Showing papers on "Bacillus anthracis published in 1993"
TL;DR: It is found that when mice were specifically depleted of macrophages by silica injections, they became resistant to the toxin, implying that systemic shock and death from anthrax result primarily from the effects of high levels of cytokines, principally IL-1, produced by macrophage that have been stimulated by the anthrax lethal toxin.
Abstract: Bacillus anthracis, the causative agent of anthrax, produces systemic shock and death in susceptible animals, primarily through the action of its lethal toxin. This toxin, at high concentrations, induces lysis of macrophages in vitro but shows little or no effect on other cells. We found that when mice were specifically depleted of macrophages by silica injections, they became resistant to the toxin. Sensitivity could be restored by coinjection of toxin-sensitive cultured macrophages (RAW 264.7 cells) but not by coinjection of other cell lines tested. These results implied that macrophages mediate the action of lethal toxin in vivo and led us to investigate their role in death of the mammalian host. Sublytic concentrations of lethal toxin, orders of magnitude lower than those required to induce lysis of RAW 264.7 cells, were found to induce these cells to express interleukin 1 (IL-1) and tumor necrosis factor in vitro. Passive immunization against IL-1 or injection of an IL-1 receptor antagonist protected mice from toxin challenge, whereas anti-tumor necrosis factor provided little, if any, protection. These results imply that systemic shock and death from anthrax result primarily from the effects of high levels of cytokines, principally IL-1, produced by macrophages that have been stimulated by the anthrax lethal toxin.
308 citations
TL;DR: Results demonstrate that the atxA product activates not only transcription of pag but also that of cya and lef, and plays a role in the induction of anthrax toxin gene expression by bicarbonate.
Abstract: The 184-kb Bacillus anthracis plasmid pXO1, which is required for virulence, contains three genes encoding the protein components of anthrax toxin, cya (edema factor gene), lef (lethal factor gene), and pag (protective antigen gene). Expression of the three proteins is induced by bicarbonate or serum. Using a pag-lacZ transcriptional construct to measure pag promoter activity, we cloned in Bacillus subtilis a gene (atxA) whose product acts in trans to stimulate anthrax toxin expression. Deletion analysis located atxA on a 2.0-kb fragment between cya and pag. DNA sequencing identified one open reading frame encoding 476 amino acids with a predicted M(r) of 55,673, in good agreement with the value of 53 kDa obtained by in vitro transcription-translation analysis. The cloned atxA gene complemented previously characterized Tn917 insertion mutants UM23 tp29 and UM23 tp32 (J. M. Hornung and C. B. Thorne, Abstr. 91st Gen. Meet. Am. Soc. Microbiol. 1991, abstr. D-121, p. 98), which are deficient in synthesis of all three toxin proteins. These results demonstrate that the atxA product activates not only transcription of pag but also that of cya and lef. beta-Galactosidase synthesis from the pag-lacZ transcriptional fusion construct introduced into an insertion mutant (UM23 tp62) which does not require bicarbonate for toxin synthesis indicated that additional regulatory genes other than atxA play a role in the induction of anthrax toxin gene expression by bicarbonate.
151 citations
TL;DR: The results suggest that macrophage resistance is due to a defect at a stage occurring after toxin internalization, and that A/J macrophages may lack the putative lethal factor target in the cytosol or be defective in the further processing or activation of lethal factor inThe cytosols or in endocytic vesicles.
