Natural Dissemination of Bacillus anthracis Spores in Northern Canada

Soil samples were collected from around fresh and year-old bison carcasses and areas not associated with known carcasses in Wood Buffalo National Park during an active anthrax outbreak in the summer of 2001. Sample selection with a grid provided the most complete coverage of a site. Soil samples were screened for viable Bacillus anthracis spores via selective culture, phenotypic analysis, and PCR. Bacillus anthracis spores were isolated from 28.4% of the samples. The highest concentrations of B. anthracis spores were found directly adjacent to fresh carcasses and invariably corresponded to locations where the soil had been saturated with body fluids escaping the carcass through either natural body orifices or holes torn by scavengers. The majority of positive samples were found within 2 m of both year-old and fresh carcasses and probably originated from scavengers churning up and spreading the body fluid-saturated soil as they fed. Trails of lesser contamination radiating from the carcasses probably resulted from spore dissemination through adhesion to scavengers and through larger scavengers dragging away disarticulated limbs. Comparison of samples from minimally scavenged and fully necropsied carcass sites revealed no statistically significant difference in the level of B. anthracis spore contamination. Therefore, the immediate area around a suspected anthrax carcass should be considered substantially contaminated regardless of the condition of the carcass.     

Interactions between Bacillus anthracis and Plants May Promote Anthrax Transmission

Environmental reservoirs are essential in the maintenance and transmission of anthrax but are poorly characterized. The anthrax agent, Bacillus anthracis was long considered an obligate pathogen that is dormant and passively transmitted in the environment. However, a growing number of laboratory studies indicate that, like some of its close relatives, B. anthracis has some activity outside of its vertebrate hosts. Here we show in the field that B. anthracis has significant interactions with a grass that could promote anthrax spore transmission to grazing hosts. Using a local, virulent strain of B. anthracis, we performed a field experiment in an enclosure within a grassland savanna. We found that B. anthracis increased the rate of establishment of a native grass (Enneapogon desvauxii) by 50% and that grass seeds exposed to blood reached heights that were 45% taller than controls. Further we detected significant effects of E. desvauxii, B. anthracis, and their interaction on soil bacterial taxa richness and community composition. We did not find any evidence for multiplication or increased longevity of B. anthracis in bulk soil associated with grass compared to controls. Instead interactions between B. anthracis and plants may result in increased host grazing and subsequently increased transmission to hosts.     

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.     

Bacillus anthracis Multiplication, Persistence, and Genetic Exchange in the Rhizosphere of Grass Plants

Bacillus anthracis, the causative agent of anthrax, is known for its rapid proliferation and dissemination in mammalian hosts. In contrast, little information exists regarding the lifestyle of this important pathogen outside of the host. Considering that Bacillus species, including close relatives of B. anthracis, are saprophytic soil organisms, we investigated the capacity of B. anthracis spores to germinate in the rhizosphere and to establish populations of vegetative cells that could support horizontal gene transfer in the soil. Using a simple grass plant-soil model system, we show that B. anthracis strains germinate on and around roots, growing in characteristic long filaments. From 2 to 4 days postinoculation, approximately one-half of the B. anthracis CFU recovered from soil containing grass seedlings arose from heat-sensitive organisms, while B. anthracis CFU retrieved from soil without plants consisted of primarily heat-resistant spores. Coinoculation of the plant-soil system with spores of a fertile B. anthracis strain carrying the tetracycline resistance plasmid pBC16 and a selectable B. anthracis recipient strain resulted in transfer of pBC16 from the donor to the recipient as early as 3 days postinoculation. Our findings demonstrate that B. anthracis can survive as a saprophyte outside of the host. The data suggest that horizontal gene transfer in the rhizosphere of grass plants may play a role in the evolution of the Bacillus cereus group species.     

Bacillus anthracis Peptidoglycan Stimulates an Inflammatory Response in Monocytes through the p38 Mitogen-Activated Protein Kinase Pathway

