Roles of Small,Acid-Soluble Spore Proteins and Core Water Content in Survival of Bacillus subtilis Spores Exposed to Environmental Solar UV Radiation

Spores of Bacillus subtilis contain a number of small, acid-soluble spore proteins (SASP) which comprise up to 20% of total spore core protein. The multiple α/β-type SASP have been shown to confer resistance to UV radiation, heat, peroxides, and other sporicidal treatments. In this study, SASP-defective mutants of B. subtilis and spores deficient in dacB, a mutation leading to an increased core water content, were used to study the relative contributions of SASP and increased core water content to spore resistance to germicidal 254-nm and simulated environmental UV exposure (280 to 400 nm, 290 to 400 nm, and 320 to 400 nm). Spores of strains carrying mutations in sspA, sspB, and both sspA and sspB (lacking the major SASP-α and/or SASP-β) were significantly more sensitive to 254-nm and all polychromatic UV exposures, whereas the UV resistance of spores of the sspE strain (lacking SASP-γ) was essentially identical to that of the wild type. Spores of the dacB-defective strain were as resistant to 254-nm UV-C radiation as wild-type spores. However, spores of the dacB strain were significantly more sensitive than wild-type spores to environmental UV treatments of >280 nm. Air-dried spores of the dacB mutant strain had a significantly higher water content than air-dried wild-type spores. Our results indicate that α/β-type SASP and decreased spore core water content play an essential role in spore resistance to environmentally relevant UV wavelengths whereas SASP-γ does not.Spores of Bacillus spp. are highly resistant to inactivation by different physical stresses, such as toxic chemicals and biocidal agents, desiccation, pressure and temperature extremes, and high fluences of UV or ionizing radiation (reviewed in references 33, 34, and 48). Under stressful environmental conditions, cells of Bacillus spp. produce endospores that can stay dormant for extended periods. The reason for the high resistance of bacterial spores to environmental extremes lies in the structure of the spore. Spores possess thick layers of highly cross-linked coat proteins, a modified peptidoglycan spore cortex, a low core water content, and abundant intracellular constituents, such as the calcium chelate of dipicolinic acid and α/β-type small, acid-soluble spore proteins (α/β-type SASP), the last two of which protect spore DNA (6, 42, 46, 48, 52). DNA damage accumulated during spore dormancy is also efficiently repaired during spore germination (33, 47, 48). UV-induced DNA photoproducts are repaired by spore photoproduct lyase and nucleotide excision repair, DNA double-strand breaks (DSB) by nonhomologous end joining, and oxidative stress-induced apurinic/apyrimidinic (AP) sites by AP endonucleases and base excision repair (15, 26-29, 34, 43, 53, 57).Monochromatic 254-nm UV radiation has been used as an efficient and cost-effective means of disinfecting surfaces, building air, and drinking water supplies (31). Commonly used test organisms for inactivation studies are bacterial spores, usually spores of Bacillus subtilis, due to their high degree of resistance to various sporicidal treatments, reproducible inactivation response, and safety (1, 8, 19, 31, 48). Depending on the Bacillus species analyzed, spores are 10 to 50 times more resistant than growing cells to 254-nm UV radiation. In addition, most of the laboratory studies of spore inactivation and radiation biology have been performed using monochromatic 254-nm UV radiation (33, 34). Although 254-nm UV-C radiation is a convenient germicidal treatment and relevant to disinfection procedures, results obtained by using 254-nm UV-C are not truly representative of results obtained using UV wavelengths that endospores encounter in their natural environments (34, 42, 50, 51, 59). However, sunlight reaching the Earth''s surface is not monochromatic 254-nm radiation but a mixture of UV, visible, and infrared radiation, with the UV portion spanning approximately 290 to 400 nm (33, 34, 36). Thus, our knowledge of spore UV resistance has been constructed largely using a wavelength of UV radiation not normally reaching the Earth''s surface, even though ample evidence exists that both DNA photochemistry and microbial responses to UV are strongly wavelength dependent (2, 30, 33, 36).Of recent interest in our laboratories has been the exploration of factors that confer on B. subtilis spores resistance to environmentally relevant extreme conditions, particularly solar UV radiation and extreme desiccation (23, 28, 30, 34 36, 48, 52). It has been reported that α/β-type SASP but not SASP-γ play a major role in spore resistance to 254-nm UV-C radiation (20, 21) and to wet heat, dry heat, and oxidizing agents (48). In contrast, increased spore water content was reported to affect B. subtilis spore resistance to moist heat and hydrogen peroxide but not to 254-nm UV-C (12, 40, 48). However, the possible roles of SASP-α, -β, and -γ and core water content in spore resistance to environmentally relevant solar UV wavelengths have not been explored. Therefore, in this study, we have used B. subtilis strains carrying mutations in the sspA, sspB, sspE, sspA and sspB, or dacB gene to investigate the contributions of SASP and increased core water content to the resistance of B. subtilis spores to 254-nm UV-C and environmentally relevant polychromatic UV radiation encountered on Earth''s surface.     

Identification of Bacillus anthracis Spore Component Antigens Conserved across Diverse Bacillus cereus sensu lato Strains

We sought to identify proteins in the Bacillus anthracis spore, conserved in other strains of the closely related Bacillus cereus group, that elicit an immune response in mammals. Two high throughput approaches were used. First, an in silico screening identified 200 conserved putative B. anthracis spore components. A total of 192 of those candidate genes were expressed and purified in vitro, 75 of which reacted with the rabbit immune sera generated against B. anthracis spores. The second approach was to screen for cross-reacting antigens in the spore proteome of 10 diverse B. cereus group strains. Two-dimensional electrophoresis resolved more than 200 protein spots in each spore preparation. About 72% of the protein spots were found in all the strains. 18 of these conserved proteins reacted against anti-B. anthracis spore rabbit immune sera, two of which (alanine racemase, Dal-1 and the methionine transporter, MetN) overlapped the set of proteins identified using the in silico screen. A conserved repeat domain protein (Crd) was the most immunoreactive protein found broadly across B. cereus sensu lato strains. We have established an approach for finding conserved targets across a species using population genomics and proteomics. The results of these screens suggest the possibility of a multiepitope antigen for broad host range diagnostics or therapeutics against Bacillus spore infection.The anthrax causing bacterium Bacillus anthracis is a member of the Bacillus cereus sensu lato (s.l.)¹ group, a term given to the polyphyletic species consisting of Bacillus thuringiensis, Bacillus cereus, Bacillus mycoides, Bacillus weihenstephanensis, and Bacillus pseudomycoides (1). Genomics studies of B. cereus s.l. strains have shown a similar chromosomal gene composition within this group (2–7). Many phenotypes that distinguish B. cereus s.l. members, such as crystalline toxin production (8), emesis in humans (9), and anthrax virulence (10), are encoded by genes on large plasmids. Experimental conjugative transfer of plasmids between B. cereus s.l. strains has been demonstrated in vitro, in complex media, and in vector species (11–13). Therefore there is a concern about transfer of virulence genes between genetic backgrounds creating new pathogen lineages. In this regard, there is an emerging evidence of natural dissemination of the pXO1 and pXO2 plasmids that encode the anthrax lethal toxin and capsule, respectively. For example, B. cereus G9241 carries a pXO1 plasmid and lethal toxin genes almost identical to those in B. anthracis (6), and a B. cereus strain, which causes anthrax-like illness in African great apes, apparently contains both pXO1 and pXO2 plasmids (14).The infectious agent of most if not all human B. cereus s.l. diseases is the spore. The spore is a dormant, environmentally resistant structure that persists in nutrient- or water-limiting conditions. Anthrax infection occurs after introduction of the B. anthracis spore into a skin abrasion or via inhalation or ingestion (10). The spore germinates inside host cells, and the resulting vegetative bacteria express toxins and capsules that elicit an immune response (10, 15, 16). Formation of the B. cereus spore involves asymmetric cell division during which a copy of the genome is partitioned into each of the sister cells. The smaller cell (prespore) develops into mature endospore, and the larger cell (mother cell) contributes to the differentiation process but undergoes autolysis following its completion to release the endospore into the surrounding medium. Synthesis of cortex, coat, and exosporium are a function mainly of the mother cell. The cortex and coat layers are in close proximity to one another, whereas the exosporium tends to appear as an irregularly shaped, loosely attached, balloon-like layer (17–20). The coat and the exosporium contribute to the remarkable resistance of spores to extreme physical and chemical stresses including the exposure to extraterrestrial conditions (21, 22). Recent work on the structure, composition, assembly, and function of the spore coat and exosporium of pathogenic organisms like B. anthracis and B. cereus have highlighted the crucial link that exists between the origin of these layers (19, 23). There are differences in the appearance and thickness of the coat layers among the spores of various strains and species. In some B. thuringiensis strains, the inner coat is laminated but consists of a patchwork of striated packets, appearing either stacked or comblike, and the outer coat is granular (24), whereas in B. anthracis and other B. cereus s.l. isolates the coat appears compact (25–27). The coat layers comprise about 30% of the total proteins present in the spore (19, 28). Intraspecies variation in the structure and composition of the spore surface layers may reflect the environmental conditions under which these spores are formed (29–31).Because the spore is crucial to infection and persistence of B. anthracis and its close relatives, we undertook an investigation of its protein profile variability across the B. cereus s.l. group. Our goal in this study was to identify conserved antigenic spore proteins that may be transitioned in the future as candidates for immunodiagnostics, therapeutics, or vaccines. We used two high throughput approaches: genome-based bioinformatics analysis and comparative proteomics analysis of spores of B. cereus s.l. to select conserved targets. Our analysis revealed a list of conserved spore proteins within B. cereus but relatively few cross-reacting antigens. Two of these spore conserved antigens (Crd and MetN) have not been described previously for B. anthracis.     

