recent events have drawn attention to cases of inhalational and cutaneous anthrax associated with contaminated mail . Gastrointestinal anthrax, the disease caused by ingestion of Bacillus anthracis organisms, has rarely been reported in the United States . This review provides background information on the gastrointestinal form of the disease . We describe the clinical course of gastrointestinal anthrax, outline current therapy, review the microbiology of B anthracis, examine the epidemiology of natural outbreaks, discuss considerations regarding deliberate contamination, and summarize existing literature on the inactivation of spores present in food and water.

Biochem J, 2004 Feb 15, 378(Pt 1), 93 - 103
Acylation of SC4 dodecapeptide increases bactericidal potency against Gram-positive bacteria, including drug-resistant strains; Lockwood NA et al.; We have conjugated dodecyl and octadecyl fatty acids to the N-terminus of SC4, a potently bactericidal, helix-forming peptide 12-mer (KLFKRHLKWKII), and examined the bactericidal activities of the resultant SC4 'peptide-amphiphile' molecules . SC4 peptide-amphiphiles showed up to a 30-fold increase in bactericidal activity against Gram-positive strains (Staphylococcus aureus, Streptococcus pyogenes and Bacillus anthracis), including S . aureus strains resistant to conventional antibiotics, but little or no increase in bactericidal activity against Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) . Fatty acid conjugation improved endotoxin (lipopolysaccharide) neutralization by 3- to 6-fold . Although acylation somewhat increased lysis of human erythrocytes, it did not increase lysis of endothelial cells, and the haemolytic effects occurred at concentrations 10- to 100-fold higher than those required for bacterial cell lysis . For insight into the mechanism of action of SC4 peptide-amphiphiles, CD, NMR and fluorescence spectroscopy studies were performed in micelle and liposome models of eukaryotic and bacterial cell membranes . CD indicated that SC4 peptide-amphiphiles had the strongest helical tendencies in liposomes mimicking bacterial membranes, and strong membrane integration of the SC4 peptide-amphiphiles was observed using tryptophan fluorescence spectroscopy under these conditions; results that correlated with the increased bactericidal activities of SC4 peptide-amphiphiles . NMR structural analysis in micelles demonstrated that the two-thirds of the peptide closest to the fatty acid tail exhibited a helical conformation, with the positively-charged side of the amphipathic helix interacting more with the model membrane surface . These results indicate that conjugation of a fatty acid chain to the SC4 peptide enhances membrane interactions, stabilizes helical structure in the membrane-bound state and increases bactericidal potency.

J Microbiol Methods, 2003 Dec, 55(3), 709 - 16
Identification of foodborne bacteria by infrared spectroscopy using cellular fatty acid methyl esters; Whittaker P et al.; Identification of bacterial species by profiling fatty acid methyl esters (FAMEs) has commonly been carried out by using a 20-min capillary gas chromatographic procedure followed by library matching of FAME profiles using commercial MIDI databases and proprietary pattern recognition software . Fast GC (5 min) FAME procedures and mass spectrometric methodologies that require no lipid separation have also been reported . In this study, bacterial identification based on the rapid (2 min) infrared measurement of FAME mixtures was demonstrated . The microorganisms investigated included Gram positive bacteria Staphylococcus aureus, Listeria monocytogenes, Bacillus anthracis, and Bacillus cereus, and Gram negative bacteria from the family Enterobacteriacae: Yersinia enterocolitica, Salmonella typhimurium, Shigella sonnei, and Escherichia coli (four strains of E . coli), and non-Enterobacteriacae: Vibrio cholerae, Vibrio vulnificus, and Vibrio parahemolyticus . Foodborne bacterial mixtures of FAMEs were measured by using an attenuated total reflection (ATR)-Fourier transform infrared (FTIR) spectroscopic procedure and discriminated by multivariate analysis . Results showed that the Enterobacteriacae could be discriminated from the vibrios . The identification was at the level of species (for the Bacillus and Vibrio genera) or strains (for the E . coli species) . A series of bacterial FAME test samples were prepared and analyzed for accuracy of identification, and all were correctly identified . Our results suggest that this infrared strategy could be used to identify foodborne pathogens.

Microbiol Immunol, 2003, 47(10), 693 - 9
Sensitive and rapid quantitative detection of anthrax spores isolated from soil samples by real-time PCR; Ryu C et al.; Quantitative analysis of anthrax spores from environmental samples is essential for accurate detection and risk assessment since Bacillus anthracis spores have been shown to be one of the most effective biological weapons . Using TaqMan real-time PCR, specific primers and probes were designed for the identification of pathogenic B . anthracis strains from pag gene and cap gene on two plasmids, pXO1 and pXO2, as well as a sap gene encoded on the S-layer . To select the appropriate lysis method of anthrax spore from environmental samples, several heat treatments and germination methods were evaluated with multiplex-PCR . Among them, heat treatment of samples suspended with sucrose plus non-ionic detergent was considered an effective spore disruption method because it detected up to 10(5) spores/g soil by multiplex-PCR . Serial dilutions of B . anthracis DNA and spore were detected up to a level of 0.1 ng/ microliters and 10 spores/ml, respectively, at the correlation coefficient of 0.99 by real-time PCR . Quantitative analysis of anthrax spore could be obtained from the comparison between C(T) value and serial dilutions of soil sample at the correlation coefficient of 0.99 . Additionally, spores added to soil samples were detected up to 10(4) spores/g soil within 3 hr by real-time PCR . As a consequence, we established a rapid and accurate detection system for environmental anthrax spores using real-time PCR, avoiding time and labor-consuming preparation steps such as enrichment culturing and DNA preparation.

Appl Environ Microbiol, 2003 Nov, 69(11), 6888 - 98
Sequencing and characterization of pBM400 from Bacillus megaterium QM B1551; Scholle MD et al.; Bacillus megaterium QM B1551 plasmid pBM400, one of seven indigenous plasmids, has been labeled with a selectable marker, isolated, completely sequenced, and partially characterized . A sequence of 53,903 bp was generated, revealing a total of 50 predicted open reading frames (ORFs); 33 were carried on one strand and 17 were carried on the other . These ORFs comprised 57% of the pBM400 sequence . Besides the replicon region and a complete rRNA operon that have previously been described, several interesting genes were found, including genes for predicted proteins for cell division (FtsZ and FtsK), DNA-RNA interaction (FtsK, Int/Rec, and reverse transcriptase), germination (CwlJ), styrene degradation (StyA), and heavy metal resistance (Cu-Cd export and ATPase) . Three of the ORF products had high similarities to proteins from the Bacillus anthracis virulence plasmid pXO1 . An insertion element with similarity to the IS256 family and several hypothetical proteins similar to those from the chromosomes of other Bacillus and Lactococcus species were present . This study provides a basis for isolation and sequencing of other high-molecular-weight plasmids from QM B1551 and for understanding the role of megaplasmids in gram-positive bacteria . The genes carried by pBM400 suggest a possible role of this plasmid in the survival of B . megaterium in hostile environments with heavy metals or styrene and also suggest that there has been an exchange of genes within the gram-positive bacteria, including pathogens.

