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.     

Identification of Bacillus anthracis by Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry and Artificial Neural Networks

This report demonstrates the applicability of a combination of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) and chemometrics for rapid and reliable identification of vegetative cells of the causative agent of anthrax, Bacillus anthracis. Bacillus cultures were prepared under standardized conditions and inactivated according to a recently developed MS-compatible inactivation protocol for highly pathogenic microorganisms. MALDI-TOF MS was then employed to collect spectra from the microbial samples and to build up a database of bacterial reference spectra. This database comprised mass peak profiles of 374 strains from Bacillus and related genera, among them 102 strains of B. anthracis and 121 strains of B. cereus. The information contained in the database was investigated by means of visual inspection of gel view representations, univariate t tests for biomarker identification, unsupervised hierarchical clustering, and artificial neural networks (ANNs). Analysis of gel views and independent t tests suggested B. anthracis- and B. cereus group-specific signals. For example, mass spectra of B. anthracis exhibited discriminating biomarkers at 4,606, 5,413, and 6,679 Da. A systematic search in proteomic databases allowed tentative assignment of some of the biomarkers to ribosomal protein or small acid-soluble proteins. Multivariate pattern analysis by unsupervised hierarchical cluster analysis further revealed a subproteome-based taxonomy of the genus Bacillus. Superior classification accuracy was achieved when supervised ANNs were employed. For the identification of B. anthracis, independent validation of optimized ANN models yielded a diagnostic sensitivity of 100% and a specificity of 100%.Members of the genus Bacillus are rod-shaped bacteria that exhibit catalase activity and can be characterized as endospore-forming obligate or facultative aerobes. The genus Bacillus contains two important groups of bacteria named after B. subtilis and B. cereus. The best-characterized member of the former group is B. subtilis, a renowned model organism for genetic research. Other group members, like B. pumilis, B. licheniformis, B. atrophaeus, and B. amyloliquefaciens, exhibit a high degree of phenotypic similarity and are thus not easily distinguishable (15).The B. cereus group comprises a number of closely related bacteria, some of which interfere with human health. Bacteria classified as B. cereus are occasionally associated with food poisoning (16, 28), while B. thuringiensis is primarily an insect pathogen because of its ability to produce toxins that have been widely used for the biocontrol of insect pests (28, 30). A third member of the B. cereus group, B. anthracis, is the causative agent of anthrax and is highly relevant to human and animal health. Other members of the B. cereus group are B. mycoides, B. pseudomycoides, and B. weihenstephanensis (4, 15).B. anthracis is a possible agent in biological warfare and bioterrorism. Its applicability as a biological warfare agent was made apparent by an accidental release from a Soviet military facility in Sverdlovsk (1, 10). Also, the well-publicized mailing of B. anthracis spores in the United States, which caused 18 confirmed cases of cutaneous and inhalational anthrax and an additional 4 suspected cases of cutaneous anthrax (3, 22), demonstrated that B. anthracis may become a threat from terrorist groups (10).Rapid detection of B. anthracis may be challenging because of its great genetic similarity to other species of the B. cereus group (10) and the difficulties of phenotypic differentiation of B. cereus group members (15). There is some controversy in the literature regarding the taxonomy of the B. cereus group. Indeed, some authors state that B. anthracis, B. cereus, and B. thuringiensis are one species with various virulence plasmids for the toxin pXO1 and the capsule pXO2 of B. anthracis and the insecticidal toxin of B. thuringiensis (10, 19). Other authors do not support this opinion and suggest the presence of even more species within the group (21).Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) intact-cell mass spectrometry (ICMS) has been suggested as a rapid, objective, and reliable technique for bacterial identification (8, 13, 23, 25, 38). As a proteomic technique, ICMS of whole bacterial cells, or cell lysates, relies on the reproducible detection of microbial protein patterns and thus delivers information complementary to genotypic or phenotypic test methods. With the pattern-matching approach, microbial identification is achieved by comparing experimental mass spectra with a collection of mass spectra of known organisms. This requires the compilation of large databases of bacterial reference spectra but has the advantage that an extensive knowledge of biomarker identities is not required. Another advantage of the pattern-matching approach is that genus- and species-specific procedures or consumables are not required, i.e., the same methodology can in principle be applied to all kinds of microorganisms (multiplex advantage).It is thus believed that ICMS offers the possibility to systematically investigate the diversity of bacterial subproteomes, complementing existing methodologies of bacterial characterization. This potential and the need for a rapid, objective, and reliable microbial identification technique that does not rely on nucleic acid detection and the availability of an MS-compatible inactivation protocol for highly pathogenic biosafety level 3 microorganisms and bacterial endospores (26) prompted us to systematically study the MALDI-TOF MS profiles of Bacillus strains and to establish a database of bacterial mass spectra. In the present work, we describe strategies of spectral analysis that allow the identification and validation of group- and species-specific sets of biomarkers. Using unsupervised hierarchical cluster analysis (UHCA) and supervised artificial neural network (ANN) analysis, we also demonstrate how microbial spectra can be employed to establish an MS-based methodology for rapid, objective, and reliable identification of the target species, B. anthracis.     

