Showing papers on "Bacillus anthracis published in 2009"

Raxibacumab for the treatment of inhalational anthrax.

Abstract: Background Inhalational anthrax caused by Bacillus anthracis is associated with high mortality primarily due to toxin-mediated injury. Raxibacumab is a human IgG1λ monoclonal antibody directed against protective antigen, a component of the anthrax toxin. Methods We evaluated the efficacy of raxibacumab as a prophylactic agent and after disease onset in a total of four randomized, placebo-controlled studies conducted in rabbits and monkeys. Animals were exposed to an aerosolized target exposure of B. anthracis spores that was approximately 100 times (in the prophylactic studies) and 200 times (in the therapeutic-intervention studies) the median lethal dose. In the therapeutic-intervention studies, animals were monitored for the onset of symptoms. Animals with detectable protective antigen in serum, a significant increase in temperature, or both received a single intravenous bolus of placebo or raxibacumab at a dose of either 20 mg per kilogram of body weight or 40 mg per kilogram. The primary end point was...

What Sets Bacillus anthracis Apart from Other Bacillus Species

Abstract: Bacillus anthracis is the cause of anthrax, and two large plasmids are essential for toxicity: pXO1, which contains the toxin genes, and pXO2, which encodes a capsule. B. anthracis forms a highly monomorphic lineage within the B. cereus group, but strains of Bacillus thuringiensis and B. cereus exist that are genetically closely related to the B. anthracis cluster. During the past five years B. cereus strains that contain the pXO1 virulence plasmid were discovered, and strains with both pXO1 and pXO2 have been isolated from great apes in Africa. Therefore, the presence of pXO1 and pXO2 no longer principally separates B. anthracis from other Bacilli. The B. anthracis lineage carries a specific mutation in the global regulator PlcR, which controls the transcription of secreted virulence factors in B. cereus and B. thuringiensis. Coevolution of the B. anthracis chromosome with its plasmids may be the basis for the successful development and uniqueness of the B. anthracis lineage.

Dendritic Cell Targeting of Bacillus anthracis Protective Antigen Expressed by Lactobacillus acidophilus Protects Mice from Lethal Challenge

Abstract: Efficient vaccines potentiate antibody avidity and increase T cell longevity, which confer protection against microbial lethal challenge. A vaccine strategy was established by using Lactobacillus acidophilus to deliver Bacillus anthracis protective antigen (PA) via specific dendritic cell-targeting peptides to dendritic cells (DCs), which reside in the periphery and mucosal surfaces, thus directing and regulating acquired immunity. The efficiency of oral delivery of L. acidophilus expressing a PA-DCpep fusion was evaluated in mice challenged with lethal B. anthracis Sterne. Vaccination with L. acidophilus expressing PA-DCpep induced robust protective immunity against B. anthracis Sterne compared with mice vaccinated with L. acidophilus expressing PA-control peptide or an empty vector. Additionally, serum anti-PA titers, neutralizing PA antibodies, and the levels of IgA-expressing cells were all comparable with the historical recombinant PA plus aluminum hydroxide vaccine administered s.c. Collectively, development of this strategy for oral delivery of DC-targeted antigens provides a safe and protective vaccine via a bacterial adjuvant that may potentiate mucosal immune responses against deadly pathogens.

The Secret Life of the Anthrax Agent Bacillus anthracis: Bacteriophage-Mediated Ecological Adaptations

Abstract: Ecological and genetic factors that govern the occurrence and persistence of anthrax reservoirs in the environment are obscure. A central tenet, based on limited and often conflicting studies, has long held that growing or vegetative forms of Bacillus anthracis survive poorly outside the mammalian host and must sporulate to survive in the environment. Here, we present evidence of a more dynamic lifecycle, whereby interactions with bacterial viruses, or bacteriophages, elicit phenotypic alterations in B. anthracis and the emergence of infected derivatives, or lysogens, with dramatically altered survival capabilities. Using both laboratory and environmental B. anthracis strains, we show that lysogeny can block or promote sporulation depending on the phage, induce exopolysaccharide expression and biofilm formation, and enable the long-term colonization of both an artificial soil environment and the intestinal tract of the invertebrate redworm, Eisenia fetida. All of the B. anthracis lysogens existed in a pseudolysogenic-like state in both the soil and worm gut, shedding phages that could in turn infect non-lysogenic B. anthracis recipients and confer survival phenotypes in those environments. Finally, the mechanism behind several phenotypic changes was found to require phage-encoded bacterial sigma factors and the expression of at least one host-encoded protein predicted to be involved in the colonization of invertebrate intestines. The results here demonstrate that during its environmental phase, bacteriophages provide B. anthracis with alternatives to sporulation that involve the activation of soil-survival and endosymbiotic capabilities.