We hypothesized that the peptidoglycan component of B. anthracis may play a critical role in morbidity and mortality associated with inhalation anthrax. To explore this issue, we purified the peptidoglycan component of the bacterial cell wall and studied the response of human peripheral blood cells. The purified B. anthracis peptidoglycan was free of non-covalently bound protein but contained a complex set of amino acids probably arising from the stem peptide. The peptidoglycan contained a polysaccharide that was removed by mild acid treatment, and the biological activity remained with the peptidoglycan and not the polysaccharide. The biological activity of the peptidoglycan was sensitive to lysozyme but not other hydrolytic enzymes, showing that the activity resides in the peptidoglycan component and not bacterial DNA, RNA or protein. B. anthracis peptidoglycan stimulated monocytes to produce primarily TNFα; neutrophils and lymphocytes did not respond. Peptidoglycan stimulated monocyte p38 mitogen-activated protein kinase and p38 activity was required for TNFα production by the cells. We conclude that peptidoglycan in B. anthracis is biologically active, that it stimulates a proinflammatory response in monocytes, and uses the p38 kinase signal transduction pathway to do so. Given the high bacterial burden in pulmonary anthrax, these findings suggest that the inflammatory events associated with peptidoglycan may play an important role in anthrax pathogenesis.     

Surface Plasmon Resonance Biosensor for Detection of Bacillus anthracis, the Causative Agent of Anthrax from Soil Samples Targeting Protective Antigen

Bacillus anthracis, the causative agent of anthrax is one of the most important biological warfare agents. In this study, surface plasmon resonance (SPR) technology was used for indirect detection of B. anthracis by detecting protective antigen (PA), a common toxin produced by all live B. anthracis bacteria. For development of biosensor, a monoclonal antibody raised against B. anthracis PA was immobilized on carboxymethyldextran modified gold chip and its interaction with PA was characterized in situ by SPR and electrochemical impedance spectroscopy. By using kinetic evaluation software, K_D (equilibrium constant) and B_max (maximum binding capacity of analyte) were found to be 20 fM and 18.74, respectively. The change in Gibb’s free energy (∆G = −78.04 kJ/mol) confirmed the spontaneous interaction between antigen and antibody. The assay could detect 12 fM purified PA. When anthrax spores spiked soil samples were enriched, PA produced in the sample containing even a single spore of B. anthracis could be detected by SPR. PA being produced only by the vegetative cells of B. anthracis, confirms indirectly the presence of B. anthracis in the samples. The proposed method can be a very useful tool for screening and confirmation of anthrax suspected environmental samples during a bio-warfare like situation.     

Natural dissemination of Bacillus anthracis spores in northern Canada

Soil samples were collected from around fresh and year-old bison carcasses and areas not associated with known carcasses in Wood Buffalo National Park during an active anthrax outbreak in the summer of 2001. Sample selection with a grid provided the most complete coverage of a site. Soil samples were screened for viable Bacillus anthracis spores via selective culture, phenotypic analysis, and PCR. Bacillus anthracis spores were isolated from 28.4% of the samples. The highest concentrations of B. anthracis spores were found directly adjacent to fresh carcasses and invariably corresponded to locations where the soil had been saturated with body fluids escaping the carcass through either natural body orifices or holes torn by scavengers. The majority of positive samples were found within 2 m of both year-old and fresh carcasses and probably originated from scavengers churning up and spreading the body fluid-saturated soil as they fed. Trails of lesser contamination radiating from the carcasses probably resulted from spore dissemination through adhesion to scavengers and through larger scavengers dragging away disarticulated limbs. Comparison of samples from minimally scavenged and fully necropsied carcass sites revealed no statistically significant difference in the level of B. anthracis spore contamination. Therefore, the immediate area around a suspected anthrax carcass should be considered substantially contaminated regardless of the condition of the carcass.     

Genotype Analysis of Bacillus anthracis Strains Circulating in Bangladesh

In Bangladesh, anthrax, caused by the bacterium Bacillus anthracis, is considered an endemic disease affecting ruminants with sporadic zoonotic occurrences in humans. Due to the lack of knowledge about risks from an incorrect removal of infected carcasses, the disease is not properly monitored, and because of the socio-economic conditions, the situation is under-reported and under-diagnosed. For sensitive species, anthrax represents a fatal outcome with sudden death and sometimes bleeding from natural orifices. The most common source of infection for ruminants is ingestion of spores during grazing in contaminated pastures or through grass and water contaminated with anthrax spores. Domestic cattle, sheep and goats can also become infected through contaminated bone meal (used as feed) originating from anthrax-infected carcasses. The present investigation was conducted to isolate B. anthracis organisms from 169 samples (73 soil, 1 tissue, 4 bone and 91 bone meal samples) collected from 12 different districts of Bangladesh. The sampling was carried out from 2012 to 2015. Twelve samples resulted positive for B. anthracis. Biomolecular analyses were conducted starting from the Canonical Single Nucleotide Polymorphism (CanSNP) to analyze the phylogenetic origin of strains. The analysis of genotype, obtained through the Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) with the analysis of 15 Variable Number Tandem Repeats (VNTR), demonstrated four different genotypes: two of them were previously identified in the district of Sirajganj. The sub-genotyping, conducted with Single Nucleotide Repeats analysis, revealed the presence of eight subgenotypes. The data of the present study concluded that there was no observed correlation between imported cattle feed and anthrax occurrence in Bangladesh and that the remarkable genetic variations of B. anthracis were found in the soil of numerous outbreaks in this country.     

