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.     

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.     

Activation of the classical complement pathway by Bacillus anthracis is the primary mechanism for spore phagocytosis and involves the spore surface protein BclA

Interactions between spores of Bacillus anthracis and macrophages are critical for the development of anthrax infections, as spores are thought to use macrophages as vehicles to disseminate in the host. In this study, we report a novel mechanism for phagocytosis of B. anthracis spores. Murine macrophage-like cell line RAW264.7, bone marrow-derived macrophages, and primary peritoneal macrophages from mice were used. The results indicated that activation of the classical complement pathway (CCP) was a primary mechanism for spore phagocytosis. Phagocytosis was significantly reduced in the absence of C1q or C3. C3 fragments were found deposited on the spore surface, and the deposition was dependent on C1q and Ca(2+). C1q recruitment to the spore surface was mediated by the spore surface protein BclA, as recombinant BclA bound directly and specifically to C1q and inhibited C1q binding to spores in a dose-dependent manner. C1q binding to spores lacking BclA (ΔbclA) was also significantly reduced compared with wild-type spores. In addition, deposition of both C3 and C4 as well as phagocytosis of spores were significantly reduced when BclA was absent, but were not reduced in the absence of IgG, suggesting that BclA, but not IgG, is important in these processes. Taken together, these results support a model in which spores actively engage CCP primarily through BclA interaction with C1q, leading to CCP activation and opsonophagocytosis of spores in an IgG-independent manner. These findings are likely to have significant implications on B. anthracis pathogenesis and microbial manipulation of complement.     

Environmental Persistence of Bacillus anthracis and Bacillus subtilis Spores

There is a lack of data for how the viability of biological agents may degrade over time in different environments. In this study, experiments were conducted to determine the persistence of Bacillus anthracis and Bacillus subtilis spores on outdoor materials with and without exposure to simulated sunlight, using ultraviolet (UV)-A/B radiation. Spores were inoculated onto glass, wood, concrete, and topsoil and recovered after periods of 2, 14, 28, and 56 days. Recovery and inactivation kinetics for the two species were assessed for each surface material and UV exposure condition. Results suggest that with exposure to UV, decay of spore viability for both Bacillus species occurs in two phases, with an initial rapid decay, followed by a slower inactivation period. The exception was with topsoil, in which there was minimal loss of spore viability in soil over 56 days, with or without UV exposure. The greatest loss in viable spore recovery occurred on glass with UV exposure, with nearly a four log₁₀ reduction after just two days. In most cases, B. subtilis had a slower rate of decay than B. anthracis, although less B. subtilis was recovered initially.     

Roles of the Bacillus anthracis Spore Protein ExsK in Exosporium Maturation and Germination

