Surface-layer (S-layer) proteins sap and EA1 govern the binding of the S-layer-associated protein BslO at the cell septa of Bacillus anthracis

The Gram-positive pathogen Bacillus anthracis contains 24 genes whose products harbor the structurally conserved surface-layer (S-layer) homology (SLH) domain. Proteins endowed with the SLH domain associate with the secondary cell wall polysaccharide (SCWP) following secretion. Two such proteins, Sap and EA1, have the unique ability to self-assemble into a paracrystalline layer on the surface of bacilli and form S layers. Other SLH domain proteins can also be found within the S layer and have been designated Bacillus S-layer-associated protein (BSLs). While both S-layer proteins and BSLs bind the same SCWP, their deposition on the cell surface is not random. For example, BslO is targeted to septal peptidoglycan zones, where it catalyzes the separation of daughter cells. Here we show that an insertional lesion in the sap structural gene results in elongated chains of bacilli, as observed with a bslO mutant. The chain length of the sap mutant can be reduced by the addition of purified BslO in the culture medium. This complementation in trans can be explained by an increased deposition of BslO onto the surface of sap mutant bacilli that extends beyond chain septa. Using fluorescence microscopy, we observed that the Sap S layer does not overlap the EA1 S layer and slowly yields to the EA1 S layer in a growth-phase-dependent manner. Although present all over bacilli, Sap S-layer patches are not observed at septa. Thus, we propose that the dynamic Sap/EA1 S-layer coverage of the envelope restricts the deposition of BslO to the SCWP at septal rings.     

Secretion genes as determinants of Bacillus anthracis chain length

Bacillus anthracis grows in chains of rod-shaped cells, a trait that contributes to its escape from phagocytic clearance in host tissues. Using a genetic approach to search for determinants of B. anthracis chain length, we identified mutants with insertional lesions in secA2. All isolated secA2 mutants exhibited an exaggerated chain length, whereas the dimensions of individual cells were not changed. Complementation studies revealed that slaP (S-layer assembly protein), a gene immediately downstream of secA2 on the B. anthracis chromosome, is also a determinant of chain length. Both secA2 and slaP are required for the efficient secretion of Sap and EA1 (Eag), the two S-layer proteins of B. anthracis, but not for the secretion of S-layer-associated proteins or of other secreted products. S-layer assembly via secA2 and slaP contributes to the proper positioning of BslO, the S-layer-associated protein, and murein hydrolase, which cleaves septal peptidoglycan to separate chains of bacilli. SlaP was found to be both soluble in the bacterial cytoplasm and associated with the membrane. The purification of soluble SlaP from B. anthracis-cleared lysates did not reveal a specific ligand, and the membrane association of SlaP was not dependent on SecA2, Sap, or EA1. We propose that SecA2 and SlaP promote the efficient secretion of S-layer proteins by modifying the general secretory pathway of B. anthracis to transport large amounts of Sap and EA1.     

Structural analysis and evidence for dynamic emergence of Bacillus anthracis S-layer networks

Surface layers (S-layers), which form the outermost layers of many Bacteria and Archaea, consist of protein molecules arranged in two-dimensional crystalline arrays. Bacillus anthracis, a gram-positive, spore-forming bacterium, responsible for anthrax, synthesizes two abundant surface proteins: Sap and EA1. Regulatory studies showed that EA1 and Sap appear sequentially at the surface of the parental strain. Sap and EA1 can form arrays. The structural parameters of S-layers from mutant strains (EA1(-) and Sap(-)) were determined by computer image processing of electron micrographs of negatively stained regular S-layer fragments or deflated whole bacteria. Sap and EA1 projection maps were calculated on a p1 symmetry basis. The unit cell parameters of EA1 were a = 69 A, b = 83 A, and gamma = 106 degrees, while those of Sap were a = 184 A, b = 81 A, and gamma = 84 degrees. Freeze-etching experiments and the analysis of the peripheral regions of the cell suggested that the two S-layers have different settings. We characterized the settings of each network at different growth phases. Our data indicated that the scattered emergence of EA1 destabilizes the Sap S-layer.     

