Showing papers on "Bacillus anthracis published in 1997"

Crystal structure of the anthrax toxin protective antigen.

Abstract: Protective antigen (PA) is the central component of the three-part protein toxin secreted by Bacillus anthracis, the organism responsible for anthrax. After proteolytic activation on the host cell surface, PA forms a membrane-inserting heptamer that translocates the toxic enzymes, oedema factor and lethal factor, into the cytosol. PA, which has a relative molecular mass of 83,000 (M(r) 83K), can also translocate heterologous proteins, and is being evaluated for use as a general protein delivery system. Here we report the crystal structure of monomeric PA at 2.1 A resolution and the water-soluble heptamer at 4.5 A resolution. The monomer is organized mainly into antiparallel beta-sheets and has four domains: an amino-terminal domain (domain 1) containing two calcium ions and the cleavage site for activating proteases; a heptamerization domain (domain 2) containing a large flexible loop implicated in membrane insertion; a small domain of unknown function (domain 3); and a carboxy-terminal receptor-binding domain (domain 4). Removal of a 20K amino-terminal fragment from domain 1 allows the assembly of the heptamer, a ring-shaped structure with a negatively charged lumen, and exposes a large hydrophobic surface for binding the toxic enzymes. We propose a model of pH-dependent membrane insertion involving the formation of a porin-like, membrane-spanning beta-barrel.

Molecular evolution and diversity in Bacillus anthracis as detected by amplified fragment length polymorphism markers

Abstract: Bacillus anthracis causes anthrax and represents one of the most molecularly monomorphic bacteria known. We have used AFLP (amplified fragment length polymorphism) DNA markers to analyze 78 B. anthracis isolates and six related Bacillus species for molecular variation. AFLP markers are extremely sensitive to even small sequence variation, using PCR and high-resolution electrophoresis to examine restriction fragments. Using this approach, we examined ca. 6.3% of the Bacillus genome for length mutations and ca. 0.36% for point mutations. Extensive variation was observed among taxa, and both cladistic and phenetic analyses were used to construct a phylogeny of B. anthracis and its closest relatives. This genome-wide analysis of 357 AFLP characters (polymorphic fragments) indicates that B. cereus and B. thuringiensis are the closest taxa to B. anthracis, with B. mycoides slightly more distant. B. subtilis, B. polymyxa, and B. stearothermophilus shared few AFLP markers with B. anthracis and were used as outgroups to root the analysis. In contrast to the variation among taxa, only rare AFLP marker variation was observed within B. anthracis, which may be the most genetically uniform bacterial species known. However, AFLP markers did establish the presence or absence of the pXO1 and pXO2 plasmids and detected 31 polymorphic chromosomal regions among the 79 B. anthracis isolates. Cluster analysis identified two very distinct genetic lineages among the B. anthracis isolates. The level of variation and its geographic distribution are consistent with a historically recent African origin for this pathogenic organism. Based on AFLP marker similarity, the ongoing anthrax epidemic in Canada and the northern United States is due to a single strain introduction that has remained stable over at least 30 years and a 1,000-mile distribution.

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

Abstract: The protective effects of polyclonal antisera produced by injecting guinea pigs with protective antigen (PA), the chemical anthrax vaccine AVA, or Sterne spore vaccine, as well as those of toxin-neutralizing monoclonal antibodies (MAbs) produced against PA, lethal factor, and edema factor, were examined in animals infected with Bacillus anthracis spores. Only the anti-PA polyclonal serum significantly protected the guinea pigs from death, with 67% of infected animals surviving. Although none of the MAbs was protective, one PA MAb caused a significant delay in time to death. Our findings demonstrate that antibodies produced against only PA can provide passive protection against anthrax infection in guinea pigs.