The Bacillus anthracis spore is the causative agent of the disease anthrax. The outermost structure of the B. anthracis spore, the exosporium, is a shell composed of approximately 20 proteins. The function of the exosporium remains poorly understood and is an area of active investigation. In this study, we analyzed the previously identified but uncharacterized exosporium protein ExsK. We found that, in contrast to other exosporium proteins, ExsK is present in at least two distinct locations, i.e., the spore surface as well as a more interior location underneath the exosporium. In spores that lack the exosporium basal layer protein ExsFA/BxpB, ExsK fails to encircle the spore and instead is present at only one spore pole, indicating that ExsK assembly to the spore is partially dependent on ExsFA/BxpB. In spores lacking the exosporium surface protein BclA, ExsK fails to mature into high-molecular-mass species observed in wild-type spores. These data suggest that the assembly and maturation of ExsK within the exosporium are dependent on ExsFA/BxpB and BclA. We also found that ExsK is not required for virulence in murine and guinea pig models but that it does inhibit germination. Based on these data, we propose a revised model of exosporium maturation and assembly and suggest a novel role for the exosporium in germination.During starvation, bacteria of the genus Bacillus differentiate into dormant, highly robust cell types called spores, thereby preserving their genomes during stressful and nutrient-poor conditions (10). Spores can withstand extremely harsh environmental insults, including toxic chemicals, UV radiation, and heat (31). When conditions again become favorable for cell survival, spores can return to vegetative cell growth through a process called germination (17, 18, 31, 49). Spores are formed in an approximately 8-h process during which the developing spore first forms as a compartment (the forespore) contained within the surrounding cell (the mother cell) (34). Ultimately, the mother cell envelope lyses, releasing the mature spore into the environment.Spores from all Bacillus species have similar architectures. At the spore interior is the core, which houses the spore chromosome. Surrounding the core is an inner membrane encased in a specialized peptidoglycan called the cortex and finally a series of outer layers that vary significantly among species (10). In some species, including Bacillus subtilis, the outermost structure is a protective layer called the coat, which guards the spore against reactive small molecules, degradative enzymes, and predation by other microbes (11, 17, 20, 38). Spores of other species, including the pathogens Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis and the nonpathogenic bacteria Bacillus megaterium and Bacillus odysseyi, have an additional structure called the exosporium, which surrounds the coat (24, 32, 47). The exosporium is composed of two structural units: the basal layer, which is a shell of proteins forming a hexagonal array, and a nap of hairlike protrusions extending outward from the basal layer (2, 32). A major component of the nap (and of the spore surface) is the collagen-like protein BclA (40, 43). The proteins that comprise the outer structures (the coat and exosporium) are synthesized in the mother cell cytoplasm, from which location they assemble onto the spore surface to form their respective structures (11).The function of the exosporium is poorly understood. Previous studies have implicated its contribution to germination, resistance to host cells and other stresses, adhesion to inert surfaces, and interactions with epithelial cells and macrophages (1, 6, 7, 13, 33, 41, 48; G. Chen, A. Driks, K. Tawfiq, M. Mallozzi, and S. Patil, submitted for publication). In most cases, however, the roles of individual exosporium proteins in each of these functions remain unclear, in part because the location of each protein within the exosporium is largely unknown.Interestingly, it appears that the exosporium is not essential for virulence of B. anthracis in several animal models (5, 7, 12, 13). Nonetheless, it is possible that in natural infections the exosporium plays a significant role. Because it is involved in attachment, the exosporium is also likely to have a significant impact on the persistence of B. anthracis spores in the environment.To gain insight into the molecular basis of exosporium assembly and function, we studied a previously identified but otherwise uncharacterized exosporium protein, ExsK. Using immunofluorescence microscopy (IFM), we found that ExsK is asymmetrically distributed on the surfaces of mature spores and is also present beneath the exosporium. In the absence of ExsFA/BxpB, ExsK was restricted to one spore pole, suggesting that the encirclement of the spore by ExsK depends on ExsFA/BxpB. Western blot analysis indicated that in mature spores ExsK is present in high-molecular-mass complexes, the formation of which is BclA dependent. Although ExsK is not required for several spore resistance properties or virulence, we found that it is required for normal germination. Our results provide a deeper understanding of the composition, function, and assembly of the B. anthracis exosporium and show that proteins comprising outer-spore structures can have multiple locations.     

Detection of B. anthracis Spores and Vegetative Cells with the Same Monoclonal Antibodies

Bacillus anthracis, the causative agent of anthrax disease, could be used as a biothreat reagent. It is vital to develop a rapid, convenient method to detect B. anthracis. In the current study, three high affinity and specificity monoclonal antibodies (mAbs, designated 8G3, 10C6 and 12F6) have been obtained using fully washed B. anthracis spores as an immunogen. These mAbs, confirmed to direct against EA1 protein, can recognize the surface of B. anthracis spores and intact vegetative cells with high affinity and species-specificity. EA1 has been well known as a major S-layer component of B. anthracis vegetative cells, and it also persistently exists in the spore preparations and bind tightly to the spore surfaces even after rigorous washing. Therefore, these mAbs can be used to build a new and rapid immunoassay for detection of both life forms of B. anthracis, either vegetative cells or spores.     

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.     

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.     