Species-Specific Peptide Ligands for the Detection of Bacillus anthracis Spores

Currently available detectors for spores of Bacillus anthracis, the causative agent of anthrax, are inadequate for frontline use and general monitoring. There is a critical need for simple, rugged, and inexpensive detectors capable of accurate and direct identification of B. anthracis spores. Necessary components in such detectors are stable ligands that bind tightly and specifically to target spores. By screening a phage display peptide library, we identified a family of peptides, with the consensus sequence TYPXPXR, that bind selectively to B. anthracis spores. We extended this work by identifying a peptide variant, ATYPLPIR, with enhanced ability to bind to B. anthracis spores and an additional peptide, SLLPGLP, that preferentially binds to spores of species phylogenetically similar to, but distinct from, B. anthracis. These two peptides were used in tandem in simple assays to rapidly and unambiguously identify B. anthracis spores. We envision that these peptides can be used as sensors in economical and portable B. anthracis spore detectors that are essentially free of false-positive signals due to other environmental Bacillus spores.     

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.     

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.     

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.     

Characterization of Bacillus anthracis Persistence In Vivo

Pulmonary exposure to Bacillus anthracis spores initiates inhalational anthrax, a life-threatening infection. It is known that dormant spores can be recovered from the lungs of infected animals months after the initial spore exposure. Consequently, a 60-day course antibiotic treatment is recommended for exposed individuals. However, there has been little information regarding details or mechanisms of spore persistence in vivo. In this study, we investigated spore persistence in a mouse model. The results indicated that weeks after intranasal inoculation with B. anthracis spores, substantial amounts of spores could be recovered from the mouse lung. Moreover, spores of B. anthracis were significantly better at persisting in the lung than spores of a non-pathogenic Bacillus subtilis strain. The majority of B. anthracis spores in the lung were tightly associated with the lung tissue, as they could not be readily removed by lavage. Immunofluorescence staining of lung sections showed that spores associated with the alveolar and airway epithelium. Confocal analysis indicated that some of the spores were inside epithelial cells. This was further confirmed by differential immunofluorescence staining of lung cells harvested from the infected lungs, suggesting that association with lung epithelial cells may provide an advantage to spore persistence in the lung. There was no or very mild inflammation in the infected lungs. Furthermore, spores were present in the lung tissue as single spores rather than in clusters. We also showed that the anthrax toxins did not play a role in persistence. Together, the results suggest that B. anthracis spores have special properties that promote their persistence in the lung, and that there may be multiple mechanisms contributing to spore persistence.     

Role of Visible Light-Activated Photocatalyst on the Reduction of Anthrax Spore-Induced Mortality in Mice

Background

Photocatalysis of titanium dioxide (TiO₂) substrates is primarily induced by ultraviolet light irradiation. Anion-doped TiO₂ substrates were shown to exhibit photocatalytic activities under visible-light illumination, relative environmentally-friendly materials. Their anti-spore activity against Bacillus anthracis, however, remains to be investigated. We evaluated these visible-light activated photocatalysts on the reduction of anthrax spore-induced pathogenesis.

Methodology/Principal Findings

Standard plating method was used to determine the inactivation of anthrax spore by visible light-induced photocatalysis. Mouse models were further employed to investigate the suppressive effects of the photocatalysis on anthrax toxin- and spore-mediated mortality. We found that anti-spore activities of visible light illuminated nitrogen- or carbon-doped titania thin films significantly reduced viability of anthrax spores. Even though the spore-killing efficiency is only approximately 25%, our data indicate that spores from photocatalyzed groups but not untreated groups have a less survival rate after macrophage clearance. In addition, the photocatalysis could directly inactivate lethal toxin, the major virulence factor of B. anthracis. In agreement with these results, we found that the photocatalyzed spores have tenfold less potency to induce mortality in mice. These data suggest that the photocatalysis might injury the spores through inactivating spore components.

