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Bacillus anthracis surface: Capsule and S-layer
Abstract
Two abundant surface proteins, EA1 and Sap, are components of the Bacillus anthracis surface layer (S-layer). Their corresponding genes have been cloned, shown to be clustered on the chromosome and sequenced. EA1 and Sap each possess three 'S-layer homology' motifs. Single and double disrupted mutants were constructed. EA1 and Sap were co-localized at the cell surface of both the non-capsulated and capsulated bacilli. When present, the capsule is exterior to, and completely covers, the S-layer proteins, which form an array beneath it. Nevertheless, the presence of these proteins is not required for normal capsulation of the bacilli. Thus both structures are compatible, and yet neither is required for the correct formation of the other. Bacillus anthracis has, therefore, a very complex cell wall organization for a gram-positive bacterium.
... The N-terminal domain (no more than 200 residues) of an S-layer protein usually harbors three SLH motifs, each containing a conserved sequence of 10–15 residues. The sequence was first revealed in the S-protein of Acetogenium kivui [28] and later was found in Clostridium thermocellum [29] , Thermus thermophi- lus [30], Bacillus anthracis (a causal agent of anthrax) [31], and numerous other Gram-positive bacteria [27]. Interestingly, the SLH motif has homologs in the Cterminal region of some exoenzymes associated with cells of Gram-positive bacteria. ...
... Membranes based on S-layers are more promising for studying the S-layer characteristics, e.g., the pore size as dependent on the ionic strength or pH in solution [57] or the effects of chemical modification on the hydrophilic or hydrophobic properties of S-layers [61]. S-LAYERS AS VACCINES Although the S-layer is contained in the envelope of numerous human and animal pathogens, including anthrax-causing B. anthracis [31] , its role as a virulence factor was demonstrated only for Gram-negative bacteria of the genus Campylobacter [62] . These bacteria cause acute intestinal disorders, which account for about 15% of the total intestinal disorder incidence in humans. ...
Many bacteria and archaea have a crystalline surface layer (S-layer), which overlies the cell envelope. S-layers each consist of one protein or glycoprotein species. Protein subunits of the S-layer noncovalently interact with each other and with the underlying cell-envelope component. On average, the S-layer lattice has pores of 2–6 nm and is 5–10 nm high. Isolated S-layer proteins recrystallize to form two-dimensional crystalline structures in solution, on a solid support, and on planar lipid membranes. Owing to this unique property, S-layers have a broad range of applications. This review focuses on the structural features and applications of S-layers and their proteins, with special emphasis on their use in nanobiotechnology.
... After germination, the vegetative cells express a range of putative and known adhesins that may promote attachment to host cells including S-layer proteins (EA1, Sap and BslA), teichoic acid, collagen-and fibronectin-binding proteins and the poly-γ-D-glutamic acid capsule. 74,76,77 Inhibition of attachment of vegetative cells of a pXO1 pXO2 double-cured strain to the A549 cell line using oligosaccharides was greatest in the case of GalNacβ1-3Gal, Galβ1-4GlcNAc, GalNAcβ1-4Gal and dextran sulphate (56-71%). 29 Adhesion was not mediated by the BslA S-layer protein or capsule, both of which are encoded on the absent pXO1. ...
... 29 Adhesion was not mediated by the BslA S-layer protein or capsule, both of which are encoded on the absent pXO1. 74,76 F. tularensis clinically presents as a range of manifestations, however the most serious is pneumonic infection. Recently, some cell-surface associated adhesins have been identified. ...
The specter of intentional release of pathogenic microbes and their toxins is a real threat. This article reviews the literature on adhesins of biothreat agents, their interactions with oligosaccharides and the potential for anti-adhesion compounds as an alternative to conventional therapeutics. The minimal binding structure of ricin has been well characterised and offers the best candidate for successful anti-adhesion therapy based on the Galβ1-4GlcNAc structure. The botulinum toxin serotypes A-F bind to a low number of gangliosides (GT1b, GQ1b, GD1a and GD1b) hence it should be possible to determine the minimal structure for binding. The minimal disaccharide sequence of GalNAcβ1-4Gal found in the gangliosides asialo-GM1 and asialo-GM2 is required for adhesion for many respiratory pathogens. Although a number of adhesins have been identified in bacterial biothreat agents such as Yersinia pestis, Bacillus anthracis, Francisella tularensis, Brucella species and Burkholderia pseudomallei, specific information regarding their in vivo expression during pneumonic infection is lacking. Limited oligosaccharide inhibition studies indicate the potential of GalNAcβ1-4Gal, GalNAcβ-3Gal and the hydrophobic compound, para-nitrophenol as starting points for the rational design of generic anti-adhesion compounds. A cocktail of multivalent oligosaccharides based on the minimal binding structures of identified adhesins would offer the best candidates for anti-adhesion therapy.
... There is very limited information concerning B. anthracis in vivo-expressed pathogenicity-related genes other than the genes coding for the bacterial toxins LT and ET and for the biosynthesis of the antiphagocytic capsule. The S-layer proteins have been found to be expressed during infection, based on the massive presence of anti-S-layer antibodies in infected animals (24). In addition, in vivo expression of several individual genes or operons has been suggested on the basis of the observation that disruption of these genes influences the virulence of the bacteria; in a murine model of infection in which the attenuated Sterne strain was used, it was shown that the LD 50 and/or the mean time to death were altered by disruption of three phospholipases C encoded by the plcB, smcA, and plcA loci (37), a cell-wall D-alanine esterification system encoded by the dltABCD operon (22), the siderophore anthrachelin encoded by the asbA operon (13), and possibly two of the four B. anthracis superoxide dismutases encoded by the sod-15 and sodA1 loci (63). ...
... This represents 50% of the 22 SLH proteins encoded in the genome of B. anthracis. Sap and EA1, the proteins constituting the S-layer, have been found to be immunogenic (24), yet in vivo expression of the other proteins containing SLH domains, which are not part of the S-layer, has not been described. We documented previously that SLH domains are a prevalent membrane docking modality in B. anthracis (15), and many SLH proteins, other than Sap and EA1, reside both on the bacterial surface and in the secretome (16). ...
In a previous comparative proteomic study of Bacillus anthracis examining the influence of the virulence plasmids and of various growth conditions on the composition of the bacterial secretome, we identified 64 abundantly expressed proteins (T. Chitlaru, O. Gat, Y. Gozlan, N. Ariel, and A. Shafferman, J. Bacteriol. 188:3551-3571, 2006). Using a battery of sera from B. anthracis-infected animals, in the present study we demonstrated that 49 of these proteins are immunogenic. Thirty-eight B. anthracis immunogens are documented in this study for the first time. The relative immunogenicities of the 49 secreted proteins appear to span a >10,000-fold range. The proteins eliciting the highest humoral response in the course of infection include, in addition to the well-established immunogens protective antigen (PA), Sap, and EA1, GroEL (BA0267), AhpC (BA0345), MntA (BA3189), HtrA (BA3660), 2,3-cyclic nucleotide diesterase (BA4346), collagen adhesin (BAS5205), an alanine amidase (BA0898), and an endopeptidase (BA1952), as well as three proteins having unknown functions (BA0796, BA0799, and BA0307). Of these 14 highly potent secreted immunogens, 11 are known to be associated with virulence and pathogenicity in B. anthracis or in other bacterial pathogens. Combining the results reported here with the results of a similar study of the membranal proteome of B. anthracis (T. Chitlaru, N. Ariel, A. Zvi, M. Lion, B. Velan, A. Shafferman, and E. Elhanany, Proteomics 4:677-691, 2004) and the results obtained in a functional genomic search for immunogens (O. Gat, H. Grosfeld, N. Ariel, I. Inbar, G. Zaide, Y. Broder, A. Zvi, T. Chitlaru, Z. Altboum, D. Stein, S. Cohen, and A. Shafferman, Infect. Immun. 74:3987-4001, 2006), we generated a list of 84 in vivo-expressed immunogens for future evaluation for vaccine development, diagnostics, and/or therapeutic intervention. In a preliminary study, the efficacies of eight immunogens following DNA immunization of guinea pigs were compared to the efficacy of a PA DNA vaccine. All eight immunogens induced specific high antibody titers comparable to the titers elicited by PA; however, unlike PA, none of them provided protection against a lethal challenge (50 50% lethal doses) of virulent B. anthracis strain Vollum spores.
... Au cours de son cycle infectieux, B. anthracis exprime différents systèmes d'acquisition du fer afin de chélater le fer présent chez l'hôte. De part l'architecture particulière de sa paroi qui comprend une couche S (43,99,197,198), cette bactérie est considérée comme un modèle pour l'étude des mécanismes d'acquisition du fer chez les bactéries appartenant au groupe B. cereus. ...
Au cours des interactions hôte-pathogène, la lutte pour le fer peut conditionner l'évolution de l'infection. Afin de s'adapter à la faible quantité de fer libre présente chez l'hôte, les bactéries ont développé diverses stratégies leur permettant d'acquérir cet élément vital. Chez Bacillus cereus, une bactérie pathogène opportuniste de l'homme, la protéine IlsA (Iron-regulated leucine rich surface protein), qui contient entre autres des domaines NEAT et LRR, a été identifiée. Il s'agit d'une protéine de surface impliquée dans l'acquisition du fer des hémoprotéines et de la ferritine. Dans un premier temps, nous avons caractérisé la fonction d'IlsA et de son domaine NEAT ainsi que leur implication dans la liaison à l'hème et l'hémoglobine. Nos résultats indiquent qu'IlsA capte l'hème via son domaine NEAT avec une stœchiométrie 1:1 et que ce domaine est capable d'extraire l'hème de l'hémoglobine. Par ailleurs, l'interaction entre IlsA et IsdC, une des protéines du système Isd (Iron surface determinant) décrit chez d'autres bactéries à Gram-positif, a été analysée. Suite à un contact direct entre ces deux protéines, nous avons démontré que l'hème était transféré d'holo-IlsA vers apo-IsdC. Dans un second temps, nous avons étudié les mécanismes permettant l'assimilation du fer à partir de la ferritine. Nous avons ainsi montré qu'IlsA pouvait interagir avec plusieurs types de ferritine avec une stœchiométrie de 24 molécules d'IlsA pour une ferritine. Ceci suggère qu'IlsA se lie à chaque sous-unité de la ferritine. De plus, en présence de sidérophores, cette interaction conduit à une augmentation de la libération du fer contenu dans la ferritine. Finalement, une souche de B. cereus qui ne produit plus de bacillibactine, un des deux sidérophores synthétisés par cette bactérie, n'est plus capable d'utiliser la ferritine comme source de fer pour sa croissance. De plus, sa virulence chez l'insecte est atténuée. Ces données indiquent qu'IlsA et la bacillibactine sont toutes les deux impliquées dans l'acquisition du fer de la ferritine. Ces travaux mettent donc en évidence l'interaction qui existe entre IlsA et les protéines de surface d'une part et les sidérophores d'autre part. Ils apportent également de nouveaux éléments permettant de mieux comprendre les mécanismes d'adaptation mis en œuvre chez l'hôte par B. cereus.Mots clés : Fer, Bacillus cereus, IlsA, NEAT, IsdC, hème, hémoglobine, ferritine, sidérophore.
... These findings on EA1 based vaccines advocate that immunization with EA1 rather than AVA may further increase the chance of successful antibiotic treatment. Further, as EA1 is a major S-layer component of both vegetative cells and spore preparations of B. anthracis [27,34], the authors hypothesize that immunity to EA1 may protect against both spore and bacteria, even though the same is not addressed through present investigation. Thus, studies of these DNA constructs against lethal spore challenge are warranted in the future. ...