Molecular characterization of the Bacillus anthracis main S‐layer component: evidence that it is the major cell‐associated antigen

Abstract: Summary Bacillus anthracis, the aetiological agent of anthrax, is a Gram-positive spore-forming bacterium. The cell wall of vegetative cells of B. anthracis is surrounded by an S-layer. An array remained when sap, a gene described as encoding an S-layer component, was deleted. The remaining S-layer component, termed EA1, is chromosomally encoded. The gene encoding EA1 (eag) was obtained on two overlapping fragments in Escherichia coli and shown to be contiguous to the sap gene. The EA1 amino acid sequence, deduced from the eag nucleotide sequence, shows classical S-layer protein features (no cysteine, only 0.1% methionine, 10% lysine, and a weakly acidic pi). Similar to Sap and other Gram-positive surface proteins, EA1 has three 'S-layer-homology’motifs immediately downstream from a signal peptide. Single- and double-disrupted mutants were constructed. EA1 and Sap were co-localized at the cell surface of the wild-type bacilli. However, EA1 was more tightly bound than Sap to the bacteria. Electron microscopy studies and in vivo experiments with the constructed mutants showed that EA1 constitutes the main lattice of the B. anthracis S-layer, and is the major cell-associated antigen.

The anthrax toxin activator gene atxA is associated with CO2-enhanced non-toxin gene expression in Bacillus anthracis

Abstract: The Bacillus anthracis toxin genes, cya, lef, and pag, can be viewed as a regulon, in which transcription of all three genes is activated in trans by the same regulatory gene, atxA, in response to the same signal, CO2. In atxA+ strains, toxin gene expression is increased 5- to 20-fold in cells grown in 5% CO2 relative to cells grown in air. CO2-enhanced toxin gene transcription is not observed in atx4-null mutants. Here, we used two independent techniques to obtain evidence for additional CO2-induced atxA-regulated genes. First, total protein preparations from atxA4+ and atxA isolates grown in 5% CO2 and in air were examined by two-dimensional electrophoresis. Comparison of the resulting protein patterns indicated that synthesis of non-toxin proteins is influenced by growth in elevated CO2 and the toxin gene regulator, atxA. Second, we generated random transcriptional lacZ fusions in B. anthracis with transposon Tn917-LTV3. Transposon-insertion libraries were screened for mutants expressing CO2-enhanced atxA-dependent beta-galactosidase activity. DNA sequence analysis of transposon insertion sites in 17 mutants carrying CO2- and atxA-regulated fusions revealed 10 mutants carrying independent insertions on the 185-kb toxin plasmid pXO1 which did not map to the toxin genes. The tcr-lacZ fusion mutants (tcr for toxin coregulated) were Tox+, indicating that these genes may not be involved in anthrax toxin gene activation. Our data indicate a clear association of atxA with CO2-enhanced gene expression in B. anthracis and provide evidence that atxA regulates genes other than the structural genes for the anthrax toxin proteins.

AtxA activates the transcription of genes harbored by both Bacillus anthracis virulence plasmids

Abstract: Fully virulent Bacillus anthracis bacilli are encapsulated and toxinogenic. These bacteria carry two plasmids, pXO1 and pXO2, encoding toxins and capsule synthetic-enzymes (capB, C, A, dep), respectively. The pXO1 plasmid strongly enhances capsule formation. This influence was studied by analysing the expression of a capB–lacZ fusion in various backgrounds. The β-galactosidase activities were similar in a ΔatxA strain and a pXO1 cured strain. Moreover, the capB–lacZ expression level could be restored, in a pXO1 cured strain, by addition of atxA in trans. Thus, we conclude that the pXO1 influence on capsule synthesis is mediated by AtxA, the pXO1-encoded trans-activator of the toxin gene expression.

Intracytoplasmic delivery of listeriolysin O by a vaccinal strain of Bacillus anthracis induces CD8-mediated protection against Listeria monocytogenes.

