BslA,the S‐layer adhesin of B. anthracis,is a virulence factor for anthrax pathogenesis

Microbial pathogens use adhesive surface proteins to bind to and interact with host tissues, events that are universal for the pathogenesis of infectious diseases. A surface adhesin of Bacillus anthracis, the causative agent of anthrax, required to mediate these steps has not been discovered. Previous work identified BslA, an S‐layer protein, to be necessary and sufficient for adhesion of the anthrax vaccine strain, Bacillus anthracis Sterne, to host cells. Here we asked whether encapsulated bacilli require BslA for anthrax pathogenesis in guinea pigs. Compared with the highly virulent parent strain B. anthracis Ames, bslA mutants displayed a dramatic increase in the lethal dose and in mean time‐to‐death. Whereas all tissues of animals infected with B. anthracis Ames contained high numbers of bacilli, only few vegetative forms could be recovered from internal organs of animals infected with the bslA mutant. Surface display of BslA occurred at the poles of encapsulated bacilli and enabled the binding of vegetative forms to host cells. Together these results suggest that BslA functions as the surface adhesin of the anthrax pathogen B. anthracis strain Ames.     

Comparative analysis of virulence factors secreted by Bacillus anthracis Sterne at host body temperature

Aims: For the analysis of virulence factors produced and secreted by Bacillus anthracis vegetative cells during mammalian host infection, we evaluated the secretome of B. anthracis Sterne exposed to host‐specific factors specifically to host body temperature. Methods and Results: We employed a comparative proteomics‐based approach to analyse the proteins secreted by B. anthracis Sterne under host‐specific body temperature conditions. A total of 17 proteins encoded on a single chromosome and the pXO1 plasmid were identified by peptide mass fingerprinting. Multiple algorithms were used to predict the secretion mechanisms of the detected proteins in B. anthracis. Conclusions: Several putative virulence factors and known factors responsible for sporulation were differentially regulated, including CodY, pXO1‐130 and BA1952, revealing insights into temperature cues in the B. anthracis secretome. Significance and Impact of the Study: This study identified temperature‐regulated proteins. Further studies aimed at understanding the physical and functional roles of these proteins in infection and control by elevated temperatures will contribute to detection, diagnostics and prophylaxis.     

Modulation of the Bacillus anthracis Secretome by the Immune Inhibitor A1 Protease

The Bacillus anthracis secretome includes protective antigen, lethal factor, and edema factor, which are the components of anthrax toxin, and other proteins with known or potential roles in anthrax disease. Immune inhibitor A1 (InhA1) is a secreted metalloprotease that is unique to pathogenic members of the Bacillus genus and has been associated with cleavage of host proteins during infection. Here, we report the effect of InhA1 on the B. anthracis secretome. Differential in-gel electrophoresis of proteins present in culture supernatants from a parent strain and an isogenic inhA1-null mutant revealed multiple differences. Of the 1,340 protein spots observed, approximately one-third were less abundant and one-third were more abundant in the inhA1 secretome than in the parent strain secretome. Proteases were strongly represented among those proteins exhibiting a 9-fold or greater change. InhA1 purified from a B. anthracis culture supernatant directly cleaved each of the anthrax toxin proteins as well as an additional secreted protease, Npr599. The conserved zinc binding motif HEXXH of InhA1 (HEYGH) was critical for its proteolytic activity. Our data reveal that InhA1 directly and indirectly modulates the form and/or abundance of over half of all the secreted proteins of B. anthracis. The proteolytic activity of InhA1 on established secreted virulence factors, additional proteases, and other secreted proteins suggests that this major protease plays an important role in virulence not only by cleaving mammalian substrates but also by modulating the B. anthracis secretome itself.     

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.     

