Cell Wall Of Gram-positive Bacteria Research Articles

Curvature of Synthetic and Natural Surfaces Is an Important Target Feature in Classical Pathway Complement Activation

The binding of Abs to microbial surfaces followed by complement activation constitutes an important line of defense against infections. In this study, we have investigated the relationship between complement activation and the binding of human IgM Abs to surfaces with different curvatures. IgM Abs to dextran were shown to activate complement potently on dextran-coated particles having a diameter around 250 nm, whereas larger (600 nm) particles were less potent activators. This selectivity regarding particle dimension was also found for complement activation by colloidal substances of microbial origin. Peptidoglycan (PGN) is the major chemical component in the cell wall of Gram-positive bacteria. Fragments of purified PGN with sizes of approximately 100 nm promoted complement activation effectively through the classical pathway. By contrast, larger or smaller fragments of PGN did not activate complement strongly. A careful analysis of PGN fragments released during planctonic growth of Staphylococcus aureus showed that these include curvatures that would permit strong IgM-mediated complement activation, whereas the curvature of intact cells would be less effective for such activation. Consistently, we found that the suspended PGN fragments were strong activators of complement through the classical pathway. We suggest that these fragments act as decoy targets for complement activation, providing protection for S. aureus against the host immune response to infection.

The effect of nebivolol on the production of nitric oxide induced by bacterial lipopolysaccharide and peptidoglycan in mice

Nitric oxide (NO) plays a pivotal role in main- taining balance of physiological events in many systems including the autonomic, cardiovas- cular, hematological, and pulmonary systems. Lipopolysaccharide (LPS) and peptidoglycan (PGN), components of the outer cell membranes of Gram-negative bacteria and cell walls of Gram-positive bacteria respectively, are in- criminated in NO-induced septic shock. Ne- bivolol is a third generation β1- adrenoceptor blocker with a vasodilatory property attributed to enhanced availability of nitric oxide and re- duction of cellular oxidative stress through an unknown mechanism. The current study ex- plored the hypothesis that if nebivolol enhances the availability of NO, pretreatment with ne- bivolol may enhance production of NO in re- sponse to subsequent treatment with LPS and PGN, an observation that may have relevance in clinical septic shock. Groups of female BALB/c mice each containing 12 mice (6-8 weeks old) were injected intraperitoneally with LPS (30 µg/mouse), PGN (100 µg/mouse), nebivolol (0.25 µg/g, 0.35 µg/g, 0.7 µg/g), LPS and nebivolol (0.25 µg/g), LPS and nebivolol (0.35 µg/g), LPS and nebivolol (0.7 µg/g), PGN and nebivolol (0.25 µg/g), PGN and nebivolol (0.35µg/g). One group of mice was injected with saline and an- other served as control. Three mice from each group were bled 1, 3, 6 and 9 hours post-injec- tion, the blood was pooled and the nitrite serum levels, reflecting NO concentration, were de- termined using Greiss reagent. The following results were obtained: 1) Treatment with saline did not induce NO production; 2) LPS induced NO production to a maximal limit of 545% at 9 hours as compared to treatment with saline; 3) PGN did not induce NO production; 4) nebivolol at most doses and periods (7 out of 10 deter- minations) increased NO production over a range of 18-110% as compared to treatment with saline; 5) Nebivolol enhanced LPS-induced production of NO by 58% at a dose of 0.7 µg/gm at 9 hours. It is concluded that nebivolol in- duces NO production. At low doses nebivolol initially appeared to have a suppressive or no effect on NO production induced by LPS. In- crease in the dose of nebivolol resulted in augmentation of LPS-induced production of NO. PGN, in the dose tested, did not have an effect on NO production.

Structural characterization of teichoic acids from Lactobacillus brevis

Teichoic acids are a major constituent of the cell wall of Gram-positive bacteria. Structural characterization of lipoteichoic and teichoic acids isolated from Lactobacillus brevis was undertaken using 1D and 2D NMR experiments as well as chemical methodology. Compositional analysis indicated the presence of high amounts of glycerol, glucose, and alanine. In the case of LTA octadecenoic acid was also detected. The basic LTA/WTA structure was identified as 1,3-poly(glycerol phosphate) nonstoichiometrically substituted at C-2 of the glycerol residues with d-Ala or α- d-Glc. In the case of LTA a higher amount of Ala could be detected and partial alanylation at position C-6 of the Glc could also be observed.