Observations on the Inactivation Efficacy of a MALDI-TOF MS Chemical Extraction Method on Bacillus anthracis Vegetative Cells and Spores

A chemical (ethanol; formic acid; acetonitrile) protein extraction method for the preparation of bacterial samples for matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) identification was evaluated for its ability to inactivate bacterial species. Initial viability tests (with and without double filtration of the extract through 0.2 μM filters), indicated that the method could inactivate Escherichia coli MRE 162 and Klebsiella pneumoniae ATCC 35657, with or without filtration, but that filtration was required to exclude viable, avirulent, Bacillus anthracis UM23CL2 from extracts. Multiple, high stringency, viability experiments were then carried out on entire filtered extracts prepared from virulent B. anthracis Vollum vegetative cells and spores ranging in concentration from 10⁶-10⁸cfu per extract. B. anthracis was recovered in 3/18 vegetative cell extracts and 10/18 spore extracts. From vegetative cell extracts B. anthracis was only recovered from extracts that had undergone prolonged Luria (L)-broth (7 day) and L-agar plate (a further 7 days) incubations. We hypothesise that the recovery of B. anthracis in vegetative cell extracts is due to the escape of individual sub-lethally injured cells. We discuss our results in view of working practises in clinical laboratories and in the context of recent inadvertent releases of viable B. anthracis.     

Identification of the Immunogenic Spore and Vegetative Proteins of Bacillus anthracis Vaccine Strain A16R

Immunoproteomics was used to screen the immunogenic spore and vegetative proteins of Bacillus anthracis vaccine strain A16R. The spore and vegetative proteins were separated by 2D gel electrophoresis and transferred to polyvinylidene difluoride membranes, and then western blotting was performed with rabbit immune serum against B.anthracis live spores. Immunogenic spots were cut and digested by trypsin. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was performed to identify the proteins. As a result, 11 and 45 immunogenic proteins were identified in the spores and vegetative cells, respectively; 26 of which have not been reported previously. To verify their immunogenicity, 12 of the identified proteins were selected to be expressed, and the immune sera from the mice vaccinated by the 12 expressed proteins, except BA0887, had a specific western blot band with the A16R whole cellular lytic proteins. Some of these immunogenic proteins might be used as novel vaccine candidates themselves or for enhancing the protective efficacy of a protective-antigen-based vaccine.     

Decontamination Options for Bacillus anthracis-Contaminated Drinking Water Determined from Spore Surrogate Studies