Abstract: Anthrax lethal toxin, which consists of two proteins, protective antigen and lethal factor, is cytolytic for macrophages. Macrophages from different mouse strains were found to vary in their sensitivities to toxin. C3H mouse macrophages lysed by lethal factor concentrations of 0.001 micrograms/ml were 100,000 times more sensitive than those from resistant A/J mice. We analyzed various stages of the intoxication process to determine the basis for this resistance. Direct binding studies with radioiodinated protective antigen revealed that the affinity (Kd, approximately 0.5 nM) and number of receptors per cell (25,000 to 33,000) were the same in sensitive and resistant cells. Proteolytic activation of protective antigen by a cell surface protease and subsequent binding of lethal factor were also the same in both sensitive and resistant macrophages. Resistant A/J macrophages were not cross-resistant to other toxins and a virus which, like lethal toxin, require vesicular acidification for activity, implying that resistance is not due to a defect in vesicular acidification. When introduced into the cytosol by osmotic lysis of pinosomes, lethal factor in the absence of protective antigen was cytolytic for the sensitive macrophages while resistant cells were unaffected. Thus, lethal factor by itself possesses the toxic activity of lethal toxin. These results suggest that macrophage resistance is due to a defect at a stage occurring after toxin internalization. A/J macrophages may lack the putative lethal factor target in the cytosol or be defective in the further processing or activation of lethal factor in the cytosol or in endocytic vesicles.
143 citations
TL;DR: Although the biological functions of the dep gene are unknown, it is possible that low‐molecular‐weight, diffusible polyglutamates produced through the action of the Dep gene may act to inhibit host defence mechanisms.
Abstract: Summary
Bacillus anthracis produces a gamma-linked poly-D-glutamic acid capsule that is essential for virulence. A 6.2 kb fragment of B. anthracis DNA (cap), when present in Escherichia coli, produces a capsular polymer that is immunologically identical to that produced by B. anthracis. By immunodiffusion analysis of E. coli strains carrying varying portions of the cap region, we identified a novel gene (dep) responsible for degradation of the capsular polymer of B. anthracis. The simultaneous presence of the cap region and the dep gene caused production of low-molecular-weight, degraded capsular polymer both in E. coli and in B. anthracis, whereas the cap region atone caused production of a high-molecular-weight capsule. The dep gene mapped immediately downstream of the cap region within a 1.8 kb fragment and was transcribed in the same direction. This fragment was sequenced and a 1401 bp open reading frame (ORF) was found that is predicted to encode a peptide with molecular weight of 51460. By in vitro transcription-translation analysis, this ORF was shown to be the dep gene product. The deduced amino acid sequence of the dep product has sequence similarity to E. coli and mammalian γ-glutamyltranspeptidase (GGT). However, the Dep protein did not have GGT activity. The Dep protein appears to be an enzyme that catalyses the hydrolysis of the poly-D-glutamic acid capsule. Although the biological functions of the dep gene are unknown, it is possible that low-molecular-weight, diffusible polyglutamates produced through the action of the dep gene may act to inhibit host defence mechanisms.
120 citations
TL;DR: In this article, the authors used gas chromatography-mass spectrometry (GCMS) to identify Bacillus anthracis and Bacillus cereus, two closely related pathogenic organisms that are difficult to differentiate phenotypically or genotypically.
Abstract: Bacillus anthracis and Bacillus cereus are closely related pathogenic organisms that are difficult to differentiate phenotypically or genotypically. It is well known that vegetative and spore forms of bacilli are quite distinct both morphologically and chemically, but spore-specific chemical markers allowing these species to be distinguished have not been previously described. By using gas chromatography-mass spectrometry, vegetative cells and spores of the two species were shown to exhibit distinct carbohydrate profiles. Profiles of vegetative B. anthracis typically contained high levels of galactose but did not contain galactosamine, whereas B. cereus contained galactosamine and generally low levels of galactose. Spore cultures exhibited unique carbohydrate profiles compared with those of vegetative cultures. B. anthracis spore profiles contained rhamnose alone, whereas B. cereus spore profiles contained rhamnose and fucose. Additionally, two spore-specific O-methylated methylpentoses were discovered. Both B. anthracis and B. cereus spores contained 3-O-methyl rhamnose, whereas B. cereus spores also contained 2-O-methyl rhamnose. Carbohydrate profiling is demonstrated to be a powerful tool for differentiating the two closely related species. Differentiation does not depend on whether organisms are in the vegetative or spore stage of growth.
97 citations
TL;DR: Using a simple, two-step protocol, double mutant strains of B. anthracis producing only one toxin component have been constructed and characterization of the mutant strains indicated that they produced the expected single toxin protein.
