Showing papers on "Bacillus anthracis published in 1996"

Identification and characterization of Bacillus anthracis by multiplex PCR analysis of sequences on plasmids pXO1 and pXO2 and chromosomal DNA

Abstract: Bacillus anthracis can be identified on the basis of the detection of virulence factor genes located on two plasmids, pXO1 and pXO2. Thus isolates lacking both pXO1 and pXO2 are indistinguishable from closely related B. cereus group bacteria. We developed a multiplex PCR assay for characterization of B. anthracis isolates, and simultaneous confirmation of the species identity independent of plasmid content. The assay amplifies lef, cya, pag (pXO1) and cap (pXO2) genes, and a B. anthracis specific chromosomal marker, giving an easy-to-read profile. This system unambiguously identified virulent (pXO1+/2+) and avirulent (pXO1+/2−, pXO1−/2+ and pXO1−/2−) strains of B. anthracis and distinguished ‘anthrax-like’ strains from other B. cereus group bacteria.

Identification of a region of genetic variability among Bacillus anthracis strains and related species.

Abstract: The identification of a region of sequence variability among individual isolates of Bacillus anthracis as well as the two closely related species, Bacillus cereus and Bacillus mycoides, has made a sequence-based approach for the rapid differentiation among members of this group possible. We have identified this region of sequence divergence by comparison of arbitrarily primed (AP)-PCR "fingerprints" generated by an M13 bacteriophage-derived primer and sequencing the respective forms of the only polymorphic fragment observed. The 1,480-bp fragment derived from genomic DNA of the Sterne strain of B. anthracis contained four consecutive repeats of CAATATCAACAA. The same fragment from the Vollum strain was identical except that two of these repeats were deleted. The Ames strain of B. anthracis differed from the Sterne strain by a single-nucleotide deletion. More than 150 nucleotide differences separated B. cereus and B. mycoides from B. anthracis in pairwise comparisons. The nucleotide sequence of the variable fragment from each species contained one complete open reading frame (ORF) (designated vrrA, for variable region with repetitive sequence), encoding a potential 30-kDa protein located between the carboxy terminus of an upstream ORF (designated orf1) and the amino terminus of a downstream ORF (designated lytB). The sequence variation was primarily in vrrA, which was glutamine- and proline-rich (30% of total) and contained repetitive regions. A large proportion of the nucleotide substitutions between species were synonymous. vrrA has 35% identity with the microfilarial sheath protein shp2 of the parasitic worm Litomosoides carinii.

Anthrax toxin-mediated delivery of a cytotoxic T-cell epitope in vivo

Abstract: The protective antigen (PA) component of anthrax toxin mediates entry of the toxin's lethal factor (LF) and edema factor into the cytosolic compartment of mammalian cells. The amino-terminal domain of LF (LFn; 255 amino acids) binds LF to PA, and when fused to heterologous proteins, the LFn domain delivers such proteins to the cytoplasm in the presence of PA. In the current study, we fused a 9-amino acid cytotoxic T-lymphocyte (CTL) epitope (LLO91-99) from an intracellular pathogen, Listeria monocytogenes, to LFn and measured the ability of the resulting LFn-LLO91-99 fusion protein to stimulate a CTL response against the epitope in BALB/c mice. As little as 300 fmol of fusion could stimulate a response. The stimulation was PA-dependent and occurred with the peptide fused to either the amino terminus or the carboxyl terminus of LFn. Upon challenge with L. monocytogenes, mice previously injected with LFn-LLO91-99 and PA showed a reduction of colony-forming units in spleen and liver, relative to nonimmunized control mice. These results indicate that anthrax toxin may be useful as a CTL-peptide delivery system for research and medical applications.

Characterization of Lethal Factor Binding and Cell Receptor Binding Domains of Protective Antigen of Bacillus anthracis using Monoclonal Antibodies

Abstract: Lethal toxin from Bacillus anthracis is composed of protective antigen (PA) and lethal factor (LF). Anti-PA mAbs that neutralized lethal toxin activity, either in vivo or in vitro identified three non-overlapping antigenic regions on PA. Two distinct antigenic regions were recognized by the four mAbs that neutralized lethal toxin activity by inhibiting the binding of 125I-LF to cell-bound PA. Mapping showed that one mAb, 1G3PA63, recognized an epitope on a 17 kDa fragment located between amino acid residues Ser-168 and Phe-314. The three other mAbs, 2D3PA, 2D5PA and 10D2PA, recognized an epitope between amino acids Ile-581 and Asn-601. A single antigenic region was recognized by the three mAbs, 3B6PA, 14B7PA and 10E10PA63, that inhibited binding of 125I-PA to cells. This region was located between amino acids Asp-671 and Ile-721. These results confirm previously defined functional domains of PA and suggest that LF may interact with two different sites on PA to form lethal toxin.

