Morphology of the type strain of Bacillus anthracis EY 3169T=ATCC 14578T grown either aerobically or anaerobically on agar plates--observation by light and laser microscopes

Growth characteristics including cell-arrangement of the type strain of Bacillus anthracis EY 3169T=ATCC 14578T grown on agar plates in level 3 laboratory were observed by both light and laser microscopes. Small daughter colonies appeared on parent colonies grown on 5% sheep blood or chocolate agar plates after 12 days incubation at room temperature. Daughter colonies, stained by Wirtz-Conklin method, were composed with vegetative cells and spores. Growth of daughter colonies might be supported by the debris of cells in the parent colony. Colonies grown under anaerobic conditions were flat with smooth edges, and the cells neither formed chains of any length, nor produced any spores after 25 days incubation at room temperature. It was thought that spores of B. anthracis were produced at the terminal stage of individual cell life instead of under unfavorable conditions for the organism. Air is needed for spore formation and cell-chain formation. More nutrients, probably amino acids, are needed for anaerobic growth rather than aerobic.     

Specific intoxication in anthrax infection

The pathomorphological picture of experimental B. anthracis infection in white rats (strain Fisher-344) essentially corresponds to experimental anthracic intoxication with very moderately pronounced morphological manifestation of B. anthracis invasion. This indicates that specific anthracic intoxication is an essential component of the pathological process in B. anthracis infection.     

Human neutrophils kill Bacillus anthracis

Bacillus anthracis spores cause natural infections and are used as biological weapons. Inhalation infection with B. anthracis, the etiological agent of anthrax, is almost always lethal, yet cutaneous infections usually remain localized and resolve spontaneously. Neutrophils are typically recruited to cutaneous but seldom to other forms of anthrax infections, raising the possibility that neutrophils kill B. anthracis. In this study we infected human neutrophils with either spores or vegetative bacteria of a wild-type strain, or strains, expressing only one of the two major virulence factors. The human neutrophils engulfed B. anthracis spores, which germinated intracellularly and were then efficiently killed. Interestingly, neutrophil killing was independent of reactive oxygen species production. We fractionated a human neutrophil granule extract by high-performance liquid chromatography and identified alpha-defensins as the component responsible for B. anthracis killing. These data suggest that the timely recruitment of neutrophils can control cutaneous infections and possibly other forms of B. anthracis infections, and that alpha-defensins play an important role in the potent anti-B. anthracis activity of neutrophils.     

Identification of chromosomally encoded membranal polypeptides of Bacillus anthracis by a proteomic analysis: prevalence of proteins containing S-layer homology domains

Bacillus anthracis is the causative agent of anthrax disease. Improvement of existing anthrax vaccines, which are currently based on the administration of Protective Antigen (the highly immunogenic nontoxic subunit of the bacterial toxin) may entail other bacterial immunogenic elements, part of which are predicted to reside on the surface of bacterial cells. In the present study, membranal proteins extracted from a stationary-phase culture of a nonvirulent B. anthracis strain, devoid of the native virulence plasmids pXO1 and pXO2, were separated by two-dimensional electrophoresis (2-DE) and a characteristic protein map was defined. The proteomic analysis allowed matrix-assisted laser desorption/ionization-time of flight mass spectrometry-assisted identification of 86 protein spots which represent the product of 30 individual open reading frames (ORF). Among these, a prevalent class of proteins was the S-layer proteins (which were found to represent more than 75% of the B. anthracis membranal fraction) and proteins containing S-layer homology (SLH)-membranal localization domains. Five novel SLH proteins, previously inferred only from bioinformatic ORF analysis (draft genome sequence), were identified and one was shown to be a highly abundant membranal protein. Western blots of the 2-DE gels were probed with sera from convalescent rabbits and guinea pigs infected with virulent B. anthracis (Vollum strain). This analysis revealed that B. anthracis immune animals exhibit antibodies against at least 14 distinct membranal proteins present in the 2-DE map, establishing that these proteins are expressed in vivo and are able to elicit an immune response. The identification of the protein components of the B. anthracis membranal fraction, as well as the establishment of their potential immunogenicity, underscore the strength of the proteomic approach for identifying molecules which may serve for further analysis of immune and protective abilities.     