A Novel Spore Protein,ExsM, Regulates Formation of the Exosporium in Bacillus cereus and Bacillus anthracis and Affects Spore Size and Shape

Bacillus cereus spores are assembled with a series of concentric layers that protect them from a wide range of environmental stresses. The outermost layer, or exosporium, is a bag-like structure that interacts with the environment and is composed of more than 20 proteins and glycoproteins. Here, we identified a new spore protein, ExsM, from a β-mercaptoethanol extract of B. cereus ATCC 4342 spores. Subcellular localization of an ExsM-green fluorescent protein (GFP) protein revealed a dynamic pattern of fluorescence that follows the site of formation of the exosporium around the forespore. Under scanning electron microscopy, exsM null mutant spores were smaller and rounder than wild-type spores, which had an extended exosporium (spore length for the wt, 2.40 ± 0.56 μm, versus that for the exsM mutant, 1.66 ± 0.38 μm [P < 0.001]). Thin-section electron microscopy revealed that exsM mutant spores were encased by a double-layer exosporium, both layers of which were composed of a basal layer and a hair-like nap. Mutant exsM spores were more resistant to lysozyme treatment and germinated with higher efficiency than wild-type spores, and they had a delay in outgrowth. Insertional mutagenesis of exsM in Bacillus anthracis ΔSterne resulted in a partial second exosporium and in smaller spores. In all, these findings suggest that ExsM plays a critical role in the formation of the exosporium.Bacillus cereus and Bacillus anthracis are closely related members of the Bacillus cereus group (47). Although B. cereus is mainly an apathogenic organism, certain isolates can cause two different types of food poisoning, emetic syndrome and diarrheal disease (18). The emetic syndrome is caused by ingestion of cereulide, a heat-resistant toxin produced by vegetative cells contaminating the food (30), while the diarrheal disease occurs when spores germinate in the intestinal tract. Spores are also the infective agent in anthrax, a disease caused by B. anthracis (64).B. cereus and B. anthracis differentiate into spores when faced with nutrient deprivation. The spore is a dormant cell type that can remain viable for decades until favorable conditions induce germination and the resumption of vegetative growth. The remarkable resistance properties of the spore result from its unique architecture, consisting of a series of concentric protective layers (51). The spore core contains the genetic material and is surrounded by the cortex, a thick layer of modified peptidoglycan that promotes a highly dehydrated state. Encasing the core and the cortex, the coat is a multilayer protein shell that provides mechanical and chemical resistance. In addition, both the cortex and coat contribute to spore germination (17). Separated from the coat by an interspace, the exosporium encloses the rest of the spore, and it is composed of an inner basal layer and an outer hair-like nap (25).Being the most external layer of the spore, the exosporium interacts directly with the environment and as such provides a semipermeable barrier that may exclude large molecules, like antibodies and hydrolytic enzymes (3, 23, 24, 54). However, the exosporium does not appear to contribute to the typical resistance properties of the spore (6, 35, 60). Also, the exosporium is not necessary in anthrax pathogenesis when tested under laboratory conditions (7, 27, 59), although it is able to down-modulate the innate immune response to spores and mediate adhesion to host tissues (4, 8, 43, 44). The exosporium may also help the spore avoid premature germination in unsustainable environments, since it contains two enzymes, alanine racemase (Alr) and inosine hydrolase (Iunh), that can inactivate low quantities of the germinants l-alanine and inosine, respectively (6, 48, 55, 61). However, regulation of germination by the exosporium is poorly understood. Mutation of exosporial proteins has resulted in only negligible and inconsistent germination phenotypes (2, 5, 27, 28, 52, 54).The exosporium is composed of at least 20 proteins and glycoproteins in tight or loose association (48, 53, 57, 61, 65). These proteins are synthesized in the mother cell and always start self-assembly at the forespore pole near the middle of the mother cell, concurrently with the cortex and coat formation (42). Exosporium assembly is discontinuous and starts with a synthesis of a substructure known as the cap, which likely contains only a subset of the proteins present in the exosporium (55). After cap formation, construction of the rest of the exosporium requires the expression of ExsY (6). BclA is the main component of the hair-like nap on the external side of the exosporium, and it is linked to the basal layer through interaction with ExsFA/BxpB (54, 58). In addition, CotE participates in the correct attachment of the exosporium to the spore (27).Despite these findings, exosporium assembly continues to be a poorly understood process, and many questions remain regarding its composition and the regulation of its synthesis. In this study, we characterized a new spore protein, ExsM, which plays a key role in assembly of the exosporium. In B. cereus, inactivation of exsM resulted in spores with an unusual double-layer exosporium, and a similar phenotype was also observed in B. anthracis exsM null mutant spores. Finally, double-layer exosporium spores allowed us to study the role of the exosporium in germination and outgrowth.     

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.     

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 Silicon Layer Supports Acid Resistance of Bacillus cereus Spores

Silicon (Si) is considered to be a “quasiessential” element for most living organisms. However, silicate uptake in bacteria and its physiological functions have remained obscure. We observed that Si is deposited in a spore coat layer of nanometer-sized particles in Bacillus cereus and that the Si layer enhances acid resistance. The novel acid resistance of the spore mediated by Si encapsulation was also observed in other Bacillus strains, representing a general adaptation enhancing survival under acidic conditions.Silicon (Si), the second-most-abundant element in the earth''s crust, is an important mineral for living organisms; it acts as a component of the outer skeleton of diatomaceous protozoans (1), as a trace element to help animal bone and tooth development (5), and as an element in plants that enhances their tissue strength and disease resistance (8, 9). These organisms take up silicate from the environment and accumulate it as silica that is formed from highly concentrated silicate (27). In 1980, relatively high concentrations of Si were observed at the spore coat region of Bacillus cereus and Bacillus megaterium spores by an analysis using scanning transmission electron microscopy (STEM) (14, 23). However, due to the low resolution and relatively weak signal, the precise localization of Si was not determined. On the other hand, the Si contents of Bacillus coagulans and Bacillus subtilis spores were reported to be almost absent or under the detection limit (4, 24). Some bacteriologists familiar with these data consider the presence of Si an anomaly (17). The presence of Si in bacterial spores (specifically, the spores of Bacillus anthracis) again became the focus of attention when anthrax spores were mailed to U.S. senators in the fall of 2001 (17). The Senate anthrax spores could be easily dispersed as single spores when the container was opened. The investigators considered that coating spores with silica might be involved in preventing spores from sticking to each other (17). Thus, if silica is normally absent from spores, its presence in B. anthracis spores suggested that they had been weaponized (17). Subsequent analysis convinced the investigators that the Si was a natural occurrence (3). However, since silica-rich and -poor spores of the same bacterial strain have never been compared, any relationship between naturally accumulated silica and spore dispersion remained hypothetical.In the present study, we screened for the bacterium that takes up the largest amount of silicate from among a number of strains isolated from paddy field soil in order to study Si uptake, clarify the localization of Si, and reveal the roles of Si in bacteria. The effect of silica on spore dispersion was also discussed.     

Proteomic and Genomic Characterization of Highly Infectious Clostridium difficile 630 Spores