Anthrax, by Bacillus anthracis, remains a dreadful fatal hazard worldwide. The presently used anthrax vaccines are plagued by numerous issues that limit their widespread use. As an immunization approach targeting both extracellular antigens and toxins of B. anthracis may achieve better sterile immunity, the present investigation designed a bicistronic secretory anti-anthrax DNA vaccine targeting immune response against toxin and cells. The efficacy of the vaccine was compared with monocistronic DNA vaccines and the currently used anthrax vaccine. For this, mice were immunized with the developed vaccine containing pag (encoding protective antigen to block toxin) and eag genes (encoding EA1 to target cells) of B. anthracis through DNA-prime/Protein-boost (D/P) and DNA prime/DNA-boost (D/D) approaches. There was a >2 and > 5 fold increase in specific antibody level by D/D and D/P approaches respectively, on 42nd days post-immunization (dpi). Serum cytokine profiling showed that both Th1 and Th2 immune responses were elicited, with more Th2 responses in D/P strategy. More importantly, challenge with 100 times LD50 of B. anthracis at 42nd dpi exhibited maximum cumulative survival (83.33 %) by bicistronic D/P approach. Remarkably, immunization with EA1 delayed mortality onset in infection. The study forms the first report on complement-dependent bactericidal activity of antiEA1 antibodies. In short, co-immunization of PA and EA1 through the developed bicistronic DNA vaccine would be an effective immunization approach in anthrax vaccination. Further, D/P strategy could enhance vaccine-induced immunity against B. anthracis. Altogether, the study generates certain critical insights having direct applications in next-generation vaccine development against anthrax.
... Elevated (5% or greater) quantities of CO 2 and added bicarbonate or serum help in increasing capsule production. Capsule is present outside of S-layer, and this layer is not essential for attachment of capsule to cell surface (Fouet et al. 1999). In absence of capsule, the virulence of B. anthracis isolates is reduced, and such isolates are often used as vaccine candidates to elicit immunity in livestock and humans in some countries. ...
Probiotics are defined as live microorganisms that confer health benefits on the host when consumed in adequate amounts. They maintain proper microbial balance in gastrointestinal tract by strain-specific health-promoting activities like their antimicrobial, immunomodulation, anti-inflammatory, and anticarcinogenic attributes. A number of reports on anticarcinogenic effects exerted by probiotics against various types of cancer are available in literature. The anticarcinogenic properties of probiotics have been reported in various cancers and are attributable to a number of mechanisms at molecular level. The mechanisms underlying anti-cancerous effect include enhanced immune response in host, probiotic binding of potential carcinogens and their degradation, changes in the intestinal microflora, secretion of antitumorigenic compounds by probiotics in the colon leading to metabolic changes of colonic microflora, alteration in physicochemical environment of the colon, etc. The studies have been reviewed as to the in vivo and in vitro mechanisms underlying the antiproliferative effects exerted by probiotic lactic acid bacteria (LABs). However, for the therapy to reach the clinic, research in larger datasets needs to be carried out taking into consideration strain specificity of the effective probiotic or derivatives and their effect on the microbiome of the patients along with envisaged side effects. Further to establish the precise mechanisms by which LAB and its various entities inhibit cancer, carefully designed epidemiological studies are required. It purports to be a field which can yield novel drug entities with few or no side effects.
... The fully virulent form of B. anthracis is described by the presence of two extra-chromosomal plasmids -pXO1 and pXO2 [7]. The genes encoding the tripartite anthrax toxin componentsprotective antigen (PA), lethal factor (LF) and edema factor (EF), are present on the pXO1 plasmid [9], whereas the genes encoding the biosynthetic operon of the poly-gamma-D-glutamate capsule is located on the pXO2 plasmid [10,11]. The toxin component PA is responsible for binding to the mammalian cell surface and delivering the other two components, either LF (a zinc metalloprotease) or EF (an adenylate cyclase) into the host cell [12]. ...
Small RNAs (sRNAs) are important modulators of gene expression in bacteria. Regulation by sRNAs is yet to be established in Bacillus anthracis. Here, regulation and RNA binding properties of Hfq-like RNA chaperones in B. anthracis are investigated.
Transcript levels were measured by RT-PCR. Proteins were cloned, purified and their ability to bind sRNA was seen by EMSA. Regulators of Hfq1 were identified by in silico analysis and validated by EMSA. Conserved sRNAs were identified by homology search and their ability to bind Hfq1 was seen by EMSA. Residues crucial for sRNA binding were identified by mutational studies.
hfq1 and hfq3 showed expression during exponential phase on BHI medium, while hfq2 showed no expression. Hfq1 and Hfq3 formed hexamer and sRNA-Hfq complex, not Hfq2. In silico prediction and EMSA confirmed AbrB binding to the promoter of Hfq1. Homology search identified 7 sRNAs in B. anthracis. The sRNA CsfG showed binding to Hfq1 via residues Y24, F29, Q30, R32, K56 and H57.
Hfq1 and Hfq3 can function as RNA chaperones in B. anthracis. The transition phase repressor AbrB might be responsible for growth dependent expression of Hfq1. The sporulation specific sRNA CsfG binds to Hfq1 via its distal surface and requires an intact hexameric structure for forming CsfG-Hfq1 complex.
This is the first report demonstrating the regulation and functional properties of Hfq-like RNA chaperones in B. anthracis, and paves the path towards establishing sRNA based regulation in B. anthracis.
Copyright © 2015 Elsevier B.V. All rights reserved.
... No cytosolic proteins were identified, which is consistent with the recognized role of NEAT proteins in nutrient acquisition from the extracellular environment. Some proteins also possessed S-layer homology (SLH) domains that enable non-covalent anchoring of a protein to an S-layer, a crystalline structure that surrounds the capsule of various bacteria during certain growth conditions [49,50,51]. Once such example is the previously characterized B. anthracis BslK protein, which possesses a single heme-binding NEAT domain, and three SLH-domains ( Figure 2, Table S1; [26]). ...
Iron is essential for bacterial survival, being required for numerous biological processes. NEAr-iron Transporter (NEAT) domains have been studied in pathogenic Gram-positive bacteria to understand how their proteins obtain heme as an iron source during infection. While a 2002 study initially discovered and annotated the NEAT domain encoded by the genomes of several Gram-positive bacteria, there remains a scarcity of information regarding the conservation and distribution of NEAT domains throughout the bacterial kingdom, and whether these domains are restricted to pathogenic bacteria. This study aims to expand upon initial bioinformatics analysis of predicted NEAT domains, by exploring their evolution and conserved function. This information was used to identify new candidate domains in both pathogenic and nonpathogenic organisms. We also searched metagenomic datasets, specifically sequence from the Human Microbiome Project. Here, we report a comprehensive phylogenetic analysis of 343 NEAT domains, encoded by Gram-positive bacteria, mostly within the phylum Firmicutes, with the exception of Eggerthella sp. (Actinobacteria) and an unclassified Mollicutes bacterium (Tenericutes). No new NEAT sequences were identified in the HMP dataset. We detected specific groups of NEAT domains based on phylogeny of protein sequences, including a cluster of novel clostridial NEAT domains. We also identified environmental and soil organisms that encode putative NEAT proteins. Biochemical analysis of heme binding by a NEAT domain from a protein encoded by the soil-dwelling organism Paenibacillus polymyxa demonstrated that the domain is homologous in function to NEAT domains encoded by pathogenic bacteria. Together, this study provides the first global bioinformatics analysis and phylogenetic evidence that NEAT domains have a strong conservation of function, despite group-specific differences at the amino acid level. These findings will provide information useful for future projects concerning the structure and function of NEAT domains, particularly in pathogens where they have yet to be studied.
... It is an effective pathogen because the infectious agent of anthrax is the metabolically dormant and highly resistant spore. Upon ingestion by a professional phagocytic cell (e.g., activated macrophage), the spore undergoes germination and outgrowth to generate a vegetative cell that is capable of reproduction within the infected host, as it produces plasmid-encoded toxins and protective capsule material for evading immune capture and destruction (Fouet et al. 1999; Koehler 2009; Moayeri and Leppla 2009; Tournier et al. 2009). Germination and outgrowth in the macrophage takes place within a hostile environment, made so by the phagocyte's oxidative burst, which generates a toxic combination of reactive oxygen species (ROS), nitric oxide (NO), and hypochlorous acid (HOCl), as well as phospholipase, and antimicrobial peptides (Piris-Gimenez et al. 2005; Dawson and Liu 2008; Welkos et al. 2011). ...
Spx of Bacillus subtilis is a redox-sensitive protein, which, under disulfide stress, interacts with RNA polymerase to activate genes required for maintaining thiol homeostasis. Spx orthologs are highly conserved among low %GC Gram-positive bacteria, and often exist in multiple paralogous forms. In this study, we used B. anthracis Sterne, which harbors two paralogous spx genes, spxA1 and spxA2, to examine the phenotypes of spx null mutations and to identify the genes regulated by each Spx paralog. Cells devoid of spxA1 were sensitive to diamide and hydrogen peroxide, while the spxA1 spoxA2 double mutant was hypersensitive to the thiol-specific oxidant, diamide. Bacillus anthracis Sterne strains expressing spxA1DD or spxA2DD alleles encoding protease-resistant products were used in microarray and quantitative real-time polymerase chain reaction (RT-qPCR) analyses in order to uncover genes under SpxA1, SpxA2, or SpxA1/SpxA2 control. Comparison of transcriptomes identified many genes that were upregulated when either SpxA1DD or SpxA2DD was produced, but several genes were uncovered whose transcript levels increased in only one of the two SpxADD-expression strains, suggesting that each Spx paralog governs a unique regulon. Among genes that were upregulated were those encoding orthologs of proteins that are specifically involved in maintaining intracellular thiol homeostasis or alleviating oxidative stress. Some of these genes have important roles in B. anthracis pathogenesis, and a large number of upregulated hypothetical genes have no homology outside of the B. cereus/thuringiensis group. Microarray and RT-qPCR analyses also unveiled a regulatory link that exists between the two spx paralogous genes. The data indicate that spxA1 and spxA2 are transcriptional regulators involved in relieving disulfide stress but also control a set of genes whose products function in other cellular processes.
... Bacterial surface (S) layers are proteinaceous cell envelope structures ubiquitously found in Gram-positive and Gramnegative bacterial species and in Archaea (Sára and Sleytr 2000). When present, they form the outermost layer of the cell, being occasionally covered only by capsules (Fouet et al. 1999). S-layers are composed of numerous identical (glyco)protein subunits, 40–200 kDa in molecular weight, which form a two-dimensional, regular and highly porous array with oblique (p1, p2), square (p4) or hexagonal (p3, p6) symmetry. ...
Bacterial surface (S) layers are the outermost proteinaceous cell envelope structures found on members of nearly all taxonomic groups of bacteria and Archaea. They are composed of numerous identical subunits forming a symmetric, porous, lattice-like layer that completely covers the cell surface. The subunits are held together and attached to cell wall carbohydrates by non-covalent interactions, and they spontaneously reassemble in vitro by an entropy-driven process. Due to the low amino acid sequence similarity among S-layer proteins in general, verification of the presence of an S-layer on the bacterial cell surface usually requires electron microscopy. In lactobacilli, S-layer proteins have been detected on many but not all species. Lactobacillus S-layer proteins differ from those of other bacteria in their smaller size and high predicted pI. The positive charge in Lactobacillus S-layer proteins is concentrated in the more conserved cell wall binding domain, which can be either N- or C-terminal depending on the species. The more variable domain is responsible for the self-assembly of the monomers to a periodic structure. The biological functions of Lactobacillus S-layer proteins are poorly understood, but in some species S-layer proteins mediate bacterial adherence to host cells or extracellular matrix proteins or have protective or enzymatic functions. Lactobacillus S-layer proteins show potential for use as antigen carriers in live oral vaccine design because of their adhesive and immunomodulatory properties and the general non-pathogenicity of the species.