Isolation of a specific chromosomic DNA sequence of Bacillus anthracis and its possible use in diagnosis.

Abstract: A 277-bp long DNA fragment, Ba813, was isolated from an avirulent Bacillus anthracis strain 7700 genomic library. Two oligonucleotides derived from the Ba813 sequence were used as primers in polymerase chain reaction tests on genomic DNA from 28 Bacillus anthracis and from 33 heterologous bacteria strains. A specific, 152-bp long DNA fragment was amplified only when Bacillus anthracis DNA was used as the target. The amplified product was analysed by non-radioactive sandwich hybridisation in microtiter plates using two oligonucleotides. The capture oligonucleotide C1 was covalently linked onto aminated wells of microtiter plates. The detection oligonucleotide D3 was labelled with biotine. The hybrid molecules were detected by avidine conjugated with alkaline phosphatase and chromogenic substrate. Amplification of Ba813 sequence may provide the basis for rapid and reliable assay for the detection and identification of Bacillus anthracis.

Efficacy of a standard human anthrax vaccine against Bacillus anthracis aerosol spore challenge in rhesus monkeys

Abstract: In conducting research using animals, the investigators adhered to the “Guide for the Care and Use of Laboratory Animals,” prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (NIH Publication No. 8623, revised 1985). The views, opinions and/or findings contained in this publication are those of the authors and should not be construed as an official Department of the Army position, policy or decision unless so designated by other documentation.

Differential influence of the two Bacillus anthracis plasmids on regulation of virulence gene expression.

Abstract: Fully virulent Bacillus anthracis bacilli are encapsulated and toxinogenic. These bacteria contain two plasmids, pXO1 and pXO2, carrying genes coding for toxins (pag, lef, and cya) and for capsule synthetic enzymes (capB, capC, capA, and dep), respectively. A transcriptional fusion between the capB regulatory region and the lacZ reporter gene was constructed to study the regulation of capsule synthesis. A single copy of this fusion was inserted into the cap region of pXO2. The influence of environmental factors on the capB-lacZ fusion expression was initially analyzed in a pXO1-negative background: bicarbonate but not temperature induced the transcription from the capB promoter. A strain carrying the recombinant pXO2 and (delta)pag pXO1 was constructed for transregulatory studies. The pXO1 plasmid strongly enhanced capsule formation without modifying the bicarbonate-dependent induction level. A (delta)cap pXO2 was transduced into a strain containing pXO1 harboring a pag-lacZ transcriptional fusion (19). pXO2 showed no influence on the toxin gene transcription.

Cytotoxic effects of anthrax lethal toxin on macrophage-like cell line J774A.1.

Abstract: The cytotoxic effects of anthrax lethal toxin purified from an avirulent strain were examined on mouse macrophage-like J774A.1 cells. Cell death induced by high concentration of purified lethal toxin had the characteristics of necrosis. At lower concentrations, the toxin caused no morphological change and most of the cells were viable. Interestingly, apoptotic cells were observed when the cells were preincubated with a serine/threonine phosphatase inhibitor, calyculin A, and then exposed to a toxin concentration of 0.1 microg/ml. This is the first report that lethal toxin of the anthrax bacillus can induce both necrosis and apoptosis and that protein phosphatases are implicated in the regulation of bacterial toxin-induced apoptosis.

The cytotoxic activity of Bacillus anthracis lethal factor is inhibited by leukotriene A4 hydrolase and metallopeptidase inhibitors.