Physiol Genomics, 2003 Dec 16, 16(1), 19 - 23
Identification of genomic islands in the genome of Bacillus cereus by comparative analysis with Bacillus anthracis; Zhang R et al.; Horizontal gene transfer has been recognized as a universal event throughout bacterial evolution . The availability of both complete genome sequences of Bacillus cereus and B . anthracis provides the possibility to perform comparative analysis based on their genomes . By using a windowless method to display the distribution of the genomic GC content of B . cereus and B . anthracis, we have found three genomic islands in the genome of B . cereus, i.e., BCGI-1, BCGI-2, and BCGI-3, respectively, which are absent in the genome of B . anthracis . All the genomic islands have abrupt changes in GC content compared with that of surrounding regions . BCGI-1 has many conserved features of genomic islands, e.g., a Val-tRNA gene is utilized as the integration site, and a site-specific recombinase gene is located at the 3' end . BCGI-2 has a large percentage of phage protein, suggesting a phage-related recombination is involved . BCGI-3 contains a ferric anguibactin transport system, which is likely to be involved in the iron transport that enables the bacterium to overcome the iron limitation in the host . In addition, BCGI-3 also contains a cluster of genes related to lantibiotics, which may play a role during the evolution of the genome . Furthermore, the integrations of the genomic islands, BCGI-1 and BCGI-3, result in deletions of DNA sequence fragments; therefore, such integrations lead to both gene gain and gene loss simultaneously.

J Nutr Sci Vitaminol (Tokyo), 2003 Aug, 49(4), 297 - 9
Bacteriocidal activity of garlic powder against Bacillus anthracis; Sasaki J et al.; The antibacterial activity of garlic powder was examined against Bacillus anthracis using agar plate cultivation and test tube methods . On the agar plate test, 1-5% garlic powder inhibited the growth of B . anthracis and Escherichia coli O157 used as references . A 1% water solution of garlic powder in the test tube method killed B . anthracis at 10(7) cfu/mL within 3 h of treatment at room temperature . A number of intestinal bacteria in a BALB/c mouse decreased after the oral administration of 1 mL of 1%, garlic powder solution once a day for 3 d . These results suggest that the oral administration of garlic powder is effective against pathogenic bacteria invasion into the intestine as an infection.

J Bacteriol, 2003 Nov, 185(22), 6633 - 9
Purification and characterization of the PcrA helicase of Bacillus anthracis; Naqvi A et al.; PcrA is an essential helicase in gram-positive bacteria, and a gene encoding this helicase has been identified in all such organisms whose genomes have been sequenced so far . The precise role of PcrA that makes it essential for cell growth is not known; however, PcrA does not appear to be necessary for chromosome replication . The pcrA gene was identified in the genome of Bacillus anthracis on the basis of its sequence homology to the corresponding genes of Bacillus subtilis and Staphylococcus aureus, with which it shares 76 and 72% similarity, respectively . The pcrA gene of B . anthracis was isolated by PCR amplification and cloning into Escherichia coli . The PcrA protein was overexpressed with a His6 fusion at its amino-terminal end . The purified His-PcrA protein showed ATPase activity that was stimulated in the presence of single-stranded (ss) DNA (ssDNA) . Interestingly, PcrA showed robust 3'-->5' as well as 5'-->3' helicase activities, with substrates containing a duplex region and a 3' or 5' ss poly(dT) tail . PcrA also efficiently unwound oligonucleotides containing a duplex region and a 5' or 3' ss tail with the potential to form a secondary structure . DNA binding experiments showed that PcrA bound much more efficiently to oligonucleotides containing a duplex region and a 5' or 3' ss tail with a potential to form a secondary structure than to those with ssDNAs or duplex DNAs with ss poly(dT) tails . Our results suggest that specialized DNA structures and/or sequences represent natural substrates of PcrA in biochemical processes that are essential for the growth and survival of gram-positive organisms, including B . anthracis.

Anal Chem, 2003 Nov 1, 75(21), 5825 - 34
Electrogenerated chemiluminescence . 72 . Determination of immobilized DNA and C-reactive protein on Au(111) electrodes using tris(2,2'-bipyridyl)ruthenium(II) labels; Miao W et al.; Anodic electrogenerated chemiluminescence (ECL) with tri-n-propylamine (TPrA) as a coreactant was used to determine DNA and C-reactive protein (CRP) by immobilizations on Au(111) electrodes using tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)(3)(2+)) labels . A 23-mer synthetic single-stranded (ss) DNA derived from the Bacillus anthracis with an amino-modified group at the 5' end position was covalently attached to the Au(111) substrate precoated with a self-assembled thiol monolayer of 3-mercaptopropanoic acid (3-MPA) in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) and then hybridized with a target ssDNA tagged with Ru(bpy)(3)(2+) ECL labels . Similarly, biotinylated anti-CRP species were immobilized effectively onto the Au(111) substrate precovered with a layer of avidin linked covalently via the reaction between avidin and a mixed thiol monolayer of 3-MPA and 16-mercaptohexadecanoic acid on Au(111) in the presence of EDAC and N-hydroxysuccinimide . CRP and anti-CRP tagged with Ru(bpy)(3)(2+) labels were then conjugated to the surface layer . ECL responses were generated from the modified electrodes described above by immersing them in a TPrA-containing electrolyte solution . A series of electrode treatments, including blocking free -COOH groups with ethanol amine, pinhole blocking with bovine serum albumin, washing with EDTA/NaCl/Tris buffer, and spraying with inert gases, were used to reduce the nonspecific adsorption of the labeled species . The ECL peak intensity was linearly proportional to the analyte CRP concentration over the range 1-24 microg/mL . CRP concentrations of two unknown human plasma/serum specimens were measured by the standard addition method based on this technique.

Med Sci Monit, 2003 Nov, 9(11), RA276 - 83
Anthrax--an overview; Oncu S et al.; Anthrax, a disease of mammals (including humans), is caused by a spore-forming Gram-positive bacilli called Bacillus anthracis . Anthrax is one of the oldest threats to humanity, and remains endemic in animals in many parts of the world . The incidence of anthrax has decreased in developed countries, but it remains a considerable health problem in developing countries . The disease is transmitted to humans by contact with sick animals or their products, such as wool, skin, meat etc . Capsular polypeptide and anthrax toxin are the principal virulence factors of B . anthracis . Anthrax toxin consists of three proteins called protective antigen, edema factor, and lethal factor, each of which is nontoxic but acts synergistically . Human anthrax has three major clinical forms: cutaneous, inhalational, and gastrointestinal . The diagnosis is easily established in cutaneous cases, characterized by black eschar . Severe intoxication and collapse during the course of bronchopneumonia or hemorrhagic enteritis should prompt suspicion of anthrax . Treatment with antibiotics is mandatory . If untreated, anthrax in all forms can lead to septicemia and death . Recently, considerable attention has been focused on the potential for B . anthracis to be used in acts of biological terrorism . The ease of laboratory production and its dissemination via aerosol led to its adoption by terrorists, as shown by recent events in the USA . A good knowledge of anthrax, its epidemiology, pathogenesis, clinical forms and potential as a biological weapon is essential for timely prevention and treatment . This review summarizes the current knowledge on anthrax.

J Emerg Med, 2003 Oct, 25(3), 271 - 6
Lymphocytic vasculitis associated with the anthrax vaccine: case report and review of anthrax vaccination; Muniz AE; Anthrax is caused by the spore-forming bacteria Bacillus anthracis . It occurs naturally, but recently has been manufactured as a biological warfare agent . This makes prophylaxis for anthrax an urgent concern and efforts are ongoing for the production of an efficient and safe vaccine . Side effects to the current anthrax vaccine are usually minor and mainly consist of local skin reactions . Occasionally an unusual complication may occur; a case of a patient with lymphocytic vasculitis temporally associated with the anthrax vaccine is reported.