... Furthermore, the data may indicate that the Sap and EA1 proteins are either synthesized and rapidly degraded, or that HtrA has a direct or indirect role in promoting Sap and EA1 translation from existing RNA messages. A complete disappearance of the S-layer was previously observed only when both of the adjacent sap and eag genes were disrupted (Mesnage et al., 1997;Fouet et al., 1999;Fouet and Mesnage, 2002) or in strains in which genes encoding for functions governing the pyruvylation of cell wall polysaccharide were mutated, resulting in the inability of the Sap or EA1 monomers to anchor and multimerize at the bacterial cell wall (Mesnage et al., 2000;. ...
We demonstrate that disruption of the htrA (high temperature requirement A) gene in either the virulent Bacillus anthracis Vollum (pXO1(+) , pXO2(+) ), or in the ΔVollum (pXO1(-), pXO2(-), nontoxinogenic and noncapsular) strains, affect significantly the ability of the resulting mutants to withstand heat, oxidative, ethanol and osmotic stress. The ΔhtrA mutants manifest altered secretion of several proteins, as well as complete silencing of the abundant extracellular starvation-associated neutral protease A (NprA). VollumΔhtrA bacteria exhibit delayed proliferation in a macrophage infection assay, and despite their ability to synthesize the major B. anthracis toxins LT (lethal toxin) and ET (oedema toxin) as well as the capsule, show a decrease of over six orders of magnitude in virulence (lethal dose 50% = 3 × 10(8) spores, in the guinea pig model of anthrax), as compared with the parental wild-type strain. This unprecedented extent of loss of virulence in B. anthracis, as a consequence of deletion of a single gene, as well as all other phenotypic defects associated with htrA mutation, are restored in their corresponding trans-complemented strains. It is suggested that the loss of virulence is due to increased susceptibility of the ΔhtrA bacteria to stress insults encountered in the host. On a practical note, it is demonstrated that the attenuated Vollum ΔhtrA is highly efficacious in protecting guinea pigs against a lethal anthrax challenge.
... Surface (S) layers are cell envelope structures ubiquitously found in Gram-positive and Gram-negative bacterial species as well as in Archaea . They form the outermost proteinaceous layer on the cell and are sometimes covered only by capsules (Fouet et al., 1999). S-layers are composed of numerous identical (glyco)protein subunits, 40-200 kDa in molecular weight, which completely cover the cell surface, forming a crystalline, two-dimensional, regular and porous array with oblique (p1, p2), square (p4) or hexagonal (p3, p6) symmetry. ...
Bacterial surface (S) layers are proteinaceous arrays found on the surface of hundreds of bacterial species, including several species of lactobacilli. They are composed of numerous identical, non-covalently bound subunits, which completely cover the cell surface forming a symmetric, porous, lattice-like structure. Several functions for S-layers have been found, but no common one probably exists. S-layer proteins have a wide application potential in nanobiotechnology as well as in health-related applications such as vaccine design. In this work, the structure and function of the S-layer protein SlpA of Lactobacillus brevis ATCC 8287 and the expression of the slpA gene were studied. SlpA was identified as a two-domain protein, in which the N-terminal domain is responsible for binding to the cell wall and the C-terminal domain for forming the regular polymer. The domain organization is thus reversed compared with other hitherto characterized Lactobacillus S-layer proteins. Conserved carbohydrate binding motifs were identified in the N-terminal, positively charged amino acid sequences of SlpA and five other Lactobacillus brevis S-layer proteins. The component in the cell wall interacting with SlpA was shown to be something other than teichoic or lipoteichoic acid, in contrast to the cell wall receptors of S-layer proteins previously characterized in lactobacilli. The structure of the C-terminal self-assembly domain was studied in more detail using cysteine scanning mutagenesis and targeted chemical modification. Importantly for the potential future applications of SlpA as a display vehicle of foreign peptides, four amino acid segments with high surface accessibility in the assembled form of SlpA were detected. The 46 mutated residues could be grouped according to their location in the lattice: in the protein interior, on the inner surface of the lattice, on the outer surface of the lattice and on the subunit interface or the pore region of the lattice. L. brevis ATCC 8287 very efficiently adheres to cultured human epithelial cells representing the human gut, bladder and blood vessels, while the removal of the S-layer abolishes the binding. This binding was shown to be mediated by SlpA by using flagellum display. Hybrid flagella carrying fragments from the N-terminal part of SlpA bound to epithelial cells and to fibronectin, while flagella carrying the C-terminal part were unable to bind. The smallest fragment conferring binding to Int 407 cells comprised amino acids 66-215 in mature SlpA. The gene encoding SlpA is preceded by two promoters. By separating them on reporter plasmids, both of the promoters were shown to be used in L. brevis in all growth phases. More upstream region was needed for the full activity of the upstream promoter than for the downstream promoter. The promoter activities seen at the reporter enzyme level were also seen at the mRNA level, suggesting transcriptional rather than translational regulation of slpA. Three potential regulatory motifs were identified in the upstream region of slpA. Both promoters retained their activities under selected conditions mimicking the intestinal environment in vitro. Laktobasilleja esiintyy paitsi hapatetuissa elintarvikkeissa, myös laajalti luonnossa ja ihmisten ja eläinten luontaisten mikrobistojen osina, ja eräillä niistä on terveyttä edistäviä eli probioottisia ominaisuuksia. Monien laktobasillienkin pinnalla on ns. pintakerrosproteiinien muodostama kuori. Se muodostuu lukuisista samanlaisista, säännöllisesti järjestäytyneistä proteiinimolekyyleistä, jotka muodostavat levymäisen, huokoisen, bakteerisolun pinnan kokonaan peittävän rakenteen. Pintakerrosproteiinit on helppo irrottaa bakteerien pinnalta, minkä jälkeen niillä on taipumus muodostaa itsestään uudestaan säännöllisiä rakenteita. Pintakerrosproteiinien luontaisia tehtäviä ei useinkaan tunneta. Niillä on kuitenkin lukuisia mahdollisia sovellutuksia nanobiotekniikassa, molekyylibiologisessa tutkimuksessa ja rokotekehittelyssä. Tässä työssä selvitettiin molekyyligeneettisin menetelmin Lactobacillus brevis ATCC 8287 bakteerin pintakerrosproteiinimolekyylin (SlpA) rakennetta. Molekyylissä osoitettiin olevan kaksi selkeästi erotettavaa aluetta, joista toinen tarvitaan kiinnittymiseen alla olevaan bakteerisolun soluseinään ja toinen säännöllisen pintakerrosrakenteen muodostumiseen. Nämä alueet sijaitsivat L. brevis bakteerin pintakerrosproteiinissa eri järjestyksessä toisiinsa nähden kuin laktobasillien aiemmin kuvatuissa pintakerrosproteiineissa. Myös se bakteerin soluseinän hiilihydraattirakenne, johon SlpA kiinnittyy, osoitettiin erilaiseksi. SlpA-proteiinimolekyylistä kartoitettiin myös sen yksittäisten aminohappojen sijaintia kohdennettujen mutaatioiden ja kemiallisen muokkauksen avulla: 46 aminohapolle osoitettiin sijainti joko molekyylin sisällä, pintakerroksen ulkopinnalla, pintakerroksen sisäpinnalla tai pintakerrosmolekyylien kosketuspinnassa tai huokosen pinnassa. Työssä osoitettiin lisäksi, että SlpA välittää L. brevis ATCC 8287 bakteerien kiinnittymistä viljeltyihin ihmisen epiteelisoluihin sekä fibronektiiniin, joka on solujen välisessä aineessa esiintyvä proteiini. Tämän osoittamiseen käytettiin flagella-display-menetelmää. Kiinnittymisominaisuus sijaitsi proteiinissa samalla alueella kuin soluseinään sitoutumiskyky. SlpA-proteiinin geeniä edeltää kaksi promoottorialuetta. Aiemmista tiedoista poiketen niiden molempien osoitettiin tehokkaasti ohjaavan slpA-geenin ilmenemistä laboratoriokasvatuksen aikana, ja geenin ilmenemisen mahdollisen säätelyn osoitettiin perustuvan geeniluennan säätelyyn. Tätä tukevia tuloksia saatiin myös slpA-geeniä edeltävän DNA:n sekvenssin analysoinnista. Suolisto-olosuhteita matkivat laboratoriokasvatusolosuhteet eivät merkittävästi vähentäneet slpA-geenin promoottorialueiden hyvin tehokasta toimintaa. Työn havainnoilla on suuri merkitys esim. kehitettäessä SlpA-proteiinista rokotekuljettajaa käytettäväksi suun kautta annettavissa, eläviin L. brevis-soluihin perustuvissa rokotteissa, tai SlpA-proteiinin mahdollisissa nanobioteknologisissa sovelluksissa.
... The heme-bound hemophores form an active complex with IsdC that facilitates heme transfer to the cell envelope. Alternatively, B. anthracis may utilize BslK, which is noncovalently bound to the external surface of the cell wall via its SLH domains (22,51), for heme transfer in a rapid manner to IsdC via protein-protein interactions. Holo-IsdC next passes the iron-porphyrin to membrane transporters for delivery into the cell cytoplasm. ...
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.
... In nature, B. anthracis is known to have a complex cell wall architecture that includes a thick peptidoglycan, a polysaccharide associated with the PGN, S-layer proteins, and a poly-Dglutamic acid-containing capsule (9,10). Since the capsule forms the outermost layer of the cell wall, it is possible that the PGN may not be accessible to the immune cells of the host. ...
During advanced stages of inhalation anthrax, Bacillus anthracis accumulates at high levels in the bloodstream of the infected host. This bacteremia leads to sepsis during late-stage anthrax;
however, the mechanisms through which B. anthracis-derived factors contribute to the pathology of infected hosts are poorly defined. Peptidoglycan, a major component of the
cell wall of Gram-positive bacteria, can provoke symptoms of sepsis in animal models. We have previously shown that peptidoglycan
of B. anthracis can induce the production of proinflammatory cytokines by cells in human blood. Here, we show that biologically active peptidoglycan
is shed from an active culture of encapsulated B. anthracis strain Ames in blood. Peptidoglycan is able to bind to surfaces of responding cells, and internalization of peptidoglycan
is required for the production of inflammatory cytokines. We also show that the peptidoglycan traffics to lysosomes, and lysosomal
function is required for cytokine production. We conclude that peptidoglycan of B. anthracis is initially bound by an unknown extracellular receptor, is phagocytosed, and traffics to lysosomes, where it is degraded
to a product recognized by an intracellular receptor. Binding of the peptidoglycan product to the intracellular receptor causes
a proinflammatory response. These findings provide new insight into the mechanism by which B. anthracis triggers sepsis during a critical stage of anthrax disease.
... Bacillus anthracis, the causal agent of anthrax, is a Gram-positive, spore-forming bacterium with an S-layer below a poly -D-glutamic acid capsule (Mesnage et al., 1998). Two simultaneously synthesized S-layer proteins, named EA1 (extracellular antigen 1) and Sap (surface array protein) have been characterized (Fouet et al., 1997). In the amino-terminal region, each protein has three motifs of about 50 residues following the signal peptide. ...
Many surface proteins of Gram-positive bacteria contain motifs, about 50 amino acids long, called S-layer homology (SLH) motifs. Bacillus anthracis, the causal agent of anthrax, synthesizes two S-layer proteins, each with three SLH motifs towards the amino-terminus. We used biochemical and genetic approaches to investigate the involvement of these motifs in cell surface anchoring. Proteinase K digestion produced polypeptides lacking these motifs, and stable three-motif polypeptides were produced in Escherichia coli that were able to bind the B. anthracis cell walls in vitro, demonstrating that the three SLH motifs were organized into a cell surface anchoring domain. We also determined the function of these SLH domains by constructing chimeric genes encoding the SLH domains fused to the normally secreted levansucrase of Bacillus subtilis. Cell fractionation and electron microscopy studies showed that each three-motif domain was sufficient for the efficient anchoring of levansucrase onto the cell surface. Proteins consisting of truncated SLH domains fused to levansucrase were unstable and associated poorly with the cell surface. Surface-exposed levansucrase retained its enzymatic and antigenic properties.