Abstract: The facultative intracellular pathogen Listeria monocytogenes secretes a 58-kDa hemolysin, listeriolysin O (LLO), that allows bacteria to access the cytoplasm and to multiply inside infected cells. LLO is also a protective Ag required for the development of specific immunity. We studied the capacity of a new bacterial vector, derived from an attenuated strain of Bacillus anthracis, to deliver in vivo LLO and to induce protection against L. monocytogenes infection. The hly gene encoding LLO was fused to a B. anthracis regulatory region induced in vivo and was integrated into a resident plasmid of this bacterium. This recombinant strain secreted a functional LLO in vitro and inside phagosomes of bone marrow macrophages. This LLO production enabled the conversion of the extracellular replicating B. anthracis into an intracytoplasmic bacterium. LLO+ B. anthracis thus mimicked the intracellular behavior of L. monocytogenes in macrophages. Specific protection of mice against lethal doses of L. monocytogenes was induced by immunization with LLO+ B. anthracis. The immunity was mediated by CD8+ T lymphocytes and was associated with the activation of LLO-specific MHC class I-restricted CD8+ CTL, able to recognize the immunodominant H-2d-restricted epitope 91-99 of LLO. This study, therefore, suggests that intracytoplasmic delivery of LLO by B. anthracis is sufficient to induce a MHC class I-restricted CD8-mediated protection against L. monocytogenes. The LLO+ B. anthracis recombinant strain represents a potential vector for delivering foreign Ags involved in CD8-mediated protective responses.

Immunological and functional comparison between Clostridium perfringens iota toxin, C. spiroforme toxin, and anthrax toxins

Abstract: Clostridium perfringens iota and C. spiroforme toxins consist of two separate proteins. One is the binding component and the other the enzymatic component. The two toxins secreted by Bacillus anthracis are composed of binary combinations of three proteins: protective antigen, lethal factor, and edema factor. As shown by Western blotting and ELISA, the binding component of anthrax toxin shares common epitopes with that of iota toxin and C. spiroforme toxin which are closely related immunologically. However, no functional complementation was observed between iota toxin and anthrax toxin components. The binding components can form toxins active on macrophages only in combination with their respective enzymatic components. Agents which prevent acidification of endosomes do not have the same effects on anthrax toxin activity as they do on iota and C. spiroforme toxins. Therefore, the mechanisms of entry into the cells are presumably different. Since the binding components of anthrax toxins and iota toxin share a conserved putative translocation domain, these binding components could have a common mode of insertion into the cell membranes.

Extraordinary Case Report: Cutaneous Anthrax

Abstract: Anthrax is a very rare disease in the United Kingdom. It is caused by the spore-forming bacterium Bacillus anthracis. Humans become infected when they come into contact with infected animals or their products. Cutaneous anthrax, the most common form of the disease, accounts for 95% of cases, and the disease usually developing on exposed sites. We present a patient who developed cutaneous disease after exposure to untreated leather. Owing to the initial clinical information, the biopsy specimen was misinterpreted as representing a severe acute insect bite reaction. The subsequent involvement by the Department of Microbiology established the correct diagnosis. Because today the disease is so rare in Europe and the United States, sporadic cases of anthrax are easily overlooked as the diagnosis often is not considered. Cutaneous anthrax should be considered in any patient with a painless ulcer with vesicles, edema, and a history of exposure to animals or animal products.

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

Abstract: The Bacillus anthracis toxinogenic Sterne strain is currently used as a live veterinary vaccine against anthrax. The capacity of a toxin-deficient derivative strain to produce a heterologous antigen by using the strong inducible promoter of the B. anthracis pag gene was investigated. The expression of the foreign gene ibp, encoding the Ib component of iota toxin from Clostridium perfringens, was analyzed. A pag-ibp fusion was introduced by allelic exchange into a toxin-deficient Sterne strain, thereby replacing the wild-type pag gene. This recombinant strain, called BAIB, was stable and secreted large quantities of Ib protein in induced culture conditions. Mice given injections of live BAIB spores developed an antibody response specific to the Ib protein. The pag-ibp fusion was therefore functional both in vitro and in vivo. Moreover, the immunized animals were protected against a challenge with C. perfringens iota toxin or with the homologous Clostridium spiroforme toxin. The protective immunity was mediated by neutralizing antibodies. In conclusion, B. anthracis is promising for the development of live veterinary vaccines.

