Specificity of the action of lysoamidase on Staphylococcus aureus 209P cell walls

Specificity of Staphylococcus aureus 209P cell wall hydrolysis by the L1 and L2-bacteriolytic enzymes from lysoamidase lytic complex was studied. L1-peptidase was shown to display both glycyl-glycine endopeptidase and N-acetylmuramyl-L-alanine amidase enzymatic activities on the S. aureus peptidoglycan molecule, whereas L2-peptidase acts as N-acetylmuramyl-L-alanine amidase.     

The effect of bacteriolytic preparation lysoamidase on Staphylococcus aureus 209P cells

The effect of the bacteriolytic preparation "Lysoamidase" on Staphylococcus aureus 299 P was studied. The maximum activity of the preparation was observed at pH 8.0 ionic strength 0.01-0.02 M and 50-60 degrees of the incubation medium. The electron microscopic examination revealed that "Lysoamidase" hydrolyzed the cell wall in one or several points with the following osmotic shock and extrusion of the cytoplasm. In an isotonic solution (1 M sucrose) "Lysoamidase" caused protoplast formation.     

Isolation of Staphylococcus aureus protoplasts using lysoamidase

The effect of the bacteriolytic enzyme preparation, lysoamidase, on Staphylococcus aureus 209P cells was studied. The protoplast formation was examined by spectrophotometric, biochemical and electron microscopic methods. Optimal conditions for isolation of S. aureus protoplasts were chosen. The susceptibility of S. aureus cells to lysoamidase depended on the culture age: the maximum effect was observed in the logarithmic growth phase. The protoplast yield was 80% when 1 M sucrose was used as an osmotic stabilizer. Lysoamidase caused local disruptures of the staphylococcus cell walls, which resulted in the formation of osmotically fragile spheroplasts and the release of protoplasts into the medium. The protoplasts obtained could retain 85-90% of the respiration activity and were able of cell wall regeneration.     

Cross-linked peptidoglycan mediates lysostaphin binding to the cell wall envelope of Staphylococcus aureus

Staphylococcus simulans bv. staphylolyticus secretes lysostaphin, a bacteriocin that cleaves pentaglycine cross bridges in the cell wall of Staphylococcus aureus. The C-terminal cell wall-targeting domain (CWT) of lysostaphin is required for selective binding of this bacteriocin to S. aureus cells; however, the molecular target for this was unknown. We used purified green fluorescent protein fused to CWT (GFP-CWT) to reveal species-specific association of the reporter with staphylococci. GFP-CWT bound S. aureus cells as well as purified peptidoglycan sacculi. The addition of cross-linked murein, disaccharides linked to interconnected wall peptides, blocked GFP-CWT binding to staphylococci, whereas murein monomers or lysostaphin-solubilized cell wall fragments did not. S. aureus strain Newman variants lacking the capacity for synthesizing polysaccharide capsule (capFO), poly-N-acetylglucosamine (icaAC), lipoprotein (lgt), cell wall-anchored proteins (srtA), or the glycolipid anchor of lipoteichoic acid (ypfP) bound GFP-CWT similar to wild-type staphylococci. A tagO mutant strain, defective in the synthesis of polyribitol wall teichoic acid attached to the cell wall envelope, displayed increased GFP-CWT binding. In contrast, a femAB mutation, reducing both the amount and the length of peptidoglycan cross-linking (monoglycine cross bridges), showed a dramatic reduction in GFP-CWT binding. Thus, the CWT domain of lysostaphin directs the bacteriocin to cross-linked peptidoglycan, which also serves as the substrate for its glycyl-glycine endopeptidase domain.     