Transposon mutagenesis of Bacillus anthracis strain Sterne using Bursa aurealis

Bacillus anthracis, a spore forming Gram-positive microbe, is the causative agent of anthrax. Although plasmid encoded factors such as lethal toxin (LeTx), edema toxin (EdTx), and gamma-poly-d-glutamic acid (PGA) capsule are known to be required for disease pathogenesis, B. anthracis genes that enable spore invasion, phagosomal escape and macrophage replication are still unknown. To establish transposon mutagenesis as a tool for the characterization of anthrax genes, we employed the mariner-based mini-transposon Bursa aurealis in B. anthracis strain Sterne 7702. B. aurealis carrying an erythromycin resistance cassette and its cognate transposase were delivered by transformation of two plasmids. B. aurealis transposition can be selected for by temperature shift to prevent plasmid replication and by screening colonies for erythromycin resistance. Using inverse polymerase chain reaction, DNA fragments of 129 random erythromycin-resistant transposon mutants were amplified and submitted to DNA sequence analysis. These studies demonstrate that B. aurealis inserts randomly into the genome of B. anthracis and can therefore be employed for finding genes involved in virulence.     

Synthesis of lipoteichoic acids in Bacillus anthracis

Lipoteichoic acid (LTA), a glycerol phosphate polymer, is a component of the envelope of Gram-positive bacteria that has hitherto not been identified in Bacillus anthracis, the causative agent of anthrax. LTA synthesis in Staphylococcus aureus and other microbes is catalyzed by the product of the ltaS gene, a membrane protein that polymerizes polyglycerol phosphate from phosphatidyl glycerol. Here we identified four ltaS homologues, designated ltaS1 to -4, in the genome of Bacillus anthracis. Polyglycerol phosphate-specific monoclonal antibodies were used to detect LTA in the envelope of B. anthracis strain Sterne (pXO1(+) pXO2(-)) vegetative forms. B. anthracis mutants lacking ltaS1, ltaS2, ltaS3, or ltaS4 did not display defects in growth or LTA synthesis. In contrast, B. anthracis strains lacking both ltaS1 and ltaS2 were unable to synthesize LTA and exhibited reduced viability, altered envelope morphology, aberrant separation of vegetative forms, and decreased sporulation efficiency. Expression of ltaS1 or ltaS2 alone in B. anthracis as well as in other microbes was sufficient for polyglycerol phosphate synthesis. Thus, similar to S. aureus, B. anthracis employs LtaS enzymes to synthesize LTA, an envelope component that promotes bacterial growth and cell division.     

Bacillus anthracis sortase A (SrtA) anchors LPXTG motif-containing surface proteins to the cell wall envelope

Cell wall-anchored surface proteins of gram-positive pathogens play important roles during the establishment of many infectious diseases, but the contributions of surface proteins to the pathogenesis of anthrax have not yet been revealed. Cell wall anchoring in Staphylococcus aureus occurs by a transpeptidation mechanism requiring surface proteins with C-terminal sorting signals as well as sortase enzymes. The genome sequence of Bacillus anthracis encodes three sortase genes and eleven surface proteins with different types of cell wall sorting signals. Purified B. anthracis sortase A cleaved peptides encompassing LPXTG motif-type sorting signals between the threonine (T) and the glycine (G) residues in vitro. Sortase A activity could be inhibited by thiol-reactive reagents, similar to staphylococcal sortases. B. anthracis parent strain Sterne 34F(2), but not variants lacking the srtA gene, anchored the collagen-binding MSCRAMM (microbial surface components recognizing adhesive matrix molecules) BasC (BA5258/BAS4884) to the bacterial cell wall. These results suggest that B. anthracis SrtA anchors surface proteins bearing LPXTG motif sorting signals to the cell wall envelope of vegetative bacilli.     

