HtrA is a major virulence determinant of Bacillus anthracis

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.     

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.     

Recognition of heteropolysaccharide alginate by periplasmic solute-binding proteins of a bacterial ABC transporter

Alginate is a heteropolysaccharide that consists of β-D-mannuronate (M) and α-L-guluronate (G). The Gram-negative bacterium Sphingomonas sp. A1 directly incorporates alginate into the cytoplasm through the periplasmic solute-binding protein (AlgQ1 and AlgQ2)-dependent ABC transporter (AlgM1-AlgM2/AlgS-AlgS). Two binding proteins with at least four subsites strongly recognize the nonreducing terminal residue of alginate at subsite 1. Here, we show the broad substrate preference of strain A1 solute-binding proteins for M and G present in alginate and demonstrate the structural determinants in binding proteins for heteropolysaccharide recognition through X-ray crystallography of four AlgQ1 structures in complex with saturated and unsaturated alginate oligosaccharides. Alginates with different M/G ratios were assimilated by strain A1 cells and bound to AlgQ1 and AlgQ2. Crystal structures of oligosaccharide-bound forms revealed that in addition to interaction between AlgQ1 and unsaturated oligosaccharides, the binding protein binds through hydrogen bonds to the C4 hydroxyl group of the saturated nonreducing terminal residue at subsite 1. The M residue of saturated oligosaccharides is predominantly accommodated at subsite 1 because of the strict binding of Ser-273 to the carboxyl group of the residue. In unsaturated trisaccharide (ΔGGG or ΔMMM)-bound AlgQ1, the protein interacts appropriately with substrate hydroxyl groups at subsites 2 and 3 to accommodate M or G, while substrate carboxyl groups are strictly recognized by the specific residues Tyr-129 at subsite 2 and Lys-22 at subsite 3. Because of this substrate recognition mechanism, strain A1 solute-binding proteins can bind heteropolysaccharide alginate with different M/G ratios.     

A collagen-like surface glycoprotein is a structural component of the Bacillus anthracis exosporium

Bacillus anthracis, the aetiological agent of anthrax, is a Gram-positive spore-forming bacterium. The exosporium is the outermost integument surrounding the mature spore. Here, we describe the purification and the characterization of an immunodominant protein of the spore surface. This protein was abundant, glycosylated and part of the exosporium. The amino-terminal sequence was determined and the corresponding gene was identified. It encodes a protein of 382 amino acid residues, the central part of which contains a region of GXX motifs presenting similarity to mammalian collagen proteins. Thus, this collagen-like surface protein was named BclA (for Bacillus collagen-like protein of anthracis). BclA was absent from vegetative cells; it was detected only in spores and sporulating cells. A potential promoter, dependent on the sigma factor sigma(K), which is required for a variety of events late in sporulation, was found upstream from the bclA gene. A bclA deletion mutant was constructed and analysed. Electron microscopy studies showed that BclA is a structural component of the filaments covering the outer layer of the exosporium.     

Structural analysis of a putative aminoglycoside N-acetyltransferase from Bacillus anthracis

For the last decade, worldwide efforts for the treatment of anthrax infection have focused on developing effective vaccines. Patients that are already infected are still treated traditionally using different types of standard antimicrobial agents. The most popular are antibiotics such as tetracyclines and fluoroquinolones. While aminoglycosides appear to be less effective antimicrobial agents than other antibiotics, synthetic aminoglycosides have been shown to act as potent inhibitors of anthrax lethal factor and may have potential application as antitoxins. Here, we present a structural analysis of the BA2930 protein, a putative aminoglycoside acetyltransferase, which may be a component of the bacterium's aminoglycoside resistance mechanism. The determined structures revealed details of a fold characteristic only for one other protein structure in the Protein Data Bank, namely, YokD from Bacillus subtilis. Both BA2930 and YokD are members of the Antibiotic_NAT superfamily (PF02522). Sequential and structural analyses showed that residues conserved throughout the Antibiotic_NAT superfamily are responsible for the binding of the cofactor acetyl coenzyme A. The interaction of BA2930 with cofactors was characterized by both crystallographic and binding studies.     

Maturation of Streptococcus pneumoniae lipoproteins by a type II signal peptidase is required for ABC transporter function and full virulence

Cell surface lipoproteins are important for the full virulence of several bacterial pathogens, including Streptococcus pneumoniae. Processing of prolipoproteins seems to be conserved among different bacterial species, and requires type II signal peptidase (Lsp) mediated cleavage of the N-terminal signal peptide to form the mature lipoprotein. Lsp has been suggested as a target for new antibiotic therapies, but at present there are only limited data on the function of Lsp for Gram-positive bacterial pathogens. We have investigated the function and role during disease pathogenesis of the S. pneumoniae Lsp, which, blast searches suggest, is encoded by the gene Sp0928. Expression of Sp0928 protected Escherichia coli against the Lsp antagonist globomycin, and proteomics and immunoblot analysis demonstrated that deletion of Sp0928 prevented processing of S. pneumoniae prolipoproteins to mature lipoproteins. These data strongly suggest that Sp0928 encodes the S. pneumoniae Lsp. However, immunoblots of membrane-associated proteins, immunoelectron microscopy and flow cytometry assays all confirmed that in the absence of Lsp, immature lipoproteins were still attached to the cell surface. Despite preservation of lipoprotein attachment to the cell membrane, loss of S. pneumoniae Lsp resulted in several phenotypes associated with impaired lipoprotein function and reduced S. pneumoniae replication in animal models of infection.     