Capillary morphogenesis protein-2 is the major receptor mediating lethality of anthrax toxin in vivo.

Abstract: Anthrax toxin, a major virulence factor of Bacillus anthracis, gains entry into target cells by binding to either of 2 von Willebrand factor A domain-containing proteins, tumor endothelium marker-8 (TEM8) and capillary morphogenesis protein-2 (CMG2). The wide tissue expression of TEM8 and CMG2 suggest that both receptors could play a role in anthrax pathogenesis. To explore the roles of TEM8 and CMG2 in normal physiology, as well as in anthrax pathogenesis, we generated TEM8- and CMG2-null mice and TEM8/CMG2 double-null mice by deleting TEM8 and CMG2 transmembrane domains. TEM8 and CMG2 were found to be dispensable for mouse development and life, but both are essential in female reproduction in mice. We found that the lethality of anthrax toxin for mice is mostly mediated by CMG2 and that TEM8 plays only a minor role. This is likely because anthrax toxin has approximately 11-fold higher affinity for CMG2 than for TEM8. Finally, the CMG2-null mice are also shown to be highly resistant to B. anthracis spore infection, attesting to the importance of both anthrax toxin and CMG2 in anthrax infections.

Identification of Bacillus anthracis by using matrix-assisted laser desorption ionization-time of flight mass spectrometry and artificial neural networks.

Abstract: This report demonstrates the applicability of a combination of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) and chemometrics for rapid and reliable identification of vegetative cells of the causative agent of anthrax, Bacillus anthracis. Bacillus cultures were prepared under standardized conditions and inactivated according to a recently developed MS-compatible inactivation protocol for highly pathogenic microorganisms. MALDI-TOF MS was then employed to collect spectra from the microbial samples and to build up a database of bacterial reference spectra. This database comprised mass peak profiles of 374 strains from Bacillus and related genera, among them 102 strains of B. anthracis and 121 strains of B. cereus. The information contained in the database was investigated by means of visual inspection of gel view representations, univariate t tests for biomarker identification, unsupervised hierarchical clustering, and artificial neural networks (ANNs). Analysis of gel views and independent t tests suggested B. anthracis- and B. cereus group-specific signals. For example, mass spectra of B. anthracis exhibited discriminating biomarkers at 4,606, 5,413, and 6,679 Da. A systematic search in proteomic databases allowed tentative assignment of some of the biomarkers to ribosomal protein or small acid-soluble proteins. Multivariate pattern analysis by unsupervised hierarchical cluster analysis further revealed a subproteome-based taxonomy of the genus Bacillus. Superior classification accuracy was achieved when supervised ANNs were employed. For the identification of B. anthracis, independent validation of optimized ANN models yielded a diagnostic sensitivity of 100% and a specificity of 100%.

Four Superoxide Dismutases Contribute to Bacillus anthracis Virulence and Provide Spores with Redundant Protection from Oxidative Stress

Abstract: The Bacillus anthracis genome encodes four superoxide dismutases (SODs), enzymes capable of detoxifying oxygen radicals. That two of these SODs, SOD15 and SODA1, are present in the outermost layers of the B. anthracis spore is indicated by previous proteomic analyses of the exosporium. Given the requirement that spores must survive interactions with reactive oxygen species generated by cells such as macrophages during infection, we hypothesized that SOD15 and SODA1 protect the spore from oxidative stress and contribute to the pathogenicity of B. anthracis. To test these theories, we constructed a double-knockout (Δsod15 ΔsodA1) mutant of B. anthracis Sterne strain 34F2 and assessed its lethality in an A/J mouse intranasal infection model. The 50% lethal dose of the Δsod15 ΔsodA1 strain was similar to that of the wild type (34F2), but surprisingly, measurable whole-spore SOD activity was greater than that in 34F2. A quadruple-knockout strain (Δsod15 ΔsodA1 ΔsodC ΔsodA2) was then generated, and as anticipated, spore-associated SOD activity was diminished. Moreover, the quadruple-knockout strain, compared to the wild type, was attenuated more than 40-fold upon intranasal challenge of mice. Spore resistance to exogenously generated oxidative stress and to macrophage-mediated killing correlated with virulence in A/J mice. Allelic exchange that restored sod15 and sodA1 to their wild-type state restored wild-type characteristics. We conclude that SOD molecules within the spore afford B. anthracis protection against oxidative stress and enhance the pathogenicity of B. anthracis in the lung. We also surmise that the presence of four SOD alleles within the genome provides functional redundancy for this key enzyme.