Am J Pathol, 2003 Nov, 163(5), 1901 - 10
The critical role of pathology in the investigation of bioterrorism-related cutaneous anthrax; Shieh WJ et al.; Cutaneous anthrax is a rare zoonotic disease in the United States . The clinical diagnosis traditionally has been established by conventional microbiological methods, such as culture and gram staining . However, these methods often yield negative results when patients have received antibiotics . During the bioterrorism event of 2001, we applied two novel immunohistochemical assays that can detect Bacillus anthracis antigens in skin biopsy samples even after prolonged antibiotic treatment . These assays provided a highly sensitive and specific method for the diagnosis of cutaneous anthrax, and were critical in the early and rapid diagnosis of 8 of 11 cases of cutaneous anthrax during the outbreak investigation . Skin biopsies were obtained from 10 of these 11 cases, and histopathological findings included various degrees of ulceration, hemorrhage, edema, coagulative necrosis, perivascular inflammation, and vasculitis . Serology was also an important investigation tool, but the results required several weeks because of the need to test paired serum specimens . Other tests, including culture, special stains, and polymerase chain reaction assay, were less valuable in the diagnosis and epidemiological investigation of these cutaneous anthrax cases . This report underscores the critical role of pathology in investigating potential bioterrorism events and in guiding epidemiological studies, a role that was clearly demonstrated in 2001 when B . anthracis spores were intentionally released through the United States postal system.

Am J Pathol, 2003 Nov, 163(5), 1735 - 41
Susceptibility to anthrax lethal toxin is controlled by three linked quantitative trait loci; McAllister RD et al.; Anthrax lethal toxin (LT) is the principal virulence factor associated with lethal pathologies following infection with Bacillus anthracis . Macrophages are the primary effector cells mediating lethality since macrophage-depleted mice are resistant to LT challenge . Recently, Ltxs1, the gene controlling differential susceptibility of murine macrophages to cytolysis following in vitro exposure to LT, was identified as Kif1c . To directly assess the in vivo role of Kif1c alleles in mortality, we studied a panel of interval-specific recombinant congenic lines carrying various segments of central chromosome 11 derived from LT-resistant DBA/2 mice on the LT-susceptible BALB/c background . The results of this study reveal that mortality is controlled by three linked quantitative trait loci (QTL): Ltxs1/Kif1c (42-43 cM), Ltxs2 (35-37 cM), and Ltxs3 (45-47 cM) . The Ltxs3 interval encompasses Nos2, which is an attractive candidate gene for Ltxs3 . In this regard, we demonstrate that selective, pharmacologically based inhibition of Nos2 activity in vivo partially overrides genetic resistance to LT and that Nos2 expression as determined by reverse transcription-polymerase chain reaction differs significantly between DBA/2 and BALB/c macrophages . Additionally, to recapitulate dominant resistance to mortality as seen in (BALB/c x DBA/2) F(1) hybrids, DBA/2 alleles are required at all three QTL.

Indian J Med Res, 2003 Mar, 117, 111 - 8
Comparative evaluation of protective antigen produced from Bacillus anthracis & Escherichia coli; Sastry KS et al.; BACKGROUND & OBJECTIVE: Anthrax has been reported from almost every country and India is endemic for this disease . There is considerable under reporting of the disease because of lack of microbiological facilities and diagnostic reagents . In India only conventional methods which have limitations, are being used to diagnose the disease . Hence the aim of this study was to isolate and purify protective antigen (PA) using different protocols and to use this PA for detection of anti-PA antibodies from sera samples . METHODS: Protective antigen was isolated and purified from the Sterne strain of Bacillus anthracis . B . anthracis lacking pXO1 and pXO2 transformed with pYS5 (B . anthracis pYS5) and recombinant Escherichia coli transformed with pQE30 containing PA gene using hydroxyapatite (HA), Q-sepharose fast protein liquid chromatography (FPLC) and nickel-nitrilotriacetic acid (Ni-NTA) chromatographic methods, respectively . A mixture of PA and edema factor (EF) was injected subcutaneously into rabbits to test the biological activity of PA . The immunogenicity of PA was tested by inoculating the protein into rabbits along with adjuvant . Using this PA, 20 bovine sera samples (pre- and post-vaccinated) were tested by Western blotting (WB) for the presence of anti-PA antibodies . RESULTS: The 83 kDa PA protein was obtained from all the bacteria with the yields of 13, 50 and 9.0 mg/l from Sterne B . anthracis, B . anthracis pYS5 and recombinant Esch . coli, respectively . Formation of edematous ulcers at the site of PA+EF injection clearly confirmed the retention of biological activity of the proteins . Of the 10 post-vaccination sera tested, 9 showed clear positive by WB whereas none of the pre-vaccination sera showed the reaction . INTERPRETATION & CONCLUSION: The purified PA preparations obtained in the present study may possibly be utilized for detection of anti-PA antibodies in the sera of anthrax patients for timely diagnosis of the disease and, might also be tested for their efficacy and use as human anthrax vaccine.

Infect Immun, 2003 Nov, 71(11), 6591 - 606
Phosphatidylcholine-specific phospholipase C and sphingomyelinase activities in bacteria of the Bacillus cereus group; Pomerantsev AP et al.; Bacillus anthracis is nonhemolytic, even though it is closely related to the highly hemolytic Bacillus cereus . Hemolysis by B . cereus results largely from the action of phosphatidylcholine-specific phospholipase C (PC-PLC) and sphingomyelinase (SPH), encoded by the plc and sph genes, respectively . In B . cereus, these genes are organized in an operon regulated by the global regulator PlcR . B . anthracis contains a highly similar cereolysin operon, but it is transcriptionally silent because the B . anthracis PlcR is truncated at the C terminus . Here we report the cloning, expression, purification, and enzymatic characterization of PC-PLC and SPH from B . cereus and B . anthracis . We also investigated the effects of expressing PlcR on the expression of plc and sph . In B . cereus, PlcR was found to be a positive regulator of plc but a negative regulator of sph . Replacement of the B . cereus plcR gene by its truncated orthologue from B . anthracis eliminated the activities of both PC-PLC and SPH, whereas introduction into B . anthracis of the B . cereus plcR gene with its own promoter did not activate cereolysin expression . Hemolytic activity was detected in B . anthracis strains containing the B . cereus plcR gene on a multicopy plasmid under control of the strong B . anthracis protective antigen gene promoter or in a strain carrying a multicopy plasmid containing the entire B . cereus plc-sph operon . Slight hemolysis and PC-PLC activation were found when PlcR-producing B . anthracis strains were grown under anaerobic-plus-CO(2) or especially under aerobic-plus-CO(2) conditions . Unmodified parental B . anthracis strains did not demonstrate obvious hemolysis under the same conditions.

J Biol Chem, 2004 Jan 2, 279(1), 436 - 43 Epub 2003 Oct 21.
IsdG and IsdI, heme-degrading enzymes in the cytoplasm of Staphylococcus aureus; Skaar EP et al.; Staphylococcus aureus requires iron for growth and utilizes heme as a source of iron during infection . Staphylococcal surface proteins capture hemoglobin, release heme from hemoglobin and transport this compound across the cell wall envelope and plasma membrane into the bacterial cytoplasm . Here we show that Staphylococcus aureus isdG and isdI encode cytoplasmic proteins with heme binding properties . IsdG and IsdI cleave the tetrapyrrol ring structure of heme in the presence of NADPH cytochrome P450 reductase, thereby releasing iron . Further, IsdI complements the heme utilization deficiency of a Corynebacterium ulcerans heme oxygenase mutant, demonstrating in vivo activity of this enzyme . Although Staphylococcus epidermidis, Listeria monocytogenes, and Bacillus anthracis encode homologues of IsdG and IsdI, these proteins are not found in other bacteria or mammals . Thus, it appears that bacterial pathogens evolved different strategies to retrieve iron from scavenged heme molecules and that staphylococcal IsdG and IsdI represent examples of bacterial heme-oxygenases.