Abstract: SUMMARY: The two protein exotoxins secreted by Bacillus anthvacis are composed of three distinct components: protective antigen (PA), lethal factor (LF), and (o)edema factor (EF). We have developed a genetic strategy that permits us selectively to inactivate each of the genes coding for PA, EF or LF. This strategy involved the deletion of a portion of the structural gene and the insertion of an antibiotic resistance cassette. With this technique, double mutant strains of B. anthracis producing only one toxin component have been constructed. Characterization of the mutant strains indicated that they produced the expected single toxin protein. Using a simple, two-step protocol, we have purified PA, LF and EF to homogeneity from culture supernatants. These three mutant strains are potentially powerful tools for studying the individual effect of each toxin component in vitro and in vivo.
67 citations
TL;DR: It was possible to amplify the 288-bp DNA segment from samples containing very few bacteria, as few as only 1 sporeforming unit, indicating that the PCR detection method developed in this study will permit the monitoring of B. anthracis contamination in the environment.
Abstract: Bacillus anthracis is a soil pathogen capable of causing anthrax. To establish a method for specifically detecting B. anthracis for practical applications, such as for the inspection of slaughterhouses, the cap region, which is essential for encapsulation in B. anthracis, was used in a DNA hybridization study by polymerase chain reaction (PCR). Oligonucleotide primers were designed to amplify a 288-bp DNA fragment within the capA gene by PCR. The amplified DNA sequence specifically hybridized to the DNA of B. anthracis but not to that of other bacterial strains tested. Since this PCR-based method efficiently and specifically detected the capA sequence of bacteria in blood and spleen samples of mice within 8 h after the administration of live B. anthracis, this PCR system could be used for practical applications. By using lysis methods in preparing the samples for PCR, it was possible to amplify the 288-bp DNA segment from samples containing very few bacteria, as few as only 1 sporeforming unit, indicating that the PCR detection method developed in this study will permit the monitoring of B. anthracis contamination in the environment.
67 citations
TL;DR: Bordetella pertussis and Bacillus anthracis each produce a virulence-associated, calmodulin-dependent adenylate cyclase toxin, which generates increased levels of cyclic AMP in eukaryotic cells.
57 citations
TL;DR: A non-radioactive hybridization and detection system based on digoxigenin-11-dUTP was developed that could differentiate between virulent and avirulent strains of Bacillus anthracis.
Abstract: Two DNA probes and a number of oligonucleotide probes were designed from the virulence factor genes of Bacillus anthracis. These probes were tested for specificity against 52 B. anthracis strains and 233 Bacillus strains encompassing 23 other species. A rapid slot blotting technique was used for screening the large numbers of isolates involved. All probes tested appeared to be specific for B. anthracis under high stringency conditions. These probes could differentiate between virulent and avirulent strains. The probes were also applied to the detection of B. anthracis in routine environmental and clinical samples. A non-radioactive hybridization and detection system based on digoxigenin-11-dUTP was developed.
31 citations
TL;DR: The inducible macrolide-lincosamide-streptogramin B resistance determinant, ermJ, from Bacillus anthracis 590 was cloned in Escherichia coli CSH26, suggesting that ermK-like genes have been distributed in Bacillus strains by transposition.
Abstract: Summary: The inducible macrolide–lincosamide–streptogramin B resistance determinant, ermJ, from Bacillus anthracis 590 was cloned in Escherichia coli CSH26. The DNA sequence of ermJ was similar to that of ermK or ermD from B. licheniformis, suggesting that ermK-like genes have been distributed in Bacillus strains by transposition. Expression of ermJ was achieved in a B. subtilis minicell system, and the rRNA methyltransferase product of ermJ was purified. The molecular mass of the enzyme was 58 kDa, and it was concluded to be a homodimer. Its biochemical characteristics were different from those of ermC methyltransferase.
28 citations
TL;DR: The patient, a 63-year-old male Hispanic from southwest Texas, was a sheep shearer and had a recent history of dissecting sheep that had died suddenly, and experienced an illness characterized by left arm pain and edema, with cellulitis and lymphadenopathy.
