Showing papers on "Bacillus anthracis published in 1989"
TL;DR: It was found that the capsule of B. anthracis conferred strong resistance to phagocytosis upon the bacterial host, and these three cistrons appeared to be membrane-associated enzymes mediating the polymerization of D-glutamic acid via the membrane.
Abstract: By using genetic complementation tests with various in vitro-constructed mutants with mutations in the cap region (which is essential for encapsulation in Bacillus anthracis), we identified three cistrons, capB, capC, and capA, in this order of arrangement. Minicell analysis revealed that these cistrons produce proteins of 44, 16, and 46 kilodaltons, respectively. The complete nucleotide sequence of 3,244 base pairs covering the whole cap region was determined and revealed the existence of the three open reading frames of capB (397 amino acid residues; molecular weight, 44,872), capC (149 amino acid residues; molecular weight, 16,522), and capA (411 amino acid residues; molecular weight, 46,420) arranged in the order predicted by complementation tests. These three cistrons were all transcribed in the same direction from promoters unique to each cistron. Judging from the predicted amino acid sequence of the three proteins and from their localization and their sensitivity to various physicochemical treatments, they appeared to be membrane-associated enzymes mediating the polymerization of D-glutamic acid via the membrane. Capsular peptides immunologically identical to that of B. anthracis were found in B. subtilis, B. megaterium, and B. licheniformis, but no sequence homologous to the cap region was found in any of these bacilli other than B. anthracis. Using strains of B. anthracis with or without insertional inactivation of the cap region, we found that the capsule of B. anthracis conferred strong resistance to phagocytosis upon the bacterial host.
331 citations
TL;DR: Deleted PA is expected to have normal immunogenicity, but would not combine with trace amounts of LF and EF to cause toxicity and be useful as a replacement for the PA that is the major active ingredient in the current human anthrax vaccine.
164 citations
TL;DR: The nucleotide sequence of the Bacillus anthracis lethal factor (LF) gene (lef) has been determined and the codon usage of the LF gene reflects its high A + T content.
135 citations
TL;DR: The data demonstrate that the requirements for intoxication by the AC toxins from B. anthracis and B. pertussis are fundamentally different and provide a perspective for new approaches to study the entry processes.
111 citations
TL;DR: The results suggest that resistant cells either lack the intracellular target of LF or fail to process LF to an active form, and suggests that protective antigen may also be required at a stage subsequent to endocytosis.
109 citations
TL;DR: The large toxin plasmid, pXO1, found in virulent strains of B. anthracis, was required for stimulation of transcription of pag by bicarbonate, suggesting the existence of a trans-acting factor that is involved in the activation of pag transcription.
Abstract: Bicarbonate is required for production of the major virulence factors, the toxins and capsule, of Bacillus anthracis. In this study we examined the basis for stimulation of production of protective antigen (PA), a central component of the two anthrax toxins encoded by plasmid pXO1. RNA prepared from B. anthracis grown in media with and without added bicarbonate was probed for PA mRNA. Data showed that bicarbonate was required for increased transcription of the PA gene (pag) in minimal medium. Transcription of pag was low in rich medium and could not be stimulated by the addition of bicarbonate. To characterize further the factors required for transcriptional regulation of pag, the promoter region of pag was fused to the chloramphenicol acetyltransferase gene (cat-86) of vector pPL703 and transformed by electroporation into pXO1+ (Tox+) and pXO1- (Tox-) strains of B. anthracis. Analysis of chloramphenicol acetyltransferase produced by the pag-cat-86 fusion in each of these backgrounds confirmed the results obtained by hybridization. Data obtained with this fusion also revealed that the large toxin plasmid, pXO1, found in virulent strains of B. anthracis, was required for stimulation of transcription of pag by bicarbonate. This result suggests the existence of a trans-acting factor that is involved in the activation of pag transcription.
107 citations
TL;DR: Differences in neutralizing activity of monoclonal antibodies were related to their relative affinity and epitope specificity and the type of assay.
