Showing papers on "Bacillus anthracis published in 1994"

Anthrax toxin lethal factor contains a zinc metalloprotease consensus sequence which is required for lethal toxin activity.

Abstract: Summary Comparison of the anthrax toxin lethal factor (LF) amino acid sequence with sequences in the Swiss protein database revealed short regions of similarity with the consensus zinc-binding site, HEXXH, that is characteristic of metalloproteases. Several protease inhibitors, including bestatin and captopril, prevented intoxication of macrophages by lethal toxin. LF was fully inactivated by site-directed mutagenesis that substituted Ala for either of the residues (H-686 and H-690) implicated in zinc binding. Similarly, LF was inactivated by substitution of Cys for E-687, which is thought to be an essential part of the catalytic site. In contrast, replacement of E-720 and E-721 with Ala had no effect on LF activity. LF bound 65Zn both in solution and on protein blots. The 65Zn binding was reduced for several of the LF mutants. These data suggest that anthrax toxin LF is a zinc metallopeptidase, the catalytic function of which is responsible for the lethal activity observed in cultured cells and in animals.

The three Bacillus anthracis toxin genes are coordinately regulated by bicarbonate and temperature.

Abstract: The two Bacillus anthracis toxins are composed of three proteins, protective antigen, lethal factor, and edema factor. The structural genes for these three components are located on the virulence plasmid pXO1. We constructed transcriptional fusions between the regulatory region of each of these genes and lacZ. Each construct was then inserted as a single copy at the corresponding toxin gene locus on pXO1, resulting in three isogenic strains. Two environmental factors, bicarbonate and temperature, were found to induce beta-galactosidase synthesis in each recombinant strain. Furthermore, the transcription of the three toxin genes appears to be coordinately regulated.

Fusions of anthrax toxin lethal factor with shiga toxin and diphtheria toxin enzymatic domains are toxic to mammalian cells.

Abstract: To investigate the ability of anthrax toxin lethal factor (LF) to translocate foreign proteins into the cytosol of eukaryotic cells and to characterize the structural requirements of this process, fusion proteins containing a portion of LF and the catalytic domains of either diphtheria toxin or Shiga toxin were constructed. Previous work showed that residues 1 to 254 of anthrax toxin lethal factor (LF1-254) are sufficient for binding to the protective antigen component of the toxin and that portions of Pseudomonas exotoxin A fused to LF1-254 are efficiently translocated to the cytosol of eukaryotic cells (N. Arora and S. H. Leppla, J. Biol. Chem. 268:3334-3341, 1993). In this study, it was found that fusion proteins containing the ADP-ribosylation domain of diphtheria toxin fused at either the amino end or the carboxyl end of LF1-254 are highly toxic to Chinese hamster ovary (CHO) cells, indicating that translocation does not strictly require that the amino terminus of LF be free. A fusion protein containing the ribosome-inactivating A1 subunit of Shiga toxin fused to the carboxyl terminus of LF1-254 was also highly toxic for CHO cells. All fusion proteins were toxic only when administered with the anthrax toxin protective antigen component. The data show that the combination of protective antigen and LF fusion proteins can efficiently import polypeptides from diverse bacterial sources to the cytosol of eukaryotic cells and that LF fusion proteins may have the passenger polypeptides fused at either the amino terminus or the carboxyl terminus of LF1-254. These LF fusion proteins could potentially be used as components of a therapeutic agent when the destruction of certain types of cells is desired (e.g., in treating cancer).

Information on which to base assessments of risk from environments contaminated with anthrax spores.

Abstract: Although there has been a considerable amount of research conducted into Bacillus anthracis, the causative agent of anthrax, the data are widely disseminated in the scientific literature and are therefore not always easy to assimilate. In view of continuing concern about potential anthrax contamination in environmental materials and sites, this review brings together the currently available information relating to the health hazards from B. anthracis. The relevance of the available information for risk assessment purposes is assessed.