Rapid detection methods for <Emphasis Type="Italic">Bacillus anthracis</Emphasis> in environmental samples: a review

Bacillus anthracis is a Gram-positive, spore-forming bacterium, which causes anthrax, an often lethal disease of animals and humans. Although the disease has been well studied since the nineteenth century, it has witnessed a renewed interest during the past decade, due to its use as a bioterrorist agent in the fall of 2001 in the USA. A number of techniques aimed at rapidly detecting B. anthracis, in environmental samples as well as in point-of-care settings for humans suspected of exposure to the pathogen, are now available. These technologies range from culture-based methods to portable DNA amplification devices. Despite recent developments, specific identification of B. anthracis still remains difficult because of its phenotypic and genotypic similarities with other Bacillus species. Accordingly, many efforts are being made to improve the specificity of B. anthracis identification. This mini-review discusses the current challenges around B. anthracis identification, not only in reach-back laboratories but also in the field (in operational conditions).     

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”.     

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.     

Immunoproteomically identified GBAA_0345, alkyl hydroperoxide reductase subunit C is a potential target for multivalent anthrax vaccine

Anthrax is caused by the spore‐forming bacterium Bacillus anthracis, which has been used as a weapon for bioterrorism. Although current vaccines are effective, they involve prolonged dose regimens and often cause adverse reactions. High rates of mortality associated with anthrax have made the development of an improved vaccine a top priority. To identify novel vaccine candidates, we applied an immunoproteomics approach. Using sera from convalescent guinea pigs or from human patients with anthrax, we identified 34 immunogenic proteins from the virulent B. anthracis H9401. To evaluate vaccine candidates, six were expressed as recombinant proteins and tested in vivo. Two proteins, rGBAA_0345 (alkyl hydroperoxide reductase subunit C) and rGBAA_3990 (malonyl CoA‐acyl carrier protein transacylase), have afforded guinea pigs partial protection from a subsequent virulent‐spore challenge. Moreover, combined vaccination with rGBAA_0345 and rPA (protective antigen) exhibited an enhanced ability to protect against anthrax mortality. Finally, we demonstrated that GBAA_0345 localizes to anthrax spores and bacilli. Our results indicate that rGBAA_0345 may be a potential component of a multivalent anthrax vaccine, as it enhances the efficacy of rPA vaccination. This is the first time that sera from patients with anthrax have been used to interrogate the proteome of virulent B. anthracis vegetative cells.     

LytR-CpsA-Psr Enzymes as Determinants of Bacillus anthracis Secondary Cell Wall Polysaccharide Assembly

Bacillus anthracis, the causative agent of anthrax, replicates as chains of vegetative cells by regulating the separation of septal peptidoglycan. Surface (S)-layer proteins and associated proteins (BSLs) function as chain length determinants and bind to the secondary cell wall polysaccharide (SCWP). In this study, we identified the B. anthracis lcpD mutant, which displays increased chain length and S-layer assembly defects due to diminished SCWP attachment to peptidoglycan. In contrast, the B. anthracis lcpB3 variant displayed reduced cell size and chain length, which could be attributed to increased deposition of BSLs. In other bacteria, LytR-CpsA-Psr (LCP) proteins attach wall teichoic acid (WTA) and polysaccharide capsule to peptidoglycan. B. anthracis does not synthesize these polymers, yet its genome encodes six LCP homologues, which, when expressed in S. aureus, promote WTA attachment. We propose a model whereby B. anthracis LCPs promote attachment of SCWP precursors to discrete locations in the peptidoglycan, enabling BSL assembly and regulated separation of septal peptidoglycan.     

Detection technologies for Bacillus anthracis: Prospects and challenges

Bacillus anthracis is a Gram-positive, spore-forming bacterium representing the etiological agent of acute infectious disease anthrax, a lethal but rare disease of animals and humans in nature. With recent use of anthrax as a bioweapon, a number of techniques have been recently developed and evaluated to facilitate its rapid detection of B. anthracis in the environment as well as in point-of-care settings for humans suspected of exposure to the pathogen. Complex laboratory methods for B. anthracis identification are required since B. anthracis has similarities with other Bacillus species and its existence in both spore and vegetative forms. This review discusses current challenges and various improvements associated with anthrax agent detection.     

Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples

A real-time polymerase chain reaction (PCR) assay was developed for rapid identification of Bacillus anthracis in environmental samples. These samples often harbor Bacillus cereus bacteria closely related to B. anthracis, which may hinder its specific identification by resulting in false positive signals. The assay consists of two duplex real-time PCR: the first PCR allows amplification of a sequence specific of the B. cereus group (B. anthracis, B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pseudomycoides, and Bacillus mycoides) within the phosphoenolpyruvate/sugar phosphotransferase system I gene and a B. anthracis specific single nucleotide polymorphism within the adenylosuccinate synthetase gene. The second real-time PCR assay targets the lethal factor gene from virulence plasmid pXO1 and the capsule synthesis gene from virulence plasmid pXO2. Specificity of the assay is enhanced by the use of minor groove binding probes and/or locked nucleic acids probes. The assay was validated on 304 bacterial strains including 37 B. anthracis, 67 B. cereus group, 54 strains of non-cereus group Bacillus, and 146 Gram-positive and Gram-negative bacteria strains. The assay was performed on various environmental samples spiked with B. anthracis or B. cereus spores. The assay allowed an accurate identification of B. anthracis in environmental samples. This study provides a rapid and reliable method for improving rapid identification of B. anthracis in field operational conditions.     

Surface display of the 20-kDa N-terminal fragment of anthrax protective antigen based on attenuated recombinant <Emphasis Type="Italic">Bacillus anthracis</Emphasis>

Extracellular antigen 1 (EA1), a major component of the Bacillus anthracis surface layer (S-layer), was used as a fusion partner for the expression of heterologous antigen. A recombinant B. anthracis strain was constructed by integrating a translational fusion harboring the DNA fragments encoding the cell wall–targeting domain of the S-layer protein EA1 and the 20-kDa N-terminal fragment of anthrax protective antigen (PA20) into the chromosome. A thermosensitive plasmid expressing Cre recombinase was introduced at a permissive temperature to remove the antibiotic marker. Cre recombinase action at the loxP sites excised the spectinomycin resistance cassette. The final derivative strains were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, Western blot analysis, and immunofluorescence analysis. PA20 was successfully expressed on the S-layer of the recombinant antibiotic marker-free strain. Guinea pigs were immunized with the attenuated recombinant B. anthracis strain, and the bacilli elicited a humoral response to PA20. This antibiotic marker-free strain and the correlative experiment method may have potential applications for the generation of a live attenuated anthrax vaccine.     

Characterization of an Environmental Strain of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> from a Hot Spring in Western Himalayas

Bacillus anthracis, the etiological agent of anthrax, is responsible for a serious and often fatal disease of mammalian livestock and humans and is an important biological warfare agent. Bacillus sp. AKG was isolated from a hot spring in western Himalayas and species-specific primers targeting gyrB gene identified the strain as B. anthracis within cereus-group. Cloning, sequencing, and phylogenetic analysis of the partial gyrB sequence from strain AKG indicated a close affiliation with B. anthracis and a few recently isolated strains of B. thuringiensis (e.g., strain Al Hakam and serovar konkukian). Phylogenetic analysis of two other housekeeping genes, clpC and gdpD yielded similar results. This observation is further substantiated by phylogenetic reconstruction using concatenated sequences (1680 bases) of the three genes (gyrB, clpC, and gdpD). Phenotypic features indicated a non-anthracis affiliation for the strain AKG. A novel strategy to distinguish among strains of B. anthracis, B. cereus, and B. thuringiensis based on whole proteome comparison was developed and tested for the identification of this environmental strain. Proteome comparison was used to establish the identity of this unknown environmental strain. Group of replicate 2DE gels for whole cell proteome were generated for each of the three species and strain AKG. Protein spots unique to each group and those showing match between the groups, in a pair-wise comparison, indicated strain AKG as a member of B. thuringiensis. This strategy can be used to assign strains of B. cereus group to their respective species.     