Regulation of the bacterial cell wall: isolation and characterization of peptidoglycan mutants of Staphylococcus aureus

Temperature-sensitive mutants of Staphylococcus aureus H which require 1.0 m NaCl for growth at 42 C can be divided into two major classes. Most of the mutants (class A) do not accumulate nucleotide precursors of cell wall biosynthesis in the absence of salt at the nonpermissive temperatures, whereas the class B mutants accumulate these precursors. The most extensively studied mutant RUS 1 (carrying peg-1) is defective in biosynthesis of peptidoglycan at the nonpermissive conditions as evidenced by: (i) reduced incorporation of cell wall precursors into peptidoglycan; (ii) accumulation of the nucleotide, uridine diphosphate (UDP) muramyl-l-alanyl-d-glutamic acid; (iii) reduced specific activity of UDP N-acetylmuramyl (MurNAc)-l-alanyl-d-glutamate: l-lysine ligase (EC 6.3.2.7); and (iv) an increased susceptibility to lysis with sodium dodecyl sulfate. Addition of 1.0 m NaCl reverses these defects with the exception of the specific activity of UDP-MurNAc-l-alanyl-d-glutamate: l-lysine ligase. Nevertheless, the structure of the cell wall is normal at the nonpermissive conditions if 1.0 m NaCl is present. An alteration in the binding of a fluorescent dye, 8-anilino-1-napthalene-4-sulfonic acid at the nonpermissive conditions in the absence of 1.0 m NaCl suggests that there may also be defects in the membrane in this strain.     

Cell wall active antibiotics reduce chromosomal DNA fragmentation by peptidoglycan hydrolysis in Staphylococcus aureus

Lysostaphin digestion of peptidoglycan (PG) from Staphylococcus aureus resulted in chromosomal DNA fragmentation by released DNase, as directly visualized in situ on isolated nucleoids. Nevertheless, DNA digestion was partially prevented by previous incubation with antibiotics that inhibit PG synthesis. This inhibitory effect was much more remarkable with glycopeptides vancomycin and mainly teicoplanin than with beta-lactams cloxacillin and ceftazidime. Therefore, inhibition of PG chain elongation has a more significant inhibition of DNA degradation than inhibition of PG cross-linking, possibly due to a reduction in DNase storage at the cell wall.     

Direct observation of Staphylococcus aureus cell wall digestion by lysostaphin

The advent of Staphylococcus aureus strains that are resistant to virtually all antibiotics has increased the need for new antistaphylococcal agents. An example of such a potential therapeutic is lysostaphin, an enzyme that specifically cleaves the S. aureus peptidoglycan, thereby lysing the bacteria. Here we tracked over time the structural and physical dynamics of single S. aureus cells exposed to lysostaphin, using atomic force microscopy. Topographic images of native cells revealed a smooth surface morphology decorated with concentric rings attributed to newly formed peptidoglycan. Time-lapse images collected following addition of lysostaphin revealed major structural changes in the form of cell swelling, splitting of the septum, and creation of nanoscale perforations. Notably, treatment of the cells with lysostaphin was also found to decrease the bacterial spring constant and the cell wall stiffness, demonstrating that structural changes were correlated with major differences in cell wall nanomechanical properties. We interpret these modifications as resulting from the digestion of peptidoglycan by lysostaphin, eventually leading to the formation of osmotically fragile cells. This study provides new insight into the lytic activity of lysostaphin and offers promising prospects for the study of new antistaphylococcal agents.     

Vancomycin derivative with damaged D-Ala-D-Ala binding cleft binds to cross-linked peptidoglycan in the cell wall of Staphylococcus aureus

Des-N-methylleucyl-4-(4-fluorophenyl)benzyl-vancomycin (DFPBV) retains activity against vancomycin-resistant pathogens despite its damaged d-Ala-d-Ala binding cleft. Using solid-state nuclear magnetic resonance (NMR), a DFPBV binding site in the cell walls of whole cells of Staphylococcus aureus has been identified. The cell walls were labeled with d-[1-(13)C]alanine, [1-(13)C]glycine, and l-[epsilon-(15)N]lysine. Internuclear distances from (19)F of the DFPBV to the (13)C and (15)N labels of the cell-wall peptidoglycan were determined by rotational-echo double-resonance (REDOR) NMR. The (13)C{(19)F} and (15)N{(19)F} REDOR spectra show that, in situ, DFPBV binds to the peptidoglycan as a monomer with its vancosamine hydrophobic side chain positioned near a pentaglycyl bridge. This result suggests that the antimicrobial activity of other vancosamine-modified glycopeptides depends upon both d-Ala-d-Ala stem-terminus recognition (primary binding site) and stem-bridge recognition (secondary binding site).     