Utilization of the rpoB gene as a specific chromosomal marker for real-time PCR detection of Bacillus anthracis

The potential use of Bacillus anthracis as a weapon of mass destruction poses a threat to humans, domesticated animals, and wildlife and necessitates the need for a rapid and highly specific detection assay. We have developed a real-time PCR-based assay for the specific detection of B. anthracis by taking advantage of the unique nucleotide sequence of the B. anthracis rpoB gene. Variable region 1 of the rpoB gene was sequenced from 36 Bacillus strains, including 16 B. anthracis strains and 20 other related bacilli, and four nucleotides specific for B. anthracis were identified. PCR primers were selected so that two B. anthracis-specific nucleotides were at their 3' ends, whereas the remaining bases were specific to the probe region. This format permitted the PCR reactions to be performed on a LightCycler via fluorescence resonance energy transfer (FRET). The assay was found to be specific for 144 B. anthracis strains from different geographical locations and did not cross-react with other related bacilli (175 strains), with the exception of one strain. The PCR assay can be performed on isolated DNA as well as crude vegetative cell lysates in less than 1 h. Therefore, the rpoB-FRET assay could be used as a new chromosomal marker for rapid detection of B. anthracis.     

Chemical and immunological properties of B. anthracis arginase and its metabolic involvement

An arginase isolated from a capsulated Bacillus anthracis strain was highly purified and crystallized. The chemical and immunological characteristics of this enzyme re described. Some very important properties differ from those of another bacterial arginase, i.e. Staphylococcus aureus arginase, described in a previous paper (Soru et al. (2)). The two arginases have different crystallization forms, different molecular weight, Km, thermostability, Arrhenius activation energy. They have another N-terminal group and are immunologically strictly specific. These differences point to distinct proteins. The fact that two arginases of different origin are structurally non-identical suggests that they may be involved in different metabolic processes. Staphylococcal arginase was shown to participate in a complete ureogenetic cycle, for it also possesses the other enzymes of the cycle (Soru et al. (2)). Except arginase, no other enzyme of this cycle was identified in the capsulated B. anthracis strain. Arginase may be involved in another metabolic pathway, one that is important for the strain, such as the synthesis of glutamic acid, since the capsular material of the strain is a polymer gamma-linked polyglutamic acid, mainly configuration D (Ivanovic and Bruckner (20)). The fact that the N-terminal residue of B. anthracis arginase is a tetramer containing glutamic acid together with proline (in addition to alanine and glycine) suggests that arginase may participate as a regulatory enzyme in the synthesis of glutamic acid from proline via ornithine and arginine, respectively. This pathway is found in many bacteria. The proline oxidase system, which is supposed to catalyse the conversion of proline to glutamic acid, is under study now in Bacillus anthracis strains.     

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

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.     

Colonial and Cellular Polymorphism in Xenorhabdus luminescens

A highly polymorphic Xenorhabdus luminescens strain was isolated. The primary form of X. luminescens was luminescent and nonswarming and produced a yellow pigment and antimicrobial substances. The primary form generated a secondary form that had a distinct orange pigmentation, was weakly luminescent, and did not produce antimicrobial substances. Both the primary and secondary forms generated a set of colony variants at frequencies that exceeded normal rates for spontaneous mutation. The variant forms include nonswarming and swarming forms that formed large colonies and a small-colony (SC) form. The primary and secondary forms generated their SC forms at frequencies of between 1 and 14% and 1 and 2%, respectively. The SC forms were distinct from their parental primary and secondary forms in colony and cellular morphology and in protein composition. The cellular morphology and protein patterns of the nonswarming and swarming colony variants were all very similar. The DNA fingerprints of all forms were similar. Each SC-form colony reverted at high frequency to the form from which it was derived. The proportion of parental-type cells in the SC-form colonies varied with age, with young colonies containing as few as 0.0002% parental-type cells. The primary-to-secondary switch was stable, but all the other colony forms were able to switch at high frequencies to the alternative colony phenotypes.     