Clostridium difficile, a major cause of antibiotic-associated diarrhea, produces highly resistant spores that contaminate hospital environments and facilitate efficient disease transmission. We purified C. difficile spores using a novel method and show that they exhibit significant resistance to harsh physical or chemical treatments and are also highly infectious, with <7 environmental spores per cm² reproducibly establishing a persistent infection in exposed mice. Mass spectrometric analysis identified ∼336 spore-associated polypeptides, with a significant proportion linked to translation, sporulation/germination, and protein stabilization/degradation. In addition, proteins from several distinct metabolic pathways associated with energy production were identified. Comparison of the C. difficile spore proteome to those of other clostridial species defined 88 proteins as the clostridial spore “core” and 29 proteins as C. difficile spore specific, including proteins that could contribute to spore-host interactions. Thus, our results provide the first molecular definition of C. difficile spores, opening up new opportunities for the development of diagnostic and therapeutic approaches.Clostridium difficile is a gram-positive, spore-forming, anaerobic bacterium that can asymptomatically colonize the intestinal tracts of humans and other mammals (3, 30, 39). Antibiotic treatment can result in C. difficile overgrowth and can lead to clinical disease, ranging from diarrhea to life-threatening pseudomembranous colitis, particularly in immunocompromised hosts (2, 4, 7). In recent years, C. difficile has emerged as the major cause of nosocomial antibiotic-induced diarrhea, and it is frequently associated with outbreaks (21, 22). A contributing factor is that C. difficile can be highly infectious and difficult to contain, especially when susceptible patients are present in the same hospital setting (13).Person-to-person transmission of C. difficile is associated with the excretion of highly resistant spores in the feces of infected patients, creating an environmental reservoir that can confound many infection control measures (29, 44). Bacterial spores, which are metabolically dormant cells that are formed following asymmetric cell division, normally have thick concentric external layers, the spore coat and cortex, that protect the internal cytoplasm (15, 42). Upon germination, spores lose their protective external layers and resume vegetative growth (24, 27, 36). Bacillus spores and the spores of most Clostridium species germinate in response to amino acids, carbohydrates, or potassium ions (24, 36). In contrast, C. difficile spores show an increased level of germination in response to cholate derivatives found in bile (40, 41). Thus, spores are well adapted for survival and dispersal under a wide range of environmental conditions but will germinate in the presence of specific molecular signals (24, 36).While the spores of a number of Bacillus species, such as Bacillus subtilis and Bacillus anthracis, and those of other Clostridium species, such as Clostridium perfringens (15, 20), have been well characterized, research on C. difficile spores has been relatively limited. A greater understanding of C. difficile spore biology could be exploited to rationalize disinfection regimes, molecular diagnostics, and the development of targeted treatments such as vaccines. Here we describe a novel method to isolate highly purified C. difficile spores that maintain their resistance and infectious characteristics, thus providing a unique opportunity to study C. difficile spores in the absence of vegetative cells. A thorough proteomic and genomic analysis of the spore provides novel insight into the unique composition and predictive biological properties of C. difficile spores that should underpin future research into this high-profile but poorly understood pathogen.     

Sequence Motifs and Proteolytic Cleavage of the Collagen-Like Glycoprotein BclA Required for Its Attachment to the Exosporium of Bacillus anthracis

Bacillus anthracis spores are enclosed by an exosporium comprised of a basal layer and an external hair-like nap. The filaments of the nap are composed of trimers of the collagen-like glycoprotein BclA. The attachment of essentially all BclA trimers to the exosporium requires the basal layer protein BxpB, and both proteins are included in stable high-molecular-mass exosporium complexes. BclA contains a proteolytically processed 38-residue amino-terminal domain (NTD) that is essential for basal-layer attachment. In this report, we identify three NTD submotifs (SM1a, SM1b, and SM2, located within residues 21 to 33) that are important for BclA attachment and demonstrate that residue A20, the amino-terminal residue of processed BclA, is not required for attachment. We show that the shortest NTD of BclA—or of a recombinant protein—sufficient for high-level basal-layer attachment is a 10-residue motif consisting of an initiating methionine, an apparently arbitrary second residue, SM1a or SM1b, and SM2. We also demonstrate that cleavage of the BclA NTD is necessary for efficient attachment to the basal layer and that the site of cleavage is somewhat flexible, at least in certain mutant NTDs. Finally, we propose a mechanism for BclA attachment and discuss the possibility that analogous mechanisms are involved in the attachment of many different collagen-like proteins of B. anthracis and closely related Bacillus species.Bacillus anthracis, a Gram-positive, rod-shaped, aerobic bacterium, is the causative agent of anthrax (17). When vegetative cells of B. anthracis are starved for certain essential nutrients, they form dormant spores that can survive in harsh soil environments for many years (12, 19). Spore formation starts with asymmetric septation that divides the starved vegetative cell into two genome-containing compartments, a mother cell compartment and a smaller forespore compartment. The mother cell then engulfs the forespore and surrounds it with three protective layers: a cortex composed of peptidoglycan, a closely apposed proteinaceous coat, and a loosely fitting exosporium (11). After a spore maturation stage, the mother cell lyses and releases the mature spore. When spores encounter an aqueous environment containing nutrients, they can germinate and grow as vegetative cells (18). Anthrax is typically caused by contact with spores (17).The outermost layer of B. anthracis spores, the exosporium, has been studied intensively in recent years because it is both the first point of contact with the immune system of an infected host and the target of new detectors for agents of bioterrorism (21, 28, 32). The exosporium of B. anthracis and closely related pathogenic species, such as Bacillus cereus and Bacillus thuringiensis, is a prominent structure consisting of a paracrystalline basal layer and an external hair-like nap (1, 9). The filaments of the nap are formed by trimers of the collagen-like glycoprotein BclA (2, 29). Recent studies suggest that BclA plays a major role in pathogenesis by directing spores to professional phagocytic cells, a critical step in disease progression (4, 21). The basal layer is composed of approximately 20 different proteins (23, 25, 26), several of which have been shown to play key roles in exosporium assembly (3, 13, 27). One of these proteins is BxpB (also called ExsFA) (25, 30, 34), which is required for the attachment of approximately 98% of spore-bound BclA to the basal layer (26, 30). Residual BclA attachment requires the basal layer protein ExsFB, a paralog of BxpB (30).BclA contains three distinct domains: a 38-residue amino-terminal domain (NTD), a central collagen-like region containing a strain-specific number of XXG (mostly PTG) repeats, and a 134-residue carboxyl-terminal domain (CTD) (25, 29, 31). The CTD apparently functions as the major nucleation site for trimerization of BclA (24), and CTD trimers form the globular distal ends of the filaments in the nap (2). The highly extended collagen-like region is extensively glycosylated (5), and its length determines the depth of the nap (2, 31). The NTD is the site of attachment of BclA to the basal layer, and deletion of the NTD prevents this attachment (2). The NTD is normally proteolytically processed to remove the first 19 amino acids, and it is this mature form of BclA that is attached to the basal layer (25, 29). In an earlier report, we suggested that NTD processing of BclA is required for basal-layer attachment, perhaps through a direct covalent linkage to BxpB (26).Recently, Thompson and Stewart identified conserved 11-residue sequences in the NTDs of BclA and the minor B. anthracis collagen-like glycoprotein BclB and showed that these sequences are involved in the incorporation of BclA and BclB into the exosporium. These investigators used a truncated BclA NTD that lacked residues 2 through 19 but included the conserved 11-amino-acid sequence to target enhanced green fluorescent protein (EGFP) to the surface of the developing forespore (33). Thompson and Stewart also reported that cleavage of the BclA NTD occurred after its association with the forespore and suggested that this cleavage was involved indirectly in the attachment process. Actual cleavage sites were not determined in these studies, however. We have performed related studies of the attachment of BclA to the exosporium that provide a more detailed and somewhat different view of this process. In our studies, which are reported here, we identified short segments, or submotifs, of the BclA NTD that can be arranged in different combinations to produce 10-amino-acid motifs sufficient for tight attachment of BclA, and probably most proteins, to the exosporium basal layer. Additionally, we present direct evidence showing that BclA NTD cleavage is required for efficient attachment to the basal layer and that selection of the cleavage site can be somewhat flexible. Finally, we discuss a possible mechanism for BclA attachment and the likelihood that similar mechanisms are used for attachment of many different collagen-like proteins of B. anthracis and closely related Bacillus species.     

Factors Affecting Variability in Time between Addition of Nutrient Germinants and Rapid Dipicolinic Acid Release during Germination of Spores of Bacillus Species