Abstract: In December 1988 an indigenous case of cutaneous anthrax was identified in Texas. The patient, a 63-year-old male Hispanic from southwest Texas, was a sheep shearer and had a recent history of dissecting sheep that had died suddenly. He experienced an illness characterized by left arm pain and edema. A necrotic lesion developed on his left forearm, with cellulitis and lymphadenopathy. After treatment with oral and intravenous penicillins, the patient fully recovered. Western blot testing revealed a fourfold or greater rise in antibody titer to Bacillus anthracis protective antigen and lethal factor. This represents the first case of indigenous anthrax in Texas in more than 20 years.
Journal Article•
TL;DR: The therapeutic effect of benzylpenicillin in the treatment of the experimental infection induced by the B. anthracis strain producing penicillinase was estimated and the efficacy was shown to depend on the time of the beginning of the antibiotic therapy.
Abstract: Interaction of the cells of Bacillus anthracis strain CH-7 with benzylpenicillin was studied. The cells of strain CH-7 were shown to contain the penicillinase gene in the repressed state. Spontaneous derepression of the gene at a rate of 10(-8) resulting in the synthesis of penicillinase was observed. Penicillinase was synthesized constitutionally and its synthesis did not depend on the presence of benzylpenicillin in the cultivation medium. The therapeutic effect of benzylpenicillin in the treatment of the experimental infection induced by the B. anthracis strain producing penicillinase was estimated. The efficacy was shown to depend on the time of the beginning of the antibiotic therapy. When the clinical signs of the infection were evident in the animals contaminated with the penicillinase-producing strain of B. anthracis, their treatment with the mean daily doses of benzylpenicillin failed.
TL;DR: Anthrax in the Sudan was first recognized in 1917 and was then annually reported nation-wide affecting many kinds of domestic animals and wildlife, hence the disease was controlled in quarantines where it was predominant.
Abstract: Anthrax in the Sudan was first recognized in 1917 (1) and was then annually reported nation-wide affecting many kinds of domestic animals and wildlife (2). In 1946 (3), it was decided to vaccinate all export animals, hence the disease was controlled in quarantines where it was predominant. Mass vaccination of animals in the field was started in 1951 (4), however the disease is still sporadically occurring throughout the country. Similarly, it was found widely spread in West Africa (19, 20).
Journal Article•
TL;DR: The RifR mutation in B. anthracis did not result in cross resistance to penicillins, cephalosporins, tetracyclines, aminoglycosides, macrolides and chloramphenicol.
Abstract: Formation of spontaneous RifR mutants was detected in the populations of various strains of Bacillus anthracis (STI-1, Sterne and CH-7) at a rate of 10(-8) per 1 CFU. The levels of the rifampicin resistance in the mutants were different, the MIC ranged from 16 to 512 micrograms/ml. The clones of the RifR population of the virulent strain CH-7 were heterogeneous in the morphological properties of the colonies and cells, the capacity for the synthesis of the toxin and capsule, the sporulation and virulence. The heterogeneity did not correlate with the levels of the antibiotic resistance. Among the clones of the RifR population there were detected deletion variants by the capacity for the synthesis of the toxin and capsule along with the complete ones. The rifampicin therapy of the infection caused by the complete clone was not efficient. The RifR mutation in B. anthracis did not result in cross resistance to penicillins, cephalosporins, tetracyclines, aminoglycosides, macrolides and chloramphenicol.
TL;DR: An enzyme-linked immunosorbent assay for anthrax antibody in white-tailed deer was developed and used to evaluate a vaccination study and compare sera from hunter-killed deer in anthrax endemic and non-endemic areas.
Abstract: An enzyme-linked immunosorbent assay for anthrax antibody in white-tailed deer (Odocoileus virginianus) was developed and used to evaluate a vaccination study and compare sera from hunter-killed deer in anthrax endemic and non-endemic areas. Deer subcutaneously vaccinated with anthrax avirulent spore vaccine developed specific antibody to protective antigen (PA) which was significantly higher than the non-vaccinated controls at 30, 60, 90, and 240 days post-vaccination. There was no difference between the levels of antibody to PA between deer in anthrax endemic and non-endemic areas.