Monoclonal antibodies for Bacillus anthracis spore detection and functional analyses of spore germination and outgrowth

All members of the Bacillus genus produce endospores as part of their life cycle; however, it is not possible to determine the identity of spores by casual or morphological examination. The 2001 anthrax attacks demonstrated a need for fast, dependable methods for detecting Bacillus anthracis spores in vitro and in vivo. We have developed a variety of isotypes and specificities of mAbs that were able to distinguish B. anthracis spores from other Bacillus spores. The majority of Abs were directed toward BclA, a major component of the exosporium, although other components were also distinguished. These Abs did not react with vegetative forms. Some Abs distinguished B. anthracis spores from spores of distantly related species in a highly specific manner, whereas others discriminated among strains that are the closest relatives of B. anthracis. These Abs provide a rapid and reliable means of identifying B. anthracis spores, for probing the structure and function of the exosporium, and in the analysis of the life cycle of B. anthracis.     

Phenotypic and genotypic characterization of B.t.LDC-391 strain that produce cytocidal proteins against human cancer cells

An indigenous Bacillus thuringiensis strain B.t.LDC-391 producing cytocidal proteins against human colon cancer cell line, HCT-116, was subjected to phenotypic and genotypic characterization to evaluate its relatedness to B.anthracis. The morphological features of this strain were meta-analyzed with data of other parasporin and insecticidal protein producing Bacillus thuringiensis strains. The conventional biochemical analysis and antibiotic sensitivity test proved it as an ampicillin resistant which is a salient feature, absent in B.anthracis Ames. PCR analysis showed the absence of cyt and parasporin related genes in the genome of B.t.LDC-391. But the strain was positive for cap gene. The sequencing and bio-informatic analysis of cap gene and 16S rDNA of B.t.LDC-391 placed it closer to B.thuringiensis and revealed significant divergence from that of any B.anthracis strain. However our strain lacked β- hemolysis on human erythrocytes which is a common feature of B.anthracis strains and parasporin producers.     

Identification of Bacillus anthracis by polyclonal antibodies against extracted vegetative cell antigens

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.     

Sequence analysis of the genes encoding for the major virulence factors of Bacillus anthracis vaccine strain 'Carbosap'

AIMS: This study was performed to analyse the molecular characteristics of genes encoding for the major virulence factors in Bacillus anthracis vaccine strain 'Carbosap' compared with the wild B. anthracis strain, to evaluate the basis of attenuation. METHODS AND RESULTS: The molecular characteristics of the B. anthracis 'Carbosap' vaccine strain, used as vaccine in Italy, were analysed in comparison with a B. anthracis virulent strain. Despite the presence of the two virulence plasmids pXO1 and pXO2, the 'Carbosap' strain proved to be protective for cattle. The presence of the regulatory genes atxA and pagR and the gerX operon, known to be involved in the virulence, was verified. In addition, all genes were sequenced. The results showed that no molecular differences between 'Carbosap' and the virulent strain were evident. CONCLUSIONS: The results of this study indicate that the attenuation of the 'Carbosap' vaccine strain is not due to the lack of virulence genes or to modifications occurring on the sequence of these genes. Therefore, other virulence factors, still unknown, could be involved in the pathogenic mechanisms. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper adds new information regarding the molecular characteristics of the vaccine strain 'Carbosap' and highlights the need to better understand the virulence factors involved in the pathogenicity of B. anthracis strains.     

Correlation of the virulence of Bacillus anthracis with expression of signs, coded for by chromosomal genes

Clonal analysis of Bacillus anthracis strains showed heterogeneity of the majority of populations by the proteolytic, hemolytic, and pigment-adsorbing activities. Phenotypes isolated within each population were subdivided into 4 subpopulations. The appearance of sub-populations of virulent and vaccine strains (B. anthracis Sterne, Zenkowskii) was due to the ability of clones adsorbing Congo red and possessing high activities of proteolytic and hemolytic enzymes to dissociate with the formation of phenotypes characterized by different expression of proteolysis, hemolysis, pigment adsorption, pigment production, and sporulation signs. A characteristic feature of vaccine strains of B. anthracis CTIl, Wright, and Pasteur is a relative homogeneity of their populations consisting mainly of cells with low activities of proteolytic, hemolytic, and pigment-adsorbing enzymes. Study of the heterogeneity of di-, mono-, and plasmid-free derivatives of B. anthracis showed that the process of phenotype formation did not depend on the plasmids proper. Evaluation of the virulence of clones isolated from highly virulent B. anthracis strain 81/1 showed that the processes associated with loss of sporulation capacity notably decrease the virulence for laboratory animals.     