Differentiation of Bacillus anthracis from other Bacillus cereus group bacteria with the PCR

Abstract: Variation among isolates of Bacillus anthracis was examined by using restriction fragmentation patterns and the PCR performed with arbitrary and sequence-specific oligonucleotide primers. The patterns were compared with the patterns generated from strains of closely related species belonging to the “Bacillus cereus group” of bacteria, including B. cereus, Bacillus thuringiensis, and Bacillus mycoides. All B. anthracis profiles were identical for each of 18 restriction enzymes, each of 10 arbitrary PCR primers, and a repetitive extragenic palindrome-specific PCR primer. The PCR profiles generated with a coliphage M13-based primer exhibited slight pattern variation in a 400- to 500-bp band region. The B. anthracis profiles were unique compared with the profiles of the other species examined. In these other species, strain-to-strain variations were observed. Our results showed that isolates of B. anthracis are almost completely homogeneous, indicating a clonal lineage, and are distinct from other members of the B. cereus group and that B. anthracis, as a species in its own right, may have evolved only relatively recently.

Formaldehyde Solution Effectively Inactivates Spores of Bacillus anthracis on the Scottish Island of Gruinard

Abstract: Gruinard Island was heavily contaminated with the spores of virulent Bacillus anthracis during biological weapons trials in World War II However, an extensive survey in 1979 showed that most of the island was not contaminated In the early 1980s, a more intensive survey revealed that the contamination was largely confined to the top 8 cm of the soil in a 26-ha area of the 211-ha island Small-scale tests showed that the spores could be inactivated by drenching the soil with fluid biocides A solution of 5% formaldehyde in seawater applied by surface spray to each square meter of ground was shown to be the most effective treatment and was utilized for large-scale decontamination of the affected areas Following this treatment, extensive sampling revealed that most of the spores of B anthracis had been inactivated Isolated pockets of surviving spores were treated further A flock of sheep was then allowed to graze over the entire island for 5 months; none contracted anthrax

Identification of Capsule-Forming Bacillus Anthracis Spores with the PCR and a Novel Dual-Probe Hybridization Format

Abstract: : Anthrax is a fatal infection of humans and livestock that is caused by the gram-positive bacterium Bacillus anthracis The virulent strains of B anthracis are encapsulated and toxigenic In this paper we describe the development of a PCR technique for identifying spores of B anthracis Two 20- mer oligonucleotide primers specific for the capB region of 60-MDa plasmid pXO2 were used for amplification The amplification products were detected by using biotin- and fluorescein-labeled probes in a novel dual-probe hybridization format Using the combination of PCR amplification and dual-probe hybridization, we detected two copies of the bacterial genome Because the PCR assay could detect a minimum of 100 unprocessed spores per PCR mixture, we attempted to facilitate the release of DNA by comparing the effect of limited spore germination with the effect of mechanical spore disruption prior to PCR amplification The two methods were equally effective and allowed us to identify signal spore of B anthracis in PCR mixtures

Zinc content of the Bacillus anthracis lethal factor

Abstract: We present evidence that the anthrax toxin lethal factor binds multiple zinc atoms. Results from atomic adsorption spectroscopy indicate that lethal factor contains approximately three zinc atoms per toxin molecule. Lethal factor treated with EDTA and o-phenanthroline contained a similar number of zinc atoms, indicating that all three zinc atoms are tightly bound to the protein. Lethal factor contains the highly conserved zinc-binding consensus sequence. HExxH, that is present in all known zinc metalloproteases. In addition, lethal factor contains an inverted form of this motif, HxxDH, which may also be involved in zinc binding. Using a large array of protease model substrates, however, we were unable to detect an endogenous protease activity for lethal factor.

Protein synthesis is required for expression of anthrax lethal toxin cytotoxicity.