ThedltOperon ofBacillus cereusIs Required for Resistance to Cationic Antimicrobial Peptides and for Virulence in Insects

The dlt operon encodes proteins that alanylate teichoic acids, the major components of cell walls of gram-positive bacteria. This generates a net positive charge on bacterial cell walls, repulsing positively charged molecules and conferring resistance to animal and human cationic antimicrobial peptides (AMPs) in gram-positive pathogenic bacteria. AMPs damage the bacterial membrane and are the most effective components of the humoral immune response against bacteria. We investigated the role of the dlt operon in insect virulence by inactivating this operon in Bacillus cereus, which is both an opportunistic human pathogen and an insect pathogen. The Delta dlt(Bc) mutant displayed several morphological alterations but grew at a rate similar to that for the wild-type strain. This mutant was less resistant to protamine and several bacterial cationic AMPs, such as nisin, polymyxin B, and colistin, in vitro. It was also less resistant to molecules from the insect humoral immune system, lysozyme, and cationic AMP cecropin B from Spodoptera frugiperda. Delta dlt(Bc) was as pathogenic as the wild-type strain in oral infections of Galleria mellonella but much less virulent when injected into the hemocoels of G. mellonella and Spodoptera littoralis. We detected the dlt operon in three gram-negative genera: Erwinia (Erwinia carotovora), Bordetella (Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), and Photorhabdus (the entomopathogenic bacterium Photorhabdus luminescens TT01, the dlt operon of which did not restore cationic AMP resistance in Delta dlt(Bc)). We suggest that the dlt operon protects B. cereus against insect humoral immune mediators, including hemolymph cationic AMPs, and may be critical for the establishment of lethal septicemia in insects and in nosocomial infections in humans.

Peptidoglycan Enhances IL-6 Production in Human Synovial Fibroblasts via TLR2 Receptor, Focal Adhesion Kinase, Akt, and AP-1- Dependent Pathway

Peptidoglycan (PGN), the major component of the cell wall of Gram-positive bacteria, activates the innate immune system of the host and induces the release of cytokines and chemokines. We investigated the signaling pathway involved in IL-6 production stimulated by PGN in rheumatoid arthritis synovial fibroblasts. PGN caused concentration- and time-dependent increases in IL-6 production. PGN-mediated IL-6 production was attenuated by TLR2 small interfering RNA and nucleotide-binding oligomerization domain 2 small interfering RNA. Pretreatment with PI3K inhibitor (Ly294002 and wortmannin), Akt inhibitor, and AP-1 inhibitor (tanshinone IIA) also inhibited the potentiating action of PGN. PGN increased the focal adhesion kinase (FAK), PI3K, and Akt phosphorylation. Stimulation of rheumatoid arthritis synovial fibroblast cells with PGN increased the accumulation of phosphorylated c-Jun in the nucleus, AP-1-luciferase activity, and c-Jun binding to the AP-1 element on the IL-6 promoter. PGN mediated an increase in the accumulation of phosphorylated c-Jun in the nucleus, AP-1-luciferase activity, and c-Jun binding to AP-1 element was inhibited by Ly294002, Akt inhibitor, and FAK mutant. Our results suggest that PGN increased IL-6 production in human synovial fibroblasts via the TLR2 receptor/FAK/PI3K/Akt and AP-1 signaling pathway.

Modulation of muramyl dipeptide stimulation of cytokine production by blood components

Muramyl dipeptide (MDP) is the minimal active fragment of peptidoglycan of the cell wall of Gram-positive bacteria, with potential beneficial effects as a vaccine adjuvant. Peptidoglycans and MDP are recognized by the intracellular receptor NOD2 (nucleotide-binding oligomerization domain 2), leading to production of proinflammatory cytokines. In the present study, it is shown that, despite stimulatory effects on isolated human mononuclear cells, MDP does not stimulate production of tumour necrosis factor-alpha, interleukin-1beta or interleukin-6 in a whole-blood assay. However, MDP retains synergistic effects on lipopolysaccharide-induced cytokines in whole blood. Screening tests of NOD2 function based on whole-blood stimulation should therefore employ strategies based on the synergistic effects of MDP on Toll-like receptor-induced cytokine production. Plasma was not responsible for the inhibition of MDP in whole blood. The inhibition of MDP stimulation was dependent upon cellular components, with erythrocyte-derived haemoglobin and neutrophils collaborating in the inhibition of MDP effects in whole blood.