Conclusion/Significance

Photocatalysis induced injuries of the spores might be more important than direct killing of spores to reduce pathogenicity in the host.     

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.     

Germination and Amplification of Anthrax Spores by Soil-Dwelling Amoebas

While anthrax is typically associated with bioterrorism, in many parts of the world the anthrax bacillus (Bacillus anthracis) is endemic in soils, where it causes sporadic disease in livestock. These soils are typically rich in organic matter and calcium that promote survival of resilient B. anthracis spores. Outbreaks of anthrax tend to occur in warm weather following rains that are believed to concentrate spores in low-lying areas where runoff collects. It has been concluded that elevated spore concentrations are not the result of vegetative growth as B. anthracis competes poorly against indigenous bacteria. Here, we test an alternative hypothesis in which amoebas, common in moist soils and pools of standing water, serve as amplifiers of B. anthracis spores by enabling germination and intracellular multiplication. Under simulated environmental conditions, we show that B. anthracis germinates and multiplies within Acanthamoeba castellanii. The growth kinetics of a fully virulent B. anthracis Ames strain (containing both the pX01 and pX02 virulence plasmids) and vaccine strain Sterne (containing only pX01) inoculated as spores in coculture with A. castellanii showed a nearly 50-fold increase in spore numbers after 72 h. In contrast, the plasmidless strain 9131 showed little growth, demonstrating that plasmid pX01 is essential for growth within A. castellanii. Electron and time-lapse fluorescence microscopy revealed that spores germinate within amoebal phagosomes, vegetative bacilli undergo multiplication, and, following demise of the amoebas, bacilli sporulate in the extracellular milieu. This analysis supports our hypothesis that amoebas contribute to the persistence and amplification of B. anthracis in natural environments.     

In vivo murine and in vitro M-like cell models of gastrointestinal anthrax

Bacillus anthracis is the causative agent of anthrax and is acquired by three routes of infection: inhalational, gastrointestinal and cutaneous. Gastrointestinal (GI) anthrax is rare, but can rapidly result in severe, systemic disease that is fatal in 25%–60% of cases. Disease mechanisms of GI anthrax remain unclear due to limited numbers of clinical cases and the lack of experimental animal models. Here, we developed an in vivo murine model of GI anthrax where spore survival was maximized through the neutralization of stomach acid followed by an intragastric administration of a thiabendazole paste spore formulation. Infected mice showed a dose-dependent mortality rate and pathological features closely mimicking human GI anthrax. Since Peyer's patches in the murine intestine are the primary sites of B. anthracis growth, we developed a human M (microfold)-like-cell model using a Caco-2/Raji B-cell co-culturing system to study invasive mechanisms of GI anthrax across the intestinal epithelium. Translocation of B. anthracis spores was higher in M-like cells than Caco-2 monolayers, suggesting that M-like cells may serve as an initial entry site for spores. Here, we developed an in vivo murine model of GI anthrax and an in vitro M-like cell model that could be used to further our knowledge of GI anthrax pathogenesis.     

Mechanisms of NK Cell-Macrophage Bacillus anthracis Crosstalk: A Balance between Stimulation by Spores and Differential Disruption by Toxins

NK cells are important immune effectors for preventing microbial invasion and dissemination, through natural cytotoxicity and cytokine secretion. Bacillus anthracis spores can efficiently drive IFN-γ production by NK cells. The present study provides insights into the mechanisms of cytokine and cellular signaling that underlie the process of NK-cell activation by B. anthracis and the bacterial strategies to subvert and evade this response. Infection with non-toxigenic encapsulated B. anthracis induced recruitment of NK cells and macrophages into the mouse draining lymph node. Production of edema (ET) or lethal (LT) toxin during infection impaired this cellular recruitment. NK cell depletion led to accelerated systemic bacterial dissemination. IFN-γ production by NK cells in response to B. anthracis spores was: i) contact-dependent through RAE-1-NKG2D interaction with macrophages; ii) IL-12, IL-18, and IL-15-dependent, where IL-12 played a key role and regulated both NK cell and macrophage activation; and iii) required IL-18 for only an initial short time window. B. anthracis toxins subverted both NK cell essential functions. ET and LT disrupted IFN-γ production through different mechanisms. LT acted both on macrophages and NK cells, whereas ET mainly affected macrophages and did not alter NK cell capacity of IFN-γ secretion. In contrast, ET and LT inhibited the natural cytotoxicity function of NK cells, both in vitro and in vivo. The subverting action of ET thus led to dissociation in NK cell function and blocked natural cytotoxicity without affecting IFN-γ secretion. The high efficiency of this process stresses the impact that this toxin may exert in anthrax pathogenesis, and highlights a potential usefulness for controlling excessive cytotoxic responses in immunopathological diseases. Our findings therefore exemplify the delicate balance between bacterial stimulation and evasion strategies. This highlights the potential implication of the crosstalk between host innate defences and B. anthracis in initial anthrax control mechanisms.     