Abstract: Anthrax lethal toxin, which is composed of two proteins, i.e., protective antigen and lethal factor, is cytolytic to mouse peritoneal macrophages and the macrophage-like cell line J774A.1. After exposure of cells to lethal toxin, inhibition of protein synthesis occurred only slightly before the onset of cytolysis. Thus, cell death did not appear to be due to inhibition of protein synthesis. However, prior treatment of J774A.1 cells with cycloheximide or puromycin, which inhibited protein synthesis, protected them completely against lethal toxin-induced cytolysis, which suggested that continuous protein synthesis is required for the expression of lethal toxin activity. Inhibition of protein synthesis had no appreciable effect on the binding of protective antigen to the cell surface receptor or on proteolytic cleavage of surface-bound protective antigen. Furthermore, inhibition of protein synthesis did not alter the uptake of toxin, which suggested that protein synthesis is required at a later stage of the intoxication process. The protection provided by inhibition of protein synthesis was effective, even up to 1 h after exposure to anthrax lethal toxin. The increased uptake of calcium observed in cells exposed to lethal toxin did not occur when they were protected by blocking protein synthesis. Identifying the protein(s) synthesized during the intoxication process may help to understand the mechanism of cell death produced by anthrax lethal toxin.

Improved methods for the detection of Bacillus anthracis spores by the polymerase chain reaction

Abstract: Polymerase chain reactions (PCRs) for the capsule and oedema factor genes of Bacillus anthracis were used to assess methods for detecting B. anthracis spores. Untreated spore preparations were found to contain significant amounts of extracellular template DNA which probably accounted for observed amplification from these preparations without spore lysis. Germination of spores with suitable media allowed the detection of less than 10 spores in a PCR test. Mechanical disruption of spores with glass or zirconia beads yielded similar results to germination but in a much shorter time. The techniques described should improve the detection by PCR of B. anthracis and other sporulating bacteria.

Recombinant Bacillus anthracis strains unable to produce the lethal factor protein or edema factor protein

Abstract: A recombinant strain of B anthracis is characterized in that it can induce the production of protective antibodies against virulent strains of B anthracis in a human or animal host, and characterized also by the mutation of the pX01 plasmid of at least one given gene coding for a protein which causes a toxic effect of B anthracis, wherein said mutation leads to the deletion of all or part of said gene which codes for the protein causing the toxic effect, and to the insertion of a DNA cassette at said gene's deletion site in pX01, whereby the strain thereby modified may be selected and a back mutation of the recombinant strain may be prevented, and wherein the gene thereby mutated is thereafter either unable to produce the protein causing the toxic effect for which it codes, or able to code for a truncated protein which has lost its toxic properties The use of such a strain in immunogenic compositions is also described