Abstract: The lethal factor of Bacillus anthracis is central to the pathogenesis of anthrax. Its mechanism of action is still unknown. Recently, on the basis of sequence similarities, we suggested that lethal factor might act similarly to leukotriene A4 hydrolase (LTA4), a bifunctional enzyme also endowed with a metallopeptidase activity. Here we show that some inhibitors of the LTA4 hydrolase and metallopeptidase activities of LTA4 hydrolase also affect the cytotoxicity of the anthrax lethal factor on macrophage cell lines, without interfering with the ability of the lethal factor to enter cells. These results support the proposal that anthrax lethal factor might display in the cytosol of intoxicated cells a peptidase activity similar to that of LTA4 hydrolase.

φ20, A Temperate Bacteriophage Isolated from Bacillus anthracis Exists as a Plasmidial Prophage

Abstract: This study describes the isolation of temperate B. anthracis phages, from 4 out of 20 B. anthracis strains screened, by use of the inducing agents mitomycin C and UV light. Phage φ20 isolated from B. anthracis Sterne 34F2 (pXO1+ pXO2−) was shown to have double-stranded DNA of size 48756 bp and a restriction site map showing nine sites for enzymes BamHI, BglII, and SstI is included. The φ20 genome was found to exist as a plasmidial prophage and the phage itself to have a polyhedral head of diameter 65 nm and tail 217 nm long and 15 nm wide.

The Anthrax Toxin

Abstract: Anthrax is a disease known since antiquity1 and one of the first bacterial infections whose etiology was definitively established. The disease is caused by the Gram-positive, aerobic, spore-forming Bacillus anthracis, first isolated in 1877 by Robert Koch.2 The study of anthrax led to the establishment of Koch’s postulates, a set of criteria for identifying an organism as the causative agent of a specific infection.2 Louis Pasteur’s use of heat-inactivated anthrax cultures to immunize against the disease is generally credited as the first instance of a bacterial vaccine.3 Anthrax is primarily a disease of herbivorous animals, particularly sheep and cattle.4 Humans may acquire the disease from infected animals, typically as a cutaneous infection characterized by black pustules5 (whence the naming of the disease after the Greek word for “coal”). The pulmonary infection known as wool-sorter’s disease results from the inhalation of anthrax spores, often as a result of handling contaminated raw wool, hides or animal hair, and can lead to death within days.6–8 Though increasingly rare in human populations, anthrax remains of interest for several reasons, including its continuing incidence in animal populations,9 interest in improving the efficacy of the human vaccine,4,10 the threat of its use as a weapon of biological warfare (see ref. 11), its potential applications in the development of new therapeutic strategies such as targeted toxins,12 and as an experimental system for studying molecular pathogenesis.13

Behavior of heterologous recombinant plasmid pCET in cells of Bacillus anthracis

Abstract: Recombinant plasmid pCET was constructed in vivo in cells of enteric and hay bacillus, on the basis of plasmids pC194, and pBC16. Plasmid pCET inherits marker genes of antibiotic resistance from parental plasmids. Anthrax cells were transformed by the recombinant plasmid developed. The behavior of this plasmid was studied in vegetative Bacillus anthracis cells, which did not pass through the sporulation stage and were cultivated at temperatures permissive for the replicon of plasmid pE194. Under these conditions, plasmid pCET was shown to replicate autonomously, regardless of the host chromosome, and to retain its structure, irrespective of the recipient strain. In this case, the phenotype of transformants fully corresponded to the genotype of plasmids inherited. Elevation of the cultivation temperature of strains Bac, anthracis (pCET) up to 44 degrees C led to the elimination of plasmid pCET from cells of anthrax microbe under conditions nonselective for plasmid pCET and its integration with the host chromosome under selective conditions. The frequency of plasmid pCET integration into the chromosome was approximately 10(-1) for all Bac. anthracis strains studied. In population of vegetative cells of strains Bac. anthracis (pCET), which passed through the sporulation stage under selective for plasmid pCET conditions, DNA of plasmid pCET was detected only in the state integrated with the chromosome. Irrespective of the reasons leading to the integration of plasmid pCET into the Bac. anthracis chromosome, all strains inheriting this DNA within their own genome lost the resistance to tetracycline observed in strains with the extrachromosomal plasmid location. Genome amplification of plasmid pCET in the chromosome of Bac. anthracis was detected.

Bacterial toxins deliver the goods.