Total Synthesis of N-Acetylglucosamine-1,6-anhydro-N-acetylmuramylpentapeptide and Evaluation of Its Turnover by AmpD from Escherichia coli

The bacterial cell wall is recycled extensively during the course of cell growth. The first recycling event involves the catalytic action of the lytic transglycosylase enzymes, which produce an uncommon 1,6-anhydropyranose moiety during separation of the muramyl residues from the peptidoglycan, the major constituent of the cell wall. This product, an N-acetyl-beta-D-glucosamine-(1-->4)-1,6-anhydro-N-acetyl-beta-D-muramylpeptide, is either internalized to initiate the recycling process or diffuses into the milieu to cause stimulation of the pro-inflammatory responses by the host. We report the total syntheses of N-acetyl-beta-D-glucosamine-(1-->4)-1,6-anhydro-N-acetyl-beta-D-muramyl-L-Ala-gamma-D-Glu-meso-DAP-D-Ala-D-Ala (compound 1, the product of lytic transglycosylase action on the cell wall of gram-negative bacteria) and N-acetyl-beta-D-glucosamine-(1-->4)-1,6-anhydro-N-acetyl-beta-D-muramyl-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala (compound 2, from lytic transglycosylase action on the cell wall of gram-positive bacteria). The syntheses were accomplished in 15 linear steps. Compound 1 is shown to be a substrate of the AmpD enzyme of the gram-negative bacterium Escherichia coli, an enzyme that removes the peptide from the disaccharide scaffold in the early cytoplasmic phase of cell wall turnover.

The Listeria monocytogenes Sortase-B Recognizes Varied Amino Acids at Position 2 of the Sorting Motif

Sortases are bacterial enzymes that anchor surface proteins covalently to the peptidoglycan upon cleavage of a motif located at their C-terminal end. Motifs recognized by sortases of the class-B (SrtB) are defined by the consensus sequence NP(Q/K)(T/S)(N/G/S)(D/A). Evidence supporting this consensus is limited to IsdC of Staphylococcus aureus and Bacillus anthracis, cleaved at motifs NPQTN and NPKTG, respectively. In Listeria monocytogenes, StrB has two substrates, Lmo2185 and Lmo2186, containing NAKTN and NKVTN (or the overlapping sequence NPKSS) as putative sorting motifs. Some of these motifs do not match the consensus, because they lack either proline (P) at position 2 or glutamine/lysine (Q/K) at position 3. Here, we identified NPKSS as a sorting motif of Lmo2186 by monitoring anchoring to peptidoglycan of chimeras lacking each of its two predicted motifs. Motif-swapping experiments confirmed that NPKSS, but not NKVTN, could replace NAKTN for anchoring of an Lmo2185 chimera. Residue substitutions in the NPKSS sequence revealed the essentiality of proline at position 2 for recognition of this particular motif. Lysine at position 3 was however dispensable. Deletion of NAKTN, on the other hand, abrogated SrtB-mediated anchoring of the Lmo2185 chimera. NAKTN, therefore, represents an exception to the rule of a conserved proline in position 2 of the sorting motif. Taken together, our data indicate that proline is not absolutely required for substrate recognition by sortases of the class-B. In addition, they prove the capacity of a single sortase, as SrtB of L. monocytogenes, to recognize varied amino acids at position 2 of the sorting motif.

Differential immunostimulatory effects of Gram-positive bacteria due to their lipoteichoic acids

Lipoteichoic acid (LTA) is a major immunostimulating component in the cell wall of Gram-positive bacteria as lipopolysaccharide of Gram-negative bacteria. However, LTA is expressed on not only pathogenic but also nonpathogenic Gram-positive bacteria. In order to examine whether the immunostimulating potentials of Gram-positive bacteria are correlated with their LTAs, we prepared highly pure LTAs from Staphylococcus aureus (pathogenic), Bacillus subtilis (non-pathogenic), or Lactobacillus plantarum (beneficial). When a murine macrophage cell-line, RAW 264.7, was stimulated with heat-killed bacteria, both S. aureus and B. subtilis induced nitric oxide (NO) production in a dose-dependent manner while L. plantarum showed a minimal induction. Interestingly, purified LTAs from S. aureus and B. subtilis, but not from L. plantarum, were able to induce the production of NO. The differential inflammatory potentials of LTAs coincided with their abilities to activate Toll-like receptor 2 (TLR2), which is known to recognize Gram-positive bacteria and LTA, and transcription factors NF-κB and AP-1. Similar results were obtained with the expression of cytokines related to inflammation by RAW 264.7 and human peripheral blood mononuclear cells as well. The ability of LTA to induce TNF-α and NO production was abolished when the LTAs were treated with 0.2 N NaOH. Collectively, we suggest that the immunostimulating potentials of Gram-positive bacteria differ due to their LTAs with differential potencies in the stimulation of TLR2.