The simultaneous nutrient germination of hundreds of individual wild-type spores of three Bacillus species and a number of Bacillus subtilis strains has been measured by two new methods, and rates of release of the great majority of the large pool of dipicolinic acid (DPA) from individual spores of B. subtilis strains has been measured by Raman spectroscopy with laser tweezers. The results from these analyses and published data have allowed a number of significant conclusions about the germination of spores of Bacillus species as follows. (i) The time needed for release of the great majority of a Bacillus spore''s DPA once rapid DPA release had begun (ΔT_release) during nutrient germination was independent of the concentration of nutrient germinant used, the level of the germinant receptors (GRs) that recognize nutrient germinants used and heat activation prior to germination. Values for ΔT_release were generally 0.5 to 3 min at 25 to 37°C for individual wild-type spores. (ii) Despite the conclusion above, germination of individual spores in populations was very heterogeneous, with some spores in wild-type populations completing germination ≥15-fold slower than others. (iii) The major factor in the heterogeneity in germination of individual spores in populations was the highly variable lag time, T_lag, between mixing spores with nutrient germinants and the beginning of ΔT_release. (iv) A number of factors decrease spores'' T_lag values including heat activation, increased levels of GRs/spore, and higher levels of nutrient germinants. These latter factors appear to affect the level of activated GRs/spore during nutrient germination. (v) The conclusions above lead to the simple prediction that a major factor causing heterogeneity in Bacillus spore germination is the number of functional GRs in individual spores, a number that presumably varies significantly between spores in populations.Spores of various Bacillus species are metabolically dormant and can survive for years in this state (30). However, spores constantly sense their environment, and if appropriate small molecules termed germinants are present, spores can rapidly return to life in the process of germination followed by outgrowth (25, 29, 30). The germinants that most likely trigger spore germination in the environment are low-molecular-weight nutrient molecules, the identities of which are strain and species specific, including amino acids, sugars, and purine nucleosides. Metabolism of these nutrient germinants is not needed for the triggering of spore germination. Rather, these germinants are recognized by germinant receptors (GRs) located in the spore''s inner membrane that recognize their cognate germinants in a stereospecific manner (17, 24, 25, 29). Spores have a number of such GRs, with three functional GRs in Bacillus subtilis spores and even more in Bacillus anthracis, Bacillus cereus, and Bacillus megaterium spores (6, 29, 30). Binding of nutrient germinants to some single GRs is sufficient to trigger spore germination, for example the triggering of B. subtilis spore germination by binding of l-alanine or l-valine to the GerA GR. However, many GRs cooperate such that binding of germinants by ≥2 different GRs is needed to trigger germination (2, 29): for example, the triggering of B. subtilis spore germination by the binding of components of a mixture of l-asparagine, d-glucose, d-fructose, and K⁺ ions (AGFK) to the GerB and GerK GRs. The binding of nutrient germinants to GRs triggers subsequent events in germination, although how this is accomplished is not known.The first readily measured biochemical event after addition of nutrient germinants to Bacillus spores is the rapid release of the spore''s large depot (∼10% of spore dry weight) of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) plus its chelated divalent cations, predominantly Ca²⁺ (Ca-DPA), from the spore core (25, 29). Ca-DPA release then results in the activation of two redundant cortex-lytic enzymes (CLEs), CwlJ and SleB, which hydrolyze the spore''s peptidoglycan cortex layer (16, 22, 27, 29). CwlJ is activated by Ca-DPA as it is released from the spore while SleB is activated only after most DPA is released (17, 20, 22, 26, 27). Cortex hydrolysis ultimately allows the spore core to expand and take up more water, raising the core water content from the 35 to 45% of wet weight in the dormant spore to the 80% of wet weight characteristic of growing cells. Full hydration of the spore core then allows enzyme action, metabolism, and macromolecular synthesis to resume in the now fully germinated spore.Germination of spores in populations is very heterogeneous, with some spores germinating rapidly and some extremely slowly (4, 5, 9, 11, 13-15, 19, 26, 31, 32). Where it has been studied, the reason for this heterogeneity has been suggested to be due to a variable lag period (T_lag) between the time of mixing spores with a germinant and the time at which rapid DPA release begins, since once rapid DPA release begins, the time required for release of almost all DPA as well as for subsequent cortex hydrolysis is generally rather short compared to T_lag values in individual spores (5, 11, 13-15, 19, 26, 31, 32). The times required for DPA release and cortex hydrolysis are also similar in wild-type spores with both very short and long T_lag values (5, 15, 19, 27). The reasons for the variability in T_lag times between individual spores in populations are not known, although there are reports that both activation of spores for germination by a sublethal heat treatment (heat activation) as well as increasing concentrations of nutrient germinants can shorten T_lag values (12, 14, 15, 18, 32). However, there has been no detailed study of the causes of the variability in T_lag values between very large numbers of individual spores in populations.In order to study the heterogeneity in spore germination thoroughly, methods are needed to follow the germination of hundreds of individual spores over several hours. Initial studies of the germination of individual spores examined a single spore in a phase-contrast microscope and followed the germination of this spore by changes in the core''s refractive index due to DPA release and core swelling (14, 15, 32, 34). However, this method is labor-intensive for gathering data with hundreds of individual spores. More recently, confocal microscopy and then surface adsorption and optical tweezers have been used to capture single spores, and germination events have been followed by methods such as Raman spectroscopy to directly measure DPA release, as well as phase-contrast microscopy and elastic light scattering (3, 5, 9, 10, 19, 26). While the latter recent advances have allowed accumulation of much information about germination, collection of this type of data for large numbers of individual spores is still labor-intensive, although use of dual optical traps (35) and perhaps multiple traps in the future may alleviate this problem. However, phase-contrast microscopy plus appropriate computer software has recently allowed the monitoring of many hundreds of individual spores for several hours, with automated assessment of various changes in the cells during the period of observation (19). In the present work, we have used both phase-contrast and differential interference contrast (DIC) microscopy to monitor the germination of many hundreds of individual spores of three Bacillus species adhered on either an agarose pad or a glass coverslip for 1 to 2 h. This work, as well as examination of times needed for release of most DPA once rapid DPA release has begun during germination of individual spores under a variety of conditions, has allowed detailed examination of the effects of heat activation, nutrient germinant concentration, GR numbers per spore, and individual CLEs on spore germination heterogeneity and on values of T_lag for individual spores.     

Superdormant Spores of Bacillus Species Germinate Normally with High Pressure,Peptidoglycan Fragments,and Bryostatin

Superdormant spores of Bacillus cereus and Bacillus subtilis germinated just as well as dormant spores with pressures of 150 or 500 MPa and with or without heat activation. Superdormant B. subtilis spores also germinated as well as dormant spores with peptidoglycan fragments or bryostatin, a Ser/Thr protein kinase activator.Spores of Bacillus species are formed in sporulation, a process that is generally triggered by starvation for one or more nutrients (13, 19). These spores are metabolically dormant and extremely resistant to a large variety of environmental stresses, including heat, radiation, and toxic chemicals, and as a consequence of these properties, these spores can remain viable in their dormant state for many years (13, 18, 19). However, spores are constantly sensing their environment, and if nutrients return, the spores can rapidly return to growth through the process of spore germination (17). Spore germination is generally triggered by specific nutrients that bind to nutrient germinant receptors, with this binding alone somehow triggering germination. However, spore germination can also be triggered by many non-nutrient agents, including cationic surfactants such as dodecylamine, a 1:1 complex of Ca²⁺ with pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA], a major spore small molecule), very high pressures, specific peptidoglycan fragments, and bryostatin, an activator of Ser/Thr protein kinases (17, 19, 20). For nutrient germinants in particular, spore germination is also potentiated by a prior sublethal heat treatment termed heat activation (17).While normally the great majority of spores in populations germinate relatively rapidly in response to nutrient germinants, a small percentage of spores germinate extremely slowly. These spores that are refractory to nutrient germination have been termed superdormant spores and are a major concern for the food industry (8). Recently superdormant spores of three Bacillus species have been isolated by repeated germination of spore populations with specific nutrient germinants and isolation of remaining dormant spores (5, 6). These superdormant spores germinate extremely poorly with the nutrient germinants used in superdormant spore isolation, as well as with other nutrient germinants. All of the specific defects leading to spore superdormancy are not known, although an increased level of receptors for specific nutrient germinants decreases levels of superdormant spores obtained with the nutrients that are ligands for these receptors (5). Superdormant spores also have significantly higher temperature optima for heat activation of nutrient germination than the spore population as a whole (7).In contrast to the poor germination of superdormant spores with nutrient germinants, superdormant spores germinate normally with dodecylamine and Ca-DPA (5, 6). This is consistent with possible roles of nutrient germinant receptor levels and/or heat activation temperature optima in affecting spore superdormancy, since neither dodecylamine nor Ca-DPA triggers Bacillus spore germination through nutrient germinant receptors, and germination with these agents is also not stimulated by heat activation (11, 15, 17). However, the effects of high pressures, peptidoglycan fragments, and bryostatin, all of which almost certainly trigger spore germination by mechanisms at least somewhat different than triggering of germination by nutrients, dodecylamine, and Ca-DPA (2, 3, 11, 15, 20, 22, 23), have not been tested for their effects on superdormant spores. Consequently, we have compared the germination of dormant and superdormant spores of two Bacillus species by high-pressures, peptidoglycan fragments, and bryostatin.The spores used in this work were from Bacillus subtilis PS533 (16), a derivative of strain 168 that also carries plasmid pUB110, providing resistance to kanamycin (10 μg/ml), and Bacillus cereus T (originally obtained from H. O. Halvorson). Spores of these strains were prepared and purified as described previously (6, 10, 12). Superdormant spores of B. subtilis were prepared by germination following heat activation at 75°C for 30 min by two germination treatments at 37°C with 10 mM l-valine for 2 h, followed by isolation of remaining dormant spores, all as described previously (5, 10, 12). These superdormant spores germinated extremely poorly with 10 mM valine at 37°C, giving ≤10% germination in 2 h at 37°C, while the initial spore population exhibited >95% germination under the same conditions (data not shown). Superdormant B. cereus spores were isolated similarly, although heat activation was at 65°C for 30 min and the germinant was 5 mM inosine as described previously (6). These superdormant B. cereus spores exhibited <5% germination with inosine in 2 h at 37°C compared to the >95% germination of the initial dormant spores under the same conditions (data not shown).     