Fatal meningoencephalitis due to Bacillus anthracis

Abstract: We report the first case of fatal anthrax meningoencephalitis in Hong Kong over the past 60 years. A 13 year-old boy presented with right lower quadrant pain, diarrhoea and progressive headache. Lumbar puncture yielded gram positive bacilli initially thought to be Bacillus cereus, a contaminant. He was treated with ampicillin and cefotaxime, but died 3 days after hospitalization. The organism isolated from blood and cerebrospinal fluid was later identified as Bacillus anthracis.

Vaccine production of the bacillus anthracis protective antigen

Abstract: Methods of preparing recombinant Bacillus anthracis protective antigen or a variant or fragment thereof for use in vaccines is disclosed. The protein is expressed in a recombinant microorganism which comprises a sequence which encodes PA or said variant or fragment thereof wherein either (i) a gene of the microorganism which encodes a catabolic repressor protein and/or AbrB is inactivated, and/or (ii) wherein a region of the PA sequence which can act as a catabolic repressor binding site and/or an AbrB binding site is inactivated. Useful quantities of protein are obtainable from these organisms.

Anthrax lethal toxin-induced mitogenic response of human T-cells

Abstract: Bacillus anthracis lethal toxin (PALF) stimulated the proliferation of human peripheral blood T-cells in vitro. Activation of T-lymphocytes by PALF required the presence of monocytes and did not result from a collaborative effect between T-cells and B-cells. PALF acted directly on monocytes and independently of T-cells. The monocytes contributed to the proliferation of T-cells by secretion of mediator(s). The mitogenic activity of the lethal toxin was dependent on its metalloprotease activity.

Pyrolysis mass spectrometry studies on Bacillus anthracis, Bacillus cereus and their close relatives

Abstract: Summary Pyrolysis mass spectrometry was used to examine strains of B. anthracis, of B. cereus, of B. cereus either proven to cause emetic illness or connected with outbreaks of emetic food poisoning and of B. thuringiensis. Analysis of the data-set for all strains allowed differentiation between B. anthracis, the emetic B. cereus and B. thuringiensis but B. cereus strains could not be clearly discriminated. Removal of data for the B. thuringiensis and the emetic B. cereus strains, followed by re-analysis, allowed clear separation of the B. anthracis and B. cereus groups. Furthermore, PyMS was found to be capable of discriminating between some strains of B. anthracis, and demonstrating sub-groupings of others. This work provides further evidence of the ability of PyMS to distinguish rapidly between very closely related organisms and indicates its potential in epidemiology.

Production of highly pure l-lactic acid with bacillus group microorganism

Abstract: PROBLEM TO BE SOLVED: To profitably obtain the subject compound useful for producing biodegradable resins, foods, medicines, optical materials, etc., by culturing a Bacillus group microorganism having an ability to produce the highly optically pure L-lactic acid from a carbon source capable of being assimilated and subsequently collecting the product from the culture product. SOLUTION: A microorganism, such as Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus larvae, Bacillus lentimorbus, Bacillus popilliae or Bacillus sphaericus, having an ability to produce L-lactic acid having an optical purity of >=70% from a carbon source capable of being assimilated is cultured, and the product is collected from the culture products to obtain the objective highly pure L-lactic acid at a low cost.

Cytotoxicity of anthrax lethal factor microinjected into macrophage cells through Sendai virus envelopes.