Direct-Imaging-Based Quantification of Bacillus cereus ATCC 14579 Population Heterogeneity at a Low Incubation Temperature

Bacillus cereus ATCC 14579 was cultured in microcolonies on Anopore strips near its minimum growth temperature to directly image and quantify its population heterogeneity at an abusive refrigeration temperature. Eleven percent of the microcolonies failed to grow during low-temperature incubation, and this cold-induced population heterogeneity could be partly attributed to the loss of membrane integrity of individual cells.Bacillus cereus is a food poisoning- and food spoilage-causing organism that can be found in a large variety of foods (4, 23). There are two illnesses associated with B. cereus, namely, emetic and diarrheal intoxication (17, 24). Most of the strains related to cases or outbreaks of B. cereus food-borne poisoning were shown to be unable to grow at 7°C (1, 12). The average temperatures of domestic refrigerators have been investigated in various surveys around the world and often ranged from 5°C to 7°C, but extreme values exceeded 10°C to 12°C (5, 16). Inadequate chilling was indeed reported in various incidents of B. cereus food-borne illness (7, 8, 18, 19), pointing to the importance of appropriate refrigeration of foods contaminated with B. cereus to control its growth and toxin production in foods (9).Several studies have demonstrated that microorganisms can show diversity in their population stress response, even in an apparently homogeneous stress environment (6, 11, 21, 22). However, only very limited data describing the heterogeneity in growth performance of individual cells from food-borne pathogens cultured at low temperatures are available (10). Because inadequate chilling of food is one of the factors that contribute to the number of incidents of B. cereus food-borne illness, there is a need for better understanding of its growth performance at lowered incubation temperatures. In this study, we used the direct-imaging-based Anopore technology (6, 13-15) to quantitatively describe the population heterogeneity of B. cereus ATCC 14579 cells at 12°C. The minimum temperature for the growth of B. cereus ATCC 14579 in brain heart infusion (BHI) broth is 7.5°C (personal communication from F. Carlin), but various food-borne-associated B. cereus isolates were shown to be unable to grow at 10°C (1). Therefore, in this study, a culturing temperature of 12°C was chosen, to mimic temperature abuse of refrigerated foods. In addition, the membrane integrity of individual cells was assessed using both membrane permeant and impermeant nucleic acid dyes in order to get more insight into cellular characteristics that may contribute to heterogeneity in growth response.     

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.     