Passive administration of monoclonal antibodies to Anthrolysin O prolong survival in mice lethally infected with <Emphasis Type="Italic">Bacillus anthracis</Emphasis>

Background  

Bacillus anthracis has two major virulence factors: a tripartite toxin that produces lethal and edema toxins and a polyglutamic acid capsule. A recent report suggested that a toxin belonging to the cholesterol dependant cytolysin (CDC) family, anthrolysin O (ALO) was a new virulence factor for B. anthracis but subsequent studies have questioned its relevance in pathogenesis. In this study, we examined the immunogenicity of recombinant anthrolysin O (rALO) in mice.     

Computer-Intensive Statistical Procedures

The overall goal of this review is to summarize the current body of knowledge about the structure and function of major proteins of Bacillus anthracis and/or similar spore-forming organisms. B. anthracis is a key spore-forming biological threat agent, as well as human and animal Gram-positive bacterial pathogen. The structural information described here is limited to approximately the last 5 years. This information is then related to the role of the selected proteins in pathogenesis and in the possible development of novel vaccine and/or other antimicrobial agents against spore-forming organisms, including anthrax, a disease caused by B. anthracis.

Among spore-forming bacteria, Bacillus and Clostridium species are the predominant spore-forming bacilli that cause serious diseases. The biochemical properties and mechanism of catalysis of the novel spore germination protease that degrades small, acid-soluble proteins protecting DNA against damage, a cofactor independent phosphoglycerate mutase, NAD⁺ synthetase, and the three know B. anthracis toxins, protective antigen, lethal factor, and edema factor are described. The studies described in this work review and unify selected information critical for the prevention of microbial diseases such as anthrax. A strategy for the structure-guided development of new prophylactic and therapeutic agents is discussed.     

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, Bacillus cereus, and Bacillus thuringiensis—One Species on the Basis of Genetic Evidence

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are members of the Bacillus cereus group of bacteria, demonstrating widely different phenotypes and pathological effects. B. anthracis causes the acute fatal disease anthrax and is a potential biological weapon due to its high toxicity. B. thuringiensis produces intracellular protein crystals toxic to a wide number of insect larvae and is the most commonly used biological pesticide worldwide. B. cereus is a probably ubiquitous soil bacterium and an opportunistic pathogen that is a common cause of food poisoning. In contrast to the differences in phenotypes, we show by multilocus enzyme electrophoresis and by sequence analysis of nine chromosomal genes that B. anthracis should be considered a lineage of B. cereus. This determination is not only a formal matter of taxonomy but may also have consequences with respect to virulence and the potential of horizontal gene transfer within the B. cereus group.     

Proteomic Profiling and Identification of Immunodominant Spore Antigens of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis

Differentially expressed and immunogenic spore proteins of the Bacillus cereus group of bacteria, which includes Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis, were identified. Comparative proteomic profiling of their spore proteins distinguished the three species from each other as well as the virulent from the avirulent strains. A total of 458 proteins encoded by 232 open reading frames were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis for all the species. A number of highly expressed proteins, including elongation factor Tu (EF-Tu), elongation factor G, 60-kDa chaperonin, enolase, pyruvate dehydrogenase complex, and others exist as charge variants on two-dimensional gels. These charge variants have similar masses but different isoelectric points. The majority of identified proteins have cellular roles associated with energy production, carbohydrate transport and metabolism, amino acid transport and metabolism, posttranslational modifications, and translation. Novel vaccine candidate proteins were identified using B. anthracis polyclonal antisera from humans postinfected with cutaneous anthrax. Fifteen immunoreactive proteins were identified in B. anthracis spores, whereas 7, 14, and 7 immunoreactive proteins were identified for B. cereus and in the virulent and avirulent strains of B. thuringiensis spores, respectively. Some of the immunodominant antigens include charge variants of EF-Tu, glyceraldehyde-3-phosphate dehydrogenase, dihydrolipoamide acetyltransferase, Δ-1-pyrroline-5-carboxylate dehydrogenase, and a dihydrolipoamide dehydrogenase. Alanine racemase and neutral protease were uniquely immunogenic to B. anthracis. Comparative analysis of the spore immunome will be of significance for further nucleic acid- and immuno-based detection systems as well as next-generation vaccine development.