Five parameters were evaluated with surrogates of Bacillus anthracis spores to determine effective decontamination alternatives for use in a contaminated drinking water supply. The parameters were as follows: (i) type of Bacillus spore surrogate (B. thuringiensis or B. atrophaeus), (ii) spore concentration in suspension (10² and 10⁶ spores/ml), (iii) chemical characteristics of the decontaminant (sodium dichloro-S-triazinetrione dihydrate [Dichlor], hydrogen peroxide, potassium peroxymonosulfate [Oxone], sodium hypochlorite, and VirkonS), (iv) decontaminant concentration (0.01% to 5%), and (v) exposure time to decontaminant (10 min to 1 h). Results from 138 suspension tests with appropriate controls are reported. Hydrogen peroxide at a concentration of 5% and Dichlor or sodium hypochlorite at a concentration of 2% were highly effective at spore inactivation regardless of spore type tested, spore exposure time, or spore concentration evaluated. This is the first reported study of Dichlor as an effective decontaminant for B. anthracis spore surrogates. Dichlor''s desirable characteristics of high oxidation potential, high level of free chlorine, and a more neutral pH than that of other oxidizers evaluated appear to make it an excellent alternative. All three oxidizers were effective against B. atrophaeus spores in meeting the EPA biocide standard of greater than a 6-log kill after a 10-min exposure time and at lower concentrations than typically reported for biocide use. Solutions of 5% VirkonS and Oxone were less effective as decontaminants than other options evaluated in this study and did not meet the EPA''s efficacy standard for a biocide, although they were found to be as effective for concentrations of 10² spores/ml. Differences in methods and procedures reported by other investigators make quantitative comparisons among studies difficult.Developing a decontamination approach that can be safely and effectively applied to civilian water resources and facilities following a terrorist or catastrophic release of Bacillus anthracis spores poses many challenges. For example, if a municipal drinking water system were contaminated directly or indirectly during or after such an incident, it would be essential to assess the potential health risks posed by water consumption or other water uses (e.g., recreational and bathing) and then to apply one or more proven technologies, if deemed necessary, to decontaminate the water supply quickly and cost-effectively. Treatment of drinking water implies the use of a decontamination approach that would not pose adverse health risks to humans or result in unacceptable damage to the environment. A major obstacle in killing spores of Bacillus spp. on or in virtually any matrix is their high level of resistance to treatments such as harsh chemicals, heat, desiccation, and UV light (14, 20). Because of the substantial and widely reported resistance of Bacillus spores to inactivation, a decontaminant proven to be efficacious in killing such spores for site-specific applications is likely to be effective against all other biological warfare agents as well.Whereas nearly all biological warfare agents are intended for aerosol application, many have strong potential as waterborne threats and could inflict heavy casualties when ingested (2). B. anthracis in particular has been identified as a “probable” (12) or an actual (24) water threat. Even though the principal risk associated with the consumption of water containing B. anthracis spores would likely arise from an ingestion hazard, water used for bathing, showering, or recreational purposes might also pose cutaneous as well as aerosol exposure hazards. There is controversy regarding the long-term viability of B. anthracis in water, and experimental evidence is limited. However, according to a review of nonkinetic studies on survival of virulent strains in the environment (21), B. anthracis spores can survive from 2 to 18 years in pond water and 20 months in seawater or distilled water. B. anthracis spores have been reported by others to be stable in water for 2 years (24).Various decontamination approaches have been evaluated for efficacy against biological warfare agents, including Bacillus spores, on hard, nonporous surfaces. Recommendations by the U.S. Environmental Protection Agency (EPA) include the use of sodium hypochlorite (1:9 dilution of bleach to 5,250 to 6,000 ppm, corrected to pH 7, with a 60-min contact time at 20°C [6, 17]), and liquid chlorine dioxide with a 30-min wet contact time at 20°C (7). Liquid hydrogen peroxide/peroxyacetic acid (known as peroxy compounds and marketed as ready-to-use solutions), generally with a 15- to 20-min wet contact time and concentration as specified by the manufacturer, has also been recommended (13). Other products, such as hydrogen peroxide solution (3 to 25%) and potassium peroxymonosulfate, have been evaluated for efficacy against Bacillus spores as well (27). Although disinfectants at various concentrations have been tested previously against the spores of B. anthracis and their surrogates, wide variations in test protocols make meaningful comparisons among studies virtually impossible (9, 11, 17).In contrast to surface cleanup of spores, fewer assessments of efficacy utilizing suspension tests with the aforementioned chemicals or other methods have been reported for the decontamination of Bacillus species spores in water, and much of the published work has assessed only relatively high concentrations of spores in water. For example, one previous investigation commenced evaluations with 0.2-ml suspensions of approximately 10⁹ spores/ml of various Bacillus spp. to which 20 ml of aqueous ozone or 20 ml of hydrogen peroxide solution was added to assess sporicidal action (10), and others have reported mechanisms of deactivating B. subtilis spores prepared in concentrations of up to approximately 10⁸ spores/ml (26) and approximately 10⁹ spores/ml (17). Inactivation by chlorination of various Bacillus spp. with initial concentrations of approximately 1 × 10⁴ CFU/ml has also been tested (16). However, relatively low spore concentrations would be expected to result from dilutions following contamination of a large public water system. Therefore, it is reasonable to evaluate the effectiveness of decontaminants or other methods against even lower spore concentrations in water than have been previously assessed. In addition to assessing the parameter of Bacillus spore concentration in water, it is essential to identify the most effective commercially available chemical that will kill all the spores or minimize population growth, while considering the effects of the chemical on the environment and in humans.Several objectives served to focus our investigation. First, five potential candidate decontaminants were selected because of their relative safety and ultimate degradation in the environment without substantive adverse consequences. The five chemicals were also chosen as a way of comparing the effectiveness of available free chlorine content, pH, and oxidation potential on spore inactivation. From an evaluation of those chemical parameters, we sought to determine the most effective option for inactivating Bacillus spore surrogates suspended in water. As a second objective, we attempted to identify the lowest concentration of the selected chemicals necessary to achieve the EPA''s biocide standard of a >6-log kill. As a third objective, we wanted to assess the effect of reduced spore concentration on chemical biocide efficacy. As an important step in ascertaining an efficient, safe, and cost-effective water treatment method that could potentially provide safe water to the general population in the event of B. anthracis contamination—and limit the potential risk of contracting gastrointestinal or cutaneous anthrax as well—the following parameters were evaluated: chemical decontaminant type, chemical decontaminant concentration (0.01% to 5%), contact time of spores with chemical decontaminant (10 min to 1 h), spore type (Bacillus atrophaeus or Bacillus thuringiensis), and low versus relatively high spore concentrations (approximately 10² and 10⁶ spores/ml, respectively).Use of B. atrophaeus and B. thuringiensis spores as surrogates for B. anthracis is widely reported in the literature. For example, Szabo et al. (23) used B. atrophaeus subsp. globigii spores as a surrogate for B. anthracis to investigate the persistence and decontamination of those surrogates on corroded iron in a model drinking water system, and Rice et al. (16) used spores of B. thuringiensis as an “appropriate surrogate for spores of B. anthracis” for determining the sporicidal activity of chlorination as commonly used in drinking water treatment. Furthermore, the EPA (5) concluded that “B. globigii can serve as a conservative surrogate for B. anthracis during studies of inactivation by chlorination.”     

