Search

The pathogenesis of Bacillus anthracis depends on several virulence factors, including the anthrax toxin. Loss of the alternative sigma factor σI results in a coordinate decrease in expression of all three toxin subunits. Our observations suggest that loss of σI alters the activity of the master virulence regulator AtxA, but atxA transcription is unaffected by loss of σI. σI-containing RNA polymerase does not appear to directly transcribe either atxA or the toxin gene pagA. As in Bacillus subtilis, loss of σI in B. anthracis results in increased sensitivity to heat shock and transcription of sigI, encoding σI, is induced by elevated temperature. Encoded immediately downstream of and part of a bicistronic message with sigI is an anti-sigma factor, RsgI, which controls σI activity. Loss of RsgI has no direct effect on virulence gene expression. sigI appears to be expressed from both the σI and σA promoters, and transcription from the σA promoter is likely more significant to virulence regulation. We propose a model in which σI can be induced in response to heat shock, whilst, independently, σI is produced under non-heat-shock, toxin-inducing conditions to indirectly regulate virulence gene expression.

Bacillus anthracis, the causative agent of anthrax, is exposed to host-mediated antibacterial activities, such as reactive oxygen species (ROS), during the early stages of its disease process. The ability to resist these host-mediated stresses is an essential characteristic of a successful pathogen while it is generally assumed that non-pathogenic environmental bacteria succumb to these antimicrobial activities. In order to gain insights into the underlying mechanisms that pathogens use to resist host-mediated oxidative stress, we have compared the oxidative stress responses of B. anthracis and Bacillus subtilis, a well-studied environmental bacterium. Among the four putative catalases encoded by B. anthracis we identified KatB as the main vegetative catalase. Comparative analysis of catalase production in B. anthracis and B. subtilis in response to superoxide and peroxide stress reveals different expression profiles, even though both are regulated by the PerR repressor, which senses and responds to peroxide stress. A B. anthracis perR deletion mutant exhibits enhanced KatB activity and is hyper-resistant to peroxide stress. Superoxide dismutase A1 (SodA1) is the main contributor to the intracellular superoxide dismutase activity in vegetative cells and the gene encoding this enzyme is constitutively expressed. Although aspects of the ROS detoxifying systems of B. anthracis and B. subtilis are similar, their responses to superoxide stress are different. The observed differences are likely to reflect adaptations to specific environmental niches.

Deposition of Bacillus anthracis endospores within either the lungs or nasal passages of A/J mice after aerosol exposure was influenced by different particle sized aerosols and resulted in different infection kinetics. The infection resulting from the inhalation of endospores within a 12 μm particle aerosol was prolonged compared to that from a 1 μm particle aerosol with a mean time-to-death of 161 ± 16.1 h and 101.6 ± 10.4 h, respectively. Inhalation of endospores within 1 μm or 12 μm particle aerosols resulted in a median lethal dose of 2432 and 7656 c.f.u., respectively. Initial involvement of the upper respiratory tract lymph nodes was observed in 75-83% of mice exposed to either the 1 μm or 12 μm particle inhalational infections. Lung deposition was significantly greater after inhalation of the 1 μm particle aerosol with pronounced involvement of the mediastinal lymph node. Gastrointestinal involvement was observed only in mice exposed to 12 μm particle aerosols where bacteriological and histopathological analysis indicated primary gastritis (17%), activation of the Peyer's patches (72%) and colonization and necrosis of the mesenteric lymph nodes (67%). Terminal disease was characterized by bacteraemia in both inhalational infections with preferential dissemination to spleen, liver, kidneys and thymus. Immunization with 1 μg recombinant protective antigen vaccine was equally efficacious against B. anthracis infections arising from the inhalation of 1 and 12 μm particle aerosols, providing 73-80% survival under a suboptimum immunization schedule.

Anthrax is a zoonotic disease caused by Bacillus anthracis. The infection is associated with inflammation and sepsis, but little is known about the acute-phase response during disease and the nature of the bacterial factors causing it. In this study, we examined the levels of the acute-phase proteins (APPs) in comparative experiments using mice challenged with spores and a purified B. anthracis protease InhA as a possible factor mediating the response. A strong increase in the plasma levels of APPs such as haptoglobin and serum amyloid A was observed during infection. Protein and mRNA levels of plasminogen activator inhibitor (PAI)-1 in the liver were also increased concurrently with bacterial dissemination at 72 h post-infection. Similar effects were observed at 6 h post injection with InhA. Induction of hepatic transforming growth factor-beta1, a PAI-1 inducer, was also found in the liver of InhA-injected mice. PAI-1 elevation by InhA resulted in an increased level of urokinase-type plasminogen activator complex with PAI-1 and a decreased level of D-dimers indicating inhibition of blood fibrinolysis. These results reveal an acute liver response to anthrax infection and provide a plausible pathophysiological link between the host inflammatory response and the pro-thrombotic haemostatic imbalance in the course of disease through PAI-1 induction in the liver.