Molecular characterization of the Bacillus anthracis main S-layer component: evidence that it is the major cell-associated antigen

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.     

Distinct affinity of binding sites for S-layer homologous domains in Clostridium thermocellum and Bacillus anthracis cell envelopes

Binding parameters were determined for the SLH (S-layer homologous) domains from the Clostridium thermocellum outer layer protein OlpB, from the C. thermocellum S-layer protein SlpA, and from the Bacillus anthracis S-layer proteins EA1 and Sap, using cell walls from C. thermocellum and B. anthracis. Each SLH domain bound to C. thermocellum and B. anthracis cell walls with a different KD, ranging between 7.1 x 10(-7) and 1.8 x 10(-8) M. Cell wall binding sites for SLH domains displayed different binding specificities in C. thermocellum and B. anthracis. SLH-binding sites were not detected in cell walls of Bacillus subtilis. Cell walls of C. thermocellum lost their affinity for SLH domains after treatment with 48% hydrofluoric acid but not after treatment with formamide or dilute acid. A soluble component, extracted from C. thermocellum cells by sodium dodecyl sulfate treatment, bound the SLH domains from C. thermocellum but not those from B. anthracis proteins. A corresponding component was not found in B. anthracis.     

Characterisation of the immune response to the UK human anthrax vaccine

The UK human anthrax vaccine consists of the alum-precipitated culture supernatant of Bacillus anthracis Sterne. In addition to protective antigen (PA), the key immunogen, the vaccine also contains a number of other bacteria- and media-derived proteins. These proteins may contribute to the transient side effects experienced by some individuals and could influence the development of the PA-specific immune response. Bacterial cell-wall components have been shown to be potent immunomodulators. B. anthracis expresses two S-layer proteins, EA1 and Sap, which have been demonstrated to be immunogenic in animal studies. These are also immunogenic in man so that convalescent and post-immunisation sera contain specific antibodies to Ea1, and to a lesser extent, to Sap. To determine if these proteins are capable of modifying the protective immune response to PA, A/J mice were immunised with equivalent amounts of recombinant PA and S-layer proteins in the presence of alhydrogel. IgG isotype profiles were determined and the animals were subsequently challenged with spores of B. anthracis STI. The results suggest that there was no significant shift in IgG isotype profile and that the presence of the S-layer proteins did not adversely affect the protective immune response induced by PA.     

Role of the components of the S-layer in immunogenicity of Bacillus anthracis

The immunogenicity of proteins Sap and EA1, contained in B. anthracis S-layer, was evaluated in experiments on laboratory animals. These proteins were found to produce protective effect and could be regarded as additional immunogenic factors. The use of the newly constructed isogenic pair Sap+ and Sap- of B. anthracis strains made it possible to study the influence of Sap- mutation on the immunological properties of the causative agent of anthrax.     

BslA, a pXO1-encoded adhesin of Bacillus anthracis

The Gram-positive pathogen Bacillus anthracis causes anthrax, a fulminant and lethal infection of mammals. Two large virulence plasmids, pXO1 and pXO2, harbour genes required for anthrax pathogenesis and encode secreted toxins or provide for the poly γ- d -glutamic acid capsule. In addition to capsule, B. anthracis harbours additional cell wall envelope structures, including the surface layer (S-layer), which is composed of crystalline protein arrays. We sought to identify the B. anthracis envelope factor that mediates adherence of vegetative forms to human cells and isolated BslA ( B . anthracis S - l ayer protein A ). Its structural gene, bslA , is located on the pXO1 pathogenicity island (pXO1-90) and bslA expression is both necessary and sufficient for adherence of vegetative forms to host cells. BslA assembly into S-layers and surface exposure is presumably mediated by three N-terminal SLH domains. Twenty-three B. anthracis genes, whose products harbour similar SLH domains, may provide additional surface molecules that allow bacilli to engage cells or tissues of specific hosts during anthrax pathogenesis.     