... Thus, the high level of expression of these proteins, together with their efficient binding to the cell wall, makes this system very attractive for studying cell surface anchoring and developing new expression systems. Bacillus anthracis simultaneously synthesizes two S-layer proteins, named Extracellular Antigen 1 (EA1) and Surface array protein (Sap) (Fouet et al. , 1999. In the amino-terminal region, each protein has three motifs of about 50 residues following the signal peptide, referred to as S-layer homology (SLH) motifs by Lupas et al. (1994). ...
Bacillus anthracis synthesizes two S-layer proteins, each containing three S-layer homology (SLH) motifs towards their amino-terminus. In vitro experiments suggested that the three motifs of each protein were organized as a structural domain sufficient to bind purified cell walls. Chimeric genes encoding the SLH domains fused to the levansucrase of Bacillus subtilis were constructed and integrated on the chromosome. Cell fractionation and electron microscopy studies showed that both heterologous polypeptides were targeted to the cell surface. In addition, surface-exposed levansucrase retained its enzymatic and antigenic properties. Preliminary results concerning applications of this work are presented.
... Over the past decade, monoclonal antibodies specific to the B. anthracis cell wall polysaccharide antigen were shown to be useful in identification of B. anthracis infection (11,12). Vegetative B. anthracis cells constitutively express the galactose/N-acetylglucosamine polysaccharide cell wall antigen (13,14). In addition, during infection or growth in nutrient-rich media in an elevated CO 2 environment, B. anthracis cells produce a poly-γ-D-glutamic acid capsule, which is synthesized by the products of genes located on the pXO2 plasmid (15). ...
A two-component direct fluorescent-antibody (DFA) assay, using fluorescein-labeled monoclonal antibodies specific to the Bacillus anthracis cell wall (CW-DFA) and capsule (CAP-DFA) antigens, was evaluated and validated for rapid identification of B. anthracis. We analyzed 230 B. anthracis isolates; 228 and 229 were positive by CW-DFA and CAP-DFA assays, respectively. We also tested 56 non-B. anthracis strains; 10 B. cereus and 2 B. thuringiensis were positive by the CW-DFA assay, and 1 B. megaterium strain was positive by CAP-DFA. Analysis of the combined DFA results identified 227 of 230 B. anthracis isolates; all 56 strains of the other Bacillus spp. were negative. Both DFA assays tested positive on 14 of 26 aging clinical specimens from the 2001 anthrax outbreak investigation. The two-component DFA assay is a sensitive, specific, and rapid confirmatory test for B. anthracis in cultures and may be useful directly on clinical specimens.
... Little is known about adhesion of vegetative Bacillus anthracis cells to human tissues. Putative adhesins include the Slayer proteins (EA1 and Sap), teichoic acids, the poly-ª-Dglutamic acid capsule and fibronectin-and collagen-binding proteins (Ariel et al., 2003;Ezzell & Welkos, 1999;Fouet et al., 1999). The carbohydrate specificities displayed in this study (Table 2) were not due to interactions with the capsule, because the strain used lacked the pXO2 plasmid that encodes this virulence factor. ...
Intervention in bacterial adhesion to host cells is a novel method of overcoming current problems associated with antibiotic resistance. Antibiotic-resistant strains of bacteria that cause respiratory tract infections are a problem in hospitals and could be used in bioterrorist attacks. A range of bacterial species was demonstrated to attach to an alveolar epithelial (A549) cell line. In all cases, cell surface oligosaccharides were important in attachment, demonstrated by reduced adhesion when A549 cells were pre-treated with tunicamycin. Bacillus anthracis and Yersinia pestis displayed a restricted tropism for oligosaccharides compared to the environmental, opportunistic pathogens, Pseudomonas aeruginosa, Burkholderia cenocepacia, Burkholderia pseudomallei and Legionella pneumophila. The compound with the greatest anti-adhesion activity was p-nitrophenol. Other generic attachment inhibitors included the polymeric saccharides (dextran and heparin), GalNAcbeta1-4Gal, GalNAcbeta1-3Gal, Galbeta1-4GlcNAc and Galbeta1-3GlcNAc. Burkholderia pseudomallei attachment was particularly susceptible to oligosaccharide inhibition. Combinations of such compounds may serve as a novel generic therapeutics for respiratory tract infections.
... It should be reiterated that it is the bacilli that are replicating and secreting toxins, and eliminating bacilli would abrogate toxin production at the source. A major virulence factor of B. anthracis is its anti-phagocytic PGA capsule[7,73]. The role of a capsule in virulence has been well established for numerous bacterial species, such as Streptococcus pneumoniae and type b Haemophilus influenzae (Hib)[74,75]. ...
The successful use of Bacillus anthracis as a lethal biological weapon has prompted renewed research interest in the development of more effective vaccines against anthrax. The disease consists of three critical components: spore, bacillus, and toxin, elimination of any of which confers at least partial protection against anthrax. Current remedies rely on postexposure antibiotics to eliminate bacilli and pre- and postexposure vaccination to target primarily toxins. Vaccines effective against toxin have been licensed for human use, but need improvement. Vaccines against bacilli have recently been developed by us and others. Whether effective vaccines will be developed against spores is still an open question. An ideal vaccine would confer simultaneous protection against spores, bacilli, and toxins. One step towards this goal is our dually active vaccine, designed to destroy both bacilli and toxin. Existing and potential strategies towards potent and effective anthrax vaccines are discussed in this review.
... The basal layer contains two or more crystalline sublayers (2,12,16,43) and the hair-like nap differs in length between strains and between species (16,20,38). There is still little known about the structural components of the exosporium crystalline array, whereas the exterior components of the vegetative cell of B. anthracis have been well characterized (13). In rich medium in vitro, B. anthracis synthesizes successively one of two S-layers, one composed of SAP protein and the other composed of EA1 protein. ...
Spores of Bacillus anthracis, the etiological agent of anthrax, and the closely related species Bacillus cereus and Bacillus thuringiensis, possess an exosporium, which is the outermost structure surrounding the mature spore. It consists of a paracrystalline basal
layer and a hair-like outer layer. To date, the structural contribution of only one exosporium component, the collagen-like
glycoprotein BclA, has been described. It is the structural component of the hair-like filaments. Here, we describe two other
proteins, ExsFA and ExsFB, which are probably organized in multimeric complexes with other exosporium components, including
BclA. Single and double exsF deletion mutants were constructed and analyzed. We found that inactivation of exsF genes affects the BclA content of spores. BclA is produced by all mutants. However, it is partially and totally released
after mother cell lysis of the ΔexsFA and ΔexsFA ΔexsFB mutant strains, respectively. Electron microscopy revealed that the exsF mutant spores have defective exosporia. The ΔexsFA and ΔexsFA ΔexsFB spore surfaces are partially and totally devoid of filaments, respectively. Moreover, for all mutants, the crystalline basal
layer appeared unstable. This instability revealed the presence of two distinct crystalline arrays that are sloughed off from
the spore surface. These results indicate that ExsF proteins are required for the proper localization of BclA on the spore
surface and for the stability of the exosporium crystalline layers.
... During spore germination and outgrowth in the presence of serum and elevated levels of CO 2 , the capsule is released through openings on the spore surface (Ezzell and Abshire 1996). The capsule appears exterior to the Slayer of the vegetative cell wall, and does not require the S-layer for its attachment to the cell surface (Fouet et al. 1999). The genes controlling capsule synthesis -capB, capC and capA, which encode three membrane-associated enzymes, are present as an operon on the pXO2 plasmid (Uchida et al. 1993). ...
Anthrax is caused by the bacterium Bacillus anthracis. Although primarily a disease of animals, it can also infect man, sometimes with fatal consequences. As a result of concerns over the illicit use of this organism, considerable effort is focussed on the development of therapies capable of conferring protection against anthrax. This brief review will describe the efforts being made to address these issues.
A review of the literature and the proceedings of the sixth international conference on anthrax, held in Santa Fe, USA in 2005 shows intense activity, but there has been as yet no real progress. While effective antibiotics, antitoxins and vaccines are available, concerns over their toxicity and the emergence of resistant strains have driven the development of second-generation products. The principal target for vaccine development is Protective Antigen (PA), the nontoxic cell-binding component of anthrax lethal toxin. While the recombinant products currently undergoing human clinical trials will offer considerable advantages in terms of reduced side effects and ease of production, they would still require multiple, needle-based dosing, and the inclusion of the adjuvant alum makes them expensive to administer and stockpile. To address these issues, researchers are developing vaccine formulations, which stimulate rapid protection following needle-free injection (nasal, oral or transcutaneous), and are stable at room temperature to facilitate stockpiling and mass vaccination programs.
An array of medical countermeasures targeting B. anthracis will become available over the next 5-10 years.
The huge investment of research dollars is expected to dramatically expand the knowledge base. A better understanding of basic issues, such as survival in nature and pathogenesis in humans, will facilitate the development of new modalities to eliminate the threat posed by this organism.
... This was not the case with B. anthracis because treatment of encapsulated B. anthracis with DMSO had no apparent effect on either capsule size or the ability of MAbs to produce capsule reactions. The B. anthracis cell wall is surrounded by two abundant surface proteins, extractable antigen 1 and surface array protein, that comprise the B. anthracis S-layer (9). The capsule overlies the S-layer. ...
Bacillus anthracis is surrounded by a polypeptide capsule composed of poly-gamma-d-glutamic acid (gammaDPGA). In a previous study, we reported that a monoclonal antibody (MAb F26G3) reactive with the capsular polypeptide is protective in a murine model of pulmonary anthrax. The present study examined a library of six MAbs generated from mice immunized with gammaDPGA. Evaluation of MAb binding to the capsule by a capsular "quellung" type reaction showed a striking diversity in capsular effects. Most MAbs produced a rim type reaction that was characterized by a sharp increase followed directly by a decrease in refractive index at the capsular edge. Some MAbs produced a second capsular reaction well beneath the capsular edge, suggesting complexity in capsular architecture. Binding of MAbs to soluble gammaDPGA was assessed by a fluorescence perturbation assay in which a change in the MAb intrinsic fluorescence produced by ligand binding was used as a reporter for antigen-antibody interaction. The MAbs differed considerably in the complexity of the binding curves. MAbs producing rim type capsule reactions typically produced the more complex binding isotherms. Finally, the protective activity of the MAbs was compared in a murine model of pulmonary anthrax. One MAb was markedly less protective than the remaining five MAbs. Characteristics of the more protective MAbs included a relatively high affinity, an immunoglobulin G3 isotype, and a complex binding isotherm in the fluorescence perturbation assay. Given the relatively monotonous structure of gammaDPGA, the results demonstrate a striking diversity in the antigen binding behavior of gammaDPGA antibodies.
... S-layer homology domain proteins. The B. anthracis cell surface, in the vegetative nonencapsulated state as well as the capsulated state, is covered by a cell wall polymer, known as the surface layer (or S-layer [23,50,52,84]). Various functions have been assigned to the S-layer, ranging from shape maintenance to virulence, host recognition evasion, cell adhesion and resistance to phagocytosis (52,58,84). B. anthracis is known to synthesize two surface layer (S-layer) proteins, EA1 (extractable antigen 1) and Sap (surface array protein), which account for 5 to 10% of total cellular proteins (52). ...
Bacillus anthracis (Ames strain) chromosome-derived open reading frames (ORFs), predicted to code for surface exposed or virulence related proteins,
were selected as B. anthracis-specific vaccine candidates by a multistep computational screen of the entire draft chromosome sequence (February 2001 version,
460 contigs, The Institute for Genomic Research, Rockville, Md.). The selection procedure combined preliminary annotation
(sequence similarity searches and domain assignments), prediction of cellular localization, taxonomical and functional screen
and additional filtering criteria (size, number of paralogs). The reductive strategy, combined with manual curation, resulted
in selection of 240 candidate ORFs encoding proteins with putative known function, as well as 280 proteins of unknown function.
