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