Purification and characterization of the major surface array protein from the avirulent Bacillus anthracis Delta Sterne-1.

Many prokaryotic organisms possess surface layer (S-layer) proteins that are components of the outermost cell envelope. With immunogold labeling, it was demonstrated that the protein extractable antigen 1 (EA1) was localized on the outer surface and specifically to cell wall fragments from Bacillus anthracis which retained the S layer. When grown in rich medium under aerobic conditions, the avirulent strain Delta Sterne-1 released large amounts of EA1 into the medium. This EA1 had no higher-order structure initially but formed two-dimensional crystals under defined conditions. The released EA1 was purified in aqueous buffers with a three-step procedure and found to have a mass of 95 kDa when subjected to denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). N-terminal sequence data revealed exact identity to the first eight residues of the S-layer protein from B. thuringiensis 4045. Gel permeation chromatography of the purified EA1 under nondenaturing conditions revealed a single peak corresponding to a mass of approximately 400 kDa, suggesting that a tetramer or dimer of dimers was the primary species in solution. SDS-PAGE of EA1 purified in the absence of protease inhibitors revealed specific proteolytic processing to an 80-kDa form, which immunoreacted with polyclonal anti-EA1 antibodies. This proteolytic cleavage of EA1 to 80 kDa was duplicated with purified EA1 and the protease trypsin or pronase.     

Structure of surface layer homology (SLH) domains from Bacillus anthracis surface array protein

Surface (S)-layers, para-crystalline arrays of protein, are deposited in the envelope of most bacterial species. These surface organelles are retained in the bacterial envelope through the non-covalent association of proteins with cell wall carbohydrates. Bacillus anthracis, a Gram-positive pathogen, produces S-layers of the protein Sap, which uses three consecutive repeats of the surface-layer homology (SLH) domain to engage secondary cell wall polysaccharides (SCWP). Using x-ray crystallography, we reveal here the structure of these SLH domains, which assume the shape of a three-prong spindle. Each SLH domain contributes to a three-helical bundle at the spindle base, whereas another α-helix and its connecting loops generate the three prongs. The inter-prong grooves contain conserved cationic and anionic residues, which are necessary for SLH domains to bind the B. anthracis SCWP. Modeling experiments suggest that the SLH domains of other S-layer proteins also fold into three-prong spindles and capture bacterial envelope carbohydrates by a similar mechanism.     

The extracellular and cytoplasmic proteomes of the non-virulent Bacillus anthracis strain UM23C1-2

The recently published genome sequence of Bacillus anthracis Ames has facilitated the prediction of proteins associated with the virulence of this bacterium. The aim of this study was to define reference maps for the extracellular and cytoplasmic proteomes of the avirulent B. anthracis strain UM23C1-2 that are useful for physiological studies and the development of improved vaccines. Using 2-DE and subsequent MALDI-TOF-TOF MS, 64 proteins were identified in the extracellular proteome, only 29 of which were predicted to be exported into the culture medium. The latter included chitinases, proteases, nucleotidases, sulfatases, phosphatases and proteins of unknown function. Of the remaining proteins in the culture medium, 18 were predicted to be associated with the cell wall or anchored on the trans side of the cytoplasmic membrane while 17 other proteins lacked identifiable export signals and were predicted to be cytoplasmic proteins. Among the S-layer proteins, Sap and Eag account for 10% of the total extracellular proteome. Many of the proteins are predicted to contribute to the virulence and antigenic signature of B. anthracis. We have also studied the composition of the cytoplasmic proteome, identifying 300 distinct proteins. The most abundant cytoplasmic proteins are primarily those involved in glycolysis, amino acid metabolism, protein translation, protein folding and stress adaptation. The presence of a variety of proteases, peptidases, peptide binding proteins, as well as enzymes required for the metabolism of amino acids, suggests that B. anthracis is adapted to life in a protein-rich environment rather than the soil. We therefore speculate that proteases and peptidases could be useful targets for the development of improved vaccines. In addition, both of these B. anthracis compartment-specific proteomes can be used as reference maps to monitor changes in the production of secreted and cytosolic proteins that occur, for example, during growth in macrophages.     