Anal Chem, 2003 Jul 15, 75(14), 3446 - 50
A reusable flow-through polymerase chain reaction instrument for the continuous monitoring of infectious biological agents; Belgrader P et al.; Continuous monitoring of the environment for infectious diseases and related biowarfare agents requires the implementation of practical cost-effective methodologies that are highly sensitive and specific . One compatible method employed in clinical diagnostics is real-time polymerase chain reaction (PCR) analysis . The utility of this technique for environmental monitoring is limited, however, by the utilization of single-use consumables in commercial PCR instruments . This greatly increases mechanical complexity, because sophisticated robotic mechanisms must replenish the disposable elements . An alternative strategy develops an autonomous monitoring system consisting of reusable modules that readily interface with fluidic circuitry in a flow-through scheme . The reduced complexity should increase reliability while decreasing operating costs . In this report, we describe a reusable, flow-through PCR module that functions as one component in such a system . This module was rigorously evaluated with Bacillus anthracis genomic DNA and demonstrated high repeatability, sensitivity, and efficiency, with no evidence of sample-to-sample carryover.

Arch Hist Filoz Med, 2003, 66(2), 161 - 8
{First research work by Robert Koch on etiology of anthrax-in cooperation with Józef Knechtel, Polish apothecary}; Bednarski Z et al.; Terroristic attack on United States of America 11 September 2001 and just after many cases of anthrax spores employment as biological warfare called our attention to Robert Koch . He determined anthrax etiology and enclosed it in his first research work: Die Aetiologie der Milzbrand-Kranheit begrundet auf die Entwicklungsgeschichte des Bacillus Anthracis . The results of this research are widely described . In the scientific researches participated J . Knechtel, Pole, pharmacist, pharmacy owner in Wolsztyn . His adjacent laboratory near pharmacy was provided with microscope, camera, table and two chairs . Many slides and above mentioned article / without J . Knechtel as joint author/were the results of this findings . About cooperation Pole with R . Koch we found out from two letters doctor Brinkmann' s authorship and three reports explored by A . Skrobacki in Central Register Office in Merseburg . The objects mentioned above were delivered by J . Knechtel's widow as the gift to Institute of Infectious Diseases in Berlin in 1905 . Robert Koch' s cooperation with a Polish pharmacist was concealed . It was caused by a historic background and the policy of Prussia - an invader state in relation to Polish people . The official demonstration of cooperation with a Polish pharmacist under these circumstances could not take place.

J Bacteriol, 2003 Nov, 185(21), 6255 - 61
Morphogenesis of bacillus spore surfaces; Chada VG et al.; Spores produced by bacilli are encased in a proteinaceous multilayered coat and, in some species (including Bacillus anthracis), further surrounded by a glycoprotein-containing exosporium . To characterize bacillus spore surface morphology and to identify proteins that direct formation of coat surface features, we used atomic-force microscopy (AFM) to image the surfaces of wild-type and mutant spores of Bacillus subtilis, as well as the spore surfaces of Bacillus cereus 569 and the Sterne strain of Bacillus anthracis . This analysis revealed that the coat surfaces in these strains are populated by a series of bumps ranging between 7 and 40 nm in diameter, depending on the species . Furthermore, a series of ridges encircled the spore, most of which were oriented along the long axis of the spore . The structures of these ridges differ sufficiently between species to permit species-specific identification . We propose that ridges are formed early in spore formation, when the spore volume likely decreases, and that when the spore swells during germination the ridges unfold . AFM analysis of a set of B . subtilis coat protein gene mutants revealed three coat proteins with roles in coat surface morphology: CotA, CotB, and CotE . Our data indicate novel roles for CotA and CotB in ridge pattern formation . Taken together, these results are consistent with the view that the coat is not inert . Rather, the coat is a dynamic structure that accommodates changes in spore volume.

J Biol Chem, 2003 Dec 26, 278(52), 52425 - 31 Epub 2003 Oct 15.
Differential processing of CD4 T-cell epitopes from the protective antigen of Bacillus anthracis; Musson JA et al.; We have mapped CD4+ T-cell epitopes located in three domains of the recombinant protective antigen of Bacillus anthracis . Mouse T-cell hybridomas specific for these epitopes were generated to study the mechanisms of proteolytic processing of recombinant protective antigen for antigen presentation by bone marrow-derived macrophages . Overall, epitopes differed considerably in their processing requirements . In particular, the kinetics of presentation, ranging from 15 (fast) to 120 min (slow), suggested sequential liberation of epitopes during proteolytic processing of the intact PA molecule . Pretreatment of macrophages with ammonium chloride or inhibitors of the major enzyme families showed that T-cell responses to an epitope presented with fast kinetics were unaffected by raising endosomal pH or inhibiting cysteine or aspartic proteinases, suggesting presentation independent of lysosomal processing . In contrast, responses to epitopes presented with slower kinetics were dependent on low pH and the activity of cysteine or aspartic proteinases indicating a requirement for lysosomal processing . In addition, responses to all epitopes, whether their presentation was dependent on low pH or not, were prevented by treatment of macrophages with broad spectrum serine proteinase inhibitors . Thus, our data are consistent with a model of sequential antigen processing within the endosomal system, beginning with a pre-processing step mediated by serine or metalloproteinases prior to further processing by lysosomal enzymes . Rapidly presented epitopes seemed to require only limited proteolysis at earlier stages of endocytosis, whereas the majority of epitopes required more extensive processing by neutral proteinases followed by lysosomal enzymes.

Drug Discov Today, 2003 Oct 1, 8(19), 881 - 8
Prevention and treatment of bacterial diseases caused by bacterial bioterrorism threat agents; Greenfield RA et al.; There is general consensus that the bacterial agents or products most likely to be used as weapons of mass destruction are Bacillus anthracis, Yersinia pestis, Francisella tularensis and the neurotoxin of Clostridium botulinum . Modern supportive and antimicrobial therapy for inhalational anthrax is associated with a 45% mortality rate, reinforcing the need for better adjunctive therapy and prevention strategies . Pneumonic plague is highly contagious, difficult to recognize and is frequently fatal . Therefore, the development of vaccines against this agent is crucial . Although tularemia is associated with low mortality, the highly infectious nature of aerosolized F . tularensis poses a substantive threat that is best met by vaccine development . Safer antitoxins and a vaccine are required to meet the threat of the use of botulinum toxin as a weapon of mass destruction . In this article, the current status of research in these areas is reviewed.

J Infect Dis, 2003 Oct 15, 188(8), 1138 - 41 Epub 2003 Sep 30.
Anthrax toxin induces hemolysis: an indirect effect through polymorphonuclear cells; Wu AG et al.; Anthrax toxin can induce hemolysis in the presence of polymorphonuclear cells (PMNs), an activity primarily mediated by protective antigen, with synergic effects provided by lethal factor and edema factor . Lethal factor and edema factor, individually or in combination, are incapable of lysing red blood cells . The requirement for the presence of PMNs indicates that hemolysis associated with Bacillus anthracis infection is indirect rather than direct, as observed in many other bacterial infections.

J Clin Microbiol, 2003 Oct, 41(10), 4758 - 66
Use of denaturing high-performance liquid chromatography to identify Bacillus anthracis by analysis of the 16S-23S rRNA interspacer region and gyrA gene; Hurtle W et al.; Denaturing high-performance liquid chromatography (DHPLC) was evaluated as a method for identifying Bacillus anthracis by analyzing two chromosomal targets, the 16S-23S intergenic spacer region (ISR) and the gyrA gene . The 16S-23S ISR was analyzed by this method with 42 strains of B . anthracis, 36 strains of Bacillus cereus, and 12 strains of Bacillus thuringiensis; the gyrA gene was analyzed by this method with 33 strains of B . anthracis, 27 strains of B . cereus, and 9 strains of B . thuringiensis . Two blind panels of 45 samples each were analyzed to evaluate the potential diagnostic capability of this method . Our results show that DHPLC is an efficient method for the identification of B . anthracis.