Frontal analysis microchip capillary electrophoresis to study the binding of ligands to receptors derivatized on magnetic beads

The model binding of the glycopeptide antibiotic teicoplanin (Teic) from Actinoplanes teichomyceticus, immobilized on magnetic microspheres, to D-Ala-D-Ala terminus peptides was assessed using microchip capillary electrophoresis (MCE) with continuous frontal analysis (FA). Teic is closely related to vancomycin (Van), historically, the drug of last resort used to treat many gram-positive bacterial infections. Glycopeptide antibiotics inhibit bacterial growth by binding to the D-Ala-D-Ala terminus on the cell wall of gram-positive bacteria via hydrogen bonds, thereby preventing the enzyme-mediated cross-linking of peptidoglycan and eventual cell death. In this work direct and competitive bead-based assays in a microfluidic chip are demonstrated. The binding constants obtained using the technique are comparable with values reported in the literature.

Lipoteichoic acid from Staphylococcus aureus increases matrix metalloproteinase 9 expression in RAW 264.7 macrophages: Modulation by A2A and A2B adenosine receptors

Peptidoglycan (PEG) and lipoteichoic acid (LTA) are the main constituents of Gram-positive bacteria cell wall and are described to modulate immune functions. Increased levels of matrix metalloproteinases (MMPs) were described in endotoxemia, suggesting that they participate to tecidual damage, multiple organs failure and vascular disfunction. Staphylococcus aureus PEG is described to increase MMPs 2 and 9 levels in plasma from rat and MMP 9 secretion by human neutrophils, however, the effect of LTA on MMPs is unknown. In this work, was evaluated the modulation of MMPs 2 and 9 expression and secretion in RAW 264.7 macrophages by LTA from S. aureus. The role of A2A and A2B adenosine receptors was also investigated. LTA increased MMP 9 expression and secretion at 12 h of treatment. The modulation of MMP 9 secretion was dose dependent, with maximal effect above 1 μg/ml. The inhibitor of mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway (U0126, 10 μM) prevented LTA stimulation of MMP 9 secretion; however, the inhibitors of p38 (SB203580, 10 μM) and Jun N-terminal kinase (JNK; SP600125, 10 μM) presented any effect. A2A and A2B adenosine receptors pharmacological blockade or gene knockdown resulted in exacerbated MMP 9 secretion, while an adenosine receptors agonist inhibited LTA-stimulated MMP 9 secretion. These results suggest that LTA increased MMP 9 secretion in macrophages could be involved in complications associated to S. aureus infections. Moreover, LTA modulation of MMP 9 is dependent on MEK/ERK pathway and is regulated by A2A and A2B adenosine receptors.

Magnetic microsphere-based methods to study the interaction of teicoplanin with peptides and bacteria

Teicoplanin (teic) from Actinoplanes teichomyceticus is a glycopeptide antibiotic used to treat many gram-positive bacterial infections. Glycopeptide antibiotics inhibit bacterial growth by binding to carboxy-terminal D-Ala-D-Ala intermediates in the peptidoglycan of the cell wall of gram-positive bacteria. In this paper we report the derivatization of magnetic microspheres with teic (teic-microspheres). Fluorescence-based techniques have been developed to analyze the binding properties of the microspheres to two D-Ala-D-Ala terminus peptides. The dissociation constant for the binding of carboxyfluorescein-labeled D-Ala-D-Ala-D-Ala to teic on microspheres was established via fluorimetry and flow cytometry and was determined to be 0.5 x 10(-6) and 3.0 x 10(-6) mol L(-1), respectively. The feasibility of utilizing microparticles with fluorescence methods to detect low levels (the limit of bacterial detection was determined to be 30 colon-forming units; cfu) of gram-positive bacteria has been demonstrated. A simple microfluidic experiment is reported to demonstrate the possibility of developing microsphere-based affinity assays to study peptide-antibiotic interaction.