SpoIVA and SipL Are Clostridium difficile Spore Morphogenetic Proteins

Clostridium difficile is a major nosocomial pathogen whose infections are difficult to treat because of their frequent recurrence. The spores of C. difficile are responsible for these clinical features, as they resist common disinfectants and antibiotic treatment. Although spores are the major transmissive form of C. difficile, little is known about their composition or morphogenesis. Spore morphogenesis has been well characterized for Bacillus sp., but Bacillus sp. spore coat proteins are poorly conserved in Clostridium sp. Of the known spore morphogenetic proteins in Bacillus subtilis, SpoIVA is one of the mostly highly conserved in the Bacilli and the Clostridia. Using genetic analyses, we demonstrate that SpoIVA is required for proper spore morphogenesis in C. difficile. In particular, a spoIVA mutant exhibits defects in spore coat localization but not cortex formation. Our study also identifies SipL, a previously uncharacterized protein found in proteomic studies of C. difficile spores, as another critical spore morphogenetic protein, since a sipL mutant phenocopies a spoIVA mutant. Biochemical analyses and mutational analyses indicate that SpoIVA and SipL directly interact. This interaction depends on the Walker A ATP binding motif of SpoIVA and the LysM domain of SipL. Collectively, these results provide the first insights into spore morphogenesis in C. difficile.     

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.     

Bacillus anthracis Spore Entry into Epithelial Cells Is an Actin-Dependent Process Requiring c-Src and PI3K

Dissemination of Bacillus anthracis from the respiratory mucosa is a critical step in the establishment of inhalational anthrax. Recent in vitro and in vivo studies indicated that this organism was able to penetrate the lung epithelium by directly entering into epithelial cells of the lung; however the molecular details of B. anthracis breaching the epithelium were lacking. Here, using a combination of pharmacological inhibitors, dominant negative mutants, and colocalization experiments, we demonstrated that internalization of spores by epithelial cells was actin-dependent and was mediated by the Rho-family GTPase Cdc42 but not RhoA or Rac1. Phosphatidylinositol 3-kinase (PI3K) activity was also required as indicated by the inhibitory effects of PI3K inhibitors, wortmannin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, and a PI3K dominant negative (DN) mutant Δp85α. In addition, spore entry into epithelial cells (but not into macrophages) required the activity of Src as indicated by the inhibitory effect of Src family kinase (SFK) inhibitors, PP2 and SU6656, and specific siRNA knockdown of Src. Enrichment of PI3K and F-actin around spore attachment sites was observed and was significantly reduced by treatment with SFK and PI3K inhibitors, respectively. Moreover, B. anthracis translocation through cultured lung epithelial cells was significantly impaired by SFK inhibitors, suggesting that this signaling pathway is important for bacterial dissemination. The effect of the inhibitor on dissemination in vivo was then evaluated. SU6656 treatment of mice significantly reduced B. anthracis dissemination from the lung to distal organs and prolonged the median survival time of mice compared to the untreated control group. Together these results described a signaling pathway specifically required for spore entry into epithelial cells and provided evidence suggesting that this pathway is important for dissemination and virulence in vivo.     