Appl Environ Microbiol, 2003 Oct, 69(10), 6288 - 93
Species-specific peptide ligands for the detection of Bacillus anthracis spores; Williams DD et al.; Currently available detectors for spores of Bacillus anthracis, the causative agent of anthrax, are inadequate for frontline use and general monitoring . There is a critical need for simple, rugged, and inexpensive detectors capable of accurate and direct identification of B . anthracis spores . Necessary components in such detectors are stable ligands that bind tightly and specifically to target spores . By screening a phage display peptide library, we identified a family of peptides, with the consensus sequence TYPXPXR, that bind selectively to B . anthracis spores . We extended this work by identifying a peptide variant, ATYPLPIR, with enhanced ability to bind to B . anthracis spores and an additional peptide, SLLPGLP, that preferentially binds to spores of species phylogenetically similar to, but distinct from, B . anthracis . These two peptides were used in tandem in simple assays to rapidly and unambiguously identify B . anthracis spores . We envision that these peptides can be used as sensors in economical and portable B . anthracis spore detectors that are essentially free of false-positive signals due to other environmental Bacillus spores.

MMWR Morb Mortal Wkly Rep, 2003 Oct 3, 52(39), 937 - 8
Follow-up of deaths among U.S . Postal Service workers potentially exposed to Bacillus anthracis--District of Columbia, 2001-2002; Centers for Disease Control and Prevention (CDC); In October 2001, two letters contaminated with Bacillus anthracis spores were processed by mechanical and manual methods at the U.S . Postal Service (USPS) Brentwood Mail Processing and Distribution Center in the District of Columbia . Four postal workers at the Brentwood facility became ill with what was diagnosed eventually as inhalational anthrax; two died . The facility was closed on October 21, and postexposure prophylaxis was recommended for approximately 2,500 workers and business visitors . Subsequent reports of deaths of facility workers prompted concern about whether mortality was unusually high among workers, perhaps related to the anthrax attacks . To evaluate the rates and causes of death among workers at the Brentwood facility during October 12, 2001-October 11, 2002, CDC, in collaboration with state and local health departments, analyzed death certificate data . In addition, these data were compared with aggregate mortality data from the five USPS facilities contaminated with B . anthracis during the fall 2001 anthrax attacks . This report summarizes the results of that analysis, which indicate that rates and causes of death among Brentwood workers during the 12 months after the anthrax attacks of 2001 were not different from rates and causes of deaths that occurred during the preceding 5 years.

Proc Natl Acad Sci U S A, 2003 Oct 14, 100(21), 12426 - 31 Epub 2003 Sep 30.
Toxin-induced resistance in Bacillus anthracis lethal toxin-treated macrophages; Salles II et al.; In the current study, we show that macrophages adaptively resist anthrax lethal toxin (LT) through a toxin-activated process termed toxin-induced resistance (TIR) . TIR was triggered by pretreatment of RAW 264.7 or J774A.1 macrophages with a low dose of LT for at least 6 h, which resulted in resistance to high doses of LT for 96 h . Activation of TIR required functional toxin, because LT subunits, mutants, and heat-inactivated toxin were unable to trigger resistance . TIR macrophages were not altered in toxin receptor levels or cell cycle profiles . Treatment of TIR macrophages with high doses of LT resulted in a sustained decline in full-length mitogen-activated protein kinase kinase 2, a known target of lethal factor, and a marked reduction in diphosphorylated extracellular response kinases 1,2 for 24 h . However, despite the sustained loss of full-length mitogen-activated protein kinase kinase 2, by 48 h, TIR macrophages regained diphosphorylated extracellular response kinases 1,2, suggesting an adaptation led to recovery of this signaling pathway . TIR macrophages were also able to maintain normal levels of ubiquitinylated proteins, whereas sensitive cells show a rapid reduction in ubiquitin-modified proteins before cell death, indicating a possible alteration in proteasome activity contributed to resistance . These results provide a paradigm for toxin-cell interactions and suggest macrophages are capable of adapting to and tolerating toxic doses of LT.

Expert Rev Mol Diagn, 2003 Sep, 3(5), 605 - 16
Molecular diagnostic techniques for use in response to bioterrorism; Firmani MA et al.; The use of micro-organisms as agents of biological warfare is considered inevitable for several reasons, including ease of production and dispersion, delayed onset of symptoms, ability to cause high rates of morbidity and mortality and difficulty in diagnosis . Therefore, the clinical presentation and pathogenesis of the organisms posing the highest threat (variola major, Bacillus anthracis, Yersinia pestis, Clostridium botulinum toxin, Francisella tularensis, filoviruses, arenaviruses and Brucella species), as well as the available diagnostic techniques and treatments for such infections, will be reviewed in this article . Due to the necessity of rapid identification and diagnosis, molecular techniques have been the ongoing focus of current research . Consequently, the molecular diagnostic techniques that have recently been developed for the diseases associated with these agents will be emphasized.

J Biol Chem, 2003 Dec 5, 278(49), 49342 - 7 Epub 2003 Sep 24.
Binding of anthrax toxin to its receptor is similar to alpha integrin-ligand interactions; Bradley KA et al.; The secreted protein toxin produced by Bacillus anthracis contributes to virulence of this pathogen and can cause many of the symptoms seen during an anthrax infection, including shock and sudden death . The cell-binding component of anthrax toxin, protective antigen, mediates entry of the toxin into cells by first binding directly to the extracellular integrin-like inserted (I) domain of the cellular anthrax toxin receptor, ATR . Here we report that this interaction requires an intact metal ion-dependent adhesion site (MIDAS) in the receptor as well as the presence of specific divalent cations . Also, we demonstrate that the toxin-receptor interaction is critically dependent on the Asp-683 carboxylate group of protective antigen, which projects from the receptor binding surface . We propose that this carboxylate group completes the coordination of the MIDAS metal of ATR, mimicking integrin-ligand interactions.

Semin Respir Infect, 2003 Sep, 18(3), 134 - 45
When bioterrorism strikes: diagnosis and management of inhalational anthrax; Shafazand S; In October and November, 2001, reports of patients with inhalational anthrax reacquainted the public with this ancient disease and introduced the harsh reality of a bioterrorist act . Bacillus anthracis, a rod-shaped, spore-forming bacterium, primarily infects herbivores . Humans traditionally have acquired the disease from occupational or agricultural exposure to infected animals and animal products . Recent events saw the intentional release of anthrax spores, using the U.S . postal system as an unlikely and unwilling agent . Cutaneous disease, pulmonary disease, and gastrointestinal anthrax are the known clinical manifestations of anthrax . Inhalational anthrax has the highest mortality and is the main focus of this report.

Salud Publica Mex, 2003 Jul-Aug, 45(4), 298 - 309
{Confronting bioterrorism: Epidemiologic, clinical, and preventive aspects of smallpox}; Franco-Paredes C et al.; The worldwide eradication of smallpox, a major achievement in public health, is currently threatened by the risk of bioterrorism . The debate on the destruction of the Variola virus in the two reference laboratories of the World Health Organization has dramatically switched to the preservation of the remaining virus after the September 2001 terrorist events in the U.S . along with the intentional release of Bacillus anthracis in the U.S . The risk of intentional release of Variola virus constitutes a minimal, yet possible risk . A smallpox epidemic could have a devastating impact due to its elevated morbidity and mortality that would inflict in non-immune human population, in addition to the ensuing panic and social unrest . Therefore, the development of national preparedness and response plans along with the availability of smallpox vaccine to be used in the post-exposure phase represent a fundamental part of the preventive efforts to cope with bioterrorism . Reestablishing a preventive vaccination program was recently recommended by the Advisory Committee on Immunization Practices (ACIP) . However, the vaccine currently available has historically been associated with serious adverse reactions, even death . Thus, this recommendation has not been universally accepted . To counter an epidemic of smallpox, medical personnel in the frontline need to be prepared with updated smallpox information to identify, diagnose, isolate, and treat cases if a bioterrorist attack should occur . Herein we present an indepth review for health care personnel with relevant epidemiologic, clinical, and preventive information on smallpox.