The ABC transporter BcatrB from Botrytis cinerea exports camalexin and is a virulence factor on Arabidopsis thaliana

Arabidopsis thaliana is known to produce the phytoalexin camalexin in response to abiotic and biotic stress. Here we studied the mechanisms of tolerance to camalexin in the fungus Botrytis cinerea , a necrotrophic pathogen of A. thaliana . Exposure of B. cinerea to camalexin induces expression of BcatrB , an ABC transporter that functions in the efflux of fungitoxic compounds. B. cinerea inoculated on wild-type A. thaliana plants yields smaller lesions than on camalexin-deficient A. thaliana mutants. A B. cinerea strain lacking functional BcatrB is more sensitive to camalexin in vitro and less virulent on wild-type plants, but is still fully virulent on camalexin-deficient mutants. Pre-treatment of A. thaliana with UV-C leads to increased camalexin accumulation and substantial resistance to B. cinerea. UV-C-induced resistance was not seen in the camalexin-deficient mutants cyp79B2/B3 , cyp71A13 , pad3 or pad2 , and was strongly reduced in ups1 . Here we demonstrate that an ABC transporter is a virulence factor that increases tolerance of the pathogen towards a phytoalexin, and the complete restoration of virulence on host plants lacking this phytoalexin.     

The use of PCR to isolate a putative ABC transporter from Saccharopolyspora erythraea

Abstract The uropathogenic Escherichia coli strain J96 (04:K6) is able to produce four adherence factors [P-fimbriae ( pap and prs ), F1C-fimbriae ( foc ) and Type 1-fimbriae ( fim )], two α-hemolysins ( hfy I and II) and the cytotoxic necrotizing factor type 1 ( cnf 1). Using phenotypic test systems and genotypic analysis, it has been shown that the mutant strain J96-M1 has lost the hly II, prs and cnf 1 genes. The three virulence associated determinants are linked on one particular region on the chromosome, which is termed 'pathogenicity island II' (Pai II).     

Titratable transmembrane residues and a hydrophobic plug are essential for manganese import via the Bacillus anthracis ABC transporter MntBC-A

All extant life forms require trace transition metals (e.g., Fe^2/3+, Cu^1/2+, and Mn²⁺) to survive. However, as these are environmentally scarce, organisms have evolved sophisticated metal uptake machineries. In bacteria, high-affinity import of transition metals is predominantly mediated by ABC transporters. During bacterial infection, sequestration of metal by the host further limits the availability of these ions, and accordingly, bacterial ABC transporters (importers) of metals are key virulence determinants. However, the structure–function relationships of these metal transporters have not been fully elucidated. Here, we used metal-sensitivity assays, advanced structural modeling, and enzymatic assays to study the ABC transporter MntBC-A, a virulence determinant of the bacterial human pathogen Bacillus anthracis. We find that despite its broad metal-recognition profile, MntBC-A imports only manganese, whereas zinc can function as a high-affinity inhibitor of MntBC-A. Computational analysis shows that the transmembrane metal permeation pathway is lined with six titratable residues that can coordinate the positively charged metal, and mutagenesis studies show that they are essential for manganese transport. Modeling suggests that access to these titratable residues is blocked by a ladder of hydrophobic residues, and ATP-driven conformational changes open and close this hydrophobic seal to permit metal binding and release. The conservation of this arrangement of titratable and hydrophobic residues among ABC transporters of transition metals suggests a common mechanism. These findings advance our understanding of transmembrane metal recognition and permeation and may aid the design and development of novel antibacterial agents.     

The role of Bacillus anthracis germinant receptors in germination and virulence

Nutrient‐dependent germination of Bacillus anthracis spores is stimulated when receptors located in the inner membrane detect combinations of amino acid and purine nucleoside germinants. B. anthracis produces five distinct germinant receptors, GerH, GerK, GerL, GerS and GerX. Otherwise isogenic mutant strains expressing only one of these receptors were created and tested for germination and virulence. The GerH receptor was necessary and sufficient for wild‐type levels of germination with inosine‐containing germinants in the absence of other receptors. GerK and GerL were sufficient for germination in 50 mM L‐alanine. When mutants were inoculated intratracheally, any receptor, except for GerX, was sufficient to allow for a fully virulent infection. In contrast, when inoculated subcutaneously only the GerH receptor was able to facilitate a fully virulent infection. These results suggest that route of infection determines germinant receptor requirements. A mutant lacking all five germinant receptors was also attenuated and exhibited a severe germination defect in vitro. Together, these data give us a greater understanding of the earliest moments of germination, and provide a more detailed picture of the signals required to stimulate this process.