Bacillus anthracis physiology and genetics

Bacillus anthracis is a member of the Bacillus cereus group species (also known as the “group 1 bacilli”), a collection of Gram-positive spore-forming soil bacteria that are non-fastidious facultative anaerobes with very similar growth characteristics and natural genetic exchange systems. Despite their close physiology and genetics, the B. cereus group species exhibit certain species-specific phenotypes, some of which are related to pathogenicity. B. anthracis is the etiologic agent of anthrax. Vegetative cells of B. anthracis produce anthrax toxin proteins and a poly-d-glutamic acid capsule during infection of mammalian hosts and when cultured in conditions considered to mimic the host environment. The genes associated with toxin and capsule synthesis are located on the B. anthracis plasmids, pXO1 and pXO2, respectively. Although plasmid content is considered a defining feature of the species, pXO1- and pXO2-like plasmids have been identified in strains that more closely resemble other members of the B. cereus group. The developmental nature of B. anthracis and its pathogenic (mammalian host) and environmental (soil) lifestyles of make it an interesting model for study of niche-specific bacterial gene expression and physiology.     

Sensor domains encoded in Bacillus anthracis virulence plasmids prevent sporulation by hijacking a sporulation sensor histidine kinase

Anthrax toxin and capsule, determinants for successful infection by Bacillus anthracis, are encoded on the virulence plasmids pXO1 and pXO2, respectively. Each of these plasmids also encodes proteins that are highly homologous to the signal sensor domain of a chromosomally encoded major sporulation sensor histidine kinase (BA2291) in this organism. B. anthracis Sterne overexpressing the plasmid pXO2-61-encoded signal sensor domain exhibited a significant decrease in sporulation that was suppressed by the deletion of the BA2291 gene. Expression of the sensor domains from the pXO1-118 and pXO2-61 genes in Bacillus subtilis strains carrying the B. anthracis sporulation sensor kinase BA2291 gene resulted in BA2291-dependent inhibition of sporulation. These results indicate that sporulation sensor kinase BA2291 is converted from an activator to an inhibitor of sporulation in its native host by the virulence plasmid-encoded signal sensor domains. We speculate that activation of these signal sensor domains contributes to the initiation of B. anthracis sporulation in the bloodstream of its infected host, a salient characteristic in the virulence of this organism, and provides an additional role for the virulence plasmids in anthrax pathogenesis.     

Asporogenic recombinant producer of anthrax protective antigen

An asporogenic recombinant strain Bacillus anthracis 55ΔTPA-1(Spo⁻) producing anthrax protective antigen (PA) was obtained. The strain contains structural gene pag as a part of a hybrid replicon pUB110PA-1 and lacks determinants encoding the synthesis of main factors of anthrax pathogenicity. The level of PA production by asporogenic genetically engineered strain is approximately 80 μg/ml that is 4–5 times more than the values determined for vaccine strains B. anthracis STI-1 and B. anthracis 55. The strain preserves asporogenicity and ability to replicate the hybrid plasmid after in vitro passages. Biologically active PA was isolated from the constructed strain B. anthracis 55ΔTPA-1(Spo⁻). Double immunization of rabbits with 50 μg of the purified recombinant product provides their 100% protection from infection with 50 LD₅₀ of a highly virulent anthrax strain.     