Proteomic analysis of two-dimensional gels of a B. anthracis membrane fraction, verified the expression of some gene products. Matrix-assisted laser desorption ionization-time-of-flight
mass spectrometry analyses allowed identification of 38 spots cross-reacting with sera from B. anthracis immunized animals. These spots were found to represent eight in vivo immunogens, comprising of EA1, Sap, and 6 proteins whose
expression and immunogenicity was not reported before. Five of these 8 immunogens were preselected by the bioinformatic analysis
(EA1, Sap, 2 novel SLH proteins and peroxiredoxin/AhpC), as vaccine candidates. This study demonstrates that a combination
of the bioinformatic and proteomic strategies may be useful in promoting the development of next generation anthrax vaccine.
... A productive antibody response against surface antigens has been cited as one potential explanation for the anti-spore character of live vaccination/virulent infection [2,4,5,13,14]. SAP and EA1 are the major S-layer antigens of B. anthracis [15,16]. Additionally, the capsule synthesis machinery, consisting of the genes capA, capB, capC, and dep, forms a membrane bound complex at the surface [17]. ...
... The cell membrane and cell wall can be seen in outline, with the thin layer having a thickness of about 5-8 nm. Similar characteristic observations of the S-layer were described by others (Luckevich & Beveridge, 1989;Fouet et al., 1999;Mignot et al., 2001;Couture-Tosi et al., 2002). It can therefore be concluded that there is an S-layer on the cell surface of Bt strain CTC during the vegetative stage. ...
Bacillus thuringiensis produces various groups of active proteins, such as Cyt, Vip and Parasporin, in addition to the Cry protein. In this study we show S-layer proteins to be a new group of parasporal inclusions of B. thuringiensis. The S-layer consists of a two-dimensional lattice structure and is the outermost component of many archaeobacteria and eubacteria. The parasporal inclusion of B. thuringiensis strain CTC was found to be not a typical crystal protein encoded by the cry gene, but a proteinaceous inclusion encoded by the S-layer gene. Furthermore, the CTC-like strains (with their parasporal inclusions coded by the S-layer gene) are widely distributed and accounted for 25.4% of the B. thuringiensis strains tested. These strains constitute a new group of parasporal inclusions encoded by the S-layer gene of B. thuringiensis and shed new light on B. thuringiensis nontoxic strains.
... Un-encapsulated strains of B. anthracis are markedly attenuated in both humans and animals, despite their ability to express fully functional LT and ET complexes. Beneath the capsule is a proteinaceous S-layer, composed of two paracrystalline proteins (extractable antigen 1 [EA1] and surface array protein [SAP]) that cover the bacterial cell surface [16]. Beneath the S-layer is a peptidoglycan layer comprising the cell wall. ...
In vivo induced antigen technology (IVIAT) is an immuno-screening technique that identifies bacterial antigens expressed during infection and not during standard in vitro culturing conditions. We applied IVIAT to Bacillus anthracis and identified PagA, seven members of a N-acetylmuramoyl-L-alanine amidase autolysin family, three P60 family lipoproteins, two transporters, spore cortex lytic protein SleB, a penicillin binding protein, a putative prophage holin, respiratory nitrate reductase NarG, and three proteins of unknown function. Using quantitative real-time PCR comparing RNA isolated from in vitro cultured B. anthracis to RNA isolated from BALB/c mice infected with virulent Ames strain B. anthracis, we confirmed induced expression in vivo for a subset of B. anthracis genes identified by IVIAT, including L-alanine amidases BA3767, BA4073, and amiA (pXO2-42); the bacteriophage holin gene BA4074; and pagA (pXO1-110). The exogenous addition of two purified putative autolysins identified by IVIAT, N-acetylmuramoyl-L-alanine amidases BA0485 and BA2446, to vegetative B. anthracis cell suspensions induced a species-specific change in bacterial morphology and reduction in viable bacterial cells. Many of the proteins identified in our screen are predicted to affect peptidoglycan re-modeling, and our results support significant cell wall structural remodeling activity during B. anthracis infection. Identification of L-alanine amidases with B. anthracis specificity may suggest new potential therapeutic targets.
Institut Pasteur and Bacillus anthracis have enjoyed a relationship lasting almost 120 years, starting from its foundation and the pioneering work of Louis Pasteur in the nascent fields of microbiology and vaccination, and blooming after 1986 following the molecular biology/genetic revolution. This contribution will give a historical overview of these two research eras, taking advantage of the archives conserved at Institut Pasteur. The first era mainly focused on the production, characterisation, surveillance and improvement of veterinary anthrax vaccines; the concepts and technologies with which to reach a deep understanding of this research field were not yet available. The second period saw a new era of B. anthracis research at Institut Pasteur, with the anthrax laboratory developing a multi-disciplinary approach, ranging from structural analysis, biochemistry, genetic expression, and regulation to bacterial-host cell interactions, in vivo pathogenicity, and therapy development; this led to the comprehensive unravelling of many facets of this toxi-infection. B. anthracis may exemplify some general points on how science is performed in a given society at a given time and how a scientific research domain evolves. A striking illustration can be seen in the additive layers of regulations that were implemented from the beginning of the 21st century and their impact on B. anthracis research. B. anthracis and anthrax are complex systems that raise many valuable questions regarding basic research. One may hope that B. anthracis research will be re-initiated under favourable circumstances later at Institut Pasteur.
The Gram-positive spore-forming bacterium Bacillus anthracis is the causative agent of anthrax, a deadly disease mostly affecting wildlife and livestock, as well as representing a bioterrorism threat. Its cell surface is covered by the mutually exclusive S-layers Sap and EA1, found in early and late growth phases, respectively. Here we report the nanobody-based structural characterization of EA1 and its native lattice contacts. The EA1 assembly domain consists of 6 immunoglobulin-like domains, where three calcium-binding sites structure interdomain contacts that allow monomers to adopt their assembly-competent conformation. Nanobody-induced depolymerization of EA1 S-layers results in surface defects, membrane blebbing and cell lysis under hypotonic conditions, indicating that S-layers provide additional mechanical stability to the cell wall. Taken together, we report a complete model of the EA1 S-layer and present a set of nanobodies that may have therapeutic potential against Bacillus anthracis.
The commercial usage of probiotic Lactobacillus strains found in traditional fermented food products have been expanded due to its therapeutics potential. The purpose of this study was designed to isolate, identify, characterize, and evaluate the probiotic abilities of four selected Lactobacilli strains from Inner Mongolian cheese. Four Lactobacillus strains were aseptically isolated on previously specified de Man Rogosa media from Inner Mongolian cheese. Isolates were initially identified by Gram-staining, motility, and catalase tests. Moreover, the presumed Lactobacilli strains were further evaluated for probiotic properties including acid and bile salt tolerance, auto-aggregation, and co-aggregation assays to analyze the adhesive abilities. Further, several phylogenetic analyses were performed to discover the S-layer conserved protein motifs and theoretical protein interaction network for functional annotations. The acid and bile tolerance test were investigated under pH (2.0 & 3.0) and 0.3% bile concentration at 0, 1, 2 and 3 hours of time intervals respectively. Our findings suggested that all four selected LAB strains showed substantial increased in tolerance against acid and bile. The ability of auto-aggregation among Lactobacillus strains range from 15.94% to 70.02%. However, Lactobacillus strain 3(8) showed the highest co-aggregation phenotype with Listeria monocytogenes (54.7%), and (40.8%) with Staph. aureus while strain K showed the strongest ability with Salmonella typhi (39.34%). Phylogenetic investigations revealed the discovery of four S-layer conserved protein motifs and essential protein interaction network among selected Lactobacilli strains. These breakthroughs promote novel perspectives concerning the use of inner Mongolian cheese as a rich source of probiotic bacteria in future researches
Anthrax is an ancient disease caused by Bacillus anthracis and leads to animal and human deaths. It has imparted very important role in history of science by becoming the first bacterium to be observed under microscope, isolated in pure culture and used in attenuated vaccine and became the base of Koch’s famous postulates about germs. The bacterium contains two megaplasmids pXO1 (181 kb), encoding for three secretary toxins named as protective antigen (PA), lethal factor (LF) and edema factor (EF), and pXO2 (96 kb) encoding for anti-phagocytic capsule. The expression of genes is under the control of several cis and trans locating genetic elements and environmental factors. Present chapter provides the detailed insight to the structure and function of different toxins produced by B. anthracis bacterium including their mode of action. The lethal toxin enzymatically cleaves mitogen-activated protein kinases (MEKs), and edema toxin raises the amount of intracellular cAMP. Both toxins have important role in cellular signalling and cell survival pathways, and the same property may be exploited to cure several diseases related to propagation of cells like cancer. How different components of bacterium like toxins and receptors can be manoeuvred to find therapeutics value against cancer is being described. In summary, anthrax is a bacterium which is a life-threatening organism but tactically can be turned into life saviour.
Introduction
Historical Outline
Occurrence and Ultrastructure
Isolation and Chemical Characterization
Molecular Biology, Genetics and Biosynthesis
Assembly and Morphogenesis
Self‐assembly in vivo
Self‐assembly in vitro
Functional Aspects
Biodegradation
Production of S‐layer Proteins
Application of S‐layer Proteins
S‐layer Ultrafiltration Membranes
S‐layers as Matrix for the Immobilization of Functional Macromolecules
S‐layer‐based Dipsticks
Supramolecular Structures Generated by Oriented Recrystallization of S‐layer Fusion Proteins on Supports Precoated with SCWP
S‐layers as Templates for the Formation of Regularly Arranged Nanoparticles
S‐layers as Supporting Structures for Functional Lipid Membranes (Planar Membranes and Liposomes)
S‐layers for Vaccine Development
Outlook and Perspectives
Patents
Unlabelled:
Pathogenic bacteria sense the host environment and regulate expression of virulence-related genes. Environmental signals like temperature, bicarbonate/CO2 and glucose induce toxin production in Bacillus anthracis, but the mechanisms by which these signals contribute to virulence and overall physiological adaptation remains elusive. An integrated, systems level investigation using transcriptomics and iTRAQ-based proteomics was done to assess the effect of temperature, bicarbonate/CO2 and glucose on B. anthracis. Significant changes observed in amino acid, carbohydrate, energy and nucleotide metabolism indicates events of metabolic readjustments by environmental factors. Directed induction of genes involved in polyamine biosynthesis and iron metabolism revealed the redirection of cellular metabolite pool towards iron uptake. Protein levels of glycolytic enzymes, ptsH and Ldh along with transcripts involved in immune evasion (mprF, bNOS, Phospholipases and asnA), cell surface remodeling (rfbABCD, antABCD, and cls) and utilization of lactate (lutABC) and inositol showed constant repression under environmental perturbations. Discrepancies observed in mRNA/protein level of genes involved in glycolysis, protein synthesis, stress response and nucleotide metabolism hinted at the existence of additional regulatory layers and illustrated the utility of an integrated approach. The above findings might assist in the identification of novel adaptive strategies of B. anthracis during host associated survival and pathogenesis.
Biological significance:
In this study, the changes observed at both transcript and protein level were quantified and integrated to understand the effect of host environmental factors (host temperature, bicarbonate and glucose) in shaping the physiology and adaptive strategies of a fully virulent strain of B. anthracis for efficient survival and virulence in its host. Perturbations affecting toxin production were found to concordantly affect vital metabolic pathways and several known as well as novel virulence factors. These changes act as a valuable asset for generating testable hypotheses that can be further verified by detailed molecular and mutant studies to identify novel adaptive strategies of B. anthracis during infection. Adaptation of an integrated transcriptomics and proteomics approach also led to the identification of discrepancies between mRNA/protein levels among genes across major functional categories. Few of these discrepancies have been previously reported in literature for model organisms. However their existence in B. anthracis and that too as a result of growth perturbations have not been reported till date. These findings demonstrate a substantial role of regulatory processes post mRNA synthesis via post transcriptional, translational or protein degradation mechanisms. This article is part of a Special Issue entitled: Proteomics of non-model organisms.