Novel and Unique Diagnostic Biomarkers for Bacillus anthracis Infection

A search for bacterium-specific biomarkers in peripheral blood following infection with Bacillus anthracis was carried out with rabbits, using a battery of specific antibodies generated by DNA vaccination against 10 preselected highly immunogenic bacterial antigens which were identified previously by a genomic/proteomic/serologic screen of the B. anthracis secretome. Detection of infection biomarkers in the circulation of infected rabbits could be achieved only after removal of highly abundant serum proteins by chromatography using a random-ligand affinity column. Besides the toxin component protective antigen, the following three secreted proteins were detected in the circulation of infected animals: the chaperone and protease HtrA (BA3660), an NlpC/P60 endopeptidase (BA1952), and a protein of unknown function harboring two SH3 (Src homology 3) domains (BA0796). The three proteins could be detected in plasma samples from infected animals exhibiting 10³ to 10⁵ CFU/ml blood and also in standard blood cultures at 3 to 6 h post-bacterial inoculation at a bacteremic level as low as 10³ CFU/ml. Furthermore, the three biomarkers appear to be present only in the secretome of B. anthracis, not in those of the related pathogens B. thuringiensis and B. cereus. To the best of our knowledge, this is the first report of direct detection of B. anthracis-specific proteins, other than the toxin components, in the circulation of infected animals.The gram-positive spore-forming bacterium Bacillus anthracis is the causative agent of anthrax, a rare fatal disease which is initiated, in its most severe form, by inhalation of spores. Due to the severity of the disease, the ease of respiratory infection, and the extreme resistance of the spores to unfavorable environmental conditions, B. anthracis is considered a potential biological warfare agent (for a review, see references 8, 10, 35, 56, and 62), and in recent years, the need for novel reliable diagnostic approaches, improved vaccination strategies, novel therapeutic targets, and a better understanding of the pathogenesis of anthrax has been widely acknowledged.Inhaled B. anthracis spores are taken up by alveolar macrophages and germinate into vegetative bacilli which eventually invade the bloodstream, where they multiply massively and secrete toxins and virulence factors. Anthrax is toxinogenic in the sense that the bacterial binary exotoxin is necessary for the onset of the disease (54), yet other factors may be required for the colonization and expansion of bacteria in the host (15, 18, 31, 32, 37, 46, 65, 66, 70, 83). The toxin is composed of the following three proteins: protective antigen (PA), which mediates binding to the receptor on target cells and internalization of the toxin components (14, 74); lethal factor, a zinc protease targeting several mitogen-activated protein kinases (52); and edema factor (EF), a calmodulin-dependent adenylate cyclase (55, 57). The genes encoding the three exotoxin components are located on the native virulence plasmid pXO1. Genes encoding proteins with functions involved in the synthesis of the second major B. anthracis virulence determinant, an immunologically inert polyglutamyl capsule that protects bacteria from phagocytosis, are located on a second native virulence plasmid, pXO2 (56).In humans, the initial symptoms of inhalation anthrax are nonspecific and reminiscent of influenza-like upper respiratory tract infections. The second stage is characterized by high fever, respiratory failure, dyspnea, and shock. Unless patients are treated promptly, death occurs within 24 h of the onset of the second stage, preceded by massive bacteremia (27, 34, 73, 76). The mandatory treatment for anthrax is based on administration of antibiotics (17, 76), yet study of the disease in animal models has clearly established that the time of antibiotic administration postinfection is crucial for the effectiveness of the treatment, strongly supporting the importance of rapid diagnosis (2, 47, 48). At present, due to the severity of the disease and its rapid progression, treatment is administered to each person with confirmed contact with contaminated areas (76).Early accurate diagnosis of anthrax is complicated by the rarity of the disease and the nonspecific nature of the symptoms. Microbiologic identification of anthrax is based on the nonhemolytic nature of the typically white-gray colonies and the rod morphology of the gram-positive nonmotile bacilli detected in clinical samples or blood cultures (16, 19, 30, 73, 78). Immunofluorescence and immunohistochemistry targeted to bacterial proteins are not routinely conducted. Later in the course of the disease, seroconversion in response to the various components of the toxin may serve only as a retrospective confirmation of initial exposure. With the advent of genetic methodologies, B. anthracis in cultures inoculated with clinical and forensic samples can be detected specifically and accurately by PCR, usually designed to amplify genes located on the native virulence plasmids (30). Recently, the use of PA as a disease biomarker was suggested, owing to the presence of this protein in detectable amounts in the circulation of infected animals (53, 71). EF and lethal factor can be detected in the circulation only at later stages of infection (30).In recent years, the search for novel biomarkers of disease, including bacterial infections, has exploited the approach of global biological inspection based on functional genomic or proteomic studies (64, 85). We previously documented such global surveys, combined with a serological study of B. anthracis (5, 6, 20, 21, 22, 38, 39), for identification of vaccine and diagnostic marker candidates among extracellular (secreted or membranal) proteins. These studies indeed revealed a list of proteins that can serve as potential biomarkers, based on their immunogenicity (which probes their in vivo expression), abundance under various culture conditions, and functional relatedness to infection. In the present study, the search was extended by directly addressing the presence of bacterial secreted proteins in the circulation of B. anthracis-infected rabbits, using specific antibodies generated by DNA vaccination against the previously selected immunogenic proteins. Visualization of bacterial proteins in the circulation of infected animals was achieved only following depletion of highly abundant serum proteins by an affinity chromatography protocol. The search enabled the successful detection, in addition to PA, of three secreted proteins uniquely expressed by B. anthracis, i.e., HtrA (BA3660), the BA1952 endopeptidase, and a protein of unknown function (BA0796). All of these proteins are potential virulence-related factors. This is the first communication of the presence of B. anthracis secreted proteins other than the bacterial toxin in the circulation of infected animals, and their identification strongly supports the validity of the reductional screening approach for selection of disease biomarkers.     

Biofilm Formation and Cell Surface Properties among Pathogenic and Nonpathogenic Strains of the Bacillus cereus Group

Biofilm formation by 102 Bacillus cereus and B. thuringiensis strains was determined. Strains isolated from soil or involved in digestive tract infections were efficient biofilm formers, whereas strains isolated from other diseases were poor biofilm formers. Cell surface hydrophobicity, the presence of an S layer, and adhesion to epithelial cells were also examined.The Bacillus cereus group includes B. cereus sensu stricto, B. anthracis, and B. thuringiensis, three genetically close pathogenic species. Based on genetic evidence, it has been suggested that they could represent one species (7). B. cereus sensu stricto is itself an opportunistic human pathogen occasionally found to cause various diseases such as endophthalmitis or periodontitis but is more frequently involved in gastrointestinal diseases with diarrheal or emetic syndromes (4, 12). Emetic syndromes result from the presence of cereulide, a heat-stable toxin produced in food before ingestion, whereas diarrheal syndromes require survival of the bacterium in the host digestive tract. B. thuringiensis is an insect pathogen, and B. anthracis causes anthrax, a lethal human disease.The persistent contamination of industrial food processing systems by B. cereus (12) may facilitate its involvement in gastroenteritis. This persistence is due to spores, which may survive pasteurization, heating, and gamma-ray irradiation (9, 13), and to biofilms, which have been shown to be highly resistant to cleaning procedures (18). Biofilms are also suspected to be involved in bacterial pathogenicity, as they may form on host epithelia (15).In this study, we wanted to test whether biofilm formation by species of the B. cereus group could be connected to the pathogenicity of the bacterium. For this purpose, we screened a collection of 102 pathogenic (diarrheal, emetic, and oral diseases) and nonpathogenic strains of B. cereus and B. thuringiensis for their capability to form biofilms. As adhesion to inert or living surfaces is a prerequisite for biofilm formation, we have investigated relationships within our collection of strains between biofilm formation and cell surface hydrophobicity, the presence of an S-layer, or adhesion to epithelial cells.     