不同品系小鼠对炭疽芽胞杆菌弱毒株芽胞的敏感性研究

通过比较四种品系小鼠对炭疽芽胞杆菌（Bacillus anthracis）（简称炭疽杆菌）弱毒株芽胞的敏感性，确定炭疽杆菌弱毒株芽胞攻毒合适的动物模型。采用炭疽杆菌弱毒株A16Q1（pXO1-、pXO2+）和A16PI2（pXO1+、pXO2-）的芽胞对四种品系小鼠（DBA/2、KM、ICR和BALB/c）进行腹腔攻毒，记录小鼠死亡时间，计算LD₅₀、绘制存活曲线并统计分析。运用较敏感的KM小鼠研究不同canSNP基因型毒素缺陷株（含pXO2拷贝数不同）芽胞的毒力差异。利用更为敏感的DBA/2小鼠评价S-层蛋白BA3338对荚膜缺陷株芽胞毒力的影响。结果表明，在四种品系小鼠中，毒素缺陷株芽胞的毒力均高于荚膜缺陷株芽胞的毒力。DBA/2小鼠对炭疽杆菌弱毒株芽胞的剂量依赖关系最好，最为敏感，其次是KM小鼠，而ICR小鼠和BALB/c小鼠对炭疽杆菌弱毒株芽胞不敏感。确定了DBA/2小鼠和KM小鼠在炭疽杆菌弱毒株芽胞研究中的适用性。使用KM小鼠评价了不同canSNP基因型炭疽杆菌芽胞的毒力差异，结果表明，不同canSNP基因型炭疽杆菌由于所含pXO2质粒拷贝数的差异导致芽胞的毒力不同。使用DBA/2小鼠评价了S-层蛋白BA3338缺失对炭疽杆菌芽胞毒力的影响，表明BA3338基因的缺失导致炭疽杆菌芽胞毒力降低。     

Toxin-Independent Virulence of Bacillus anthracis in Rabbits

The accepted paradigm states that anthrax is both an invasive and toxinogenic disease and that the toxins play a major role in pathogenicity. In the guinea pig (GP) model we have previously shown that deletion of all three toxin components results in a relatively moderate attenuation in virulence, indicating that B. anthracis possesses an additional toxin-independent virulence mechanism. To characterize this toxin-independent mechanism in anthrax disease, we developed a new rabbit model by intravenous injection (IV) of B. anthracis encapsulated vegetative cells, artificially creating bacteremia. Using this model we were able to demonstrate that also in rabbits, B. anthracis mutants lacking the toxins are capable of killing the host within 24 hours. This virulent trait depends on the activity of AtxA in the presence of pXO2, as, in the absence of the toxin genes, deletion of either component abolishes virulence. Furthermore, this IV virulence depends mainly on AtxA rather than the whole pXO1. A similar pattern was shown in the GP model using subcutaneous (SC) administration of spores of the mutant strains, demonstrating the generality of the phenomenon. The virulent strains showed higher bacteremia levels and more efficient tissue dissemination; however our interpretation is that tissue dissemination per se is not the main determinant of virulence whose exact nature requires further elucidation.     

The Bacillus anthracis Protein MprF Is Required for Synthesis of Lysylphosphatidylglycerols and for Resistance to Cationic Antimicrobial Peptides

During inhalational anthrax, Bacillus anthracis survives and replicates in alveolar macrophages, followed by rapid invasion into the host's bloodstream, where it multiplies to cause heavy bacteremia. B. anthracis must therefore defend itself from host immune functions encountered during both the intracellular and the extracellular stages of anthrax infection. In both of these niches, cationic antimicrobial peptides are an essential component of the host's innate immune response that targets B. anthracis. However, the genetic determinants of B. anthracis contributing to resistance to these peptides are largely unknown. Here we generated Tn917 transposon mutants in the ΔANR strain (pXO1⁻ pXO2⁻) of B. anthracis and screened them for altered protamine susceptibility. A protamine-sensitive mutant identified carried the transposon inserted in the BA1486 gene encoding a putative membrane protein homologous to MprF known in several gram-positive pathogens. A mutant strain with the BAS1375 gene (the orthologue of BA1486) deleted in the Sterne 34F2 strain (pXO1⁺ pXO2⁻) of B. anthracis exhibited hypersusceptibility not only to protamine but also to α-helical cathelicidin LL-37 and β-sheet defensin human neutrophil peptide 1 compared to the wild-type Sterne strain. Analysis of membrane lipids using isotopic labeling demonstrated that the BAS1375 deletion mutant is unable to synthesize lysinylated phosphatidylglycerols, and this defect is rescued by genetic complementation. Further, we determined the structures of these lysylphosphatidylglycerols by using various mass spectrometric analyses. These results demonstrate that in B. anthracis a functional MprF is required for the biosynthesis of lysylphosphatidylglycerols, which is critical for resistance to cationic antimicrobial peptides.     