A Bacillus anthracis S-Layer Homology Protein That Binds Heme and Mediates Heme Delivery to IsdC

The sequestration of iron by mammalian hosts represents a significant obstacle to the establishment of a bacterial infection. In response, pathogenic bacteria have evolved mechanisms to acquire iron from host heme. Bacillus anthracis, the causative agent of anthrax, utilizes secreted hemophores to scavenge heme from host hemoglobin, thereby facilitating iron acquisition from extracellular heme pools and delivery to iron-regulated surface determinant (Isd) proteins covalently attached to the cell wall. However, several Gram-positive pathogens, including B. anthracis, contain genes that encode near iron transporter (NEAT) proteins that are genomically distant from the genetically linked Isd locus. NEAT domains are protein modules that partake in several functions related to heme transport, including binding heme and hemoglobin. This finding raises interesting questions concerning the relative role of these NEAT proteins, relative to hemophores and the Isd system, in iron uptake. Here, we present evidence that a B. anthracis S-layer homology (SLH) protein harboring a NEAT domain binds and directionally transfers heme to the Isd system via the cell wall protein IsdC. This finding suggests that the Isd system can receive heme from multiple inputs and may reflect an adaptation of B. anthracis to changing iron reservoirs during an infection. Understanding the mechanism of heme uptake in pathogenic bacteria is important for the development of novel therapeutics to prevent and treat bacterial infections.Pathogenic bacteria need to acquire iron to survive in mammalian hosts (12). However, the host sequesters most iron in the porphyrin heme, and heme itself is often bound to proteins such as hemoglobin (14, 28, 85). Circulating hemoglobin can serve as a source of heme-iron for replicating bacteria in infected hosts, but the precise mechanisms of heme extraction, transport, and assimilation remain unclear (25, 46, 79, 86). An understanding of how bacterial pathogens import heme will lead to the development of new anti-infectives that inhibit heme uptake, thereby preventing or treating infections caused by these bacteria (47, 68).The mechanisms of transport of biological molecules into a bacterial cell are influenced by the compositional, structural, and topological makeup of the cell envelope. Gram-negative bacteria utilize specific proteins to transport heme through the outer membrane, periplasm, and inner membrane (83, 84). Instead of an outer membrane and periplasm, Gram-positive bacteria contain a thick cell wall (59, 60). Proteins covalently anchored to the cell wall provide a functional link between extracellular heme reservoirs and intracellular iron utilization pathways (46). In addition, several Gram-positive and Gram-negative bacterial genera also contain an outermost structure termed the S (surface)-layer (75). The S-layer is a crystalline array of protein that surrounds the bacterial cell and may serve a multitude of functions, including maintenance of cell architecture and protection from host immune components (6, 7, 18, 19, 56). In bacterial pathogens that manifest an S-layer, the “force field” function of this structure raises questions concerning how small molecules such as heme can be successfully passed from the extracellular milieu to cell wall proteins for delivery into the cell cytoplasm.Bacillus anthracis is a Gram-positive, spore-forming bacterium that is the etiological agent of anthrax disease (30, 33). The life cycle of B. anthracis begins after a phagocytosed spore germinates into a vegetative cell inside a mammalian host (2, 40, 69, 78). Virulence determinants produced by the vegetative cells facilitate bacterial growth, dissemination to major organ systems, and eventually host death (76-78). The release of aerosolized spores into areas with large concentrations of people is a serious public health concern (30).Heme acquisition in B. anthracis is mediated by the action of IsdX1 and IsdX2, two extracellular hemophores that extract heme from host hemoglobin and deliver the iron-porphyrin to cell wall-localized IsdC (21, 45). Both IsdX1 and IsdX2 harbor near iron transporter domains (NEATs), a conserved protein module found in Gram-positive bacteria that mediates heme uptake from hemoglobin and contributes to bacterial pathogenesis upon infection (3, 8, 21, 31, 44, 46, 49, 50, 67, 81, 86). Hypothesizing that B. anthracis may contain additional mechanisms for heme transport, we provide evidence that B. anthracis S-layer protein K (BslK), an S-layer homology (SLH) and NEAT protein (32, 43), is surface localized and binds and transfers heme to IsdC in a rapid, contact-dependent manner. These results suggest that the Isd system is not a self-contained conduit for heme trafficking and imply that there is functional cross talk between differentially localized NEAT proteins to promote heme uptake during infection.     

Characterization of Wet-Heat Inactivation of Single Spores of Bacillus Species by Dual-Trap Raman Spectroscopy and Elastic Light Scattering

Dual-trap laser tweezers Raman spectroscopy (LTRS) and elastic light scattering (ELS) were used to investigate dynamic processes during high-temperature treatment of individual spores of Bacillus cereus, Bacillus megaterium, and Bacillus subtilis in water. Major conclusions from these studies included the following. (i) After spores of all three species were added to water at 80 to 90°C, the level of the 1:1 complex of Ca²⁺ and dipicolinic acid (CaDPA; ∼25% of the dry weight of the spore core) in individual spores remained relatively constant during a highly variable lag time (T_lag), and then CaDPA was released within 1 to 2 min. (ii) The T_lag values prior to rapid CaDPA release and thus the times for wet-heat killing of individual spores of all three species were very heterogeneous. (iii) The heterogeneity in kinetics of wet-heat killing of individual spores was not due to differences in the microscopic physical environments during heat treatment. (iv) During the wet-heat treatment of spores of all three species, spore protein denaturation largely but not completely accompanied rapid CaDPA release, as some changes in protein structure preceded rapid CaDPA release. (v) Changes in the ELS from individual spores of all three species were strongly correlated with the release of CaDPA. The ELS intensities of B. cereus and B. megaterium spores decreased gradually and reached minima at T₁ when ∼80% of spore CaDPA was released, then increased rapidly until T₂ when full CaDPA release was complete, and then remained nearly constant. The ELS intensity of B. subtilis spores showed similar features, although the intensity changed minimally, if at all, prior to T₁. (vi) Carotenoids in B. megaterium spores'' inner membranes exhibited two changes during heat treatment. First, the carotenoid''s two Raman bands at 1,155 and 1,516 cm⁻¹ decreased rapidly to a low value and to zero, respectively, well before T_lag, and then the residual 1,155-cm⁻¹ band disappeared, in parallel with the rapid CaDPA release beginning at T_lag.Bacterial spores of Bacillus species are formed in sporulation and are metabolically dormant and extremely resistant to a variety of harsh conditions, including heat, radiation, and many toxic chemicals (37). Since spores of these species are generally present in foodstuffs and cause food spoilage and food-borne disease (37, 38), there has long been interest in the mechanisms of both spore resistance and spore killing, especially for wet heat, the agent most commonly used to kill spores. The killing of dormant spores by wet heat generally requires temperatures about 40°C higher than those for the killing of growing cells of the same strain (37, 43). A number of factors influence spore wet-heat resistance, with a major factor being the spore core''s water content, as spores with higher core water content are less wet-heat resistant than are spores with lower core water (15, 25). The high level of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) and the types of its associated divalent cations, predominantly Ca²⁺, that comprise ∼25% of the dry weight of the core also contribute to spore wet-heat resistance, although how low core water content and CaDPA protect spores against wet heat is not known. The protection of spore DNA against depurination by its saturation with a group of α/β-type small, acid-soluble spore proteins also contributes to spore wet-heat resistance (14, 23, 33, 37).Despite knowledge of a number of factors important in spore wet-heat resistance, the mechanism for wet-heat killing of spores is not known. Wet heat does not kill spores by DNA damage or oxidative damage (35, 37). Instead, spore killing by this agent is associated with protein denaturation and enzyme inactivation (2, 7, 44), although specific proteins for which damage causes spore death have not been identified. Wet-heat treatment also often results in the release of the spore core''s large depot of CaDPA. The mechanism for this CaDPA release is not known but is presumably associated with the rupture of the spore''s inner membrane (7). In addition, the relationship between protein denaturation and CaDPA release is not clear, although recent work suggests that significant protein denaturation can occur prior to CaDPA release (7). Almost all information on spore killing by moist heat has been obtained with spore populations, and essentially nothing is known about the behavior of individual spores exposed to potentially lethal temperatures in water. Given the likely heterogeneity of spores in populations, in particular in their wet-heat resistances (16, 18, 39, 40), it could be most informative to analyze the behavior of individual spores exposed to high temperatures in water.Raman spectroscopy is widely used in biochemical studies, as this technique has high sensitivity and responds rapidly to subtle changes in molecule structure (1, 22, 31). In addition, when Raman spectroscopy is combined with confocal microscopy and optical tweezers, the resultant laser tweezers Raman spectroscopy (LTRS) allows the nondestructive, noninvasive detection of biochemical processes at the single-cell level (9, 10, 19, 46). Indeed, LTRS has been used to analyze the DPA level and the germination of individual Bacillus spores (5, 19, 30). In order to obtain information more rapidly, dual- and multitrap laser tweezers have been developed to allow multiple individual cells or particles to be analyzed simultaneously (11, 13, 24, 27), and the dual trap has been used to measure the hydrodynamic cross-correlations of two particles (24). In addition to Raman scattering, the elastic light scattering (ELS) from trapped individual cells also provides valuable information on cell shape, orientation, refractive index, and morphology (12, 45) and has been used to monitor spore germination dynamics as well (30).In this work, we report studies of wet-heat treatment of individual spores of three different Bacillus species by dual-trap LTRS and ELS. A number of important processes related to wet-heat inactivation of spores, including CaDPA release and protein denaturation, and the correlation between these processes were investigated by monitoring changes in Raman scattering at CaDPA-, protein structure-, and phenylalanine-specific bands and changes in ELS intensity.     