The S-layer homology domain as a means for anchoring heterologous proteins on the cell surface of Bacillus anthracis

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.     

The Capsule and S-Layer: Two Independent and Yet Compatible Macromolecular Structures in Bacillus anthracis

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 anthracis, a gram-positive spore-forming bacterium, is the causative agent of anthrax. This disease, to which many animals, including humans, are susceptible, involves toxemia and septicemia. In the mammalian host, B. anthracis bacilli synthesize two toxins (lethal and edema toxins) (31) and a capsule (18) encoded by two large plasmids, pXO1 and pXO2, respectively (12, 21). The capsule is composed of poly-γ-d-glutamic acid and has antiphagocytic properties (13, 31, 37). Although unusual, a similar capsule is also found on Bacillus licheniformis bacilli (9). In the absence of pXO2 or the inducer bicarbonate, the cell does not produce a capsule and the cell wall appears layered. These layers are composed of fragments displaying a highly patterned ultrastructure (10, 16). This type of cell surface is now referred to as the surface layer (S-layer).S-layers are present on the surfaces of many archaea and bacteria (for reviews, see references 29 and 30). Most are formed by noncovalent, entropy-driven assembly of a single (glyco)protein protomer on the bacterial surface, giving rise to proteinaceous paracrystalline layers. Generally, a single S-layer is present, constituting 5 to 10% of total cell protein. Its synthesis is thus presumably energy consuming for the bacterium. Numerous bacteria have S-layers, suggesting that they play important roles in the interaction between the cell and its environment. Various functions have been proposed for S-layers, including shape maintenance and molecular sieving, and they can serve in phage fixation. The S-layer may be a virulence factor, protecting pathogenic bacteria against complement killing, facilitating binding of bacteria to host molecules, or enhancing their ability to associate with macrophages (for reviews, see references 27 and 29).Some bacteria, such as cyanobacteria or Azotobacter spp., possess both a capsule and an S-layer; however, to our knowledge, their structural relationships have not been analyzed through simultaneous genetic and cytologic studies. Both of these features have been independently described for the surface of the pathogenic bacterium B. anthracis. The components of the B. anthracis S-layer are two abundant surface proteins, EA1 and Sap (6, 20). Previous analyses of the B. anthracis S-layer used plasmid-cured strains; consequently, the interaction, if any, between the capsule and the S-layer could not be studied. Temporal or environmental regulation could be such that only one or the other structure is ever present at the cell surface. However, we show that S-layer proteins are synthesized under conditions where the bacilli are capsulated. We determined the localizations of capsule and S-layer components and analyzed whether the S-layer is necessary for proper capsulation. Finally, the assembly of the S-layer proteins in a two-dimensional crystal was examined in the presence of the capsule.     

The SLH-domain protein BslO is a determinant of Bacillus anthracis chain length

The Gram-positive pathogen Bacillus anthracis grows in characteristic chains of individual, rod-shaped cells. Here, we report the cell-separating activity of BslO, a putative N-acetylglucosaminidase bearing three N-terminal S-layer homology (SLH) domains for association with the secondary cell wall polysaccharide (SCWP). Mutants with an insertional lesion in the bslO gene exhibit exaggerated chain lengths, although individual cell dimensions are unchanged. Purified BslO complements this phenotype in trans, effectively dispersing chains of bslO-deficient bacilli without lysis and localizing to the septa of vegetative cells. Compared with the extremely long chain lengths of csaB bacilli, which are incapable of binding proteins with SLH-domains to SCWP, bslO mutants demonstrate a chaining phenotype that is intermediate between wild-type and csaB. Computational simulation suggests that BslO effects a non-random distribution of B. anthracis chain lengths, implying that all septa are not equal candidates for separation.     