Inhalational anthrax is the most severe form of anthrax. It has been shown in small-animal and non-human primate models that relatively large pools of ungerminated Bacillus anthracis spores can remain within the alveolar spaces for days to weeks post-inhalation or until transported to areas more favourable for germination and bacillary outgrowth. In this study, spores of the Ames strain that were exposed to germination-inducing media prior to intranasal delivery were significantly less infectious than spores delivered in either water or germination-inhibitory medium. The effect of manipulating the germination potential of these spores within the lungs of infected mice by exogenous germination-altering media was examined. The data suggested that neither inducing germination nor inhibiting germination of spores within the lungs protected mice from the ensuing infection. Germination-altering strategies could, instead, significantly increase the severity of disease in a mouse model of inhalational anthrax when implemented in vivo. It was shown that germination-altering strategies, in this study, were not beneficial to the infected host and are impractical as in vivo countermeasures.

Bacterial spores possess a series of concentrically arranged protective structures that contribute to dormancy, survival and, ultimately, germination. One of these structures, the coat, is present in all spores. In Bacillus anthracis, however, the spore is surrounded by an additional, poorly understood, morphologically complex structure called the exosporium. Here, we characterize three previously discovered exosporium proteins called ExsFA (also known as BxpB), ExsFB (a highly related paralogue of exsFA/bxpB) and IunH (similar to an inosine-uridine-preferring nucleoside hydrolase). We show that in the absence of ExsFA/BxpB, the exosporium protein BclA accumulates asymmetrically to the forespore pole closest to the midpoint of the sporangium (i.e. the mother-cell-proximal pole of the forespore), instead of uniformly encircling the exosporium. ExsFA/BxpB may also have a role in coat assembly, as mutant spore surfaces lack ridges seen in wild-type spores and have a bumpy appearance. ExsFA/BxpB also has a modest but readily detected effect on germination. Nonetheless, an exsFA/bxpB mutant strain is fully virulent in both intramuscular and aerosol challenge models in Guinea pigs. We show that the pattern of localization of ExsFA/BxpB-GFP is a ring, consistent with a location for this protein in the basal layer of the exosporium. In contrast, ExsFB-GFP fluorescence is a solid oval, suggesting a distinct subcellular location for ExsFB-GFP. We also used these fusion proteins to monitor changes in the subcellular locations of these proteins during sporulation. Early in sporulation, both fusions were present throughout the mother cell cytoplasm. As sporulation progressed, GFP fluorescence moved from the mother cell cytoplasm to the forespore surface and formed either a ring of fluorescence, in the case of ExsFA/BxpB, or a solid oval of fluorescence, in the case of ExsFB. IunH-GFP also resulted in a solid oval of fluorescence. We suggest the interpretation that at least some ExsFB-GFP and IunH-GFP resides in the region between the coat and the exosporium, called the interspace.

The significance of Bacillus anthracis as an agent of bioterrorism has been well established. An understanding of both the pathogenesis and the host response is required to elucidate approaches to more rapidly detect and effectively prevent or treat anthrax. Current vaccine strategies are focused primarily on production of antibodies against the protective antigen components of the anthrax toxins, which are secreted by the bacilli. A better understanding of the dynamic morphology of the dormant and germinating spore and its interaction with the host immune system could be important in developing an optimally efficacious anthrax vaccine. A spore-associated protein was identified that was specific to the Bacillus cereus group of bacteria and referred to as spore opsonization-associated antigen A (SoaA). Immuno-electron microscopy localized this protein to the area of the cortex beneath the coat of the dormant spore. Although our data suggested that SoaA was found below the coat layers of the ungerminated spore, SoaA was involved in the interaction of spores with macrophages shortly after infection. To investigate further the specific properties of the SoaA protein, the soaA gene was inactivated in the B. anthracis Ames strain. The SoaA protein in the Ames strain of B. anthracis increased the phagocytic uptake of the spores in the presence of anti-spore antibodies. Unlike the wild-type strain, the mutant soaA : : Kan strain was not readily opsonized by anti-spore antibodies. While the mutant spores retained characteristic resistance properties in vitro and virulence in vivo, the soaA : : Kan mutant strain was significantly less suited for survival in vivo when competed against the wild-type Ames strain.