Detection of anthrax toxin by an ultrasensitive immunoassay using europium nanoparticles.

Abstract: We developed a europium nanoparticle-based immunoassay (ENIA) for the sensitive detection of anthrax protective antigen (PA). The ENIA exhibited a linear dose-dependent pattern within the detection range of 0.01 to 100 ng/ml and was approximately 100-fold more sensitive than enzyme-linked immunosorbent assay (ELISA). False-positive results were not observed with serum samples from healthy adults, mouse plasma without PA, or plasma samples collected from mice injected with anthrax lethal factor or edema factor alone. For the detection of plasma samples spiked with PA, the detection sensitivities for ENIA and ELISA were 100% (11/11 samples) and 36.4% (4/11 samples), respectively. The assay exhibited a linear but qualitative correlation between the PA injected and the PA detected in murine blood (r = 0.97731; P < 0.0001). Anthrax PA was also detected in the circulation of mice infected with spores from a toxigenic Sterne-like strain of Bacillus anthracis, but only in the later stages of infection. These results indicate that the universal labeling technology based on europium nanoparticles and its application may provide a rapid and sensitive testing platform for clinical diagnosis and laboratory research.

CD14-Mac-1 interactions in Bacillus anthracis spore internalization by macrophages

Abstract: Anthrax, a potentially lethal disease of animals and humans, is caused by the Gram-positive spore-forming bacterium Bacillus anthracis. The outermost exosporium layer of B. anthracis spores contains an external hair-like nap formed by the glycoprotein BclA. Recognition of BclA by the integrin Mac-1 promotes spore uptake by professional phagocytes, resulting in the carriage of spores to sites of spore germination and bacterial growth in distant lymphoid organs. We show that CD14 binds to rhamnose residues of BclA and acts as a coreceptor for spore binding by Mac-1. In this process, CD14 induces signals involving TLR2 and PI3k that promote inside-out activation of Mac-1, thereby enhancing spore internalization by macrophages. As observed with mice lacking Mac-1, CD14−/− mice are also more resistant than wild-type mice to infection by B. anthracis spores. Additionally, after B. anthracis spore challenge of CD14−/− mice, interference with the CD14-mediated signaling pathways results in increased mortality. Our results show that the binding and uptake of B. anthracis spores by phagocytic cells is a dynamic process and involves multiple receptors and signaling pathways.

The global regulator CodY regulates toxin gene expression in Bacillus anthracis and is required for full virulence.

Abstract: In gram-positive bacteria, CodY is an important regulator of genes whose expression changes upon nutrient limitation and acts as a repressor of virulence gene expression in some pathogenic species. Here, we report the role of CodY in Bacillus anthracis, the etiologic agent of anthrax. Disruption of codY completely abolished virulence in a toxinogenic, noncapsulated strain, indicating that the activity of CodY is required for full virulence of B. anthracis. Global transcriptome analysis of a codY mutant and the parental strain revealed extensive differences. These differences could reflect direct control for some genes, as suggested by the presence of CodY binding sequences in their promoter regions, or indirect effects via the CodY-dependent control of other regulatory proteins or metabolic rearrangements in the codY mutant strain. The differences included reduced expression of the anthrax toxin genes in the mutant strain, which was confirmed by lacZ reporter fusions and immunoblotting. The accumulation of the global virulence regulator AtxA protein was strongly reduced in the mutant strain. However, in agreement with the microarray data, expression of atxA, as measured using an atxA-lacZ transcriptional fusion and by assaying atxA mRNA, was not significantly affected in the codY mutant. An atxA-lacZ translational fusion was also unaffected. Overexpression of atxA restored toxin component synthesis in the codY mutant strain. These results suggest that CodY controls toxin gene expression by regulating AtxA accumulation posttranslationally.