Identification of the initial binding sites of α s2-casein f(183–207) and effect on bacterial membranes and cell morphology

The aim of this work was to identify the initial binding sites to the bacterial membranes of the antimicrobial peptide α s2-casein f(183–207) and also to acquire further insight into membrane permeabilization of this peptide. Furthermore, cell morphology was studied by transmission electron microscopy. In all the experiments, bovine LFcin was employed as a comparison. Results showed that initial binding sites of α s2-casein f(183–207) peptide were lipoteichoic acid in Gram-positive bacteria and lipopolysaccharide in Gram-negative. The peptide was able to permeabilize the outer and inner membranes. Moreover, the α s2-casein peptide f(183–207) generated pores in the outer membrane of Gram-negative bacteria and in the cell wall of Gram-positive bacteria. In the Gram-negative bacteria, f(183–207) originated cytoplasm condensation, and in the Gram-positive bacteria the cytoplasmic content leaked into the extracellular medium. Furthermore, the experiments of inner and outer membrane permeabilization performed with LFcin-B showed that this peptide also has the ability to permeabilize both the inner and outer membranes.

Are gram‐positive bacteria capable of electron transfer across their cell wall without an externally available electron shuttle?

The extensive contributions by Terry Beveridge to our understanding of the differences in cell wall organization with respect to structure, chemistry and compartmentalization between gram-positive and gram-negative bacteria are summarized. These contributions greatly aided in conceptualization of recent discoveries concerning electron export and import across cell walls of some gram-negative bacteria. Although electron export and import across the cell wall by any gram-positive has not been documented so far, Beveridge's observations and concepts concerning cell walls of gram-positive bacteria suggest potential mechanisms by which such electron transfer may occur.

Presentation of lipoteichoic acid potentiates its inflammatory activity

Lipoteichoic acid (LTA) is a major immunostimulatory molecule in the cell wall of Gram-positive bacteria. Adhesion of LTA to a polystyrene surface drastically increased its immunostimulatory potency in human whole blood in comparison to soluble LTA, although only 1% of the LTA had bound, as determined using rhodamine-labelled LTA. The release of the proinflammatory cytokines IL-1 β, TNF and IL-6 and the chemokines IL-8 and G-CSF was increased 2- to 10-fold, but IL-10 release was unaltered. This presentation effect was not shared by lipopolysaccharide (LPS) or other toll-like receptor 2 agonists and was less pronounced in polypropylene vessels. LTA did not induce cytokine release in silicone-coated borosilicate vessels, but covalent coupling of LTA to polystyrene beads restored cytokine induction in these vessels, indicating that presentation of LTA on a surface is in fact essential for its immunostimulatory potency. This novel aspect of presentation as a factor in the recognition of LTA may reflect the physiological situation in the bacterial cell wall, where LTA is anchored in the bacterial membrane and projects through the peptidoglycan. In practical terms, contamination of medical devices with components of Gram-positive bacteria may pose an underestimated inflammatory risk.

Role of Toll-like receptor 2 in innate resistance to Group B Streptococcus

The Gram-positive bacterium Group B Streptococcus (GBS) is an important cause of serious neonatal and adult infections. Toll-like receptor 2 (TLR2) recognizes components of the cell wall of Gram-positive bacteria and is critical for defense against certain invasive pathogens. In GBS, penicillin-binding protein 1a (PBP1a), encoded by ponA, is required for virulence. PBPs participate in cell wall synthesis and in previous studies; the absence of PBP1a was shown to result in subtle changes in the cell wall ultrastructure. Here, we examine the role of TLR2 in defense against GBS infection and the impact of mutation of ponA on TLR2-mediated host responses. We demonstrate TLR2-recognition of both wild-type (WT) GBS and the ponA mutant in vitro. TLR2 −/− mice were significantly more susceptible than WT mice to infection with either strain of GBS, indicating a crucial role for TLR2 in defense against GBS. Additionally, the ponA mutant was severely attenuated for virulence in both strains of mice. The mutation in ponA did not affect cytokine expression by WT or TLR2 −/− mice. These data indicate that TLR2 is required for host defense against GBS and this response is unaffected by the absence of PBP1a and the resultant changes in cell wall ultrastructure.