Comparative proteome analysis of <Emphasis Type="Italic">Bacillus anthracis</Emphasis> with pXO1 plasmid content

Bacillus anthracis the causative agent of anthrax, is an important pathogen among the Bacillus cereus group of species because of its physiological characteristics and its importance as a biological warfare agent. Tripartite anthrax toxin proteins and a poly-D-glutamic acid capsule are produced by B. anthracis vegetative cells during mammalian hosts infection and when cultured in conditions that are thought to mimic the host environment. To identify the factors regulating virulence in B. anthracis the whole cell proteins were extracted from two B. anthracis strains and separated by narrow range immobilized pH gradient (IPG) strips (pH 4–7), followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins that were differentially expressed were identified by the peptide fingerprinting using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). A total of 23 proteins were identified as being either upregulated or downregulated in the presence or absence of the virulence plasmid pXO1. Two plasmid encoded proteins and 12 cellular proteins were identified and documented as potential virulence factors.     

The Bicarbonate Transporter Is Essential for Bacillus anthracis Lethality

In the pathogenic bacterium Bacillus anthracis, virulence requires induced expression of the anthrax toxin and capsule genes. Elevated CO₂/bicarbonate levels, an indicator of the host environment, provide a signal ex vivo to increase expression of virulence factors, but the mechanism underlying induction and its relevance in vivo are unknown. We identified a previously uncharacterized ABC transporter (BAS2714-12) similar to bicarbonate transporters in photosynthetic cyanobacteria, which is essential to the bicarbonate induction of virulence gene expression. Deletion of the genes for the transporter abolished induction of toxin gene expression and strongly decreased the rate of bicarbonate uptake ex vivo, demonstrating that the BAS2714-12 locus encodes a bicarbonate ABC transporter. The bicarbonate transporter deletion strain was avirulent in the A/J mouse model of infection. Carbonic anhydrase inhibitors, which prevent the interconversion of CO₂ and bicarbonate, significantly affected toxin expression only in the absence of bicarbonate or the bicarbonate transporter, suggesting that carbonic anhydrase activity is not essential to virulence factor induction and that bicarbonate, and not CO₂, is the signal essential for virulence induction. The identification of this novel bicarbonate transporter essential to virulence of B. anthracis may be of relevance to other pathogens, such as Streptococcus pyogenes, Escherichia coli, Borrelia burgdorferi, and Vibrio cholera that regulate virulence factor expression in response to CO₂/bicarbonate, and suggests it may be a target for antibacterial intervention.     

The genome sequence of Bacillus cereus ATCC 10987 reveals metabolic adaptations and a large plasmid related to Bacillus anthracis pXO1

We sequenced the complete genome of Bacillus cereus ATCC 10987, a non-lethal dairy isolate in the same genetic subgroup as Bacillus anthracis. Comparison of the chromosomes demonstrated that B.cereus ATCC 10987 was more similar to B.anthracis Ames than B.cereus ATCC 14579, while containing a number of unique metabolic capabilities such as urease and xylose utilization and lacking the ability to utilize nitrate and nitrite. Additionally, genetic mechanisms for variation of capsule carbohydrate and flagella surface structures were identified. Bacillus cereus ATCC 10987 contains a single large plasmid (pBc10987), of ~208 kb, that is similar in gene content and organization to B.anthracis pXO1 but is lacking the pathogenicity-associated island containing the anthrax lethal and edema toxin complex genes. The chromosomal similarity of B.cereus ATCC 10987 to B.anthracis Ames, as well as the fact that it contains a large pXO1-like plasmid, may make it a possible model for studying B.anthracis plasmid biology and regulatory cross-talk.     

Bacillus anthracis secretome time course under host-simulated conditions and identification of immunogenic proteins

Background  

The secretion time course of Bacillus anthracis strain RA3R (pXO1⁺/pXO2^-) during early, mid, and late log phase were investigated under conditions that simulate those encountered in the host. All of the identified proteins were analyzed by different software algorithms to characterize their predicted mode of secretion and cellular localization. In addition, immunogenic proteins were identified using sera from humans with cutaneous anthrax.     