Convergent evolution of diverse Bacillus anthracis outbreak strains toward altered surface oligosaccharides that modulate anthrax pathogenesis

Bacillus anthracis, a spore-forming gram-positive bacterium, causes anthrax. The external surface of the exosporium is coated with glycosylated proteins. The sugar additions are capped with the unique monosaccharide anthrose. The West African Group (WAG) B. anthracis have mutations rendering them anthrose deficient. Through genome sequencing, we identified 2 different large chromosomal deletions within the anthrose biosynthetic operon of B. anthracis strains from Chile and Poland. In silico analysis identified an anthrose-deficient strain in the anthrax outbreak among European heroin users. Anthrose-deficient strains are no longer restricted to West Africa so the role of anthrose in physiology and pathogenesis was investigated in B. anthracis Sterne. Loss of anthrose delayed spore germination and enhanced sporulation. Spores without anthrose were phagocytized at higher rates than spores with anthrose, indicating that anthrose may serve an antiphagocytic function on the spore surface. The anthrose mutant had half the LD₅₀ and decreased time to death (TTD) of wild type and complement B. anthracis Sterne in the A/J mouse model. Following infection, anthrose mutant bacteria were more abundant in the spleen, indicating enhanced dissemination of Sterne anthrose mutant. At low sample sizes in the A/J mouse model, the mortality of ΔantC-infected mice challenged by intranasal or subcutaneous routes was 20% greater than wild type. Competitive index (CI) studies indicated that spores without anthrose disseminated to organs more extensively than a complemented mutant. Death process modeling using mouse mortality dynamics suggested that larger sample sizes would lead to significantly higher deaths in anthrose-negative infected animals. The model was tested by infecting Galleria mellonella with spores and confirmed the anthrose mutant was significantly more lethal. Vaccination studies in the A/J mouse model showed that the human vaccine protected against high-dose challenges of the nonencapsulated Sterne-based anthrose mutant. This work begins to identify the physiologic and pathogenic consequences of convergent anthrose mutations in B. anthracis.

A study of the spontaneous loss of the spore coat monosaccharide anthrose suggests that convergent evolution in several anthrax strains towards increased pathogenicity could exacerbate global human and animal anthrax disease.     

The Germination-Specific Lytic Enzymes SleB,CwlJ1, and CwlJ2 Each Contribute to Bacillus anthracis Spore Germination and Virulence