Bacillus anthracis is a Gram-positive, spore-forming rod that causes anthrax. Culture-based methods are the gold standard for the identification of virulent B. anthracis strains but these require days for completion. The experience from the anthrax attacks in September and October of 2001 revealed the urgent need for methods that can rapidly detect this pathogen with high reliability. Because of the extensive homology among non-anthrax Bacillus sp. at the chromosomal level, rapid detection of virulent B. anthracis strains depends on markers associated with the two plasmids, pXO1 and pXO2, responsible for its virulence. Genes encoding toxins and capsules have been used as markers for pXO1 and pXO2, respectively, in methods that are designed for rapid and sensitive detection of B. anthracis DNA, such as real-time polymerase chain reaction, direct liquid phase hybridization, and DNA microarrays. A variety of platforms can be modified to suit the needs for rapid detection of B. anthracis antigens, but little is known about plasmid-encoded antigens expressed in spores. Future studies should be aimed at detecting markers for pXO1 and pXO2in viable spores.
Bacillus anthracis spores germinate to vegetative forms in host cells, and produced fatal toxins. A toxin-targeting prophylaxis blocks the effect of toxin, but may allow to grow vegetative cells which create subsequent toxemia. In this study, we examined protective effect of extractable antigen 1 (EA1), a major S-layer component of B. anthracis, against anthrax. Mice were intranasally immunized with recombinant EA1, followed by a lethal challenge of B. anthracis spores. Mucosal immunization with EA1 resulted in a significant level of anti-EA1 antibodies in feces, saliva and serum. It also delayed the onset of anthrax and remarkably decreased the mortality rate. In addition, the combination of EA1 and protective antigen (PA) protected all immunized mice from a lethal challenge with B. anthracis spores. The numbers of bacteria in tissues of EA1-immunized mice were significantly decreased compared to those in the control and PA alone-immunized mice. Immunity to EA1 might contribute to protection at the early phase of infection, i.e., before massive multiplication and toxin production by vegetative cells. These results suggest that EA1 is a novel candidate for anthrax vaccine and provides a more effective protection when used in combination with PA.
Bacillus anthracis is the etiological agent of anthrax. Although anthrax is primarily an epizootic disease; humans are at risk for contracting anthrax. The potential use of B. anthracis spores as biowarfare agent has led to immense attention. Prolonged vaccination schedule of current anthrax vaccine and variable protection conferred; often leading to failure of therapy. This highlights the need for alternative anthrax countermeasures. A number of approaches are being investigated to substitute or supplement the existing anthrax vaccines. These relied on expression of Protective antigen (PA), the key protective immunogen; in bacterial or plant systems; or utilization of attenuated strains of B. anthracis for immunization. Few studies have established potential of domain IV of PA for immunization. Other targets including the spore, capsule, S-layer and anthrax toxin components have been investigated for imparting protective immunity. It has been shown that co-immunization of PA with domain I of lethal factor that binds PA resulted in higher antibody responses. Of the epitope based vaccines, the loop neutralizing determinant, in particular; elicited robust neutralizing antibody response and conferred 97% protection upon challenge. DNA vaccination resulted in varying degree of protection and seems a promising approach. Additionally, the applicability of monoclonal and therapeutic antibodies in the treatment of anthrax has also been demonstrated. The recent progress in the direction of anthrax prophylaxis has been evaluated in this review.
Significant efforts to develop both laboratory and field-based detection assays for an array of potential biological threats started well before the anthrax attacks of 2001 and have continued with renewed urgency following. While numerous assays and methods have been explored that are suitable for laboratory utilization, detection in the field is often complicated by requirements for functionality in austere environments, where limited cold-chain facilities exist. In an effort to overcome these assay limitations for Bacillus anthracis, one of the most recognizable threats, a series of single domain antibodies (sdAbs) were isolated from a phage display library prepared from immunized llamas. Characterization of target specificity, affinity, and thermal stability was conducted for six sdAb families isolated from rounds of selection against the bacterial spore. The protein target for all six sdAb families was determined to be the S-layer protein EA1, which is present in both vegetative cells and bacterial spores. All of the sdAbs examined exhibited a high degree of specificity for the target bacterium and its spore, with affinities in the nanomolar range, and the ability to refold into functional antigen-binding molecules following several rounds of thermal denaturation and refolding. This research demonstrates the capabilities of these sdAbs and their potential for integration into current and developing assays and biosensors.
During an infection, bacterial pathogens must acquire iron from the host to survive. However, free iron is sequestered in host proteins, which presents a barrier to iron-dependent bacterial replication. In response, pathogens have developed mechanisms to acquire iron from the host during infection. Interestingly, a significant portion of the iron pool is sequestered within heme, which is further bound to host proteins such as hemoglobin. The copious amount of heme-iron makes hemoglobin an ideal molecule for targeted iron uptake during infection. While the study of heme acquisition is well represented in Gram-negative bacteria, the systems and mechanism of heme uptake in Gram-positive bacteria has only recently been investigated. Bacillus anthracis, the causative agent of anthrax disease, represents an excellent model organism to study iron acquisition processes owing to a multifaceted lifecycle consisting of intra- and extracellular phases and a tremendous replicative potential upon infection. This review provides an in depth description of the current knowledge of B. anthracis iron acquisition and applies these findings to a general understanding of how pathogenic Gram-positive bacteria transport this critical nutrient during infection.
The lethal anthrax disease is caused by spores of the gram-positive Bacillus anthracis, a member of the cereus group of bacilli. Although the disease is very rare in the Western world, development of anthrax countermeasures gains increasing attention due to the potential use of B. anthracis spores as a bio-terror weapon. Protective antigen (PA), the non-toxic subunit of the bacterial secreted exotoxin, fulfills the role of recognizing a specific receptor and mediating the entry of the toxin into the host target cells. PA elicits a protective immune response and represents the basis for all current anthrax vaccines. Anti-PA neutralizing antibodies are useful correlates for protection and for vaccine efficacy evaluation. Post exposure anti-toxemic and anti-bacteremic prophylactic treatment of anthrax requires prolonged antibiotic administration. Shorter efficient postexposure treatments may require active or passive immunization, in addition to antibiotics. Although anthrax is acknowledged as a toxinogenic disease, additional factors, other than the bacterial toxin, may be involved in the virulence of B. anthracis and may be needed for the long-lasting protection conferred by PA immunization. The search for such novel factors is the focus of several high throughput genomic and proteomic studies that are already leading to identification of novel targets for therapeutics, for vaccine candidates, as well as biomarkers for detection and diagnosis.
Bacillus anthracis, the causative agent of anthrax disease, could be used as a biothreat reagent. It is vital to develop a rapid, convenient method to detect B. anthracis. In the current study, three high affinity and specificity monoclonal antibodies (mAbs, designated 8G3, 10C6 and 12F6) have been obtained using fully washed B. anthracis spores as an immunogen. These mAbs, confirmed to direct against EA1 protein, can recognize the surface of B. anthracis spores and intact vegetative cells with high affinity and species-specificity. EA1 has been well known as a major S-layer component of B. anthracis vegetative cells, and it also persistently exists in the spore preparations and bind tightly to the spore surfaces even after rigorous washing. Therefore, these mAbs can be used to build a new and rapid immunoassay for detection of both life forms of B. anthracis, either vegetative cells or spores.
Bacillus anthracis is a Gram-positive, spore-forming bacterium representing the etiological cause of anthrax, a rare lethal disease of animals and humans. Development of anthrax countermeasures has gained increasing attention owing to the potential use of B. anthracis spores as a bioterror weapon. The various forms of infection by B. anthracis are characterized both by toxemia and septicemia, both of which are the result of spore entry into the host followed by their germination into rapidly multiplying, toxin-producing bacilli. Following the publication of the bacterial genome, proteomic studies were carried out to determine the protein composition of the spore and identify exposed vegetative (membrane-located or secreted) proteins. These studies included comparison of strains differing in their virulence, cultured under different conditions and, in some cases, were complemented by serological inspection, which addressed expression during infection of proteomically identified proteins and their immunogenicity. The proteomic approach emerged as a valuable strategy for the generation of a pool of potential B. anthracis protein targets for further evaluation in detection, diagnostics, therapy and prophylaxis, and contributed to the elucidation of some aspects of the pathogenesis of the disease.
Bacillus anthracis is a Gram positive organism possessing a complex parietal structure. An S-layer, a bi-dimensional crystalline layer, and a peptidic capsule surround the thick peptidoglycan of bacilli harvested during infection. A review of the current literature indicates that elements from each of these three structures, as well as membrane components, have been studied. So-called cell-wall secondary polymers, be they attached to the cell-wall or to the membrane play important functions, either per se or because they permit the anchoring of proteins. Some surface proteins, whichever compartment they are attached to, play, as had been hypothesized, key roles in virulence. Others, of yet unknown function, are nevertheless expressed in vivo. This review will focus on well-studied polymers or proteins and indicate, when appropriate, the mechanisms by which they are targeted to their respective locations.
Bacillus anthracis, the causal agent of anthrax, synthesizes two surface layer (S-layer) proteins, EA1 and Sap, which account for 5 to 10% of total protein and are expressed in vivo. A recombinant B. anthracis strain was constructed by integrating into the chromosome a translational fusion harboring the DNA fragments encoding the cell wall-targeting domain of the S-layer protein EA1 and tetanus toxin fragment C (ToxC). This construct was expressed under the control of the promoter of the S-layer component gene. The hybrid protein was stably expressed on the cell surface of the bacterium. Mice were immunized with bacilli of the corresponding strain, and the hybrid protein elicited a humoral response to ToxC. This immune response was sufficient to protect mice against tetanus toxin challenge. Thus, the strategy developed in this study may make it possible to generate multivalent live veterinary vaccines, using the S-layer protein genes as a cell surface display system.
Anthrax is primarily a disease of herbivores caused by gram-positive, aerobic, spore-forming Bacillus anthracis. Humans are accidental hosts through the food of animal origin and animal products. Anthrax is prevelant in most parts of the globe, and cases of anthrax have been reported from almost every country. Three forms of the disease have been recognized: cutaneous (through skin), gastrointestinal (through alimentary tract), and pulmonary (by inhalation of spores). The major virulence factors of Bacillus anthracis are a poly-D glutamic acid capsule and a three-component protein exotoxin. The genes coding for the toxin and the enzymes responsible for capsule production are carried on plasmid pXO1 and pXO2, respectively. The three proteins of the exotoxin are protective antigen (PA, 83 kDa), lethal factor (LF, 90 kDa), and edema factor (EF, 89 kDa). The toxins follow the A-B model with PA being the B moeity and LF/EF, the alternative A moeities. LF and EF are individually nontoxic, but in combination with PA form two toxins causing different pathogenic responses in animals and cultured cells. PA + LF forms the lethal toxin and PA + EF forms the edema toxin. During the process of intoxication, PA binds to the cell surface receptor and is cleaved at the sequence RKKR (167) by cell surface proteases such as furin generating a cell-bound, C-terminal 63 kDa protein (PA63). PA63 possesses a binding site to which LF or EF bind with high affinity. The complex is then internalized by receptor-mediated endocytosis. Acidification of the vesicle leads to instertion of PA63 into the endosomal membrane and translocation of LF/EF across the bilayer into the cytosol where they exert their toxic effects. EF has a calcium- and calmodulin-dependent adenylate cyclase activity. Recent reports indicate that LF is a protease that cleaves the amino terminus of mitogen-activated protein kinase kinases 1 and 2 (MAPKK1 and 2), and this cleavage inactivates MAPKK1 and thus inhibits the mitogen-activated protein kinase signal transduction pathway. We describe in detail the studies so far done on unraveling the molecular mechanisms of pathogenesis of Bacillus anthracis.