Abstract: Some toxins produced by bacteria are so potent that scientists have long considered putting them to good use. This became possible when their structures were determined through the combined use of molecular biology and structural biochemistry. The current status of targeted toxin technology is perhaps best illustrated by exotoxin A produced by Pseudomonas aeruginosa, a Gram-negative bacterium that frequently infects superficial wounds and especially bums, and causes serious lung infections in cystic fibrosis patients. Exotoxin A, one of the huge battery of virulence factors produced by P. aeruginosa, is a 66-kDa protein composed of three important domains, one required for binding to the cell surface, one for intoxication of the target cell (through ADP ribosylation of elongation factor 2 with consequent arrest of protein synthesis), and one for translocation of the intoxication domain into the cell. Toxin adsorbed to its receptor is endocytosed by means of coated pits into endosomes, reduced, and then processed [by furin proteases (1, 2)] to release the translocation and intoxication domains that are translocated into the cytosol. Only the second and third domains are crucial for translocation and toxicity; the first domain can be artificially replaced by others that allow the chimeric toxin to bind to receptors different from those to which the toxin normally binds. This seminal observation by Ira Pastan and his colleagues at the National Institutes of Health (Bethesda, MD) (3) led to the creation of chimeric toxins that kill very specific cell types (4). Some of the most impressive examples are hybrid toxins in which the receptor binding domain is the Fv region of monoclonal antibodies raised against proteins that are only present on the surface of specific cell types (the so-called single-chain immunotoxins) (5). In other chimeras, the receptor-recognition domain is a ligand that binds to a cell typespecific receptor (6). Chimeras produced by recombinant bacteria form cytoplasmic inclusion bodies that can be separated relatively easily from other proteins, denatured, and then renatured to produce fully active toxins. These \"magic bullet\" toxins will revolutionize some aspects of cancer chemotherapy. The basic technology is also likely to have a major impact on the analysis of cell and tissue development, in studies of degenerative disorders (7) and as a method for modulating gene expression, or for the treatment of genetic disorders leading to the loss of an essential protein. Most importantly, as pointed out in the article in this issue of the Proceedings from John Colliers' laboratory at Harvard Medical School (8), this and related methods of introducing proteins directly into the cytosol do not have any of the deleterious effects on cell viability that are associated with other methods, such as partial permeabilization of the plasma membrane, which are difficult to control and that induce leakage of cell contents. Many toxins produced by Gram-negative bacteria are composed of multiple subunits that assemble, prior to their release, in the periplasm between the two membranes of the cell envelope. Indeed, the high viscosity, high protein concentration, and plethora of folding catalysts in the periplasm provide an ideal environment for the assembly of multimeric protein complexes such as toxins (9). Gram-positive bacteria do not produce preassembled multisubunit toxins but some produce so-called binary toxins, the intoxication component of which interacts with its cognate receptor recognition and uptake component only after the latter has bound to its receptor on the target cell surface. The binary anthrax toxin studied by Collier's group is produced by the spore-forming Grampositive bacterium Bacillus anthracis, the highly infectious causative agent of anthrax in domestic livestock, wild animals and, occasionally, man. The bacterium usually infects minor cutaneous wounds, leading to the formation of pustules with a characteristic black eschar of dead or disorganized tissue at its center. Most of the clinical symptoms are caused by the toxin, a mixture of three proteins that act in synergy (10): edema factor (EF), a calcium and calmodulin-dependent adenylate cyclase that dramatically increases cellular cAMP levels; lethal factor (LF), which appears to be a zinc-dependent, presumably target-specific protease; and the receptor component, the so-called protective antigen (PA). PA (83 kDa) must be cleaved (again by furin proteases) to give the carboxyl terminal, active 63-kDa form (PA63), which probably remains bound to the receptor, before EF or PA can bind to it (1). The complex is then endocytosed, whereupon the acidic environment of the endosome triggers the insertion of PA into the membrane and permits the translocation of EF or LF into the cytosol. PA63 can form channels in artificial membranes at low pH, conditions that also lead to the formation of stable heptameric rings with a 12A-channel through which LF or EF are probably transported (11, 12). In support of this idea, an N-terminal fragment of EF (see below) interacts with PA63 in a voltage-dependent manner (11), and binding of LF to PA63 at low pH prevents solute and ion flux through the channel (13). Some studies indicate that both LF and EF might also penetrate into the lipid bilayer (14), but the results reported by Blanke et al. (8) indicate that any contribution they make to channel formation is negligible. How then do LF and EF recognize the same receptor (PA) in order to be internalized? The considerable sequence similarity between their N-terminal regions (15) led Stephen Leppla and his colleagues at the National Institutes of Health to propose that this could be a common receptor (PA)recognition domain. In support o-f this idea, they found that the ADP ribosylation domain of exotoxin A could be converted into a potent PA-dependent toxin by fusing it to the first 254 amino acids of LF (16). This phenomenon is not specific to the exotoxin A fragment and is independent of the end of the LF fragment to which the heterologous fragment is fused (17, 18). Collier's group previously found that the 255 N-terminal amino acids of LF were capable of inducing PA-dependent internalization of the ADP ribosylation domain of another bacterial toxin, diphtheria toxin (DT) (18). The DT fragment alone was not toxic, but the addition of a polyhistidine tag (to allow its facile purification by nickel chelate affinity chromatography) caused the reappearance of low but measurable levels of toxicity. This activity was dramatically increased when the histidines were replaced by more strongly-charged lysines, indicating that the tagged DT fragment probably binds to the surface of the target cells by electrostatic interactions and is then internalized. The observation is made even more astounding by the fact that the action of the polylysine-tagged DT