The complete genome sequence of Bacillus anthracis Ames "Ancestor"

Abstract: The pathogenic bacterium Bacillus anthracis has become the subject of intense study as a result of its use in a bioterrorism attack in the United States in September and October 2001. Previous studies suggested that B. anthracis Ames Ancestor, the original Ames fully virulent plasmid-containing isolate, was the ideal reference. This study describes the complete genome sequence of that original isolate, derived from a sample kept in cold storage since 1981.

Bacillus anthracis HssRS signalling to HrtAB regulates haem resistance during infection.

Abstract: Bacillus anthracis proliferates to high levels within vertebrate tissues during the pathogenesis of anthrax. This growth is facilitated by the acquisition of nutrient iron from host haem. However, haem acquisition can lead to the accumulation of toxic amounts of haem within B. anthracis. Here, we show that B. anthracis resists haem toxicity by sensing haem through the HssRS two-component system, which regulates expression of the haem-detoxifying transporter HrtAB. In addition, we demonstrate that B. anthracis exhibits elevated HssRS function compared with its evolutionary relative Staphylococcus aureus. Elevated haem sensing is likely required by B. anthracis due to the significant haem sensitivity exhibited by members of the genus Bacilli. We also demonstrate that B. anthracis depends on conserved residues within the previously uncharacterized sensing domain of the histidine kinase HssS for HssS function. Finally, we show that the haem- and HssRS-regulated hrtAB promoter is activated in a murine model of anthrax. These results demonstrate the evolutionary conservation of haem sensing among multiple Gram-positive bacteria and begin to provide a mechanistic explanation for the haem resistance of B. anthracis. Further, these data suggest that haem stress is experienced by bacterial pathogens during infection.

The Spore-Specific Alanine Racemase of Bacillus anthracis and Its Role in Suppressing Germination during Spore Development

Abstract: Spores of Bacillus anthracis are enclosed by an exosporium composed of a basal layer and an external hair-like nap. The nap is apparently formed by a single glycoprotein, while the basal layer contains many different structural proteins and several enzymes. One of the enzymes is Alr, an alanine racemase capable of converting the spore germinant l-alanine to the germination inhibitor d-alanine. Unlike other characterized exosporium proteins, Alr is nonuniformly distributed in the exosporium and might have a second spore location. In this study, we demonstrated that expression of the alr gene, which encodes Alr, is restricted to sporulating cells and that the bulk of alr transcription and Alr synthesis occurs during the late stages of sporulation. We also mapped two alr promoters that are differentially active during sporulation and might be involved in the atypical localization of Alr. Finally, we constructed a Δalr mutant of B. anthracis that lacks Alr and examined the properties of the spores produced by this strain. Mature Δalr spores germinate more efficiently in the presence of l-alanine, presumably because of their inability to convert exogenous l-alanine to d-alanine, but they respond normally to other germinants. Surprisingly, the production of mature spores by the Δalr mutant is defective because approximately one-half of the nascent spores germinate and lose their resistance properties before they are released from the mother cell. This phenotype suggests that an important function of Alr is to produce d-alanine during the late stages of sporulation to suppress premature germination of the developing spore.

Phage coated magnetoelastic micro-biosensors for real-time detection of Bacillus anthracis spores

Abstract: A micro-scale, freestanding, magnetoelastic biosensor coated with phage has been developed for the real-time in vitro detection of Bacillus anthracis spores. The sensor exhibits a characteristic resonance frequency upon the application of an alternating external magnetic field. It has a high sensitivity to the change in mass when spores are attached. The frequency versus mass sensitivity increases significantly with a decrease in sensor length. Spore detection is realized by measuring the resonance frequency change due to the change in mass as spores are captured onto the sensor surface. B. anthracis spore suspensions in a range of concentration levels (5 × 10 1 to 5 × 10 8 spores/ml) was tested using a 1000 μm × 200 μm × 15 μm sensor in a flowing fluid at a flow rate of 40 μl/min. The binding kinetics was analyzed based on the attachment rate. The specificity of the sensor to B. anthracis spores was examined compared with other Bacillus species.