A Bacillus thuringiensis S-layer protein involved in toxicity against Epilachna varivestis (Coleoptera: Coccinellidae)

The use of Bacillus thuringiensis as a biopesticide is a viable alternative for insect control since the insecticidal Cry proteins produced by these bacteria are highly specific; harmless to humans, vertebrates, and plants; and completely biodegradable. In addition to Cry proteins, B. thuringiensis produces a number of extracellular compounds, including S-layer proteins (SLP), that contribute to virulence. The S layer is an ordered structure representing a proteinaceous paracrystalline array which completely covers the surfaces of many pathogenic bacteria. In this work, we report the identification of an S-layer protein by the screening of B. thuringiensis strains for activity against the coleopteran pest Epilachna varivestis (Mexican bean beetle; Coleoptera: Coccinellidae). We screened two B. thuringiensis strain collections containing unidentified Cry proteins and also strains isolated from dead insects. Some of the B. thuringiensis strains assayed against E. varivestis showed moderate toxicity. However, a B. thuringiensis strain (GP1) that was isolated from a dead insect showed a remarkably high insecticidal activity. The parasporal crystal produced by the GP1 strain was purified and shown to have insecticidal activity against E. varivestis but not against the lepidopteran Manduca sexta or Spodoptera frugiperda or against the dipteran Aedes aegypti. The gene encoding this protein was cloned and sequenced. It corresponded to an S-layer protein highly similar to previously described SLP in Bacillus anthracis (EA1) and Bacillus licheniformis (OlpA). The phylogenetic relationships among SLP from different bacteria showed that these proteins from Bacillus cereus, Bacillus sphaericus, B. anthracis, B. licheniformis, and B. thuringiensis are arranged in the same main group, suggesting similar origins. This is the first report that demonstrates that an S-layer protein is directly involved in toxicity to a coleopteran pest.     

炭疽杆菌S层的研究进展

S层是广泛存在于古细菌和真细菌细胞壁最外层的结构独特、功能特殊的成分,对它的研究具有重要的意义。炭疽杆菌是引起人畜炭疽病的病原体,对其S层的研究将为深入认识该菌的生理特性及致病机制等提供坚实的基础。就近年来对炭疽杆菌S层的结构、功能特点及应用前景等方面的研究进展做一简要的综述。     

Production and cell surface anchoring of functional fusions between the SLH motifs of the Bacillus anthracis S-layer proteins and the Bacillus subtilis levansucrase

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.     

Distribution of S-layers on the surface of Bacillus cereus strains: phylogenetic origin and ecological pressure

Bacillus anthracis , Bacillus cereus and Bacillus thuringiensis have been described as members of the Bacillus cereus group but are, in fact, one species. B. anthracis is a mammal pathogen, B. thuringiensis an entomopathogen and B. cereus a ubiquitous soil bacterium and an occasional human pathogen. In two clinical isolates of B. cereus , in some B. thuringiensis strains and in B. anthracis , an S-layer has been described. We investigated how the S-layer is distributed in B. cereus , and whether phylogeny or ecology could explain its presence on the surface of some but not all strains. We first developed a simple biochemical assay to test for the presence of the S-layer. We then used the assay with 51 strains of known genetic relationship: 26 genetically diverse B. cereus and 25 non- B. anthracis of the B. anthracis cluster. When present, the genetic organization of the S-layer locus was analysed further. It was identical in B. cereus and B. anthracis . Nineteen strains harboured an S-layer, 16 of which belonged to the B. anthracis cluster. All 19 were B. cereus clinical isolates or B. thuringiensis , except for one soil and one dairy strain. These findings suggest a common phylogenetic origin for the S-layer at the surface of B. cereus strains and, presumably, ecological pressure on its maintenance.