Isolation and Characterization of Superdormant Spores of Bacillus Species

Superdormant spores of Bacillus subtilis and Bacillus megaterium were isolated in 4 to 12% yields following germination with high nutrient levels that activated one or two germinant receptors. These superdormant spores did not germinate with the initial nutrients or those that stimulated other germinant receptors, and the superdormant spores'' defect was not genetic. The superdormant spores did, however, germinate with Ca²⁺-dipicolinic acid or dodecylamine. Although these superdormant spores did not germinate with high levels of nutrients that activated one or two nutrient germinant receptors, they germinated with nutrient mixtures that activated more receptors, and using high levels of nutrient mixtures activating more germinant receptors decreased superdormant spore yields. The use of moderate nutrient levels to isolate superdormant spores increased their yields; the resultant spores germinated poorly with the initial moderate nutrient concentrations, but they germinated well with high nutrient concentrations. These findings suggest that the levels of superdormant spores in populations depend on the germination conditions used, with fewer superdormant spores isolated when better germination conditions are used. These findings further suggest that superdormant spores require an increased signal for triggering spore germination compared to most spores in populations. One factor determining whether a spore is superdormant is its level of germinant receptors, since spore populations with higher levels of germinant receptors yielded lower levels of superdormant spores. A second important factor may be heat activation of spore populations, since yields of superdormant spores from non-heat-activated spore populations were higher than those from optimally activated spores.Spores of various Bacillus species are formed in sporulation and are metabolically dormant and very resistant to environmental stress factors (21, 37). While such spores can remain in this dormant, resistant state for long periods, they can return to life rapidly through the process of germination, during which the spore''s dormancy and extreme resistance are lost (36). Spore germination has long been of intrinsic interest, and continues to attract applied interest, because (i) spores of a number of Bacillus species are major agents of food spoilage and food-borne disease and (ii) spores of Bacillus anthracis are a major bioterrorism agent. Since spores are much easier to kill after they have germinated, it would be advantageous to trigger germination of spores in foods or the environment and then readily inactivate the much less resistant germinated spores. However, this simple strategy has been largely nullified because germination of spore populations is heterogeneous, with some spores, often called superdormant spores, germinating extremely slowly and potentially coming back to life long after treatments are applied to inactivate germinated spores (8, 9, 16). The concern over superdormant spores in populations also affects decisions such as how long individuals exposed to B. anthracis spores should continue to take antibiotics, since spores could remain dormant in an individual for long periods and then germinate and cause disease (3, 11).In many species, spore germination can be increased by a prior activation step, generally a sublethal heat treatment, although the changes taking place during heat activation are not known (16). Spore germination in Bacillus species is normally triggered by nutrients such as glucose, amino acids, or purine ribosides (27, 36). These agents bind to germinant receptors located in the spore''s inner membrane that are specific for particular nutrients. In Bacillus subtilis, the GerA receptor responds to l-alanine or l-valine, while the GerB and GerK receptors act cooperatively to respond to a mixture of l-asparagine (or l-alanine), d-glucose, d-fructose and K⁺ ions (AGFK [or Ala-GFK]) (1, 27, 36). There are even more functional germinant receptors in Bacillus megaterium spores, and these respond to d-glucose, l-proline, l-leucine, l-valine, or even salts, such as KBr (6). Glucose appears to trigger germination of B. megaterium spores through either of two germinant receptors, GerU or GerVB, while l-proline triggers germination through only the GerVB receptor, and KBr germination is greatly decreased by the loss of either GerU or GerVB (6). Nutrient binding to the germinant receptors triggers the release of small molecules from the spore core, most notably the huge depot (∼10% of spore dry weight) of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) present in spores predominantly as a 1:1 diluted chelate with Ca²⁺ (Ca-DPA) (35, 36). Ca-DPA release then triggers the activation of one of two redundant cortex lytic enzymes (CLEs) that degrade the spore''s peptidoglycan cortex, and cortex degradation completes spore germination and allows progression into outgrowth and then vegetative growth (27, 33, 36).Spore germination can also be triggered by nonnutrient agents, including Ca-DPA and cationic surfactants (27, 33, 36). With B. subtilis spores, Ca-DPA triggers germination by activating one particular CLE, termed CwlJ, and bypasses the spore''s germinant receptors. Germination by the cationic surfactant dodecylamine also bypasses the germinant receptors, and this agent appears to release small molecules including Ca-DPA from the spore core either by opening a normal channel in the spore''s inner membrane for Ca-DPA and other small molecules or by creating such a channel (31, 38, 39).Almost all work on the specifics of the germination of spores of Bacillus species has focused on the majority of spores in populations, and little detailed attention has been paid to that minority of spores that either fail to germinate or germinate extremely slowly. However, it is these latter spores that are most important in unraveling the cause of superdormancy and perhaps suggesting a means to germinate and thus easily inactivate such superdormant spores. Consequently, we have undertaken the task of isolating superdormant spores from spore populations, using buoyant density centrifugation to separate dormant spores from germinated spores. The properties of these purified superdormant spores were then studied, and this information has suggested some reason(s) for spore superdormancy.     

Recovery Efficiency and Limit of Detection of Aerosolized Bacillus anthracis Sterne from Environmental Surface Samples

After the 2001 anthrax incidents, surface sampling techniques for biological agents were found to be inadequately validated, especially at low surface loadings. We aerosolized Bacillus anthracis Sterne spores within a chamber to achieve very low surface loading (ca. 3, 30, and 200 CFU per 100 cm²). Steel and carpet coupons seeded in the chamber were sampled with swab (103 cm²) or wipe or vacuum (929 cm²) surface sampling methods and analyzed at three laboratories. Agar settle plates (60 cm²) were the reference for determining recovery efficiency (RE). The minimum estimated surface concentrations to achieve a 95% response rate based on probit regression were 190, 15, and 44 CFU/100 cm² for sampling steel surfaces and 40, 9.2, and 28 CFU/100 cm² for sampling carpet surfaces with swab, wipe, and vacuum methods, respectively; however, these results should be cautiously interpreted because of high observed variability. Mean REs at the highest surface loading were 5.0%, 18%, and 3.7% on steel and 12%, 23%, and 4.7% on carpet for the swab, wipe, and vacuum methods, respectively. Precision (coefficient of variation) was poor at the lower surface concentrations but improved with increasing surface concentration. The best precision was obtained with wipe samples on carpet, achieving 38% at the highest surface concentration. The wipe sampling method detected B. anthracis at lower estimated surface concentrations and had higher RE and better precision than the other methods. These results may guide investigators to more meaningfully conduct environmental sampling, quantify contamination levels, and conduct risk assessment for humans.Anthrax, the spectrum of diseases caused by infection with Bacillus anthracis, is not considered a communicable disease but is generally acquired via environmental exposures. Many anthrax cases through history have been the result of agricultural or industrial exposure to B. anthracis spores (33). The disease most often presents itself as a cutaneous infection; however, there are both gastrointestinal and inhalational forms of the disease. Inhalational anthrax is typically rapidly fatal, even with treatment. In general, inhalation exposures require specific conditions, such as poor ventilation and activities that disturb dust containing B. anthracis spores (13).Because diagnosing anthrax in its early stages in human and animal hosts is difficult and B. anthracis spores are extremely stable in the environment, this microorganism has been investigated, developed, and deployed as a biological weapon throughout the 20th century. Use of this microorganism has seen varied success during World War I (9) and subsequently. It is generally accepted that there was an accidental release of B. anthracis spores from a weapons manufacturing or development facility in 1979 in Sverdlovsk, USSR (now Yekaterinaburg, Russia) (10, 26). In 1993, an attempt by a civilian group, Aum Shinrikyo, to use this microorganism to attack a civilian population in a Tokyo suburb did not result in any casualties (22, 28).In 2001, envelopes containing a powder formulation of B. anthracis were mailed in the United States to several individuals. These letters were the presumed cause of 22 cases of clinical anthrax, 11 inhalational and 11 cutaneous, with 5 fatalities, all of whom suffered from inhalational disease (34). According to congressional testimony, the powdered spore suspension was “easily dispersed into the air” (29). Of the 11 individuals with inhalational disease, 2 had no history of handling mail or having any other direct contact with these threat letters (11, 21). Of the remaining nine individuals, eight were thought to have been exposed through handling or processing mail (20) but may never have picked up or directly handled the actual threat letters. Thus, some individuals who contracted inhalational disease may have been exposed to aerosols that were generated from residual spore material deposited on contaminated surfaces. This conclusion was borne out by a study conducted on the scene of one contamination incident, which demonstrated that spores could be reaerosolized from surfaces during simulated office activities—e.g., paper handling, foot traffic, moving containers—after a period of no entry and no ventilation for several days (38). McCleery et al. (25) found that reaerosolization of spores is possible in postal facilities.In the mail-related instance of 2001, aerosol exposures occurred. Since spore-contaminated surfaces can become sources for aerosol generation, nonporous surfaces (walls, desks, lockers, etc.) were decontaminated to reduce risk while porous surfaces (draperies and sofas) were removed. To determine the efficacy of decontamination, contaminated buildings were first sampled for the presence of B. anthracis spores followed by treatment by a variety of techniques. Postdecontamination sampling was used to determine efficacy (37) and to assess the safety for reoccupancy.The Government Accountability Office (GAO) reported that additional methodological validation of sampling collection and analytical methods should be conducted to enhance the interpretation of negative sampling results because initial samples from two postal facilities were negative, but later samples were positive (17). The GAO (17) report defined validation as “… a formal and independently administered empirical process. For validation, the overall performance characteristics of a given method must be certified as meeting the specified requirements for intended use and as conforming with applicable standards.” Currently, there is no preexisting standard for a presumable safe level of surface contamination with B. anthracis spores that may be assessed through sampling and analysis.Development of independent standards for assessing the requirements for surface sampling methods requires an understanding of the rate at which spores leave surfaces to become entrained in aerosols, the potential for aerosol exposure by humans, and the infectivity of inhaled spores. Inhalation infectivity has been researched, but estimates of a lethal dose vary (14, 15). Bartrand et al. (5) conducted a risk analysis on the mortality of guinea pigs and rhesus monkeys exposed to B. anthracis spores and found a 50% lethal dose (LD₅₀; i.e., the dose at which 50% of subjects die) of about 100,000 spores inhaled for 1-μm particles. Limitations of relating exposure to inhalation infectivity include quantification of the ability of spores to move from stasis on a surface to entrainment as an aerosol, quantification of exposures to the resultant aerosol, uptake by humans, room size and ventilation characteristics, and exposure time. Despite these limitations, it is necessary to standardize the performance of surface sampling methods.Brown et al. evaluated wipe (6), swab (7), and vacuum (8) spore collection methods with B. atrophaeus. These studies have added significant information to the understanding of recovery efficiencies for these three sampling methods; however, sampling performance was not evaluated at very low spore surface loading concentrations. Sampling performance measures at very low surface loading of B. anthracis are needed to aid in the decision making for decontamination and other interventions (31, 38).The goal of this study was to evaluate the current CDC environmental surface sampling methods for B. anthracis (12) as slightly modified based on subsequent CDC research (19, 30). We estimated B. anthracis Sterne sampling limit of detection (LOD), recovery efficiency (RE), and measurement precision for three sampling methods (swab, wipe, and vacuum) and two surfaces (steel and carpet) by allowing spores to settle from an aerosol in a controlled environment. In addition, we compared sample analyses performed at three laboratories to determine the level of interlaboratory variability.     