Protective immunity induced by Bacillus anthracis toxin mutant strains.

Abstract: Bacillus anthracis is the etiological agent of anthrax, a disease often fatal in humans and many animals species. Fully virulent strains of this pathogen harbor two plasmids, pXO1 and pXO2, coding for the production of two toxins and D-glutamic acid polymer capsule, respectively. The two toxins, edema and lethal toxin, are secreted by B. anthracis and are composed of three distinct proteins, protective antigen (PA; 85 kDa), lethal factor (LF; 83 kDa) and edema factor (EF; 89 kDa). PA combined with LF forms the lethal toxin (Beali et al., 1962; Smith & Stoner, 1967), whereas edema toxin consists of PA and EF. Both toxins are organized according to the A-B type model (Gill, 1978). PA represents a common B component, with receptor-binding activity, and mediates entry of either LF or EF into target cells (Leppla, 1984). EF has been shown to be a calmodulin-dependent adenylate cyclase (Leppla, 1982). By sequence comparison, it has been suggested that LF is a metalloprotease (Klimpel et al., 1994).

The characteristics of the interaction of Bacillus anthracis with host phagocytes in relation to the plasmid spectrum of the causative agent

Abstract: The study revealed that after the intraperitoneal inoculation of spores of B. anthracis strains with different plasmid composition into guinea pigs the active germination of the spores both outside and inside the cells of the host occurred as early as on hour 2 of their interaction with the macroorganism. The further fate of the infective agent and the character of its interaction with peritoneal exudate cells depended on the plasmid composition of the bacilli. Thus, the presence of toxin-formation plasmid PXO1 and capsule-formation plasmid PXO2 in B. anthracis permitted the successful adaptation of these bacilli to the conditions of the macroorganism, while the absence of such plasmids made them nonviable in this environment. The presence of plasmid PXO1 in B. anthracis permitted the manifestation of their cytopathic effort on the cells and the presence of plasmid PXO2 only gave the bacilli incomplete protection from phagocytosis.

Bacillus anthracis and its possible use m alagnosls

Abstract: A 277-bp long DNA fragment, Ba813, was isolated from an avirulent Bacillus anthracis strain 7700 genomic library. Two oligonucleotides derived from the Ba8 13 sequence were used as primers in polymerase chain reaction tests on genomic DNA from 28 Bacillus ant/track and from 33 heterologous bacteria strains. A specific, 152-bp long DNA fragment was amplified only when Bacillus anthracis DNA was used as the target. The amplified product was analysed by non-radioac- tive sandwich hybridisation in microtiter plates using two oligonucleotides. The capture oligonucleotide Cl was covalently linked onto aminated wells of microtiter plates. The detection oligonucleotide D3 was labelled with biotine. The hybrid molecules were detected by avidine conjugated with alkaline phosphatase and chromogenic substrate. Amplification of Ba8 13 sequence may provide the basis for rapid and reliable assay for the detection and identification of Bacillus anfhracis.