J Appl Microbiol, 2003, 95(4), 728 - 33
Rapid and effective detection of anthrax spores in soil by PCR; Cheun HI et al.; AIMS: To detect Bacillus anthracis DNA from soil using rapid and simple procedures . METHODS AND RESULTS: Various amounts of B . anthracis Pasteur II spores were added artificially to 1 g of soil, which was then washed with ethanol and sterile water . Enrichment of the samples in trypticase soy broth was performed twice . A DNA template was prepared from the second enrichment culture using a FastPrep instrument . The template was then used for nested and real-time polymerase chain reaction (PCR) with B . anthracis-specific primers, to confirm the presence of B . anthracis chromosomal DNA and the pXO1/pXO2 plasmids . CONCLUSIONS: One cell of B . anthracis in 1 g of soil could be detected by nested and real-time PCR . The usefulness of the PCR method using field samples was also confirmed . SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that this could be a useful method for detecting anthrax-spore contaminated soil with high sensitivity . Its application could have great impact on the progress of epidemiological surveillance.

Diagn Microbiol Infect Dis, 2003 Sep, 47(1), 313 - 20
Identifying and subtyping species of dangerous pathogens by automated ribotyping; Grif K et al.; An investigation of dangerous bacterial pathogens was conducted to determine the usefulness of automated rRNA operon ribotyping (RiboPrinter system) to identify species . A total of 26 isolates comprising Bacillus anthracis, Brucella spp., Burkholderia mallei, Francisella tularensis, and Yersinia pestis were tested using restriction endonucleases EcoRI, PstI, PvuII and AseI . The main problem was that the system's database-relying on EcoRI as restriction enzyme-does not contain the essential dangerous pathogens . B . anthracis was misidentified as B . cereus and Y . pestis as Y . pseudotuberculosis . Two isolates of F . tularensis ssp . holarctica were falsely identified as Vibrio cholerae . This study underscores that riboprint patterns generated with a single restriction enzyme are not always unique for each of the species tested . Using more than one enzyme, the RiboPrinter proved to be a valuable primary typing method . Databases of commercially available systems for the identification of bacteria should include the most important dangerous pathogens.

Emerg Infect Dis, 2003 Aug, 9(8), 1013 - 5
Nonhemolytic, nonmotile gram-positive rods indicative of Bacillus anthracis; Dib EG et al.; We report a 40-year-old female patient who was admitted to the hospital because of a left ovarian mass torsion . A nonhemolytic, nonmotile Bacillus, suspicious of Bacillus anthracis, was isolated from a blood culture . We discuss the evaluation that led to the final identification of the bacterium as B . megaterium.

Emerg Infect Dis, 2003 Aug, 9(8), 909 - 14
Detecting bioterror attacks by screening blood donors: a best-case analysis; Kaplan EH; To assess whether screening blood donors could provide early warning of a bioterror attack, we combined stochastic models of blood donation and the workings of blood tests with an epidemic model to derive the probability distribution of the time to detect an attack under assumptions favorable to blood donor screening . Comparing the attack detection delay to the incubation times of the most feared bioterror agents shows that even under such optimistic conditions, victims of a bioterror attack would likely exhibit symptoms before the attack was detected through blood donor screening . For example, an attack infecting 100 persons with a noncontagious agent such as Bacillus anthracis would only have a 26% chance of being detected within 25 days; yet, at an assumed additional charge of $10 per test, donor screening would cost $139 million per year . Furthermore, even if screening tests were 99.99% specific, 1,390 false-positive results would occur each year . Therefore, screening blood donors for bioterror agents should not be used to detect a bioterror attack.

Anal Biochem, 2003 Oct 1, 321(1), 125 - 30
An enzymatic electrochemiluminescence assay for the lethal factor of anthrax; Rivera VR et al.; The lethal factor (LF) of anthrax toxin is the toxic component of the exotoxin (lethal toxin) secreted by toxic strains of Bacillus anthracis . The lethal factor is a zinc-dependent metalloprotease that specifically cleaves the mitogen-activated protein kinase kinase (MAPKK) family of enzymes . We took advantage of this substrate specificity to develop an electrochemiluminescence (ECL) peptide cleavage assay . The ECL assay uses the stable ruthenium (Ru) metal chelate that, in the presence of tripropylamine, generates a light reaction triggered by the application of an electric potential . The Ru label is specifically incorporated into the C-terminal CYS residue of a synthetic peptide (23mer) containing the MAPKK2 cleavage sequence of LF . Streptavidin-coated paramagnetic beads were the solid phase and facilitated separation and characterization of the enzymatic reaction products based upon N-terminal biotinylation of the peptide substrate . Intact peptide bound via the biotin moiety generated high signal due to the Ru label, whereas binding of the cleaved peptide fragment devoid of Ru label reduced the ECL signal . The proposed assay provides a novel opportunity for the screening of potential therapeutics against anthrax.

J Biomol Struct Dyn, 2003 Oct, 21(2), 159 - 70
MD simulations of anthrax edema factor: calmodulin complexes with mutations in the edema factor "switch a" region and docking of 3'-deoxy ATP into the adenylyl cyclase active site of wild-type and mutant edema factor variants; Zhao J et al.; Bacillus anthracis, a spore-forming infectious bacterium, produces an exotoxin, called the edema factor (EF), that functions in part by disrupting internal signalling pathways . When complexed with human host cell calmodulin (CaM), EF becomes an active adenylyl cyclase, producing the internal signal substance cyclic-AMP in an uncontrolled fashion . Recently, the crystal structures for uncomplexed EF and EF:CaM complexes in the presence and absence of a substrate analog (3'-deoxy-ATP), were reported . EF mutational studies have implicated a number of residues important in CaM binding and/or in the generation of the adenylyl cyclase active site, formed by the movements of the EF switch A, B and C regions upon CaM binding . Here we report on the results of molecular dynamics (MD) simulations on two EF:CaM complexes, one containing wild-type EF and the other containing EF in which a cluster of residues in the switch A region (L523, K525, Q526 and V529) have been mutated to alanine . The switch A mutations cause a large increase in the flexibility of the switch C region, the rupture of a number of EF-CaM interactions, an expansion of the carboxyl-terminal domain of CaM, and a change in the Ca(2+) ion binding abilities of the CaM that is in complex with EF . The results indicate the importance of the mutated switch A residues in maintaining a compact EF:CaM complex that appears to be a prerequisite for the generation of a fully-functional adenylyl cyclase active site . The effects of mutating key residues (K346, K353, H577, E588, D590 and N639) in the active site region of EF (to alanine) on the ability of EF to bind the 3'-deoxy-ATP substrate analog were also examined . Active-site residue substitutions at positions 583 (N583A) and 577 (H577A) were found to be particularly disruptive for the placement of the adenine ring moiety into the position found in the x-ray crystal structure of the ligand-protein complex.

Microbiol Immunol, 2003, 47(7), 491 - 7
Morphology of the type strain of Bacillus anthracis EY 3169T=ATCC 14578T grown either aerobically or anaerobically on agar plates--observation by light and laser microscopes; Yabuuchi E et al.; Growth characteristics including cell-arrangement of the type strain of Bacillus anthracis EY 3169T=ATCC 14578T grown on agar plates in level 3 laboratory were observed by both light and laser microscopes . Small daughter colonies appeared on parent colonies grown on 5% sheep blood or chocolate agar plates after 12 days incubation at room temperature . Daughter colonies, stained by Wirtz-Conklin method, were composed with vegetative cells and spores . Growth of daughter colonies might be supported by the debris of cells in the parent colony . Colonies grown under anaerobic conditions were flat with smooth edges, and the cells neither formed chains of any length, nor produced any spores after 25 days incubation at room temperature . It was thought that spores of B . anthracis were produced at the terminal stage of individual cell life instead of under unfavorable conditions for the organism . Air is needed for spore formation and cell-chain formation . More nutrients, probably amino acids, are needed for anaerobic growth rather than aerobic.