Superdormant Spores of Bacillus Species Have Elevated Wet-Heat Resistance and Temperature Requirements for Heat Activation

Purified superdormant spores of Bacillus cereus, B. megaterium, and B. subtilis isolated after optimal heat activation of dormant spores and subsequent germination with inosine, d-glucose, or l-valine, respectively, germinate very poorly with the original germinants used to remove dormant spores from spore populations, thus allowing isolation of the superdormant spores, and even with alternate germinants. However, these superdormant spores exhibited significant germination with the original or alternate germinants if the spores were heat activated at temperatures 8 to 15°C higher than the optimal temperatures for the original dormant spores, although the levels of superdormant spore germination were not as great as those of dormant spores. Use of mixtures of original and alternate germinants lowered the heat activation temperature optima for both dormant and superdormant spores. The superdormant spores had higher wet-heat resistance and lower core water content than the original dormant spore populations, and the environment of dipicolinic acid in the core of superdormant spores as determined by Raman spectroscopy of individual spores differed from that in dormant spores. These results provide new information about the germination, heat activation optima, and wet-heat resistance of superdormant spores and the heterogeneity in these properties between individual members of dormant spore populations.Spores of Bacillus species are formed in sporulation and are metabolically dormant and extremely resistant to a variety of stress factors (31, 32). While spores can remain dormant for long periods, if given the proper stimulus, they can rapidly “return to life” in the process of spore germination followed by outgrowth (30). Since spores are generally present in significant amounts on many foodstuffs and growing cells of a number of Bacillus species are significant agents of food spoilage and food-borne disease (32), there is continued applied interest in spore resistance and germination. While dormant spores can be killed by a treatment such as wet heat, this requires high temperatures that are costly and detrimental to food quality. Consequently, there has long been interest in triggering spore germination in foodstuffs, since germinated spores have lost the extreme resistance of dormant spores and are relatively easy to kill. However, this strategy has been difficult to apply because of the significant heterogeneity in germination rates between individual spores in populations. One reflection of this heterogeneity is the extremely variable lag times following addition of germinants but prior to initiation of germination events; while these lag times can vary from 10 to 30 min for most spores in populations, some spores have lag times of many hours or even many days (2, 12, 13, 15, 25). The spores that are extremely slow to germinate have been termed superdormant spores, and populations of superdormant spores have recently been isolated from three Bacillus species, and their germination properties characterized (9, 10). These superdormant spores germinate extremely poorly with the original germinants used to remove dormant spores from spore populations, thus allowing superdormant spore isolation, and also poorly with a number of other germinants, in particular, germinants that target nutrient germinant receptors different than those activated to isolate the superdormant spores. However, the superdormant spores germinate reasonably well with mixtures of nutrient germinants that target multiple germinant receptors. All reasons for spore superdormancy are not known, but one contributing factor is the number of nutrient germinant receptors in the spore''s inner membrane that trigger spore germination by binding to nutrient germinants (9). The levels of these receptors are most likely in the tens of molecules per spore (24), and thus stochastic variation in receptor numbers might result in some spores with such low receptor numbers that these spores germinate very poorly (23). Indeed, 20- to 200-fold elevated levels of at least one nutrient germinant receptor greatly decreases yields of superdormant spores of Bacillus subtilis (9).Spores of Bacillus species generally exhibit a requirement for an activation step in order to exhibit maximum germination (17). Usually this activation is a sublethal heat treatment that for a spore population exhibits an optimum of 60 to 100°C depending on the species. Spores are also extremely resistant to wet heat, generally requiring temperatures of 80 to 110°C to achieve rapid spore killing, with the major factor influencing the wet-heat resistance of spores of mesophilic strains being the spore core''s water content, which can be as low as 30% of wet weight as water in a fully hydrated spore (8, 19, 27, 28, 31). Invariably, increases in core water content are associated with a decrease in spore wet-heat resistance (8, 19, 22, 25). While spore populations most often exhibit log-linear kinetics of wet-heat killing, the observation of tailing in such killing curves at high levels of killing is not uncommon, suggesting there is significant heterogeneity in the wet-heat resistances of individual spores in populations (27, 28). While there has been no comparable work suggesting that there is also heterogeneity in the temperature optima for heat activation of individual spores in populations, this certainly seems possible and indeed was suggested as one cause of spore superdormancy, as yields of superdormant spores from spore populations that are not heat activated are much higher (9, 10). Consequently, the current work was initiated to test the hypothesis that superdormant spores require heat activation temperatures that are higher than those of the original dormant spores. Once this was found to be the case, the wet-heat resistance and core water content of the superdormant and original dormant spores were compared, and the environment of the spore core''s major small molecule, pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) was assessed by Raman spectroscopy of individual spores.     

Contributions of Four Cortex Lytic Enzymes to Germination of Bacillus anthracis Spores