Anthrax: early steps of the intracellular stage of infection development

It was shown that spore germination of different Bacillus anthracis strains in macrophage-like cells J774A.1 depended on the genotype of the strains. The virulent B. anthracis strains contain plasmids pXO1 and pX02 responsible for the synthesis of a toxin and a capsule, respectively. The loss of one of the plasmids results in the reduction of strain virulence. It was shown that effective survival of germinating spores in macrophages occurred in the presence of plasmid pXO1 only. The spores of the B. anthracis strains ?Ames and STI-Rif deprived of plasmid pXO1 were least adapted to passing through the intracellular stage. The B. anthracis strains 81/1 and 71/12 (carrying plasmids pXO1 and pXO2 and synthesizing the toxin and capsule) less effectively survived in the cytoplasm of macrophages than the strain STI-1 which has only the plasmid pXO1. It was found that the rate of synthesis of the capsule consisting of polymer gamma-D-glutamic acid depended on the ability of bacterial cells to escape from macrophages. In the B. anthracis strains carrying plasmid pXO2, capsule synthesis by vegetative cells was activated within macrophages that promoted a rapid escape of the vegetative cells from the macrophages. On the contrary, most of capsule-free cells of the vaccine strain STI-1 remained inside macrophages during the whole period of observation. Thus, integrated regulation of two processes, namely synthesis of the toxin components participating in the transition of the germinating cell from phagosome into cytoplasm, and synthesis of the capsule whose presence promotes rapid escape of bacterial cells from macrophages by presently unknown mechanism play the key role in anthrax development at early stages.     

Comparison of PCR-RFLP, ribotyping and ERIC-PCR for typing Bacillus anthracis and Bacillus cereus strains

PCR-RFLP analysis of the vrrA gene and cerAB gene was used to investigate the genomic diversity in 21 strains of Bacillus anthracis and 28 strains of Bacillus cereus, and was compared with results obtained by ribotyping and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) analysis. VrrA-typing divided the B. anthracis into four groups. Except for one Pasteur vaccine strain, the vrrA PCR-RFLP profiles of the B. anthracis were separated into three groups, which were different from those of the B. cereus strains. Ribotyping separated the B. anthracis isolates into seven ribotypes, and a common fragment of an approximately 850 bp band from the ERIC-PCR fingerprints separated most B. anthracis strains into two groups. VrrA/cerAB PCR-RFLP, ribotyping and ERIC-PCR generated 18, 22 and 23 types, respectively, from B. cereus strains. The results suggest that a combination of all three methods provides a high resolution typing method for B. anthracis and B. cereus. Compared with ribotyping and ERIC-PCR, PCR-RFLP is simple to perform and has potential as a rapid method for typing and discriminating B. anthracis strains from other B. cereus group bacteria.     

Immunosuppressive action of the lethal toxin of Bacillus anthracis in mice

In experiments on inbred mice infected with B. anthracis capsular strain 71/12 of Tsenkovsky's second vaccine B. anthracis lethal toxin introduced in mixture with spores has been shown to aggravate anthrax infection in CBA mice susceptible to anthrax, while producing a faint effect on the infectious process in BALB mice with hereditary resistance to anthrax. B. anthracis purified edema toxin has been found to produce a weaker aggravating effect with respect to anthrax infection than the lethal toxin. As revealed in these experiments, the capacity of the lethal toxin to suppress the activity of peritoneal macrophages in vitro is the more pronounced, the more resistant to anthrax are the mice used as the donors of these macrophages. The mechanism of hereditary immunity which may ensure resistance to infection in the presence of immunosuppression is discussed.