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.     

Regulatory Interactions of a Virulence-Associated Serine/Threonine Phosphatase-Kinase Pair in Bacillus anthracis

In the current study, we examined the regulatory interactions of a serine/threonine phosphatase (BA-Stp1), serine/threonine kinase (BA-Stk1) pair in Bacillus anthracis. B. anthracis STPK101, a null mutant lacking BA-Stp1 and BA-Stk1, was impaired in its ability to survive within macrophages, and this correlated with an observed reduction in virulence in a mouse model of pulmonary anthrax. Biochemical analyses confirmed that BA-Stp1 is a PP2C phosphatase and dephosphorylates phosphoserine and phosphothreonine residues. Treatment of BA-Stk1 with BA-Stp1 altered BA-Stk1 kinase activity, indicating that the enzymatic function of BA-Stk1 can be influenced by BA-Stp1 dephosphorylation. Using a combination of mass spectrometry and mutagenesis approaches, three phosphorylated residues, T165, S173, and S214, in BA-Stk1 were identified as putative regulatory targets of BA-Stp1. Further analysis found that T165 and S173 were necessary for optimal substrate phosphorylation, while S214 was necessary for complete ATP hydrolysis, autophosphorylation, and substrate phosphorylation. These findings provide insight into a previously undescribed Stp/Stk pair in B. anthracis.A profile of the intracellular signaling proteins that regulate transition of Bacillus anthracis from dormancy to expression of virulence factors is emerging. Like many prokaryotes, B. anthracis utilizes two-component histidine kinase systems to regulate physiological changes and the expression of virulence factors. These systems include the Spo0 histidine kinase-based phosphorelay pathway (32, 37) and the Bacillus respiratory response A and B system involved in regulating toxin expression (36). Unlike for histidine kinase systems, little is known about reversible serine/threonine phosphorylation events in B. anthracis. These systems are common to eukaryotic cells (3, 14, 25, 40) but were only recently found in prokaryotes to modulate a variety of metabolic and physiological processes (1, 2, 7, 11, 12, 15, 17, 24, 28, 35, 38). Whether reversible serine/threonine phosphorylation contributes to similar events in B. anthracis is not known.The current paradigm for prokaryotic serine/threonine kinases (STK) is based in part on the structure of PknB, a serine/threonine kinase from Mycobacterium tuberculosis that is structurally related to eukaryotic Hanks-type kinases (39). PknB autophosphorylates and is dephosphorylated by an M. tuberculosis phosphatase, PstP, in order to alter kinase activity (4). Similar to the findings for PnkB, Madec et al. identified critical autophosphorylated residues and autophosphorylated domains of PrkC, an STK from Bacillus subtilis (22), which suggested that the phosphorylation state of these residues impacts the activation of PrkC (22). These studies suggested that prokaryotic STKs exhibited activities similar to those of their eukaryotic homologs and were regulated by cognate phosphatases. Hence, studies of serine/threonine phosphatase (STP)/STK pairs may help define a core regulatory module in bacterial physiology and virulence, wherein the kinase autophosphorylates following interaction with stimuli and is subsequently downregulated by a cognate phosphatase when stimulus levels decline.Analysis of the B. anthracis genome indicates that this organism has a single phosphatase-kinase pair encoded within a putative operon. This operon, between nucleotides 3588319 and 3678099 in the genome of B. anthracis Sterne, contains eight candidate open reading frames (ORFs). Six of the potential ORFs encode proteins involved in translation and DNA metabolism, while the phosphatase-encoding ORF (stp1) and the kinase-encoding ORF (stk1) are paired at the 3′ end of this operon. Examination of the genome sequences of several other Gram-positive bacteria indicates that this putative operon and the general orientation of stp1 and stk1 are conserved among members of the Firmicutes group of bacteria. B. anthracis Stp1 (BA-Stp1) and BA-Stk1 homologs influence a variety of bacterial processes. For example, homologs of BA-Stp1 and BA-Stk1 regulate growth in Bacillus subtilis (12), cell viability and segregation in Streptococcus agalactiae (28), competence in Streptococcus pneumoniae (26), and virulence in both Streptococcus pyogenes (17) and Staphylococcus aureus (9). Although kinases homologous to BA-Stk1 influence several bacterial processes in different species, the tandem association of this kinase with a phosphatase does not vary. This observation led us to hypothesize that the phosphatase (BA-Stp1) influences Ba-Stk1 activity by dephosphorylation.In the current study, we analyzed the importance of BA-Stp1 and BA-Stk1 in the virulence of B. anthracis and assessed the biochemical interactions between these two proteins. Results from these studies indicate that this phosphatase-kinase pair contributes to the virulence of B. anthracis, as mutants lacking BA-Stp1 and BA-Stk1 exhibit decreased lethality in a mouse model of pulmonary anthrax. Furthermore, a series of biochemical analyses reveal an interaction between BA-Stk1 and BA-Stp1 where BA-Stk1 autophosphorylates in order to enhance kinase activity and is dephosphorylated by BA-Stp1 as a putative step in downregulating kinase activity as the levels of stimuli subside. Moreover, we have identified candidate serine and threonine residues that appear to modulate kinase activity. These findings provide insight into a previously undescribed serine/threonine phosphatase-kinase system in B. anthracis.     