Anthrax vaccination has become a 'hot' topic. On the one hand, fears that Iraq holds secret caches of anthrax-based weaponry, that other countries may be developing or may have developed similar devices, or that hard-line groups may make their own anthrax-based devices for bioterrorist attacks have focused official attention on the need for means of protection, principally, though, for the military. On the other hand, the unsolved issues of the Gulf War illnesses have left elements of doubt in the minds of some as to the possible role of anthrax (among other) vaccines in this syndrome, and have drawn attention to the shortage of pre-clinical, clinical, pharmacological and safety data on the existing UK and US anthrax vaccines. In the middle are those hotly debating the US and Canadian policies of mandatory anthrax immunization for military personnel or, in the case of the UK policy of voluntary immunization, simply voting with their feet. Compounding matters have been the publicized failures of the US vaccine production facility and the less publicized UK problems of supply. Meanwhile, those in genuine at-risk occupations are left unsure whether, if they can get the vaccine at all, they really want it. Despite two decades of elegant science aimed at formulating alternative vaccines to overcome all the problems of efficacy, safety and supply, such an alternative is at least five years away, and the current status is that we must live with the old vaccines or not vaccinate.
The spore is the form released in a bioterrorism attack. There is a real need for definition of new targets for Bacillus anthracis that might be incorporated into emerging biodetection technologies. Particularly of interest are macromolecules found in B. anthracis that are (1) spore-specific, (2) readily accessible on the spore surface and (3) distinct from those present in related organisms. One of the few biochemical methods to identify the spores of B. anthracis is based on the presence of rhamnose and 3-O-methyl rhamnose as determined by gas chromatography-mass spectrometry. Related organisms additionally contain 2-O-methyl rhamnose and fucose. Carbohydrates and glycoproteins of the B. cereus group of organisms and the related B. subilis group are reviewed here. It is hypothesized that the spore-specific carbohydrate is a component of the newly described glycoprotein of the exosporium of B. anthracis. Further work to define the protein and carbohydrate components of the glycoprotein of B. anthracis could be highly useful in developing new technologies for rapid biodetection.
Bacillus anthracis is the causative agent of anthrax disease. Improvement of existing anthrax vaccines, which are currently based on the administration of Protective Antigen (the highly immunogenic nontoxic subunit of the bacterial toxin) may entail other bacterial immunogenic elements, part of which are predicted to reside on the surface of bacterial cells. In the present study, membranal proteins extracted from a stationary-phase culture of a nonvirulent B. anthracis strain, devoid of the native virulence plasmids pXO1 and pXO2, were separated by two-dimensional electrophoresis (2-DE) and a characteristic protein map was defined. The proteomic analysis allowed matrix-assisted laser desorption/ionization-time of flight mass spectrometry-assisted identification of 86 protein spots which represent the product of 30 individual open reading frames (ORF). Among these, a prevalent class of proteins was the S-layer proteins (which were found to represent more than 75% of the B. anthracis membranal fraction) and proteins containing S-layer homology (SLH)-membranal localization domains. Five novel SLH proteins, previously inferred only from bioinformatic ORF analysis (draft genome sequence), were identified and one was shown to be a highly abundant membranal protein. Western blots of the 2-DE gels were probed with sera from convalescent rabbits and guinea pigs infected with virulent B. anthracis (Vollum strain). This analysis revealed that B. anthracis immune animals exhibit antibodies against at least 14 distinct membranal proteins present in the 2-DE map, establishing that these proteins are expressed in vivo and are able to elicit an immune response. The identification of the protein components of the B. anthracis membranal fraction, as well as the establishment of their potential immunogenicity, underscore the strength of the proteomic approach for identifying molecules which may serve for further analysis of immune and protective abilities.
Bacillus anthracis, the etiological agent of anthrax, is a gram-positive spore-forming bacterium. Fully virulent bacilli are toxinogenic and capsulated. Two abundant surface proteins, including the major antigen, are components of the B. anthracis surface layer (S-layer). The B. anthracis paracrystalline S-layer has previously only been found in noncapsulated vegetative cells. Here we report that the S-layer proteins are also synthesized under conditions where the poly-γ-d-glutamic acid capsule is present. Structural and immunological analyses show that the capsule is exterior to and completely covers the S-layer proteins. Nevertheless, analysis of single and double S-layer protein mutants shows that the presence of these proteins is not required for normal capsulation of the bacilli. Similarly, the S-layer proteins assemble as a two-dimensional crystal, even in the presence of the capsule. Thus, both structures are compatible, and yet neither is required for the correct formation of the other.
Bacillus brevis 47 had two protein layers (the outer and middle walls) and a peptidoglycan layer (the inner wall) and contained two major proteins with approximate molecular weights of 130,000 and 150,000 in the cell wall. Both the total and Triton-insoluble envelopes revealed a hexagonal lattice array with a lattice constant of 14.5 nm. The proteins of 130,000 and 150,000 molecular weight isolated from the Triton-insoluble envelopes were serologically different from each other and assembled in vitro on the peptidoglycan layer. A mixture of 130,000- and 150,000-molecular-weight proteins led to the formation of a five-layered cell wall structure, two layers on each side of the peptidoglycan layer, which resembled closely the Triton-insoluble envelopes. A three-layered cell wall structure, one layer on each side of the peptidoglycan layer, was reconstituted when only the 150,000-molecular-weight protein was used. Both five- and three-layered cell walls reconstituted in vitro also contained hexagonally arranged arrays with the same lattice constant as that of the total and Triton-insoluble envelopes. A mutant, strain 47-57, which was isolated as a phage-resistant colony, had a two-layered cell wall consisting of the middle and inner wall layers and contained only 150,000-molecular-weight protein as the major cell wall protein. The cell envelopes of the mutant revealed the hexagonal arrays with the same lattice constant as that of the wild-type cell envelopes. We conclude that the outer and middle wall layers consist of proteins with approximate molecular weights of 130,000 and 150,000, respectively. Furthermore, the 150,000-molecular-weight protein formed the hexagonal arrays in the middle wall layer.
The cell walls of four gliding filamentous Oscillatoriaceae species comprising three different genera were studied by freeze substitution, freeze fracturing, and negative staining. In all species, the multilayered gram-negative cell wall is covered with a complex external double layer. The first layer is a tetragonal crystalline S-layer anchored on the outer membrane. The second array is formed by parallel, helically arranged surface fibrils with diameters of 8 to 12 nm. These fibrils have a serrated appearance in cross sections. In all cases, the orientation of the surface fibrils correlates with the sense of revolution of the filaments during gliding, i.e., clockwise in both Phormidium strains and counterclockwise in Oscillatoria princeps and Lyngbya aeruginosa. The lack of longitudinal corrugations or contractions of the surface fibrils and the identical appearances of motile and nonmotile filaments suggest that this structure plays a passive screw thread role in gliding. It is hypothesized that the necessary propulsive force is generated by shear forces between the surface fibrils and the continuing flow of secreted extracellular slime. Furthermore, the so-called junctional pores seem to be the extrusion sites of the slime. In motile cells, these pores exhibit a different staining behavior than that seen in nonmotile ones. In the former, the channels of the pores are filled with electron-dense material, whereas in the latter, the channels appear comparatively empty, highly contrasting the peptidoglycan. Finally, the presence of regular surface structures in other gliding prokaryotes is considered an indication that comparable structures are general features of the cell walls of gliding microbes.
Bacillus anthracis, a gram-positive, spore-forming bacterium, is the etiological agent of anthrax. The gene coding for the S-layer protein (sap) was cloned on two contiguous fragments in Escherichia coli, and the complete sequence of the structural gene was determined. The protein, Sap, is composed of 814 residues, including a classical prokaryotic 29-amino-acid signal peptide. The mature form has a calculated molecular mass of 83.7 kDa and a molecular mass of 94 kDa on a sodium dodecyl sulfate-polyacrylamide gel. Sap possesses many charged residues, is weakly acidic, and contains only 0.9% methionine and no cysteine residues. The N-terminal region of Sap shares sequence similarities with the Acetogenium kivui S-layer protein, the Bacillus brevis middle wall protein, the Thermotoga maritima Omp alpha protein, and the Bacillus thuringiensis S-layer protein. Electron microscopy observations showed that this S-layer is not observed on B. anthracis cells in which sap has been deleted.
The three-dimensional structure of the Acetogenium kivui surface layer (S-layer) has been determined to a resolution of 1.7 nm by electron crystallographic techniques. Two independent reconstructions were made from layers negatively stained with uranyl acetate and Na-phosphotungstate. The S-layer has p6 symmetry with a center-to-center spacing of approximately 19 nm. Within the layer, six monomers combine to form a ring-shaped core surrounded by a fenestrated rim and six spokes that point towards the axis of threefold symmetry and provide lateral connectivity to other hexamers in the layer. The structure of the A. kivui S-layer protein is very similar to that of the Bacillus brevis middle wall protein, with which it shares an N-terminal domain of homology. This domain is found in several other extracellular proteins, including the S-layer proteins from Bacillus sphaericus and Thermus thermophilus, Omp alpha from Thermotoga maritima, an alkaline cellulase from Bacillus strain KSM-635, and xylanases from Clostridium thermocellum and Thermoanaerobacter saccharolyticum, and may serve to anchor these proteins to the peptidoglycan. To our knowledge, this is the first example of a domain conserved in several S-layer proteins.
Bacilli secrete numerous proteins into the environment. Many of the secretory proteins, their export signals, and their processing steps during secretion have been characterized in detail. In contrast, the molecular mechanisms of protein secretion have been relatively poorly characterized. However, several components of the protein secretion machinery have been identified and cloned recently, which is likely to lead to rapid expansion of the knowledge of the protein secretion mechanism in Bacillus species. Comparison of the presently known export components of Bacillus species with those of Escherichia coli suggests that the mechanism of protein translocation across the cytoplasmic membrane is conserved among gram-negative and gram-positive bacteria differences are found in steps preceding and following the translocation process. Many of the secretory proteins of bacilli are produced industrially, but several problems have been encountered in the production of Bacillus heterologous secretory proteins. In the final section we discuss these problems and point out some possibilities to overcome them.
Stable synthesis of the hexagonally ordered (p6) S-layer protein from the wild-type strain of Bacillus stearothermophilus PV72 could be achieved in continuous culture on complex medium only under oxygen-limited conditions when glucose was used as the sole carbon source. Depending on the adaptation of the wild-type strain to low oxygen supply, the dynamics in oxygen-induced changes in S-layer protein synthesis was different when the rate of aeration was increased to a level that allowed dissimilation of amino acids. If oxygen supply was increased at the beginning of continuous culture, synthesis of the p6 S-layer protein from the wild-type strain (encoded by the sbsA gene) was immediately stopped and replaced by that of a new type of S-layer protein (encoded by the sbsB gene) which assembled into an oblique (p2) lattice. In cells adapted to a prolonged low oxygen supply, first, low-level p2 S-layer protein synthesis and second, synchronous synthesis of comparable amounts of both types of S-layer proteins could be induced by stepwise increasing the rate of aeration. The time course of changes in S-layer protein synthesis was followed up by immunogold labelling of whole cells. Synthesis of the p2 S-layer protein could also be induced in the p6-deficient variant T5. Hybridization data obtained by applying the radiolabelled N-terminal and C-terminal sbsA fragments and the N-terminal sbsB fragment to the genomic DNA of all the three organisms indicated that changes in S-layer protein synthesis were accompanied by chromosomal rearrangement. Chemical analysis of peptidoglycan-containing sacculi and extraction and recrystallization experiments revealed that at least for the wild-type strain, a cell wall polymer consisting of N-acetylglucosamine and glucose is responsible for binding of the p6 S-layer protein to the rigid cell wall layer.