The bacterial spore cortex is critical for spore stability and dormancy and must be hydrolyzed by germination-specific lytic enzymes (GSLEs), which allows complete germination and vegetative cell outgrowth. We created in-frame deletions of three genes that encode GSLEs that have been shown to be active in Bacillus anthracis germination: sleB, cwlJ1, and cwlJ2. Phenotypic analysis of individual null mutations showed that the removal of any one of these genes was not sufficient to disrupt spore germination in nutrient-rich media. This finding indicates that these genes have partially redundant functions. Double and triple deletions of these genes resulted in more significant defects. Although a small subset of ΔsleB ΔcwlJ1 spores germinate with wild-type kinetics, for the overall population there is a 3-order-of-magnitude decrease in the colony-forming efficiency compared with wild-type spores. ΔsleB ΔcwlJ1 ΔcwlJ2 spores are unable to complete germination in nutrient-rich conditions in vitro. Both ΔsleB ΔcwlJ1 and ΔsleB ΔcwlJ1 ΔcwlJ2 spores are significantly attenuated, but are not completely devoid of virulence, in a mouse model of inhalation anthrax. Although unable to germinate in standard nutrient-rich media, spores lacking SleB, CwlJ1, and CwlJ2 are able to germinate in whole blood and serum in vitro, which may explain the persistent low levels of virulence observed in mouse infections. This work contributes to our understanding of GSLE activation and function during germination. This information may result in identification of useful therapeutic targets for the disease anthrax, as well as provide insights into ways to induce the breakdown of the protective cortex layer, facilitating easier decontamination of resistant spores.Bacillus anthracis, a gram-positive spore-forming bacterium, is the causative agent of anthrax. The dormant spore form is the infectious particle and produces three different forms of the disease depending on the route of entry into a suitable host (8). When spores enter through a skin lesion and when they are ingested, they cause cutaneous and gastrointestinal anthrax, respectively. Spores entering through the lungs cause the most severe form of the disease, inhalation anthrax, which is often fatal even with aggressive antibiotic therapy (1, 8, 34). Because true pneumonias are rarely seen in victims, it is believed that inhaled spores do not germinate in the lung but are phagocytosed by alveolar macrophages and germinate intracellularly en route to the mediastinal lymph nodes, which leads to dissemination, septicemia, toxemia, and often death (1, 34). It has been shown that the spores are able to germinate and the bacteria are able to multiply inside macrophages both in cell culture and in the lungs of challenged animals (7, 11, 28, 29).Independent of the route of infection, spore germination inside a susceptible host is essential for disease. The highly stable spore form of the bacterium can remain viable under harsh environmental conditions for many decades (32). However, a spore can form a rapidly dividing vegetative cell upon entry into a host and recognition of specific chemical signals, or germinants, through specialized germinant receptors (32). The spore cortex, a thick layer of modified peptidoglycan (PG), contributes much of the spore''s environmental resistance as it is necessary to maintain dehydration of the spore core (25). This protective barrier is broken down following the activation of germination-specific lytic enzymes (GSLEs), allowing full core rehydration and cell outgrowth (32). Experimentally, germination can also be triggered by nongerminant treatments, such as lysozyme treatment, high pressure, exogenous Ca²⁺-dipicolinic acid treatment, and treatment with cationic surfactants (32). Several of these treatments likely cause spore cortex hydrolysis, triggering spore germination. This indicates the importance of cortex degradation in the spore germination process.Bacterial cell wall PG consists of polysaccharide chains of repeating N-acetylglucosamine and N-acetylmuramic acid, joined by β(1,4) glycosidic bonds (25). This basic structure is modified in several ways in spore cortex PG. In one major modification, 50% of the muramic acid residues (alternating every other residue) are converted to muramic-δ-lactam residues (25). This modification is essential for the specificity of GSLEs for degrading the cortex and prevents degradation of the bacterial cell wall during cortex hydrolysis (21).Previous work on the role of GSLEs in Bacillus subtilis and, recently, in B. anthracis has shown that the enzymes SleB and CwlJ have partially redundant roles and are necessary together for full cortex hydrolysis and spore germination (6, 14). SleB is a lytic transglycosylase that, when activated by an unknown mechanism, hydrolyzes the bond between N-acetylmuramic acid and N-acetylglucosamine (5). In both B. subtilis and B. anthracis, the sleB gene is found in a bicistronic operon with ypeB. Although the function of YpeB is not known, deletion of ypeB prevents SleB activity in spore germination, and sleB and ypeB mutants have similar phenotypes (5). Expression of both gene products is necessary for the presence of SleB in the cortex and inner membrane of mature spores (2, 5).Although no specific enzymatic activity has been attributed to CwlJ, it is required for full germination and it shares a homologous catalytic domain with SleB (20). In B. subtilis and Bacillus cereus, cwlJ is found in an operon with gerQ. Similar to the finding that ypeB is necessary for a functional SleB protein, gerQ is required for CwlJ activity (26). The B. anthracis genome contains two homologs of cwlJ (designated cwlJ1 and cwlJ2 [14]), whereas a single copy is present in B. subtilis and B. cereus. As it is in the related species, cwlJ1 is found in an operon with gerQ, but cwlJ2 is in a different locus and is not in an operon with a gerQ homolog (14). It has been shown that CwlJ is localized to the spore coat and that it is necessary for spore germination with exogenous Ca²⁺-dipicolinic acid treatment (3, 24).GSLE activation represents a critical step in the complex process of germination. The relatively small number of genes involved and the apparent essential nature of their activity make them attractive targets for new therapeutics, as well as environmental decontamination compounds. The objective of this study was to test by using genetic analysis the role of the GSLE genes sleB, cwlJ1, and cwlJ2 in B. anthracis spore germination. Mutants lacking these three genes were tested to determine their effects on in vitro germination kinetics and colony-forming efficiency. Additionally, the virulence of these mutant strains was examined by comparing mutant and wild-type spores in an in vivo mouse model of inhalational anthrax.