The Genome of a Bacillus Isolate Causing Anthrax in Chimpanzees Combines Chromosomal Properties of B. cereus with B. anthracis Virulence Plasmids

Anthrax is a fatal disease caused by strains of Bacillus anthracis. Members of this monophyletic species are non motile and are all characterized by the presence of four prophages and a nonsense mutation in the plcR regulator gene. Here we report the complete genome sequence of a Bacillus strain isolated from a chimpanzee that had died with clinical symptoms of anthrax. Unlike classic B. anthracis, this strain was motile and lacked the four prohages and the nonsense mutation. Four replicons were identified, a chromosome and three plasmids. Comparative genome analysis revealed that the chromosome resembles those of non-B. anthracis members of the Bacillus cereus group, whereas two plasmids were identical to the anthrax virulence plasmids pXO1 and pXO2. The function of the newly discovered third plasmid with a length of 14 kbp is unknown. A detailed comparison of genomic loci encoding key features confirmed a higher similarity to B. thuringiensis serovar konkukian strain 97-27 and B. cereus E33L than to B. anthracis strains. For the first time we describe the sequence of an anthrax causing bacterium possessing both anthrax plasmids that apparently does not belong to the monophyletic group of all so far known B. anthracis strains and that differs in important diagnostic features. The data suggest that this bacterium has evolved from a B. cereus strain independently from the classic B. anthracis strains and established a B. anthracis lifestyle. Therefore we suggest to designate this isolate as “B. cereus variety (var.) anthracis”.     

A Bacillus anthracis strain deleted for six proteases serves as an effective host for production of recombinant proteins

Bacillus anthracis produces a number of extracellular proteases that impact the integrity and yield of other proteins in the B. anthracis secretome. In this study we show that anthrolysin O (ALO) and the three anthrax toxin proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF), produced from the B. anthracis Ames 35 strain (pXO1?, pXO2?), are completely degraded at the onset of stationary phase due to the action of proteases. An improved Cre-loxP gene knockout system was used to sequentially delete the genes encoding six proteases (InhA1, InhA2, camelysin, TasA, NprB, and MmpZ). The role of each protease in degradation of the B. anthracis toxin components and ALO was demonstrated. Levels of the anthrax toxin components and ALO in the supernatant of the sporulation defective, pXO1? A35HMS mutant strain deleted for the six proteases were significantly increased and remained stable over 24 h. A pXO1-free variant of this six-protease mutant strain, designated BH460, provides an improved host strain for the preparation of recombinant proteins. As an example, BH460 was used to produce recombinant EF, which previously has been difficult to obtain from B. anthracis. The EF protein produced from BH460 had the highest in vivo potency of any EF previously purified from B. anthracis or Escherichia coli hosts. BH460 is recommended as an effective host strain for recombinant protein production, typically yielding greater than 10mg pure protein per liter of culture.     

Bacillus anthracis pXO1 virulence plasmid encodes a type 1 DNA topoisomerase

The virulence plasmid pXO1 is responsible for toxin production in Bacillus anthracis. A DNA fragment from pXO1 was isolated and was shown, by sequence analysis, to contain part of a type 1 DNA topoisomerase gene. Attempts to clone the entire wild-type gene, designated topX, in Escherichia coli, were unsuccessful. In order to obtain the complete gene, it was first insertionally inactivated and then cloned in the mutated form. The deduced amino acid sequence of Topo X1 shows similarities to that of the two E. coli type 1 DNA topoisomerases. The N-terminal two-thirds of the putative B. anthracis protein exhibits strongest sequence similarity to topoisomerase III, whereas the C-terminal portion contains cysteine residues that could form three zinc-binding domains, as they do in topoisomerase I. The suggested active-site tyrosine is conserved in all three proteins. The regulation of expression from the topX promoter is modified by addition of a gyrase inhibiting antibiotic. The Topo X1 protein is likely to be involved in the stability of pXO1.