Abstract: The lethal toxin of Bacillus anthracis consists of two components, protective antigen and lethal factor. Protective antigen is cleaved after binding to cell receptors, yielding a receptor-bound fragment that binds lethal factor. Sixty-one monoclonal antibodies to the lethal factor protein have been characterized for specificity, antibody subtype, and ability to neutralize lethal toxin. Three monoclonal antibodies (10G3, 2E7, and 3F6) neutralized lethal toxin in Fisher 344 rats. However, in a macrophage cytolysis assay, monoclonal antibodies 10G3, 2E7, 10G4, 10D4, 13D10, and 1D8, but not 3F6, were found to neutralize lethal toxin. Binding studies showed that five of the monoclonal antibodies that neutralized lethal toxin in the macrophage assay (10G3, 2E7, 10G4, 10D4, and 13D10) did so by inhibiting the binding of lethal factor to the protective antigen fragment bound to cells. Monoclonal antibody 1D8, which was also able to neutralize lethal toxin activity after lethal factor was prebound to cell-bound protective antigen, only partially inhibited binding of lethal factor to protective antigen. Monoclonal antibody 3F6 did not inhibit the binding of lethal factor to protective antigen. A competitive-binding enzyme-linked immunosorbent assay showed that at least four different antigenic regions on lethal factor were recognized by these seven neutralizing hybridomas. The anomalous behavior of 3F6 suggests that it may induce a conformational change in lethal factor. Differences in neutralizing activity of monoclonal antibodies were related to their relative affinity and epitope specificity and the type of assay.
88 citations
TL;DR: It is concluded that calcium is required at several steps in the intoxication of cells by anthrax lethal toxin, including involvement of calmodulin at a step during intoxication.
Abstract: Anthrax lethal toxin, which consists of two separate proteins, protective antigen (Mr, 82,700) and lethal factor (Mr, approximately 83,000), is cytotoxic to the macrophagelike cell line J774A.1. Removal of calcium from the culture medium protected cells against the action of lethal toxin. Calcium depletion during the binding phase of intoxication afforded only partial protection. Further analysis showed that calcium removal caused some inhibition of protective antigen binding but that it had minimal effect on proteolytic conversion of protective antigen to the active 63-kilodalton fragment and that it had no effect on lethal factor binding. Cells to which lethal toxin had bound in the presence of calcium were protected when transferred to calcium-depleted culture medium, indicating a role for calcium at a postbinding stage. When ammonium chloride is present with lethal toxin, toxin accumulates in intracellular vesicles. Calcium-free medium protected these cells upon removal of the amine block, suggesting that calcium is also required at a step after internalization of lethal toxin. Calcium channel blockers inhibited 45Ca2+ uptake and protected cells against cytotoxicity. Calmodulin inhibitors also protected against the action of lethal toxin, suggesting involvement of calmodulin at a step during intoxication. We conclude that calcium is required at several steps in the intoxication of cells by anthrax lethal toxin.
77 citations
TL;DR: Examination of Tra- and Cry- derivatives of pXO12 showed that this fragment also harbored information involved in crystal formation and was adjacent to a restriction fragment containing DNA sequences carrying information required for conjugal transfer, which suggested that pBC16 was transferred by the process of donation and that the large B. anthracis plasmids were transferred bythe process of conduction.
Abstract: The self-transmissible plasmid pXO12 (112.5 kilobases [kb]), originally isolated from strain 4042A of Bacillus thuringiensis subsp. thuringiensis, codes for production of the insecticidal crystal protein (Cry+). The mechanism of pXO12-mediated plasmid transfer was investigated by monitoring the cotransfer of the tetracycline resistance plasmid pBC16 (4.2 kb) and the Bacillus anthracis toxin and capsule plasmids, pXO1 (168 kb) and pXO2 (85.6 kb), respectively. In matings of B. anthracis donors with B. anthracis and Bacillus cereus recipients, the number of Tcr transcipients ranged from 4.8 x 10(4) to 3.9 x 10(6)/ml (frequencies ranged from 1.6 x 10(-4) to 7.1 x 10(-2), and 0.3 to 0.4% of them simultaneously inherited pXO1 or pXO2. Physical analysis of the transferred plasmids suggested that pBC16 was transferred by the process of donation and that the large B. anthracis plasmids were transferred by the process of conduction. The transfer of pXO1 and pXO2 involved the transposition of Tn4430 from pXO12 onto these plasmids. DNA-DNA hybridization experiments demonstrated that Tn4430 was located on a 16.0-kb AvaI fragment of pXO12. Examination of Tra- and Cry- derivatives of pXO12 showed that this fragment also harbored information involved in crystal formation and was adjacent to a restriction fragment containing DNA sequences carrying information required for conjugal transfer.
37 citations
01 Jan 1989
TL;DR: Culture and serological results obtained during a 5-week field study on anthrax in the Etosha National Park are presented, finding vegetative forms of B. anthracis inoculated into water samples declined rapidly in number and spore forms showed no inclination to germinate.
Abstract: Culture and serological results obtained during a 5-week field study on anthrax in the Etosha National Park are presented. With one exception - a water sample from a natural fountain - Bacillus anthracis was only isolated from animal specimens or environmental samples associated with animals known to have or suspected of having died of anthrax and no environmental ""reservoir"" for the bacterium could be identified. In laboratory tests, vegetative forms of B. anthracis inoculated into water samples declined rapidly in number and spore forms showed no inclination to germinate.