J Clin Invest, 2003 Sep, 112(5), 670 - 82
Bacillus anthracis lethal toxin induces TNF-alpha-independent hypoxia-mediated toxicity in mice; Moayeri M et al.; Bacillus anthracis lethal toxin (LT) is the major virulence factor of anthrax and reproduces most of the laboratory manifestations of the disease in animals . We studied LT toxicity in BALB/cJ and C57BL/6J mice . BALB/cJ mice became terminally ill earlier and with higher frequency than C57BL/6J mice . Timed histopathological analysis identified bone marrow, spleen, and liver as major affected organs in both mouse strains . LT induced extensive hypoxia . Crisis was due to extensive liver necrosis accompanied by pleural edema . There was no evidence of disseminated intravascular coagulation or renal dysfunction . Instead, analyses revealed hepatic dysfunction, hypoalbuminemia, and vascular/oxygenation insufficiency . Of 50 cytokines analyzed, BALB/cJ mice showed rapid but transitory increases in specific factors including KC, MCP-1/JE, IL-6, MIP-2, G-CSF, GM-CSF, eotaxin, FasL, and IL-1beta . No changes in TNF-alpha occurred . The C57BL/6J mice did not mount a similar cytokine response . These factors were not induced in vitro by LT treatment of toxin-sensitive macrophages . The evidence presented shows that LT kills mice through a TNF-alpha-independent, FasL-independent, noninflammatory mechanism that involves hypoxic tissue injury but does not require macrophage sensitivity to toxin.

J Clin Invest, 2003 Sep, 112(5), 656 - 8
The host response to anthrax lethal toxin: unexpected observations; Prince AS; Bacillus anthracis, the causative agent of anthrax, is believed to induce disease and death in humans in an endotoxic shock-like manner . A comprehensive study of the effects of anthrax toxin in mice demonstrates that toxin-induced death is mediated not by cytokine release, as previously thought, but by hypoxia-induced liver failure . The study strongly suggests that the therapies developed for treatment of cytokine-mediated septic shock will not be appropriate for the treatment of anthrax.

Vaccine, 2003 Sep 8, 21(25-26), 4071 - 80
Comparison of individual and combination DNA vaccines for B . anthracis, Ebola virus, Marburg virus and Venezuelan equine encephalitis virus; Riemenschneider J et al.; Multiagent DNA vaccines for highly pathogenic organisms offer an attractive approach for preventing naturally occurring or deliberately introduced diseases . Few animal studies have compared the feasibility of combining unrelated gene vaccines . Here, we demonstrate that DNA vaccines to four dissimilar pathogens that are known biowarfare agents, Bacillus anthracis, Ebola (EBOV), Marburg (MARV), and Venezuelan equine encephalitis virus (VEEV), can elicit protective immunity in relevant animal models . In addition, a combination of all four vaccines is shown to be equally as effective as the individual vaccines for eliciting immune responses in a single animal species . These results demonstrate for the first time the potential of combined DNA vaccines for these agents and point to a possible method of rapid development of multiagent vaccines for disparate pathogens such as those that might be encountered in a biological attack.

Lab Invest, 2003 Aug, 83(8), 1201 - 9
Pathology of inhalation anthrax in cynomolgus monkeys (Macaca fascicularis); Vasconcelos D et al.; Anthrax is considered a serious biowarfare and bioterrorism threat because of its high lethality, especially by the inhalation route . Rhesus macaques (Macaca mulatta) are the most commonly used nonhuman primate model of human inhalation anthrax exposure . The nonavailability of rhesus macaques necessitated development of an alternate model for vaccine testing and immunologic studies . This report describes the median lethal dose (LD(50)) and pathology of inhalation anthrax in cynomolgus macaques (Macaca fascicularis) . Gross and microscopic tissue changes were reviewed in 14 cynomolgus monkeys that died or were killed after aerosol exposure of spores of Bacillus anthracis (Ames strain) . The LD(50) and 95% confidence intervals were 61800 (34000 to 110000) colony-forming units . The most common gross lesions were mild splenomegaly, lymph node enlargement, and hemorrhages in various organs, particularly involving the meninges and the lungs . Mediastinitis, manifested as hemorrhage or edema, affected 29% of the monkeys . Microscopically, lymphocytolysis occurred in the intrathoracic lymph nodes and spleens of all animals, and was particularly severe in the spleen and in germinal centers of lymph nodes . Hemorrhages were common in lungs, bronchial lymph nodes, meninges, gastrointestinal tract, and mediastinum . These results demonstrate that the Ames strain of B . anthracis is lethal by the inhalation route in the cynomolgus macaque . The LD(50) of the Ames strain of B . anthracis was within the expected experimental range of previously reported values in the rhesus monkey in an aerosol challenge . The gross and microscopic pathology of inhalation anthrax in the cynomolgus monkey is remarkably similar to that reported in rhesus monkeys and humans . The results of this study are important for the establishment of an alternative nonhuman primate model for evaluation of medical countermeasures against inhalational anthrax.

Di Yi Jun Yi Da Xue Xue Bao, 2003 Aug, 23(8), 816 - 9
{Preparation of gene chip probes for Bacillus anthracis protective antigen}; Ma XD et al.; OBJECTIVE: To study the method for rapid preparation of the gene chip probes for Bacillus anthracis protective antigen (pag) . METHODS: According to the sequences of pag published on PubMed, a pair of primers was designed to amplify the PA gene of Bacillus anthracis . Restriction enzyme digestion and AT clone were employed to rapidly screen out the recons of PA segments for the preparation of the gene chip probes . The DNA sequences of these segments were determined with 377 DNA Sequencer, and the resultant sequences analyzed with Bioinformatic software . RESULTS: With the designed primers, the pag 2,205 bp in length was amplified . After enzyme digestion and AT clone, 7 fragments of varied lengths were screened, which underwent analysis with the basic local alignment sequence tool (BLAST) and 377 DNA Sequencer . BLAST analysis showed that all the fragments cloned belonged to Bacillus anthracis . CONCLUSION: PCR in conjunction with restriction enzyme digestion and AT clone is rapid and simple to prepare the desired gene chip probes.

BMC Genomics . 2003 Aug 12;4(1):34.
Application of comparative genomics in the identification and analysis of novel families of membrane-associated receptors in bacteria; Anantharaman V et al.; BACKGROUND: A great diversity of multi-pass membrane receptors, typically with 7 transmembrane (TM) helices, is observed in the eukaryote crown group . So far, they are relatively rare in the prokaryotes, and are restricted to the well-characterized sensory rhodopsins of various phototropic prokaryotes . RESULTS: Utilizing the currently available wealth of prokaryotic genomic sequences, we set up a computational screen to identify putative 7 (TM) and other multi-pass membrane receptors in prokaryotes . As a result of this procedure we were able to recover two widespread families of 7 TM receptors in bacteria that are distantly related to the eukaryotic 7 TM receptors and prokaryotic rhodopsins . Using sequence profile analysis, we were able to establish that the first members of these receptor families contain one of two distinct N-terminal extracellular globular domains, which are predicted to bind ligands such as carbohydrates . In their intracellular portions they contain fusions to a variety of signaling domains, which suggest that they are likely to transduce signals via cyclic AMP, cyclic diguanylate, histidine phosphorylation, dephosphorylation, and through direct interactions with DNA . The second family of bacterial 7 TM receptors possesses an alpha-helical extracellular domain, and is predicted to transduce a signal via an intracellular HD hydrolase domain . Based on comparative analysis of gene neighborhoods, this receptor is predicted to function as a regulator of the diacylglycerol-kinase-dependent glycerolipid pathway . Additionally, our procedure also recovered other types of putative prokaryotic multi-pass membrane associated receptor domains . Of these, we characterized two widespread, evolutionarily mobile multi-TM domains that are fused to a variety of C-terminal intracellular signaling domains . One of these typified by the Gram-positive LytS protein is predicted to be a potential sensor of murein derivatives, whereas the other one typified by the Escherichia coli UhpB protein is predicted to function as sensor of conformational changes occurring in associated membrane proteins CONCLUSIONS: We present evidence for considerable variety in the types of uncharacterized surface receptors in bacteria, and reconstruct the evolutionary processes that model their diversity . The identification of novel receptor families in prokaryotes is likely to aid in the experimental analysis of signal transduction and environmental responses of several bacteria, including pathogens such as Leptospira, Treponema, Corynebacterium, Coxiella, Bacillus anthracis and Cytophaga.