Nonopsonic phagocytosis of Bacillus cereus by human polymorphonuclear leukocytes (PMNs) with particular attention to bacterial surface properties and structure was studied. Two reference strains (ATCC 14579(T) and ATCC 4342) and two clinical isolates (OH599 and OH600) from periodontal and endodontic infections were assessed for adherence to matrix proteins, such as type I collagen, fibronectin, laminin, and fibrinogen. One-day-old cultures of strains OH599 and OH600 were readily ingested by PMNs in the absence of opsonins, while cells from 6-day-old cultures were resistant. Both young and old cultures of the reference strains of B. cereus were resistant to PMN ingestion. Preincubation of PMNs with the phagocytosis-resistant strains of B. cereus did not affect the phagocytosis of the sensitive strain. Negatively stained cells of OH599 and OH600 studied by electron microscopy had a crystalline protein layer on the cell surface. In thin-sectioned cells of older cultures (3 to 6 days old), the S-layer was observed to peel off from the cells. No S-layer was detected on the reference strains. Extraction of cells with detergent followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major 97-kDa protein from the strains OH599 and OH600 but only a weak 97-kDa band from the reference strain ATCC 4342. One-day-old cultures of the clinical strains (hydrophobicity, 5.9 to 6.0%) showed strong binding to type I collagen, laminin, and fibronectin. In contrast, reference strains (hydrophobicity, -1.0 to 4.2%) as well as 6-day-old cultures of clinical strains (hydrophobicity, 19.0 to 53.0%) bound in only low numbers to the proteins. Gold-labelled biotinylated fibronectin was localized on the S-layer on the cell surface as well as on fragments of S-layer peeling off the cells of a 6-day-old culture of B. cereus OH599. Lactose, fibronectin, laminin, and antibodies against the S-protein reduced binding to laminin but not to fibronectin. Heating the cells at 84 degreesC totally abolished binding to both proteins. Benzamidine, a noncompetitive serine protease inhibitor, strongly inhibited binding to fibronectin whereas binding to laminin was increased. Overall, the results indicate that changes in the surface structure, evidently involving the S-layer, during growth of the clinical strains of B. cereus cause a shift from susceptibility to PMN ingestion and strong binding to matrix and basement membrane proteins. Furthermore, it seems that binding to laminin is mediated by the S-protein while binding to fibronectin is dependent on active protease evidently attached to the S-layer.
The presence of a regular array (RA) was demonstrated on the outer layer of the cell wall in Clostridium difficile GAI0714 by electron microscopy. The RA was composed of squarely arranged subunits with a center-to-center spacing of about 8.2 nm. The outer wall layer carrying the RA was isolated from the wall fragments of early log-phase cells by autolysis. The outer wall layer was composed of two main proteins with apparent Mrs of about 45 000 and 32 000 upon sodiumdodecylsul-fate-polyacrylamide gel electrophoresis (SDS-PAGE). Similar RAs were also present in the cell walls of the other 9 strains of C. difficile. These strains were divided into two groups on the basis of the wall protein composition: one containing Mr 45 000–47 000 and 32 000 proteins and the other containing Mr 42 000 and 38 000 proteins.
The S layer of Clostridium difficile GAI 0714, which was composed of squarely arrayed, two-subunit proteins with respective molecular weights of 32 kDa and 45 kDa, was examined for its morphological, physicochemical, and biological properties. Optical diffraction analysis of the S layer showed that the surface-arrayed rhombus had four sides of 8.1 nm with interior angles of 88-degrees. The two proteins were heterogeneous to each other in respect to the first ten N-terminal amino acid sequences except for residues No's. 4 and 9. Self-assembly of the subunit proteins into a regular array was dependent on such divalent cations as Ca2+ or Zn2+, but not Ba2+ or Mg2+. When the mixture, made of purified 32 and 45 kDa subunits in an equal concentration ratio on a protein basis was submitted to self-assembly, flattened paracrystalline sheetlike fragments were generated. However, neither sheetlike fragments nor regular arrays was observed for any self-assembled products derived from each subunit alone or their quantitatively heterologous mixtures. Intact cells of the organism adhered significantly to HeLa cells or mouse fibroblast 929 cells, whereas 8 m urea- or 4 m guanidinehydrochloride-extracted bacteria, from which both S layer protein subunits were removed, scarcely adhered to both cells. Fab fragments of anti-32 kDa or anti-45 kDa subunits antibodies effectively inhibited adhesion of the organism to HeLa cells, suggesting that the S layer had a direct role in adhesion.
Bacillus anthracis, the aetiological agent of anthrax, is a Gram-positive spore-forming bacterium. The cell wall of vegetative cells of B. anthracis is surrounded by an S-layer. An array remained when sap, a gene described as encoding an S-layer component, was deleted. The remaining S-layer component, termed EA1, is chromosomally encoded. The gene encoding EA1 (eag) was obtained on two overlapping fragments in Escherichia coli and shown to be contiguous to the sap gene. The EA1 amino acid sequence, deduced from the eag nucleotide sequence, shows classical S-layer protein features (no cysteine, only 0.1% methionine, 10% lysine, and a weakly acidic pI). Similar to Sap and other Gram-positive surface proteins, EA1 has three ‘S-layer-homology’ motifs immediately downstream from a signal peptide. Single- and double-disrupted mutants were constructed. EA1 and Sap were co-localized at the cell surface of the wild-type bacilli. However, EA1 was more tightly bound than Sap to the bacteria. Electron microscopy studies and in vivo experiments with the constructed mutants showed that EA1 constitutes the main lattice of the B. anthracis S-layer, and is the major cell-associated antigen.
Comparative Chemistry of the Rigid Cell Wall Component and its Phylogenetic Implications.- Comparative Aspects on Archaeobacterial Proteinaceous Cell Envelopes.- Evidence for the Glycoprotein Nature of Eubacterial S-Layers.- The Surface Layers of Lampropedia hyalina.- A Glycoprotein Surface Layer Covers the Pseudomurein Sacculus of the Extreme Thermophile Methanothermus fervidus.- The Structure, Chemistry and Physicochemistry of the Methanospirillum hungatei GP1 Sheath.- Ultrastructural and Biochemical Studies of the Cell Sheath of Methanothrix soehngenii.- The S-Layers of Aquaspirillum serpens.- Image Analysis of the Two Patterns from Negatively Stained A-Layers of Aeromonas salmonicida: Correction for Lattice Distortions.- The A-Layer of Aeromonas salmonicida: Organization and Functions.- Thermoresistance of A-Layer-Deficient Mutants of Aeromonas salmonicida.- The S-Layer of Pathogenic Strains of Aeromonas hydrophila.- The Three-Dimensional Structure of Bacterial Surface Layers.- Characterization of the Surface Protein of Comamonas acidovorans.- The Surface Protein of the Archaebacterium Thermoproteus tenax.- The Structure of Surface Proteins in Sulfolobus.- Structure of the Surface Glycoprotein from Halobacterium volcanii as Revealed by Electron Microscopy.- The Structure of the Surface Protein of Thermotoga maritima.- Three-Dimensional Structure of Surface Layers from Various Bacillus and Clostridium Species.- Paracrystalline Surface Layer of a Novel Bacillus sp M3198.- Three-Dimensional Structure of the Regular Tetragonal Surface Layer of Azotobacter vinelandii.- Structure and Assembly of the Oblique S-Layer Lattice of Bacillus stearothermophilus Strain NRS 2004/3a.- Charge Distribution of S-Layers and Importance of Charged Groups for Morphogenesis and Function.- Localized Insertion of New S-Layer During Growth of Bacillus stearothermophilus Strain PV72.- Crystalline Surface Layers in Anaerobic Bacteria Isolated from a Patient with Secretory Otitis Media and a Draining Ear.- Occurrence and Characterization of S-Layers in Oral Bacteria.- Ultrastructural Comparison of Bacteroides heparinolyticus and Bacteroides buccae.- S-Layers in Cyanobacteria.- Cloning and Sequencing of the S-Layer Glycoprotein Gene of Halobacterium halobium.- The Primary Structure of the HPI-Layer Polypeptide of Deinococcus radiodurans.- Characterization and Application of S-Layer Protein Gene for Production of Foreign Proteins in a Protein-Producing Bacillus brevis 47.- Permebility Properties and the Use of S-Layers for the Production of Ultrafiltration Membranes.- Comparative Studies on Synthetic and S-Layer Ultrafiltration Membranes.- Appendix Crystalline Surface Layers on Bacteria.- References.
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Sera from Hartley guinea pigs vaccinated with a veterinary live spore anthrax vaccine were compared with sera from guinea pigs vaccinated with the human anthrax vaccine, which consists of aluminum hydroxide-adsorbed culture proteins of Bacillus anthracis V770-NP-1R. Sera from animals vaccinated with the spore vaccine recognized two major B. anthracis vegetative cell-associated proteins that were either not recognized or poorly recognized by sera from animals that received the human vaccine. These proteins, termed extractable antigens 1 (EA1) and 2 (EA2), have molecular masses of 91 and 62 kilodaltons, respectively. The EA1 protein appeared to be coded by chromosomal DNA, whereas the EA2 protein was only detected in strains that possessed the pXO1 toxin plasmid. Both of the extractable antigen proteins were serologically distinct from the components of anthrax edema toxin and lethal toxin. Following vaccination with the live spore vaccine, the EA1 protein was the predominant antigen recognized, as determined by electrophoretic immunotransblots. Vaccine trials with partially purified EA1 demonstrated that it neither elicits protective antibody against anthrax nor delays time to death in guinea pigs challenged intramuscularly with virulent Ames strain spores. In addition, animals vaccinated with sterile gamma-irradiated cell walls had significant antibody titers to the N-acetylglucosamine-galactose polysaccharide of B. anthracis but were neither protected nor had a delay in time to death following challenge.
The two-layered regular surface array of Aquaspirillum serpens MW5 was removed from cell envelopes and dissociated into subunits by treatment with 6 M urea. The surface components reassembled onto an outer membrane surface and self-assembled into planar sheets in vitro in the presence of Ca2+ or Sr2+. The two layers were removed sequentially from cell envelopes by a two-step extraction procedure involving initial treatment with a high-pH buffer to remove the outermost surface layer and subsequent treatment with 6 M urea to remove the innermost layer. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the outer and inner layers of the array were composed of two proteins with molecular weights of 125,000 and 150,000, respectively. The two layers assembled sequentially; the 150,000-molecular-weight protein formed an array on an outer membrane surface, and the 125,000-molecular-weight protein required that array as a template for its in vitro assembly.
Crystalline arrays of proteinaceous subunits forming surface layers (S-layers) are one of the most commonly observed prokaryotic cell envelope structures. They are ubiquitous amongst Gram-positive and Gram-negative archeaobacteria and eubacteria and, if present, account for the major protein species produced by the cells. S-layers can provide organisms with a selection advantage by providing various functions including protective coats, molecular sieves, ion traps and structures involved in cell surface interactions. S-layers were identified as contributing to virulence when present as a structural component of pathogens. In Gram-negative archaeobacteria they are involved in determining cell shape and cell division. The crystalline arrays reveal a broad-application potential in biotechnology, vaccine development and molecular nanotechnology.
Bacillus anthracis synthesizes two S-layer proteins, each containing three S-layer homology (SLH) motifs towards their amino-terminus. In vitro experiments suggested that the three motifs of each protein were organized as a structural domain sufficient to bind purified cell walls. Chimeric genes encoding the SLH domains fused to the levansucrase of Bacillus subtilis were constructed and integrated on the chromosome. Cell fractionation and electron microscopy studies showed that both heterologous polypeptides were targeted to the cell surface. In addition, surface-exposed levansucrase retained its enzymatic and antigenic properties. Preliminary results concerning applications of this work are presented.