The dlt Operon of Bacillus cereus Is Required for Resistance to Cationic Antimicrobial Peptides and for Virulence in Insects

The dlt operon encodes proteins that alanylate teichoic acids, the major components of cell walls of gram-positive bacteria. This generates a net positive charge on bacterial cell walls, repulsing positively charged molecules and conferring resistance to animal and human cationic antimicrobial peptides (AMPs) in gram-positive pathogenic bacteria. AMPs damage the bacterial membrane and are the most effective components of the humoral immune response against bacteria. We investigated the role of the dlt operon in insect virulence by inactivating this operon in Bacillus cereus, which is both an opportunistic human pathogen and an insect pathogen. The Δdlt_Bc mutant displayed several morphological alterations but grew at a rate similar to that for the wild-type strain. This mutant was less resistant to protamine and several bacterial cationic AMPs, such as nisin, polymyxin B, and colistin, in vitro. It was also less resistant to molecules from the insect humoral immune system, lysozyme, and cationic AMP cecropin B from Spodoptera frugiperda. Δdlt_Bc was as pathogenic as the wild-type strain in oral infections of Galleria mellonella but much less virulent when injected into the hemocoels of G. mellonella and Spodoptera littoralis. We detected the dlt operon in three gram-negative genera: Erwinia (Erwinia carotovora), Bordetella (Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), and Photorhabdus (the entomopathogenic bacterium Photorhabdus luminescens TT01, the dlt operon of which did not restore cationic AMP resistance in Δdlt_Bc). We suggest that the dlt operon protects B. cereus against insect humoral immune mediators, including hemolymph cationic AMPs, and may be critical for the establishment of lethal septicemia in insects and in nosocomial infections in humans.Gram-positive bacteria are generally enclosed by cell walls consisting of macromolecular assemblies of cross-linked peptidoglycan (murein), polyanionic teichoic acids (TAs), and surface proteins (69). TAs are polymers of repeating glycerophosphate residues. They may be covalently anchored to either peptidoglycan (wall-associated TAs) or the cytoplasmic membrane via glycolipids (lipoteichoic acids [LTAs]). TAs may be involved in controlling cell shape, autolytic enzyme activity, and cation homeostasis (69). They make a significant contribution to the overall negative charge of the bacterial cell wall, attracting negatively charged compounds, including the cationic antimicrobial peptides (AMPs) of the innate humoral immune systems of higher organisms (69).Many of the gram-positive bacterial species pathogenic to humans display resistance to cationic AMPs because of a decrease in the net negative charge of bacterial cell envelopes (75). Modifications to the TAs at the bacterial surface involving the incorporation of positively charged residues, such as d-alanine, prevent cationic AMPs from reaching their target, thereby protecting the organism against these compounds. This process involves the Dlt proteins encoded by the dltABCD operon present in most of the genome sequences established to date for gram-positive bacteria (44, 58, 74). d-Alanine is incorporated into LTAs through a two-step reaction involving a d-alanine-d-alanyl carrier protein ligase (Dcl) and a d-alanyl carrier protein (Dcp), encoded by the dltA and dltC genes, respectively (18, 44, 45, 70). The dltB and dltD genes encode other proteins required for the d-alanylation of LTAs. DltD is involved in selection of the Dcp carrier protein for ligation with d-alanine (19), whereas DltB is thought to be involved in d-alanyl-Dcp secretion (69). d-Alanine may be transferred from d-alanylated LTAs to wall-associated TAs by transacylation. For many human gram-positive bacterial pathogens, dlt operon inactivation has been shown to affect bacterial resistance to cationic AMPs and virulence. Indeed, Listeria monocytogenes, Bacillus anthracis, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Lactobacillus reuteri, and group B streptococci harboring mutations in dlt genes all have a higher negative charge on the cell surface and are more susceptible to cationic AMPs of various origins (1, 34, 56, 58, 59, 77, 78, 89). The inactivation of dlt genes in these pathogenic bacterial species also decreases interactions with phagocytic and nonphagocytic cells (1, 13, 34, 78).The impact of Dlt proteins on cationic AMP resistance and virulence in insect bacterial pathogens has never before been studied, despite the major role of cationic AMPs in insect humoral immunity (9, 61). Insect bacterial pathogens also termed entomopathogenic bacteria are able to multiply in the insect hemocoel from small inocula (<10,000 viable cells) and produce fatal septicemia (8, 57). Entomopathogenic bacteria entering the hemolymph are targeted by an array of immune system mediators of both cellular and humoral reactions. The cellular response results in bacterial phagocytosis or encapsulation by circulating hemocytes, whereas the humoral response generates cationic AMPs (61). These peptides are small, inducible molecules produced in large amounts in hemolymph by the fat body (9, 26). They participate to the insect antimicrobial defense in a systemic response. Many AMP have been reported to cause damage in microbial membranes, which may ultimately lead to bacterial cell lysis (94).We investigated the role of the dlt operon in cationic AMP resistance and virulence in Bacillus cereus, a human opportunistic and insect facultative bacterial pathogen. B. cereus sensu stricto is a spore-forming gram-positive bacterium. The B. cereus sensu lato group of bacteria also includes the closely related insect pathogen Bacillus thuringiensis and the human pathogen B. anthracis. Genomic data have shown that B. thuringiensis and B. cereus have almost identical chromosomal genetic backgrounds (54, 55) but that B. thuringiensis carries a plasmid encoding entomopathogenic cytoplasmic crystalline δ-endotoxins (Cry proteins) specifically active against insect larvae upon ingestion (22, 23, 83). B. cereus can cause opportunistic food-borne gastroenteritis and local/systemic infections in immunocompromised humans (85). Both B. thuringiensis (with and without Cry toxins) and B. cereus strains are highly pathogenic when injected directly into the hemocoels of insect larvae, in which they cause lethal septicemia (46, 82, 86, 96). The occurrence, structure, and glycosylation of LTAs were studied for different Bacillus species, including B. cereus strains containing LTAs (built up of polyglycerol phosphate chains and hydrophobic anchors) and d-alanine (11, 50, 51, 62). Therefore, the presence of a dlt operon in the B. cereus 14579 genome suggests that the LTAs may be alanylated.We report here that the dlt operon of B. cereus is required for resistance to cationic AMPs of bacterial or insect origin. The dlt operon is required for full B. cereus virulence following bacterial injection into two lepidopteran insects, the caterpillar Spodoptera littoralis and the wax moth Galleria mellonella. We also detected the dlt operon in three gram-negative bacterial genera: Erwinia (Erwinia carotovora), Bordetella (Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), and Photorhabdus (the entomopathogenic bacterium Photorhabdus luminescens TT01).     