Journal Article•
TL;DR: A determinant for a protective antigen of Bacillus anthracis STI has been cloned and the hybrid plasmids obtained carrying the different fragments of the gene still code for a part of protective antigen preserving the immunological and protective properties.
Abstract: A determinant for a protective antigen (pag) of Bacillus anthracis STI has been cloned. Its expression in Escherichia coli, Bacillus subtilis and Bacillus anthracis cells has been studied. The hybrid plasmids were obtained carrying the different fragments of the gene. The plasmids pPA2 and pPA3 having the 3'-end fragment of pag deleted (the size of 1 kb) still code for a part of protective antigen preserving the immunological and protective properties.
Patent•
04 Mar 1993
TL;DR: In this article, a new oligonucleotide having high specificity as a primer for detecting Bacillus anthracis was obtained by chemically synthesizing two kinds of synthetic oligon nucleotides (MO1 and M02) with high contents of guanine and cytosine residues from 288 bases of the base sequence in the capA gene.
Abstract: PURPOSE:To obtain a new oligonucleotide having high specificity as a primer for detecting Bacillus anthracis. CONSTITUTION:This oligonucleotide is complementary to a nucleotide sequence capable of coding a capA gene of a plasmid of Bacillus anthracis and has a sequence group expressed by the formula or a complementary sequence corresponding thereto. The polynucleotide is obtained by chemically synthesizing two kinds of synthetic oligonucleotides (MO1 and M02) with high contents of guanine and cytosine residues from 288 bases of the base sequence in the capA gene of the Bacillus anthracis according to a triester method using a DNA synthesizer manufactured by Applied Biosystems, Inc. and purifying the resultant oligonucleotides with a C18 reversed phase column.
01 Oct 1993
TL;DR: In this article, the effects of a B. anthracis attack on a military operational unit following a BAC attack were investigated. And the authors concluded that the use of vaccine could ensure survivability and preserve unit effectiveness at levels in excess of 90 percent.
Abstract: : B. anthracis, the causative organism for anthrax, continues to be a biological warfare threat to U.S. forces. Exposure through inhalation is deadly unless the cause of infection is identified and treated before serious respiratory symptoms present. The objective of this study was to determine the levels of B. anthracis likely to be found on the battlefield and the potential impact of B. anthracis attacks on unit effectiveness. The levels of B. anthracis are critically dependent on the weapons, form of agent dissemination, weather conditions, and time-of-day of an attack. In assessing the effects on a military operational unit following a B. anthracis attack, various defensive measures were considered including the wearing of protective equipment, especially the protective mask, and the use of medical interventions such as vaccination and antibiotic therapy. Modeling results predicted that the use of vaccine could ensure survivability and preserve unit effectiveness at levels in excess of 90 percent. The study also provides insights into potential operational limitations imposed on a commander whose troops depend solely on antibiotic therapy and protective equipment for survival. Further, the findings illuminate some of the implications for survivability and unit effectiveness in the absence of the capability to either rapidly detect the presence of B. anthracis in the atmosphere or to easily diagnose anthrax casualties prior to the onset of symptoms.
01 Jan 1993
Dissertation•
01 Jun 1993
TL;DR: Recombinant PA was shown to induce partial protection, comparable to native PA, when both were purified and administered with adjuvant, and suggests that further work to stabilise and increase the expression of PA would be worthwhile.
Abstract: Anthrax is a disease of animals and man caused by Bacillus anthracis. The Protective Antigen (PA) of B. anthracis can induce protective immunity and is a candidate vaccine antigen, but current vaccines are not ideal. Live vaccine vectors, such as the aromatic amino acid Salmonella mutants, are capable of delivering antigens to the immune system and stimulating immune responses. The aim of this project was to develop a recombinant S. typhimurium expressing PA and to evaluate the protective immune responses generated in mice.