Anthrax: Virulence and Vaccines

Abstract: Anthrax is an infectious disease that has afflicted humans and their domestic livestock since ancient times. Although in many industrialized countries the disease is controlled by vaccination and good practices in rearing livestock, it remains a serious problem in many less developed regions of the world. The causative organism, Bacillus anthracis, is a spore-forming, rod-shaped bacterium that inhabits the soil. Although the causal relation between the organism and anthrax has been known for more than a century, since the time of Koch and Pasteur, the specific factors responsible for the virulence of the organism have been identified and characterized during the last 30 years. The precise molecular basis for virulence has been elucidated only during the past decade. Fully virulent strains of Bacillus anthracis possess two unique virulence factors: a poly-D-glutamic acid capsule that inhibits phagocytosis [1] and a tripartite toxin composed of protective antigen, edema factor, and lethal factor [2]. Capsules are produced by virulent strains of Bacillus anthracis growing in vivo and by cells grown on media containing serum or bicarbonate or both and incubated in a CO2-enriched atmosphere. The existence of an anthrax toxin was first demonstrated in 1955 in experiments that showed that injection of sterile plasma from infected guinea pigs resulted in local edema and death. Studies by American and British investigators during the ensuing decade showed that the toxin contained three separate components. The individual toxin components have no known biological effects when administered alone, but edema factor injected with protective antigen into the skin of rabbits or guinea pigs causes local edema, and protective antigen injected with lethal factor into rats causes death in as little as 60 minutes. Protective antigen, so called because its injection into experimental animals results in protective immunity, binds to cell-surface receptors to produce an uptake system that can be used by both the edema factor and lethal factor to gain access to the cytoplasm. Edema toxin (edema factor and protective antigen) and lethal toxin (lethal factor and protective antigen) thus resemble the A-B enzyme-binding structures characteristic of many well-studied bacterial toxins. After protective antigen, which is analogous to the B chain, binds to a specific membrane receptor on the surface of a eukaryotic cell, it is cleaved at a single site, exposing a binding site for the other toxin component. The membrane-bound fragment of the protective antigen then binds to the edema factor or lethal factor and mediates the entry of the active moiety into the cell. The edema factor has been found to be a calmodulin-dependent adenylate cyclase that elevates cyclic adenosine monophosphate (AMP) levels approximately 200-fold greater than normal in Chinese hamster ovary cells. Local edema, a typical sign of anthrax, may be directly related to adenylate cyclase activity associated with the edema factor. The increase in intracellular cyclic AMP caused by this toxin may lead to edema in a manner analogous to the loss of water into the intestinal lumen caused by cholera toxin, which also increases intracellular cyclic AMP [3]. Edema factor and the protective antigen also inhibit phagocytosis of anthrax bacilli by polymorphonuclear leukocytes, and this effect may further increase host susceptibility to anthrax. The dependence of edema factor activity on calmodulin, a substance found only in eukaryotic cells, suggests that the edema factor did not evolve from a bacterial enzyme but from a eukaryotic adenylate cyclase, the gene for which was adventitiously transferred into Bacillus anthracis and retained because it made the bacteria more virulent [3]. The mechanism of action of lethal factor is poorly understood, but it is lethal for many species of experimental animals and is assumed to be the major factor causing death in anthrax. No enzymatic activity has yet been associated with the lethal factor, and the nature of its intracellular target is unknown. Virulent strains of Bacillus anthracis contain two large plasmids, pX01 and pX02 [1, 4, 5]. Both plasmids are required for full pathogenicity, and strains that contain only one of these plasmids are avirulent. The plasmid pX01 encodes all three components of the anthrax toxin, and pX02 encodes the poly-D-glutamic acid capsule. These facts provide the basis for effective vaccines, the first of which was developed by Pasteur in 1881 and used in a brilliantly successful field trial in sheep at the village of Pouilly-le-Fort. The avirulent Sterne vaccine strain, which is pX01+/pX02-, produces toxin but no capsule and is used effectively as a live veterinary vaccine [6]. The nonencapsulated strain used in production of the acellular vaccine licensed for human use in the United States produces primarily protective antigen [6]. The heat-attenuated Pasteur vaccine strains form capsules but cannot produce toxin. Pasteur probably cured his strains of plasmid pX01 by heat attenuation to produce his vaccine for immunization of cows and sheep. Although the Pasteur-type vaccine provides a much lower level of protective immunity than the toxigenic vaccine strains [7], it was still good enough to save all the sheep on that fateful day at Pouilly-le-Fort. (The modest immunogenicity of Pasteur's vaccine may well have been caused by the persistence of a small proportion of pX01+/pX02+ cells, which thus remained toxigenic and fully virulent.) The genes encoding the capsule and each of the three toxin components have been cloned in Escherichia coli [8-11], and the base sequences have been determined [12-14]. The protective antigen gene has been cloned in Bacillus subtilis, and immunization with the live recombinant strain protects guinea pigs from lethal challenge with virulent Bacillus anthracis spores [15]. None of the currently available anthrax vaccines is ideal, and efforts to develop better vaccines, with the use of newer molecular methods, represent an active area of research [16, 17]. Although the Sterne vaccine is effective and safe for use in many domestic animals (cattle, sheep, pigs, camels, buffaloes, and elephants), progressive disease caused by the vaccine strain has been observed in goats and llamas. The duration of protection conferred by the Sterne vaccine is somewhat limited, and the necessity for administration by injection is a disadvantage in many developing countries. The vaccine used in humans in the United States produces high titers of antibody to protective antigen in guinea pigs, but only animals vaccinated with the Sterne strain are completely protected against challenge with highly virulent strains of Bacillus anthracis. Several different approaches are being used in the development of improved anthrax vaccines for humans. Purified protective antigen vaccines are being combined with adjuvants derived from the cell wall of the BCG strain of the tubercle bacillus [18] or with killed cells of Bordetella pertussis [19] to enhance the cellular immune response to the protective antigen. The Bacillus subtilis strain into which the protective antigen gene has been cloned, mentioned above [15], is a recombinant vaccine that does not contain the Bacillus anthracis genome. Transposon-induced mutagenesis has been used to produce mutant vaccine strains that cannot synthesize essential aromatic amino acids unavailable from the mammalian host and that thus cannot replicate for more than a few cycles in the mammalian host [16, 20]. Oral vaccines might consist of immunizing antigens from Bacillus anthracis cloned into appropriate bacterial vectors such as Salmonella species [17]. Anthrax has been known in humans and their animals for more than 7000 years, and the association between Bacillus anthracis and its hosts probably existed for millennia before it was recognized and recorded. In many countries, vaccination of animals and humans and improved methods of rearing livestock have effectively controlled the disease. Recent advances in molecular biology have allowed the elucidation of the precise mechanisms of virulence in Bacillus anthracis and give promise of even more effective vaccines. But it is difficult to imagine that the vast reservoir of Bacillus anthracis in the soil can ever be eradicated. The anthrax bacillus will surely endure; its place on our planet seems at least as secure as our own.