Identification and Validation of Specific Markers of Bacillus anthracis Spores by Proteomics and Genomics Approaches

Bacillus anthracis is the causative bacteria of anthrax, an acute and often fatal disease in humans. The infectious agent, the spore, represents a real bioterrorism threat and its specific identification is crucial. However, because of the high genomic relatedness within the Bacillus cereus group, it is still a real challenge to identify B. anthracis spores confidently. Mass spectrometry-based tools represent a powerful approach to the efficient discovery and identification of such protein markers. Here we undertook comparative proteomics analyses of Bacillus anthracis, cereus and thuringiensis spores to identify proteoforms unique to B. anthracis. The marker discovery pipeline developed combined peptide- and protein-centric approaches using liquid chromatography coupled to tandem mass spectrometry experiments using a high resolution/high mass accuracy LTQ-Orbitrap instrument. By combining these data with those from complementary bioinformatics approaches, we were able to highlight a dozen novel proteins consistently observed across all the investigated B. anthracis spores while being absent in B. cereus/thuringiensis spores. To further demonstrate the relevance of these markers and their strict specificity to B. anthracis, the number of strains studied was extended to 55, by including closely related strains such as B. thuringiensis 9727, and above all the B. cereus biovar anthracis CI, CA strains that possess pXO1- and pXO2-like plasmids. Under these conditions, the combination of proteomics and genomics approaches confirms the pertinence of 11 markers. Genes encoding these 11 markers are located on the chromosome, which provides additional targets complementary to the commonly used plasmid-encoded markers. Last but not least, we also report the development of a targeted liquid chromatography coupled to tandem mass spectrometry method involving the selection reaction monitoring mode for the monitoring of the 4 most suitable protein markers. Within a proof-of-concept study, we demonstrate the value of this approach for the further high throughput and specific detection of B. anthracis spores within complex samples.Bacillus anthracis is a highly virulent bacterium, which is the etiologic agent of anthrax, an acute and often lethal disease of animals and humans (1). According to the Centers for Disease Control and Prevention, B. anthracis is classified as a category A agent, the highest rank of potential bioterrorism agents (http://www.bt.cdc.gov/agent/agentlist-category.asp). The infectious agent of anthrax, the spore, was used as a bioterrorism weapon in 2001 in the United States when mailed letters containing B. anthracis spores caused 22 cases of inhalational and/or cutaneous anthrax, five of which were lethal (2). These events have emphasized the need for rapid and accurate detection of B. anthracis spores.Bacillus anthracis is a member of the genus Bacillus, Gram-positive, rod-shaped bacteria characterized by the ability to form endospores under aerobic or facultative anaerobic conditions (3). The genus Bacillus is a widely heterogeneous group encompassing 268 validly described species to date (http://www.bacterio.net/b/bacillus.html, last accessed on August 9^th 2013). B. anthracis is part of the B. cereus group which consists of six distinct species: B. anthracis, B. cereus, B. thuringiensis, B. mycoides, B. pseudomycoides, and B. weihenstephanensis (4, 5). The latter three species are generally regarded as nonpathogenic whereas B. cereus and B. thuringiensis could be opportunistic or pathogenic to mammals or insects (5, 6). B. cereus is a ubiquitous species that lives in soil but is also found in foods of plant and animal origin, such as dairy products (7). Its occurrence has also been linked to food poisoning and it can cause diarrhea and vomiting (6, 8). B. thuringiensis is primarily an insect pathogen, also present in soil, and often used as a biopesticide (9).B. anthracis is highly monomorphic, that is, shows little genetic variation (10), and primarily exists in the environment as a highly stable, dormant spore in the soil (1). Specific identification of B. anthracis is challenging because of its high genetic similarity (sequence similarity >99%) with B. cereus and B. thuringiensis (5, 11). The fact that these closely related species are rather omnipresent in the environment further complicates identification of B. anthracis. The main difference among these three species is the presence in B. anthracis of the two virulence plasmids pXO1 and pXO2 (1), which are responsible for its pathogenicity. pXO1 encodes a tripartite toxin (protective antigen (PA), lethal factor (LF), and edema factor (EF)) which causes edema and death (1), whereas pXO2 encodes a poly-γ-d-glutamate capsule which protects the organism from phagocytosis (1). B. anthracis identification often relies on the detection of the genes encoded by