The Gram-positive, spore-forming pathogen Bacillus anthracis is the aetiological agent of anthrax. Its main virulence factors are two toxins and an anti-phagocytic capsule. When B. anthracis is grown in laboratory culture, the highest expression of the anthrax toxin genes occurs during entry into stationary phase, suggesting that nutrient limitation is an environmental cue which induces toxin production. A common bacterial response to starvation is the so-called stringent response, in which the hyperphosphorylated guanosine nucleotide (p)ppGpp is the effector molecule. In Escherichia coli, Bacillus subtilis and other bacteria, accumulation of this molecule leads to down-regulation of stable RNA synthesis and upregulation of the expression of genes involved in survival under nutrient-poor conditions. This study focuses on the stringent response of B. anthracis. We show that in B. anthracis the relA gene is responsible for the synthesis of (p)ppGpp and the stringent down-regulation of stable RNA synthesis upon starvation for the essential amino acids isoleucine, leucine and valine. The deletion of relA did not affect the expression of the virulence gene pagA or virulence in a mouse model of infection. In contrast, spore counts upon growth and sporulation in a defined medium were approximately 10,000-fold lower for the relA deletion mutant than for the parental strain. The contribution of the stringent response to efficient sporulation of B. anthracis is notable, as this suggests that the stringent response may contribute to the persistence of B. anthracis in the natural environment.

Biofilms, communities of micro-organisms attached to a surface, are responsible for many chronic diseases and are often associated with environmental reservoirs or lifestyles. Bacillus anthracis is a Gram-positive, endospore-forming bacterium and is the aetiological agent of pulmonary, gastrointestinal and cutaneous anthrax. Anthrax infections are part of the natural lifecycle of many ruminants in North America, including cattle and bison, and B. anthracis is thought to be a central part of this ecosystem. However, in endemic areas in which humans and livestock interact, chronic cases of cutaneous anthrax are commonly reported. This suggests that biofilms of B. anthracis exist in the environment and are part of the ecology associated with its lifecycle. Currently, there are few data that account for the importance of the biofilm mode of life in B. anthracis, yet biofilms have been characterized in other pathogenic and non-pathogenic Bacillus species, including Bacillus cereus and Bacillus subtilis, respectively. This study investigated the phenotypic and functional role of biofilms in B. anthracis. The results demonstrate that B. anthracis readily forms biofilms which are inherently resistant to commonly prescribed antibiotics, and that antibiotic resistance is not solely the function of sporulation.

The Bacillus subtilis AbrB protein is a DNA-binding global regulator of a plethora of functions that are expressed during the transition from exponential growth to stationary phase and under suboptimal growth conditions. AbrB orthologues have been identified in a variety of prokaryotic organisms, notably in all species of Bacillus, Clostridium and Listeria that have been examined. Based on amino acid sequence identity in the N-terminal domains of the orthologues from B. subtilis and Bacillus anthracis, it was predicted that the proteins might display identical DNA-binding specificities. The binding of purified B. anthracis AbrB (AbrB(BA)) and purified B. subtilis AbrB (AbrB(BS)) at DNA targets of B. subtilis, B. anthracis and a synthetic origin was compared. In all cases examined, DNA-binding specificity was identical as judged by DNase I footprinting. In B. subtilis cells, the B. anthracis promoters from the atxA and abrB genes were regulated by AbrB(BS), and the B. subtilis promoter from the yxbB operon was regulated by AbrB(BA).

Spores of Bacillus anthracis, the causative agent of anthrax, possess an exosporium. As the outer surface layer of these mature spores, the exosporium represents the primary contact surface between the spore and environment/host and is a site of spore antigens. The exosporium was isolated from the endospores of the B. anthracis wild-type Ames strain, from a derivative of the Ames strain cured of plasmid pXO2(-), and from a previously isolated pXO1(-), pXO2(-) doubly cured strain, B. anthracis UM23Cl2. The protein profiles of SDS-PAGE-separated exosporium extracts were similar for all three. This suggests that avirulent variants lacking either or both plasmids are realistic models for studying the exosporium from spores of B. anthracis. A number of loosely adsorbed proteins were identified from amino acid sequences determined by either nanospray-MS/MS or N-terminal sequencing. Salt and detergent washing of the exosporium fragments removed these and revealed proteins that are likely to represent structural/integral exosporium proteins. Seven proteins were identified in washed exosporium: alanine racemase, inosine hydrolase, ExsF, CotY, ExsY, CotB and a novel protein, named ExsK. CotY, ExsY and CotB are homologues of Bacillus subtilis outer spore coat proteins, but ExsF and ExsK are specific to B. anthracis and other members of the Bacillus cereus group.