Evaluation of Bacillus subtilis strain IS53 for the production of Bacillus anthracis protective antigen

Abstract: An asporogenous strain of Bacillus subtilis, IS53, transformed with plasmid pPA102, produces the protective antigen (PA) component of the tripartite toxin of B. anthracis. Addition of yeast extract was required to support growth and PA production in all the media examined. Protective antigen expression was down-regulated during exponential growth and extracellular proteases caused marked degradation of the mature protein.

Anthrax in a dog.

Abstract: diating into a myxoid background where they often appeared as individual cells o r clusters ofcells. The first two specimens, biopsies from dog Nos. 1 and 2, had no solid or cystic areas compatible with more typical meningiomas in the dog. This led to considerable confusion and an original diagnosis of atypical choroid plexus tumor. It was only after examining the third dog that we reali7ed that these were variations of more typical meningothelial meningiomas. These tumors might also be confused with chordomas or primitive cartilaginous tumors because of the clusters of cells with occasional vacuoles and the myxoid background matrix. The immunohistochemical results ( G F A P negative, v iment in positive) are consistent with those reported in meningiomas of dogs'\" and humans.y Focal clusters of cells that stain with antibodies to keratin have been reported in meningiomas in humansy and reflect the embryological origins of the leptomeninges. In conclusion, myxoid meningiomas appear to be a rare but distinctive variant of canine meningiomas that should be recognized by veterinary pathologists.

[Delayed hypersensitivity after anthrax vaccination. I--Study of guinea pigs vaccinated against anthrax].

Abstract: To evaluate delayed hypersensitivity after anthrax vaccination, an Anthraxin skin test was performed in 682 guinea pigs at various times after immunization with veterinary unencapsulated active anthrax vaccine. Results were compared with those obtained in unimmunized control guinea pigs (n = 216), in guinea pigs that received a non-immunizing dose of live vaccine (n = 183) and in guinea pigs inoculated with inactivated vaccine (n = 120). Anthraxin skin tests were positive in the first postvaccination days. The incidence and intensity of positive tests peaked between two weeks and one month after vaccination and then gradually decreased during the first year. Study of resistance of guinea pigs to an inoculum at a lethal dose of a virulent strain of Bacillus anthracis showed a close correlation between positive tests and resistance. These findings demonstrate development of cell-mediated immunity after anthrax vaccination. The Anthraxin skin test should have practical applications for the production of vaccines and for evaluation of the immune status of vaccinated livestock [corrected].