Abstract: Lethal toxin (LT) secreted by Bacillus anthracis consists of two proteins, protective antigen (PA) and lethal factor (LF). LT causes lysis of macrophages and derived cell lines at low concentrations. PA binds to the cell surface receptors and mediates translocation of LF into cytosol of mammalian cells. Internalization of LF into cytosol by osmotic lysis of pinocytic vesicles requires high concentration of LF for cell lysis. To examine the possible cell lysis by LF at low concentration, we introduced LF directly into cytosol of J774A.1 cells through reconstituted Sendai virus envelopes. The introduction of LF lysed J774A.1 cells in a concentration dependent manner. Internalization of PA alone through virosome had no toxic effect on J774A.1 cells. In the process of cytotoxicity LF was not cleaved by cellular proteases. Unlike many protein toxins, golgi was not involved in the expression of lethal toxin activity. These results indicate that LF is the toxic component of anthrax lethal toxin and prior proteolytic processing or trafficking through golgi is not required for its activity.

Molecular mechanisms underlying bacillus anthracis infection at early stages and search for novel vaccines

Abstract: The developmental mechanisms of anthrax immunity were studied Immunization was found to generally generate specific antibodies and lysozyme Collectively, all the factors are responsible for suppressing the development of spores in the body This proves the fact that the immunity is directed not only towards the exotoxin of B anthracis, but it affects mainly the formation of vegetative cells On entering the immuned body, vegetative cells may cause B anthracis infection because antitoxic antibodies have no effect on encapsulated cells The findings indicate that any anti-anthrax vaccine strain must show a complete immunological response in the body, as well as constitute immunity to all pathogenetic factors of B anthracis

Crystallographic Studies of the Anthrax Lethal Toxin.

Abstract: : The lethal form of Anthrax results from the inhalation of anthrax spores. Death is primarily due to the effects of the lethal toxin (Protective Antigen (PA) + Lethal Factor) from the causative agent, Bacillus anthracis. All the Anthrax vaccines currently in use or under development contain or produce PA, the major antigenic component of anthrax toxin, and there is a clear need for an improved vaccine for human use. In the previous report we described the first atomic resolution structure of PA, revealing that the molecule is composed largely of beta-sheets organized into four domains. This information can be used in the design. of recombinant PA vaccines. In this report we describe additional features of the full-length PA molecule derived from further crystallographic refinement and careful examination of the structure. We compare two crystal forms of PA grown at different pH values and discuss the functional implications. A complete definition of the function of each domain must await the crystal structure of the PA63 heptamer. We have grown crystals of the heptamer under both detergent and detergent-free conditions, and made substantial progress towards the crystal structure. The mechanism of anthrax intoxication in the light of our results is reviewed.

Current status of anthrax or black fever

Abstract: Although anthrax is one of the oldest recognized infectious diseases in the world, it remains widespread particularly in tropical zones such as Africa. The impact of this major zoonoses is further enhanced by the fact that the pulmonary form can be used for biological warfare. Recently there has been a revival of interest in anthrax and research has benefited greatly from advances in molecular biology. The main factors accounting for the virulence of Bacillus anthracis have been elucidated. The author reports current data concerning pathogenesis, epidemiology and diagnosis and reviews progress made in the field of prophylaxis especially with regard to vaccines.

[Anthrax: a malady of animals and of man which hides in the earth].

Abstract: Anthrax is an infectious disease of herbivores, especially sheep and cattle, but also of horses, of pigs, of dogs, of wild animals and of humans. Bacillus anthracis causes the disease. This bacterium needs plenty of oxygen to procreate and to produce resistant spores, which remains viable in the soil during 3.5 years, at times during 15-20 years. The author tries to follow step by step the evolution of the ideas concerning the origin and the pathology as well as of the veterinarians measures against this disease during the 19th and 20th centuries. Many studies of the endemic anthrax were made from the beginning of the second half of the 19th century. Thanks to Louis Pasteur (1822-1895) in France and Robert Koch (1843-1910) in Germany, the anthrax could be identified with a soil-disease between 1870 and 1880. The statistics concerning the anthrax in the Grand-Duchy of Luxembourg compared to the geological and pedological structures of the soil, have fully confirmed the scientific findings at the end of the 19th century. Nowadays the anthrax has disappeared from our landscape thanks to the preventive inoculation and to the industrial use of the animal cadavers.