Codon-Optimized Fluorescent Proteins Designed for Expression in Low-GC Gram-Positive Bacteria

Abstract: Fluorescent proteins have wide applications in biology. However, not all of these proteins are properly expressed in bacteria, especially if the codon usage and genomic GC content of the host organism are not ideal for high expression. In this study, we analyzed the DNA sequences of multiple fluorescent protein genes with respect to codons and GC content and compared them to a low-GC gram-positive bacterium, Bacillus anthracis. We found high discrepancies for cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and the photoactivatable green fluorescent protein (PAGFP), but not GFP, with regard to GC content and codon usage. Concomitantly, when the proteins were expressed in B. anthracis, CFP- and YFP-derived fluorescence was undetectable microscopically, a phenomenon caused not by lack of gene transcription or degradation of the proteins but by lack of protein expression. To improve expression in bacteria with low genomic GC contents, we synthesized a codon-optimized gfp and constructed optimized photoactivatable pagfp, cfp, and yfp, which were in contrast to nonoptimized genes highly expressed in B. anthracis and in another low-GC gram-positive bacterium, Staphylococcus aureus. Using optimized GFP as a reporter, we were able to monitor the activity of the protective antigen promoter of B. anthracis and confirm its dependence on bicarbonate and regulators present on virulence plasmid pXO1.

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

Abstract: Bacillus anthracis is an endospore-forming gram-positive pathogen that causes the infectious disease anthrax in mammals, including humans. Infections can occur via intradermal inoculation, ingestion, or inhalation of spores (24). Although anthrax infections via the former two routes are usually self-contained, inhalational anthrax is often lethal (23). In a mouse model of inhalational anthrax, inhaled B. anthracis spores are phagocytosed by alveolar macrophages that are believed to migrate to local lymph nodes (10). During migration, the spores germinate inside the macrophage phagolysosome to give rise to vegetative bacilli. The newly formed vegetative cells lyse the phagolysosome and replicate inside the macrophage cytoplasm (6), eventually escaping from the macrophage into the bloodstream. Therefore, in order to establish a successful anthrax infection, B. anthracis must survive and replicate intracellularly inside the macrophage, as well as extracellularly in the host's blood. Upon entering the bloodstream, B. anthracis is targeted by an array of innate immune mediators circulating in the host's blood, such as the complement proteins and cellular components such as neutrophils and platelets in humans. However, inhalational anthrax infection in animals is characterized by rapid progression into systemic bacteremia and the heavy growth of B. anthracis in the bloodstream (21). This observation indicates that B. anthracis is able not only to evade complement-mediated lysis and but also to resist the antibacterial activities of innate immune cells. One important antibacterial activity of innate immune cells in the human blood relies on the production of cationic antimicrobial peptides. These peptides are present in the cytosolic granules of neutrophils, eosinophils, and platelets and are released upon contact with bacterial pathogens (18). Cationic antimicrobial peptides interact electrostatically with negatively charged cell surface molecules, such as teichoic acids and phosphatidylglycerols of gram-positive bacteria, subsequently inducing disintegration of membrane structures and ultimately causing bacterial cell death (41). Some gram-positive pathogens, however, possess resistance mechanisms, by which they change cell surface properties and avoid killing by cationic antimicrobial peptides. For example, gram-positive pathogens, such as Staphylococcus aureus (29, 30), Listeria monocytogenes (1, 37), and Streptococcus pneumoniae (16), are able to be modify teichoic acids and phospholipids with d-alanine by DltABCD and l-lysine by MprF, respectively. Since these modifications contribute to a net positive charge on the cell surface, they are believed to facilitate repulsion of the cationic peptides. Identifying the B. anthracis genes that contribute to cationic peptide resistance can elucidate the molecular basis of this virulence trait. A recent study has shown that the B. anthracis genome contains a functional dltABCD operon (7). A B. anthracis mutant strain inactivated in this operon exhibits hypersusceptibility to various cationic antimicrobial peptides, decreased survival in macrophages, and virulence attenuation in a mouse model of inhalational infection. To date, the dltABCD operon is the only genetic determinant of B. anthracis experimentally proven to contribute to cationic antimicrobial peptide resistance. In the present study, we have identified a B. anthracis gene (BA1486 in the ΔANR [pXO1−, pXO2−] strain; BAS1375 in the Sterne 34F2 [pXO1+, pXO2−] strain) whose knockout leads to hypersusceptibility to protamine, as well as to human cationic antimicrobial peptides, α-helical LL-37, and β-sheet human neutrophil peptide 1 (HNP-1). We show that inactivation of this gene results in a strain that is unable to synthesize phosphatidylglycerols modified with lysine. Our results demonstrate that the B. anthracis genome carries a functional mprF gene required for cationic antimicrobial peptide resistance.