Protein synthesis is required for the expression anthrax lethal toxin cytotoxicity

Abstract: Anthrax lethal toxin, which is composed of two proteins, i.e., protective antigen and lethal factor, is cytolytic to mouse peritoneal macrophages and the macrophage-like cell line J774A.1. After exposure of cells to lethal toxin, inhibition of protein synthesis occurred only slightly before the onset of cytolysis. Thus, cell death did not appear to be due to inhibition of protein synthesis. However, prior treatment of J774A.1 cells with cycloheximide or puromycin, which inhibited protein synthesis, protected them completely against lethal toxin-induced cytolysis, which suggested that continuous protein synthesis is required for the expression of lethal toxin activity. Inhibition of protein synthesis had no appreciable effect on the binding of protective antigen to the cell surface receptor or on proteolytic cleavage of surface-bound protective antigen. Furthermore, inhibition of protein synthesis did not alter the uptake of toxin, which suggested that protein synthesis is required at a later stage of the intoxication process. The protection provided by inhibition of protein synthesis was effective, even up to 1 h after exposure to anthrax lethal toxin. The increased uptake of calcium observed in cells exposed to lethal toxin did not occur when they were protected by blocking protein synthesis. Identifying the protein(s) synthesized during the intoxication process may help to understand the mechanism of cell death produced by anthrax lethal toxin.

Comparative evaluation of the effectiveness of fluoroquinolones in experimental anthrax infection

Abstract: Comparative antibacterial activity and protective efficacy of ciprofloxacin, pefloxacin and lomefloxacin were estimated in a model of anthrax. The MICs of the three drugs determined by the method of serial dilutions for three vaccinal strains of Bacillus anthracis were 0.5 to 1.0 microgram/ml. The protective efficacy of the chemotherapeutics in experimental anthrax induced by the spores of the vaccinal strain 71/12, Tsenkovsky was evaluated in mathematically designed four-factor experiments. It depended on the infective dose and the chemotherapy term and amounted in the protective use of the drugs in the daily doses, equivalent to those for humans, to 80-100-percent protection of the animals infected with 10 LD50 of the biological agent, to 50-80-percent protection with the use of 100 LD50, to 40-70-percent protection with the use of 1000 LD50 and to 50-90-percent protection in the therapeutic use of the fluoroquinolones. This was indicative of the fact that the fluoroquinolones were chemotherapeutically highly active in the treatment of experimental anthrax. The marked therapeutic efficacy of the fluoroquinolones and the high percentage of the animal protection after their urgent prophylactic use in a single dose are their obvious merits. The total values of the protective efficacy of the three fluoroquinolones with respect to anthrax were practically the same.

Asporogenic B anthracis expression system

Abstract: This invention relates to a bacterial expression system for production of protective antigen (PA) against bacillus anthracis . Recombinant asporogenic B. anthracits that are derived from ΔSterne-1(pPA102) and show inability to bind the dye when grown on Congo Red Agar can be screened and asporogenic strains isolated using methods of the invention. organisms of the invention lacking spore-forming function may be killed by heat shock at temperatures as low as 60° C. for 60 minutes. Hence, contamination of the environment with viable spore-forming organisms is easily avoided and decontamination is easily accomplished.

Anthrax toxin mechanisms of receptor binding and internalization

Abstract: The anthrax toxin complex contains two catalytic proteins, edema factor (EF) and lethal factor (LF), which must be translocated to the cytosol of eukaryotic cells to cause toxicity. The third protein component of the toxin, protective antigen (PA), achieves this internalization. PA binds to cell surface receptors, is cleaved after the sequence Argl64-Lysl65-Lysl66-Argl67 by a cell surface protease, and thereby exposes a binding site to which EF or LF bind with high affinity. The complexes enter endosomes, which then become acidified, causing transfer of LF and EF to the cytosol.

[A species-specific DNA probe for identifying toxic strains of anthrax pathogens].

Abstract: On the plasmid DNA pOX01 of the anthrax pathogen two BamHI fragments were localized which facilitate detection of the Bacillus anthracis strains carrying pXO1 replicon. These fragments, after complete hydrolysis of plasmid DNA by HindIII, were cloned on the vector plasmids pUC19 and pBR322 by the "shot-gun" method in Escherichia coli cells. It is shown that the 900 bp BamHI/HindIII fragment from the pZAT1 recombinant plasmid has an ability for specific hybridization with DNA of toxigenic strains of B. anthracis and could be used as species-specific anthracic DNA probe which identifies toxigenic strains of the anthrax pathogen differentiating it from the other species of Bacillus genus as well as from the bacteria of other taxonomy groups.