Tertiary structure of Staphylococcus aureus cell wall murein

The recently described scaffold model of murein architecture depicts the gram-negative bacterial cell wall as a gel-like matrix composed of cross-linked glycan strands oriented perpendicularly to the plasma membrane while peptide bridges adopt a parallel orientation (B. A. Dmitriev, F. V. Toukach, K. J. Schaper, O. Holst, E. T. Rietschel, and S. Ehlers, J. Bacteriol. 185:3458-3468, 2003). Based on the scaffold model, we now present computer simulation studies on the peptidoglycan arrangement of the gram-positive organism Staphylococcus aureus, which show that the orientation of peptide bridges is critical for the highly cross-linked murein architecture of this microorganism. According to the proposed refined model, staphylococcal murein is composed of glycan and oligopeptide chains, both running in a plane that is perpendicular to the plasma membrane, with oligopeptide chains adopting a zigzag conformation and zippering adjacent glycan strands along their lengths. In contrast to previous models of murein in gram-positive bacteria, this model reflects the high degree of cross-linking that is the hallmark of the staphylococcal cell wall and is compatible with distinguishing features of S. aureus cytokinesis such as the triple consecutive alteration of the division plane orientation and the strictly centripetal mode of septum closure.     

Electron microscopy of Staphylococcus aureus cell wall lysis

Virgilio, Rafael (Escuela de Química y Farmacia, Universidad de Chile, Santiago, Chile), C. González, Nubia Mu?oz, and Silvia Mendoza. Electron microscopy of Staphylococcus aureus cell wall lysis. J. Bacteriol. 91:2018-2024. 1966.-A crude suspension of Staphylococcus aureus cell walls (strain Cowan III) in buffer solution was shown by electron microscopy to lyse slightly after 16 hr, probably owing to the action of autolysin. The lysis was considerably faster and more intense after the addition of lysozyme. A remarkable reduction in thickness and rigidity of the cell walls, together with the appearance of many irregular protrusions in their outlines, was observed after 2 hr; after 16 hr, there remained only a few recognizable cell wall fragments but many residual particulate remnants. When autolysin was previously inactivated by trypsin, there was a complete inhibition of the lytic action of lysozyme; on the other hand, when autolysin was inactivated by heat and lysozyme was added, a distinct decrease in the thickness of the cell walls was observed, but there was no destruction of the walls. The lytic action of lysozyme, after treatment with hot 5% trichloroacetic acid, gave rise to a marked dissolution of the structure of the cell walls, which became lost against the background, without, however, showing ostensible alteration of wall outlines. From a morphological point of view, the lytic action of autolysin plus lysozyme was quite different from that of trichloroacetic acid plus lysozyme, as shown by electron micrographs, but in both cases it was very intense. This would suggest different mechanisms of action for these agents.     

Arrangement of peptidoglycan in the cell wall of Staphylococcus spp.

The arrangement of peptidoglycan in the cell wall of Staphylococcus was observed with the newly developed freeze-fracture technique, using n-octanol instead of water as the freezing medium. The replica of the trichloroacetic acid-extracted cell wall (TCA-wall) showed two areas. One of them has a concentric circular structure, a characteristic surface structure of the staphylococcal cell wall, and the other showed an irregular and rough surface. The chemical analysis of the wall revealed that the TCA-wall consisted of mostly peptidoglycan. By digesting the TCA-wall with lysozyme, the circular structures were greatly disturbed, and they disappeared after 60 min of treatment. From these observations it can be expected that the peptidoglycan is arranged in a concentric circular manner in the newly generated cell wall of Staphylococcus.     