37 citations
TL;DR: Evidence is produced that B. thuringiensis cells grown on nutrient agar possess more cross-reacting antigens than cells grown in nutrient broth, which is believed to be responsible for the disappearance of most or all of the brands in blots of these species.
Abstract: The extractable protein antigens EA1 and EA2 of Bacillus anthracis were prepared from electrophoresis transblots of SDS extracts of vegetative bacteria of the Sterne strain. Hyperimmune guinea-pig antiserum against EA2 failed to react with B. anthracis cells in immunofluorescence (IF) tests. Guinea-pig antiserum against EA1 (anti-EA1) reacted strongly in IF tests with non-encapsulated vegetative cell of 10 of 12 strains of B. anthracis and with cells of strains of B. cereus and B. thuringiensis. The unreactive B. anthracis strains were delta-Vollum-1B-1 and Texas. Encapsulated cells of B. anthracis stained poorly except for small bright regions. Absorption of anti-EA1 with cells of B. cereus NCTC 8035 and NCTC 9946 removed activity towards all B. cereus strains tested, but only partly reduced cross-reaction with B. thuringiensis strains. Absorption of anti-EA1 with B. thuringiensis 4041 removed activity towards this strain and B. cereus strains. Evidence is produced that B. thuringiensis cells grown on nutrient agar possess more cross-reacting antigens than cells grown in nutrient broth. The reaction of anti-EA1 with Bacillus spores immobilized in clumps on microscope slides was attributed to contaminating vegetative debris because well-separated individual spores failed to react. A rapid IF test was developed allowing identification of B. anthracis sampled from overnight cultures on blood plates. When sodium dodecyl sulphate extracts of B. anthracis vegetative cells were analysed on immunoblots (Western blots) by reaction with anti-EA1, a number of bands were visualized in addition to the expected 91 kiloDalton EA1 band. Prior absorption of anti-EA1 with B. cereus or B. thuringiensis cells resulted in the disappearance of most or all of the brands in blots of these species, but had less effect on blots of the B. anthracis strains. All six B. anthracis strains that were blotted including delta-Vollum-1B-1 and Texas, could thus be distinguished from B. cereus and B. thuringiensis by their differential reaction with unabsorbed and absorbed anti-EA1.
TL;DR: The data indicate that rat weight must be taken into account during in vivo assays of anthrax lethal toxin activity, in contrast to previous reports.
Abstract: Groups of Fischer 344 rats were injected intravenously with Bacillus anthracis culture supernatant containing crude anthrax toxin. Times to death of rats given identical toxin preparations varied directly with the weights of the rats (P = 0.0001). In contrast to previous reports, the data indicate that rat weight must be taken into account during in vivo assays of anthrax lethal toxin activity.
Journal Article•
TL;DR: The plasmid pXO2 determining the capsule synthesis has been shown to be transfered into the cells of different strains of Bacillus anthracis by the transducing bacteriophage CP54ant and by mobilization by pAM beta 1 replicon.
Abstract: The plasmid pXO2 determining the capsule synthesis has been shown to be transfered into the cells of different strains of Bacillus anthracis (STI-1, Sterne, KM33, KM35) by the transducing bacteriophage CP54ant and by mobilization by pAM beta 1 replicon with the frequencies, consequently, n.10(-8) and n.10(-7). The optimal parameters for the selection of clones having acquired the pXO2 plasmid have been defined. Mobilization for conjugational transfer has been demonstrated for the plasmid pXO1 coding for the production of Bacillus anthracis toxin. The dramatic increase of virulence for white mice has been registered for Bacillus anthracis strains having acquired the pXO2 plasmid replicon.
Journal Article•
TL;DR: Plasmid transduction by the mutant phage CP54ant was found to be more efficient as compared with the one by the parent phage and served as donors of the transduced plasmid for Bacillus anthracis and Bacillus thuringiensis strains.
Abstract: Possibility of plasmid transduction in Bacillus anthracis vaccine strains Sterne and STI-1 by bacteriophage CP54ant having an increased ability of adsorbtion and a shortened period of latent development in Bacillus anthracis cells has been isolated. The main parameters of plasmid transduction by the bacteriophage have been established for the plasmid pTG141 (TcR). They include the effect of multiplicity of infection, the level of UV-inactivation of bacteriophage, the presence of antiphage serum in the incubation medium. Plasmid transduction by the mutant phage CP54ant was found to be more efficient as compared with the one by the parent phage. The isolated transductants served as donors of the transduced plasmid for Bacillus anthracis and Bacillus thuringiensis strains.