UV Resistance of Bacillus anthracis Spores Revisited: Validation of Bacillus subtilis Spores as UV Surrogates for Spores of B. anthracis Sterne

Recent bioterrorism concerns have prompted renewed efforts towards understanding the biology of bacterial spore resistance to radiation with a special emphasis on the spores of Bacillus anthracis. A review of the literature revealed that B. anthracis Sterne spores may be three to four times more resistant to 254-nm-wavelength UV than are spores of commonly used indicator strains of Bacillus subtilis. To test this notion, B. anthracis Sterne spores were purified and their UV inactivation kinetics were determined in parallel with those of the spores of two indicator strains of B. subtilis, strains WN624 and ATCC 6633. When prepared and assayed under identical conditions, the spores of all three strains exhibited essentially identical UV inactivation kinetics. The data indicate that standard UV treatments that are effective against B. subtilis spores are likely also sufficient to inactivate B. anthracis spores and that the spores of standard B. subtilis strains could reliably be used as a biodosimetry model for the UV inactivation of B. anthracis spores.     

Variable lymphocyte receptor recognition of the immunodominant glycoprotein of Bacillus anthracis spores

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Highlights? VLRBs use their C-terminal LRRs and the LRRCT-loop to interact with antigen ? Sequence-related VLRBs exhibit differential recognition of their BclA epitopes ? VLR4 binds a conserved protein epitope, yet is specific for B. anthracis spores     

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.     

The spore‐associated protein BclA1 affects the susceptibility of animals to colonization and infection by Clostridium difficile

The BclA protein is a major component of the outermost layer of spores of a number of bacterial species and Clostridium difficile carries three bclA genes. Using insertional mutagenesis each gene was characterized and spores devoid of these proteins had surface aberrations, reduced hydrophobicity and germinated faster than wild‐type spores. Therefore the BclA proteins were likely major components of the spore surface and when absent impaired the protective shield effect of this outermost layer. Analysis of infection and colonization in mice and hamsters revealed that the 50% infectious dose (ID₅₀) of spores was significantly higher (2‐logs) in the bclA1^? mutant compared to the isogenic wild‐type control, but that levels of toxins (A and B) were indistinguishable from animals dosed with wild‐type spores. bclA1^? spores germinated faster than wild‐type spores yet mice were less susceptible to infection suggesting that BclA1 must play a key role in the initial (i.e. pre‐spore germination) stages of infection. We also show that the ID₅₀ was higher in mice infected with R20291, a ‘hypervirulent’ 027 strain, that carries a truncated BclA1 protein.     

CEMOVIS on a pathogen: analysis of Bacillus anthracis spores

Background information. Under conditions of starvation, bacteria of Bacillus ssp. are able to form a highly structured cell type, the dormant spore. When the environment presents more favourable conditions, the spore starts to germinate, which will lead to the release of the vegetative form in the life cycle, the bacillus. For Bacillus anthracis, the aetiological agent of anthrax, germination is normally linked to host uptake and represents an important step in the onset of anthrax disease. Morphological studies analysing the organization of the spore and the changes during germination at the electron microscopy level were only previously performed with techniques relying on fixation with aldehydes and osmium, and subsequent dehydration, which can produce artefacts. Results and conclusions. In the present study, we describe the morphology of dormant spores using CEMOVIS (Cryo‐Electron Microscopy of Vitreous Sections). Biosafety measures do not permit freezing of native spores of B. anthracis without chemical fixation. To study the influence of aldehyde fixation on the ultrastructure of the spore, we chose to analyse spores of the closely related non‐pathogen Bacillus cereus T. For none of the investigated structures could we find a difference in morphology induced by aldehyde fixation compared with the native preparations for CEMOVIS. This result legitimizes work with aldehyde‐fixed spores from B. anthracis. Using CEMOVIS, we describe two new structures present in the spore: a rectangular structure, which connects the BclA filaments with the basal layer of the exosporium, and a repetitive structure, which can be found in the terminal layer of the coat. We studied the morphological changes of the spore during germination. After outgrowth of the bacillus, coat and exosporium stay associated, and the layered organization of the coat, as well as the repetitive structure within it, remain unchanged.