Native PA was expressed at low levels in S. typhimurium. Various approaches to increasing expression were tried including changing the promoter, expressing the 63 kDa C-terminal fragment of PA as a fusion protein and expressing this fragment after a signal sequence. Altering the B. anthracis PA promoter to the E. coli lac promoter increased expression of PA which was exported with the B. anthracis signal sequence. The cytoplasmically located PA fragment was unstable and was not successfully exported by the signal sequence.
The recombinant organisms were evaluated to select one for in vivo study. Many phagemids were unstable without ampicillin selection and an attempt to stabilise one with the cer region was not successful. Two constructs were chosen for animal work.
Recombinant PA was shown to induce partial protection, comparable to native PA, when both were purified and administered with adjuvant. Mice were vaccinated intravenously with the live S. typhimurium constructs and subsequent challenged with virulent B. anthracis spores. The PA-expressing S. typhimuruon only colonised at low levels but induced partial protective responses. These protective responses occurred without detectable anti-PA antibody.
This work showed that PA expressed by S. typhimurium can induce protective responses even when only low colonisation occurs. It shows the potential for this approach and suggests that further work to stabilise and increase the expression of PA would be worthwhile.
Patent•
10 Mar 1993
TL;DR: In this paper, a new oligonucleotide was obtained for detecting Bacillus anthracis using a nucleotide sequence capable of coding a cap, which was obtained by chemical synthesis.
Abstract: PURPOSE:To obtain a new oligonucleotide useful for rapidly and simply detecting Bacillus anthracis. CONSTITUTION:This oligonucleotide is complementary to a nucleotide sequence capable of coding a capA gene of a plasmid of Bacillus anthracis and has a sequence group of the formula and a complementary sequence corresponding thereto. The oligonucleotide is obtained by chemical synthesis, etc.
Patent•
04 Mar 1993
TL;DR: In this article, a polynucleotide having high specificity as a DNA probe for detecting Bacillus anthracis was obtained by chemical synthesis, which is complementary to a nucleotide sequence capable of coding a cap.
Abstract: PURPOSE: To obtain a new polynucleotide having high specificity as a DNA probe for detecting Bacillus anthracis. CONSTITUTION: This polynucleotide is complementary to a nucleotide sequence capable of coding a capA gene of a plasmid of Bacillus anthracis and has a sequence group expressed by the formula or a complementary sequence corresponding thereto. The polynucleotide is obtained by chemical synthesis. COPYRIGHT: (C)1994,JPO&Japio
Journal Article•
01 Jan 1993
TL;DR: The toxins fit the A/B model with the 63KDa C-terminal fragment of trypsin cleaved PA being equivalent to the receptor-binding region (B moiety) which can then bind and internalize either EF or LF (alternative a moieties).
Abstract: Anthrax is an infectious disease known since antiquity. It is nearly universal in its geographic distribution and, although primarily a disease of herbivores, it can affect many species and it has important zoonotic implications. Anthrax is caused by Bacillus anthracis, a gram positive, non-motile, aerobic and facultatively anaerobic spore-forming organism. Two main virulence factors have been identified, one is a three-component protein exotoxin, termed the anthrax toxin, and the other a poly-D-glutamic acid capsule. These factors are carried on plasmids termed pXO1 and pXO2 respectively. Anthrax toxin is composed of three components, Protective Antigen (PA), Lethal Factor (LF) and Edema Factor (EF). PA is secreted as a 83 KDa protein which is cleaved in the serum, or on the cell surface, to release a 20 KDa N-terminal fragment. The remaining 63 KDa fragment binds to a specific cell surface receptor and can then bind either EF or LF. The combination of PA and LF is referred to as Lethal Toxin, and PA and EF is called Edema Toxin. This is illustrated in Figure 1. The toxins therefore fit the A/B model with the 63KDa C-terminal fragment of trypsin cleaved PA being equivalent to the receptor-binding region (B moiety) which can then bind and internalize either EF or LF (alternative a moieties). Edema Toxin causes oedema when injected intradermally in animals and EF is an adenylate cyclase. When injected intravenously in susceptible animals Lethal Toxin causes rapid death but it’s mechanism of action is unknown although it is assumed to have an enzymatic action within the cytosol.