The density of negative charge in the cell wall influences two-component signal transduction in Bacillus subtilis

The Dlt system modulates the density of negative charge in the cell wall of Gram-positive bacteria by substituting anionic polymers (wall and lipoteichoic acids) with d-alanine. The htrA and htrB genes, regulated by the CssRS two-component system (TCS) and encoding membrane-associated protein quality control proteases, were expressed at strongly decreased levels in a mutant with defective Dlt (dltD : : miniTn10) as compared to the dlt(+) wild-type strain under a secretion stress condition (hypersecretion of AmyQ alpha-amylase). The level of HtrA protein in the extracellular proteome of the dltD mutant was decreased consistently. Expression from the promoter of the liaIHGFSR (yvqIHGFEC) operon (P(liaI)) is dependent on the LiaRS TCS. The Dlt defect increased the expression from P(liaI) under two stress conditions, AmyQ hypersecretion and treatment with a cationic antimicrobial peptide (LL-37), but decreased the expression in vancomycin-treated cells. Furthermore, Dlt inactivation enhanced the expression of the YxdJK-regulated yxdL gene in LL-37-treated cells. The increased net negative charge of the cell wall seems to cause varied and opposite effects on the expression of CssRS-, LiaRS- and YxdJK-regulated genes under different stress conditions. The results suggest that TCSs which sense misfolded proteins or peptides are modulated by the density of negative charge in the cell wall. The density of negative charge on the outer surface of the cell membrane did not have a similar effect on TCSs.

Sortase A as a Novel Molecular “Stapler” for Sequence-Specific Protein Conjugation

The Sortase family of transpeptidase enzymes catalyzes sequence-specific ligation of proteins to the cell wall of Gram-positive bacteria. Here, we describe the application of recombinant Staphylococcus aureus Sortase A to attach a tagged model protein substrate (green fluorescent protein) to polystyrene beads chemically modified with either alkylamine or the in vivo Sortase A ligand, Gly-Gly-Gly, on their surfaces. Furthermore, we show that Sortase A can be used to sequence-specifically ligate eGFP to amino-terminated poly(ethylene glycol) and to generate protein oligomers and cyclized monomers using suitably tagged eGFP. We find that an alkylamine can substitute for the natural Gly3 substrate, which suggests the possibility of using the enzyme in materials applications. The highly specific and mild Sortase A-catalyzed reaction, based on small recognition tags unlikely to interfere with protein expression, thus represents a useful addition to the protein immobilization and modification tool kit.

Acceptor Substrate Selectivity and Kinetic Mechanism of Bacillus subtilis TagA

Wall teichoic acids (WTAs) are anionic polymers that coat the cell walls of Gram-positive bacteria. Because they are essential for survival or virulence in many organisms, the enzymes involved in the biosynthesis of WTAs are attractive antibiotic targets. The first committed step in the WTA biosynthetic pathway in Bacillus subtilis is catalyzed by TagA, which transfers N-acetylmannosamine (ManNAc) to the C4 hydroxyl of a membrane-anchored N-acetylglucosaminyl diphospholipid (GlcNAc-pp-undecaprenyl, lipid I) to make ManNAc-beta-(1,4)-GlcNAc-pp-undecaprenyl (lipid II). We have previously shown that TagA utilizes an alternative substrate containing a saturated C(13)H(27) lipid chain. Here we use unnatural substrates and products to establish the lipid preferences of the enzyme and to characterize the kinetic mechanism. We report that TagA is a metal ion-independent glycosyltransferase that follows a steady-state ordered Bi-Bi mechanism in which UDP-ManNAc binds first and UDP is released last. TagA shares homology with a large family of bacterial glycosyltransferases, and the work described here should facilitate structural analysis of the enzyme in complex with its substrates.

The Ectodomain of the Viral Receptor YueB Forms a Fiber That Triggers Ejection of Bacteriophage SPP1 DNA

The irreversible binding of bacteriophages to their receptor(s) in the host cell surface triggers release of the naked genome from the virion followed by transit of viral DNA to the host cell cytoplasm. We have purified, for the first time, a receptor from a Gram-positive bacterium that is active to trigger viral DNA ejection in vitro. This extracellular region ("ectodomain") of the Bacillus subtilis protein YueB (YueB780) was a 7 S elongated dimer forming a 36.5-nm-long fiber. YueB780 bound to the tail tip of bacteriophage SPP1. Although a stable receptor-phage interaction occurred between 0 and 37 degrees C, complete blocking of phage DNA release or partial ejection events were observed at temperatures below 15 degrees C. We also showed that the receptor was exposed to the B. subtilis surface. YueB differed structurally from phage receptors from Gram-negative bacteria. Its properties revealed a fiber spanning the full length of the 30-nm-thick peptidoglycan layer. The fiber is predicted to be anchored in the cell membrane through transmembrane segments. These features, highly suitable for a virus receptor in Gram-positive bacteria, are very likely shared by a large number of phage receptors.