Bioinformatics, 2003 Aug 12, 19(12), 1461 - 8
Comprehensive aligned sequence construction for automated design of effective probes (CASCADE-P) using 16S rDNA; DeSantis TZ et al.; MOTIVATION: Prokaryotic organisms have been identified utilizing the sequence variation of the 16S rRNA gene . Variations steer the design of DNA probes for the detection of taxonomic groups or specific organisms . The long-term goal of our project is to create probe arrays capable of identifying 16S rDNA sequences in unknown samples . This necessitated the authentication, categorization and alignment of the >75 000 publicly available '16S' sequences . Preferably, the entire process should be computationally administrated so the aligned collection could periodically absorb 16S rDNA sequences from the public records . A complete multiple sequence alignment would provide a foundation for computational probe selection and facilitates microbial taxonomy and phylogeny . RESULTS: Here we report the alignment and similarity clustering of 62 662 16S rDNA sequences and an approach for designing effective probes for each cluster . A novel alignment compression algorithm, NAST (Nearest Alignment Space Termination), was designed to produce the uniform multiple sequence alignment referred to as the prokMSA . From the prokMSA, 9020 Operational Taxonomic Units (OTUs) were found based on transitive sequence similarities . An automated approach to probe design was straightforward using the prokMSA clustered into OTUs . As a test case, multiple probes were computationally picked for each of the 27 OTUs that were identified within the Staphylococcus Group . The probes were incorporated into a customized microarray and were able to correctly categorize Staphylococcus aureus and Bacillus anthracis into their correct OTUs . Although a successful probe picking strategy is outlined, the main focus of creating the prokMSA was to provide a comprehensive, categorized, updateable 16S rDNA collection useful as a foundation for any probe selection algorithm.

FEMS Microbiol Lett, 2003 Jul 29, 224(2), 183 - 90
Kakadumycins, novel antibiotics from Streptomyces sp NRRL 30566, an endophyte of Grevillea pteridifolia; Castillo U et al.; An endophytic streptomycete (NRRL 30566) is described and partially characterized from a fern-leaved grevillea (Grevillea pteridifolia) tree growing in the Northern Territory of Australia . This endophytic streptomycete produces, in culture, novel antibiotics - the kakadumycins . Methods are outlined for the production and chemical characterization of kakadumycin A and related compounds . This antibiotic is structurally related to a quinoxaline antibiotic, echinomycin . Each contains, by virtue of their amino acid compositions, alanine, serine and an unknown amino acid . Other biological, spectral and chromatographic differences between these two compounds occur and are given . Kakadumycin A has wide spectrum antibiotic activity, especially against Gram-positive bacteria, and it generally displays better bioactivity than echinomycin . For instance, against Bacillus anthracis strains, kakadumycin A has minimum inhibitory concentrations of 0.2-0.3 microg x ml(-1) in contrast to echinomycin at 1.0-1.2 microg x ml(-1) . Both echinomycin and kakadumycin A have impressive activity against the malarial parasite Plasmodium falciparum with LD(50)s in the range of 7-10 ng x ml(-1) . In macromolecular synthesis assays both kakadumycin A and echinomycin have similar effects on the inhibition of RNA synthesis . It appears that the endophytic Streptomyces sp . offer some promise for the discovery of novel antibiotics with pharmacological potential.

Protein Expr Purif, 2003 Aug, 30(2), 293 - 300
Production and proteolytic assay of lethal factor from Bacillus anthracis; Kim J et al.; Bacillus anthracis is the causative agent of anthrax . The major virulence factors are a poly-D-glutamic acid capsule and three-protein component exotoxin, protective antigen (PA, 83 kDa), lethal factor (LF, 90 kDa), and edema factor (EF, 89 kDa), respectively . These three proteins individually have no known toxic activities, but in combination with PA form two toxins (lethal toxin or edema toxin), causing different pathogenic responses in animals and cultured cells . In this study, we constructed and produced rLF as a form of GST fusion protein in Escherichia coli . rLF was rapidly purified through a single affinity purification step to near homogeneity . Furthermore, we developed an in vitro immobilized proteolytic assay of LF under the condition containing full-length native substrate, MEK1, rather than short synthetic peptide . The availability of full-length substrate and of an immobilized LF assay could facilitate not only the in-depth investigation of structure-function relationship of the enzyme toward its substrate but also wide spectrum screening of inhibitor collections based on the 96-well plate system.

Expert Opin Biol Ther, 2003 Aug, 3(5), 843 - 53
Anthrax toxin: structures, functions and tumour targeting; Liu S et al.; Anthrax toxin, the major virulence factor of Bacillus anthracis, consists of three polypeptides: protective antigen (PrAg), lethal factor (LF) and oedema factor (EF) . To intoxicate mammalian cells, PrAg binds to its cellular receptors and is subsequently activated via proteolysis, yielding a carboxyl-terminal fragment which coordinately assembles to form heptamers that bind and translocate LF and EF into the cytosol to exert their cytotoxic effects . Substantial progress has been made in recent years towards the characterisation of the structure and function of anthrax toxin, and this has greatly facilitated rational drug design of antianthrax agents . There is also emerging evidence that toxins can be manipulated for cancer therapy . LF can efficiently inactivate several mitogen-activated protein kinase kinases (MAPKKs) via cleavage of their amino-terminal sequences . Consequently, antitumour effects of wild type lethal toxin were observed after treatment of mitogen-activated protein kinase (MAPK)-dependent tumours such as human melanomas . Modification of the toxin's proteolytic activation site limits its cytotoxicity to certain cell types and creates a versatile method of treatment . One approach that has successfully achieved specific tumour targeting is the alteration of the furin cleavage of PrAg so that it is not activated by furin, but, alternatively, by proteases that are highly expressed by tumour tissues, including matrix metalloproteases and urokinase.

J Environ Health, 2003 Jul-Aug, 66(1), 9 - 15, 24; quiz 27-8
Efficacy and durability of Bacillus anthracis bacteriophages used against spores; Walter MH; Antibiotics and vaccines help fight anthrax disease, but there are no anthrax spore control methods suitable for use in environments where humans are present . The work reported in this article indicates that bacteriophages may help reduce risk from anthrax spores . Dose-response studies demonstrated that higher concentrations of mixed Bacillus anthracis bacteriophages (3.5 x 10(8) plaque-forming units per milliliter) inhibited subsequent growth of bacteria when sprayed on B . anthracis spores . Phages also were tested for durability under conditions designed to simulate environments possibly encountered during mass phage production, storage, and use against anthrax spores . They remained infectious at temperatures from -20 degrees C to 37 degrees C, under filtration, aerosolization, and treatments with perspiration and blood . Phages were sensitive to temperatures over 55 degrees C and to desiccation . Ultraviolet light reduced spore viability more than phage infectivity under similar conditions . The potential for personal or environmental decontamination of anthrax spores with phages is discussed.

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