these two plasmids via nucleic acid-based assays (12–14). Nevertheless, the occasionally observed loss of the pXO2 plasmid within environmental species may impair the robustness of detection (1). In addition, in recent years a series of findings has shown that the presence of pXO1 and pXO2 is not a unique feature of B. anthracis. Indeed, Hu et al. have demonstrated that ∼7% of B. cereus/B. thuringiensis species can have a pXO1-like plasmid and ∼1.5% a pXO2-like plasmid (15). This was particularly underlined for some virulent B. cereus strains (i.e. B. cereus strains G9241, B. cereus biovar anthracis strains CA and CI) (16–20).Because of these potential drawbacks, the use of chromosome-encoded genes would be preferable for the specific detection of B. anthracis. Such genes (rpoB, gyrA, gyrB, plcR, BA5345, and BA813) have been reported as potential markers (21–25), but concerns have also been raised about their ability to discriminate B. anthracis efficiently from closely related B. cereus strains (26). Ahmod et al. have recently pointed out, by in silico database analysis, that a specific sequence deletion (indel) occurs in the yeaC gene and exploited it for the specific identification of B. anthracis (27). Nevertheless, a few B. anthracis strains (e.g. B. anthracis A1055) do not have this specific deletion and so may lead to false-negative results (27).In the last few years, protein profiling by MS, essentially based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF MS), has emerged as an alternative (or a complement) to genotypic or phenotypic methods for the fast and efficient identification of microorganisms (28, 29). Such an approach is based on the reproducible acquisition of global bacterial protein fingerprints/patterns. The combination of MS-based protein patterns and chemometric/bioinformatic tools has been demonstrated to efficiently differentiate members of the B. cereus group from other Bacillus species (30). However, the specific discrimination of B. anthracis from the closely related B. cereus and B. thuringiensis remains difficult (30). This study of Lasch and coworkers, performed on vegetative cells, identified a few ribosomal and spore proteins as being responsible for this clustering (30). Closer inspection of the data revealed that B. anthracis identification was essentially based on one particular isoform of the small acid-soluble spore protein B (SASP-B)¹ (30–34), which is exclusively expressed in spores, as the samples were shown to contain residual spores. However, the specificity of SASP-B has recently been questioned as the published genomes of B. cereus biovar anthracis CI and B. thuringiensis BGSC 4CC1 strains have been shown to share the same SASP-B isoform as B. anthracis (35). Altogether these results underline that the quest for specific markers of B. anthracis needs to be pursued.Mass spectrometry also represents a powerful tool for the discovery and identification of protein markers (36, 37). In the case of B. anthracis, this approach has more commonly been used for the comprehensive characterization of given bacterial proteomes. For example, the proteome of vegetative cells with variable plasmid contents has been extensively studied (38–40), as the proteomes of mature spores (41, 42) and of germinating spores (43, 44). Only one recent study, based on a proteo-genomic approach, was initiated to identify protein markers of B. anthracis (45). In this work, potential markers were characterized but using a very limited number of B. cereus group strains (three B. cereus and two B. thuringiensis). Moreover, this study was done on vegetative cells, whereas the spore proteome is drastically different. To our knowledge, no study has characterized and validated relevant protein markers specific to B. anthracis spores, which constitute the dissemination form of B. anthracis and are often targeted by first-line immunodetection methods (46).Here we report comparative proteomics analyses of Bacillus anthracis/cereus/thuringiensis spores, undertaken to identify proteoforms unique to B. anthracis. Preliminary identification was performed on a limited set of Bacillus species both at the peptide (after enzymatic digestion) and protein levels by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a high resolution/high mass accuracy LTQ-Orbitrap instrument. The pertinence of 11 markers was further demonstrated using proteomics and genomics approaches on a representative larger set of up to 55 different strains, including the closely related B. cereus biovar anthracis CI, CA, and B. thuringiensis 9727. Lastly, as a proof-of-concept study, we also report for four B. anthracis markers the implementation of a targeted LC-MS/MS method using selected reaction monitoring (SRM), based on the extension of a previous one focused on SASP-B (35). Preliminary results regarding method usefulness for the high throughput and accurate detection of B. anthracis spores in complex samples were also obtained and will be reported herein.     