Effect of salvin on the synthesis of macromolecules in Staphylococcus aureus 209P

The effect of salvin and its active component, carnazolic acid, on the synthesis of macromolecular compounds in the cells of S. aureus 209P was studied. It was shown that the inhibitory action of salvin on the synthesis of peptidoglycane in the culture was defined by the presence of carnazolic acid in its composition. In the bactericidal concentration, carnazolic acid was twice as less active as salvin and inhibited incorporation of labeled precursors into RNA and protein. The findings grounded the conclusion that some nonidentified components of salvin with low antimicrobial activity contained in it in insignificant quantities had an additional inhibitory effect on the process. Comparative study of salvin and antibiotics with the known mechanisms of action such as benzyl penicillin or chloramphenicol revealed a certain similarity in the action of salvin and benzyl penicillin on incorporation of labeled precursors into the macromolecular compounds of S. aureus 209P.     

Stimulation of platelet apoptosis by peptidoglycan from Staphylococcus aureus 113

Peptidoglycan (PGN), a component of bacterial cell wall and belonging to "Microbe-Associated Molecular Patterns" (MAMP) triggers host reactions contributing to the pathophysiology of infectious disease. Host cell responses to PGN exposure include apoptosis. Bacterial infections may result in activation of blood platelets and thrombocytopenia. The present study explored, whether HPLC-purified fractions of PGNs from Staphylococcus aureus 113 triggers apoptosis of platelets. To this end platelets were exposed to PGN fractions and annexin-V binding determined to depict cell membrane scrambling, DiOC6 fluorescence to estimate depolarization of mitochondrial potential, Fluo-3AM staining for intracellular Ca(2+) activity ([Ca(2+)](i)) and immunofluorescence to quantify protein abundance of active caspase-3. As a result, a 30?min exposure to monomeric fraction (mPGN) (≥50?ng/ml) was followed by annexin-V binding, paralleled by increase of [Ca(2+)](i), mitochondrial depolarization, caspase-3 activation and integrin α(IIb)β(3) upregulation. The annexin-V binding was significantly blunted by anti-TLR-2 antibodies, in absence of extracellular Ca(2+), and by pancaspase inhibitor zVAD-FMK (1?μM). In conclusion, PGN triggers apoptosis of platelets in activation-dependent manner, characterized by mitochondrial depolarization, caspase-3 activation and cell membrane scrambling.     

The effect of salvin on the growth and ultrastructure of Staphylococcus aureus 209P

Antibiotic salvin obtained from Salvia officinalis has been studied for its effect on the growth and ultrastructure of Staphylococcus aureus 209P. The antibiotic in the sub-bacteriostatic concentration considerably elongates the lag-phase (up to 11-12 h) exerting no significant effect on the growth rate of the staphylococcus population as well as it prolongs duration of the exponential phase. The analysis of electronograms of staphylococcus cells subjected to the action of salvin in the concentrations similar to the minimal inhibitory concentration (MIC), has revealed the cell thinning, inhibition and destruction of the division. The introduction of 5MIC antibiotic into the exponentially grown culture made a cell wall considerably thinner, destructing its external layer; the number of lyzed cells sharply increased. The appearance of bodies not described previously with a membrane envelope and ribosomes as well as of mesosomal structures was observed.     

Model UF-2 mutant of Staphylococcus aureus 209 P for titrating Vibrio cholera enterotoxin

The identical character of the action of crude V. cholerae enterotoxin on the anaerobic dehydrogenases of the UV-2 mutant of S. aureus 209 p and the surviving culture of Ehrlich's carcinoma has been revealed. The range of this action is linked with the concentration of the toxin and varies from the stimulation of cell dehydrogenases to their complete suppression. The rapid method for the titration of the enterotoxin in the dehydrogenase suppression test with the use of the bacterial model is proposed.