Bacterial spores remain dormant and highly resistant to environmental stress until they germinate. Completion of germination requires the degradation of spore cortex peptidoglycan by germination-specific lytic enzymes (GSLEs). Bacillus anthracis has four GSLEs: CwlJ1, CwlJ2, SleB, and SleL. In this study, the cooperative action of all four GSLEs in vivo was investigated by combining in-frame deletion mutations to generate all possible double, triple, and quadruple GSLE mutant strains. Analyses of mutant strains during spore germination and outgrowth combined observations of optical density loss, colony-producing ability, and quantitative identification of spore cortex fragments. The lytic transglycosylase SleB alone can facilitate enough digestion to allow full spore viability and generates a variety of small and large cortex fragments. CwlJ1 is also sufficient to allow completion of nutrient-triggered germination independently and is a major factor in Ca²⁺-dipicolinic acid (DPA)-triggered germination, but its enzymatic activity remains unidentified because its products are large and not readily released from the spore''s integuments. CwlJ2 contributes the least to overall cortex digestion but plays a subsidiary role in Ca²⁺-DPA-induced germination. SleL is an N-acetylglucosaminidase that plays the major role in hydrolyzing the large products of other GSLEs into small, rapidly released muropeptides. As the roles of these enzymes in cortex degradation become clearer, they will be targets for methods to stimulate premature germination of B. anthracis spores, greatly simplifying decontamination measures.The Gram-positive bacterium Bacillus anthracis is the etiologic agent of cutaneous, gastrointestinal, and inhalational anthrax (24). An anthrax infection begins when the host is infected with highly resistant, quiescent B. anthracis spores (1, 24). Within the host, the spore''s sensory mechanism recognizes chemical signals, known as germinants, and triggers germination, which leads to the resumption of metabolism (36). Spores that have differentiated into vegetative cells produce a protective capsule and deadly toxins. These virulence factors allow the bacteria to evade the host''s immune system and establish an infection resulting in septicemia, toxemia, and frequently death (24). Although vegetative cells produce virulence factors that are potentially fatal, these cells cannot initiate infections and are much more susceptible to antimicrobial treatments than spores (24). Therefore, efficient triggering of spore germination may enhance current decontamination methods.Spores are highly resistant to many environmental insults because the spore core (cytoplasm) is dehydrated, dormant, and surrounded by multiple protective layers, including a modified layer of peptidoglycan (PG) known as the cortex (36). The cortex functions to maintain dormancy and heat resistance by preventing core rehydration (9). It is composed of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) sugars (Fig. ​(Fig.1).1). Peptide side chains on the NAM residues are either involved in interstrand cross-linking, cleaved to single l-alanine side chains, or fully removed with accompanying formation of muramic-δ-lactam (2, 31, 38). After germination is initiated by either nutrient or nonnutrient germinants, the cortex is depolymerized, resulting in complete core rehydration, resumption of metabolic activity, and outgrowth (33, 36).Open in a separate windowFIG. 1.Spore PG structure and hydrolysis. The central structure shows a representative spore PG strand with alternating NAG and NAM or muramic-δ-lactam (MδL) residues and with tetrapeptide or l-Ala side chains on the NAM residues. Forked arrows originate at sites of hydrolysis by the indicated enzymes and point to muropeptide products. The indicated “aG” muropeptide names are as previously published (7, 11). SleB lytic transglycosylase activity produces muropeptides terminating in anhydro-NAM. Cleavage at adjacent NAM residues produces the tetrasaccharide aG7a or aG7b, while cleavage further apart can produce octasaccharides or larger fragments. These can be further cleaved by muramidase treatment, resulting in the production of tetrasaccharide N, which terminates in NAM. The N-acetylglucosaminidase activity of SleL produces tetrasaccharides terminating in NAG, which can be further cleaved by muramidase to trisaccharides terminating in NAM.Cortex hydrolysis is driven by autolysins called germination-specific cortex lytic enzymes (GSLEs) that recognize the cortex-specific muramic-δ-lactam residues (2, 4, 21, 32). GSLEs fall into two classes: spore cortex lytic enzymes (SCLEs), which are thought to depolymerize intact cortical PG, and cortical fragment lytic enzymes (CFLEs), which further degrade partially hydrolyzed cortex (21). Both SCLEs and CFLEs have been identified in a variety of spore-forming species, including B. anthracis (11, 18, 19), Bacillus cereus (4, 20, 26), Bacillus megaterium (8, 34), Bacillus subtilis (13, 16, 25), Bacillus thuringiensis (12), and Clostridium perfringens (5, 23). Of the four GSLEs identified in B. anthracis, CwlJ1, CwlJ2, and SleB are predicted to be SCLEs (11), whereas SleL is thought to be a CFLE (18).Recently, independent studies showed that CwlJ1 and the lytic transglycosylase SleB (Fig. ​(Fig.1)1) play partially redundant roles and that either is sufficient for spore germination and outgrowth (10, 11). However, these same studies report conflicting results concerning the role of CwlJ2 during germination. Heffron et al. found no effect of CwlJ2 on the biochemistry of cortex hydrolysis or on colony-forming efficiency of spores (11). Giebel et al. reported that loss of CwlJ2 caused a minor defect in germination kinetics and that in the absence of SleB and CwlJ1, further loss of CwlJ2 had a major effect on colony forming efficiency (10). SleL in Bacillus anthracis is proposed to be an N-acetylglucosaminidase (Fig. ​(Fig.1)1) whose role is to further degrade cortex fragments resulting from SCLE hydrolysis (18). SleL is not essential for the completion of germination but does promote the release of small muropeptides to the spore''s surrounding environment (18).This study reports the effects of multiple deletion mutations affecting GSLEs on spore germination efficiency and kinetics of cortex hydrolysis. The data confirm the dominant roles played by CwlJ1 and SleB in the initiation of cortex hydrolysis and the major role of SleL in release of small cortex fragments. A minor role of CwlJ2 in nutrient-triggered germination and the contributions of CwlJ1 and CwlJ2 to Ca²⁺-dipicolinic acid (DPA)-triggered germination were revealed.     

Penetration of the Blood-Brain Barrier by Bacillus anthracis Requires the pXO1-Encoded BslA Protein

Anthrax is a zoonotic disease caused by the gram-positive spore-forming bacterium Bacillus anthracis. Human infection occurs after the ingestion, inhalation, or cutaneous inoculation of B. anthracis spores. The subsequent progression of the disease is largely mediated by two native virulence plasmids, pXO1 and pXO2, and is characterized by septicemia, toxemia, and meningitis. In order to produce meningitis, blood-borne bacteria must interact with and breach the blood-brain barrier (BBB) that is composed of a specialized layer of brain microvascular endothelial cells (BMEC). We have recently shown that B. anthracis Sterne is capable of penetrating the BBB in vitro and in vivo, establishing the classic signs of meningitis; however, the molecular mechanisms underlying the central nervous system (CNS) tropism are not known. Here, we show that attachment to and invasion of human BMEC by B. anthracis Sterne is mediated by the pXO1 plasmid and an encoded envelope factor, BslA. The results of studies using complementation analysis, recombinant BslA protein, and heterologous expression demonstrate that BslA is both necessary and sufficient to promote adherence to brain endothelium. Furthermore, mice injected with the BslA-deficient strain exhibited a significant decrease in the frequency of brain infection compared to mice injected with the parental strain. In addition, BslA contributed to BBB breakdown by disrupting tight junction protein ZO-1. Our results identify the pXO1-encoded BslA adhesin as a critical mediator of CNS entry and offer new insights into the pathogenesis of anthrax meningitis.Bacillus anthracis, the etiologic agent of anthrax, is a gram-positive spore-forming bacterium that is commonly found in soil (29). The bacterium can infect animals and humans by ingestion, inhalation, or cutaneous inoculation of B. anthracis spores (8). Spores are taken up by resident macrophages that migrate to the lymph nodes (15). Here, the spores germinate into vegetative bacteria, multiply, and then disseminate throughout the host, causing septicemia and toxemia (8). Systemic disease can be complicated by the onset of a fulminant and rapidly fatal hemorrhagic meningitis and meningoencephalitis (27). Anthrax meningitis is associated with a high mortality rate despite intensive antibiotic therapy (24). Biopsy studies after an outbreak of inhalational anthrax and experimental studies of inhalational infection in rhesus monkeys demonstrated the presence of bacilli in the central nervous system (CNS) and pathologies consistent with suppurative and hemorrhagic meningitis in the majority of cases (1, 12). The intentional release of B. anthracis spores (19) during the 2001 bioterrorism event resulted in a case of meningitis (19), necessitating a need for a better understanding of the pathogenesis of anthrax meningitis and CNS infection.To cause meningitis, blood-borne bacteria must interact with and breach the blood-brain barrier (BBB). The majority of the BBB is anatomically represented by the cerebral microvascular endothelium; brain microvascular endothelial cells (BMEC) are joined by tight junctions and display a paucity of pinocytosis, thereby effectively limiting the passage of substances and maintaining the CNS microenvironment (4, 5). Despite its highly restrictive nature, certain bacterial pathogens are still able to penetrate the BBB and gain entry into the CNS. The presence of bacilli in the brains of patients (1, 24) and in experimental models of anthrax infection (42, 44) suggests that vegetative B. anthracis cells are able to cross the BBB to initiate meningeal inflammation and the classic pathology associated with meningitis.B. anthracis harbors two large virulence plasmids, pXO1 and pXO2 (8), which are required for full virulence, as strains lacking these plasmids are attenuated in animal models of infection (29). B. anthracis Sterne (pXO1⁺ pXO2⁻) has been utilized as a vaccine strain (41) but is still widely used in both in vitro and in vivo studies of anthrax infection since it causes lethal disease in mouse models of infection (46). Despite the crucial roles of pXO1 and pXO2 in anthrax disease pathogenesis, very few plasmid-encoded factors have been characterized. The best described are the antiphagocytic polyglutamyl capsule, encoded by biosynthetic enzymes on pXO2, and the anthrax toxin complex comprised of protective antigen, lethal factor (LF), and edema factor (EF), encoded by pXO1 (8, 29). Sequence analysis of the pXO1 plasmid revealed that the majority of plasmid-encoded factors, ∼70%, were of unknown function (31). More recently, in silico analysis identified novel pXO1-encoded proteins with immunogenic potential and relevance for pathogenesis. These included factors with putative adherent and invasive properties (2). Interestingly, two of the immunoreactive proteins were predicted surface layer (S-layer) proteins (2), one of which, B. anthracis S-layer protein A (BslA, pXO1-90), has recently been described and shown to mediate adherence of the vegetative form to host cells (20).Using in vitro and in vivo model systems, we have recently shown that B. anthracis Sterne adheres to and invades brain endothelium (44). This interaction was partially dependent on the pXO1-encoded anthrax toxins; however, the molecular mechanisms that contribute to B. anthracis penetration of the BBB are currently unknown. In this study, we investigate the role of pXO1 in B. anthracis Sterne''s interaction with brain endothelium and identify the encoded BslA adhesin as a critical mediator for BBB attachment and penetration during the pathogenesis of anthrax meningitis.