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.     

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.     

Identification of the UDP-N-Acetylglucosamine 4-Epimerase Involved in Exosporium Protein Glycosylation in Bacillus anthracis

Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by a loosely fitting exosporium composed of a basal layer and an external hair-like nap. The filaments of the nap are formed by trimers of the collagen-like glycoprotein BclA. The side chains of BclA include multiple copies of two linear rhamnose-containing oligosaccharides, a trisaccharide and a pentasaccharide. The pentasaccharide terminates with the unusual deoxyamino sugar anthrose. Both oligosaccharide side chains are linked to the BclA protein backbone through an N-acetylgalactosamine (GalNAc) residue. To identify the gene encoding the epimerase required to produce GalNAc for BclA oligosaccharide biosynthesis, three annotated UDP-glucose 4-epimerase genes of B. anthracis were cloned and expressed in Escherichia coli. The candidate proteins were purified, and their enzymatic activities were assessed. Only two proteins, encoded by the BAS5114 and BAS5304 genes (B. anthracis Sterne designations), exhibited epimerase activity. Both proteins were able to convert UDP-glucose (Glc) to UDP-Gal, but only the BAS5304-encoded protein could convert UDP-GlcNAc to UDP-GalNAc, indicating that BAS5304 was the gene sought. Surprisingly, spores produced by a mutant strain lacking the BAS5304-encoded enzyme still contained normal levels of BclA-attached oligosaccharides. However, monosaccharide analysis of the oligosaccharides revealed that GlcNAc had replaced GalNAc. Thus, while GalNAc appears to be the preferred amino sugar for the linkage of oligosaccharides to the BclA protein backbone, in its absence, GlcNAc can serve as a substitute linker. Finally, we demonstrated that the expression of the BAS5304 gene occurred in a biphasic manner during both the early and late stages of sporulation.Bacillus anthracis, the causative agent of anthrax, is a gram-positive, rod-shaped soil bacterium that forms spores when deprived of essential nutrients (15). Spore formation begins with an asymmetric septation that divides the developing cell into a smaller forespore compartment and a larger mother cell compartment, each containing a copy of the genome. 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). Mother cell lysis releases the mature spore, which is dormant and capable of surviving in harsh environments for many years (16). When spores encounter an aqueous environment containing nutrients, they can germinate and grow as vegetative cells (23).Recently, interest in B. anthracis spores has intensified in response to their use as agents of bioterrorism. Of particular interest has been the outermost exosporium layer, which serves as a semipermeable barrier excluding potentially harmful macromolecules (9, 26) and as a vital first point of contact with the immune system of an infected host (14, 18, 32). The exosporium of B. anthracis and closely related species such as Bacillus cereus and Bacillus thuringiensis is a prominent structure comprised of a paracrystalline basal layer and an external hair-like nap (2). The basal layer contains approximately 20 different proteins (22, 25), while the filaments of the nap are formed by trimers of a single collagen-like glycoprotein called BclA (4, 27). The central region of BclA contains a large number of GXX repeats, mostly GTP triplets, and this region varies in length in naturally occurring strains of B. anthracis, resulting in hair-like naps of differing lengths (24, 28). Multiple copies of two O-linked oligosaccharides, a trisaccharide and a pentasaccharide, are attached to the protein component of BclA. The pentasaccharide side chains appear to be attached to threonine residues within the central region, while the trisaccharide side chains are attached to presently undefined residues in the protein (7).The precise structure of the trisaccharide side chain has not been determined, but its sequence is 3-O-methyl-l-rhamnose-l-rhamnose-N-acetylgalactosamine (GalNAc) (7). Except for a single glycosidic linkage, the structure of the pentasaccharide is known. Its structure is 2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-β-d-glucopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→2)-l-rhamnopyranosyl-(1→?)-N-acetylgalactosamine (7). Both oligosaccharides are attached to the BclA protein backbone through GalNAc residues. The pentasaccharide sugar 2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-d-glucose, which was given the trivial name anthrose, has been found only in B. anthracis strains and a limited number of highly pathogenic strains of B. cereus and B. thuringiensis (7, 8). For that reason, anthrose has joined other exosporium components as targets for the detection of B. anthracis spores and as new targets for therapeutic intervention in anthrax (6, 26, 29).In view of the potential importance of the BclA oligosaccharides, especially the anthrose-containing pentasaccharide, we have undertaken a comprehensive study of their biosynthesis. This effort involves identifying the biosynthetic genes for the three component sugars, anthrose, rhamnose, and GalNAc, as well as the genes involved in assembling the oligosaccharides and attaching them to the protein backbone of BclA. We recently reported the identification of a four-gene anthrose biosynthetic operon (8). A four-gene rhamnose biosynthetic operon has also been identified (24). This paper describes the identification of the gene encoding the UDP-N-acetylglucosamine (GlcNAc) 4-epimerase necessary for GalNAc biosynthesis. It also describes a surprising alternative BclA oligosaccharide biosynthetic pathway, which is active only in the absence of the UDP-GlcNAc 4-epimerase. Finally, this paper reports a biphasic pattern of expression of the gene encoding this epimerase during sporulation.