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.     

Hydrolysis of a Staphylococcus aureus cell wall peptidoglycan by 209 P lysoamidase

Hydrolysis of Staphylococcus aureus 209 P cell wall peptidoglycan was accompanied by the liberation of 1.3 mol of C-terminal and 1.2 mol of N-terminal glycine per mole of Glu as well as of 0.5 mol of N-terminal and 0.3 mol of C-terminal alanine. Gel chromatography on Sephadex G-25, ion-exchange chromatography on QAE-Sephadex A-50 and paper electrophoresis of S. aureus peptidoglycan hydrolysates gave seven homogeneous fractions; these fractions were structurally defined. Lysoamidase hydrolyzed bonds Mur-Ala, Gly-Gly and Mur-GlcN in the peptidoglycan molecule. Hydrolysis of glycan chains was accompanied by the formation of large fragments, (GlcN-Mur)9 and (GlcN-Mur)28. The lytic effect of lysoamidase on S. aureus peptidoglycan is coupled with bacteriolytic enzymes of lysoamidase: acetmuramyl amidase, glycyl--glycine endopeptidase and acetyl--muramidase.     

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.     

Exotoxin S secreted by internalized Pseudomonas aeruginosa delays lytic host cell death

The Pseudomonas aeruginosa toxin ExoS, secreted by the type III secretion system (T3SS), supports intracellular persistence via its ADP-ribosyltransferase (ADPr) activity. For epithelial cells, this involves inhibiting vacuole acidification, promoting vacuolar escape, countering autophagy, and niche construction in the cytoplasm and within plasma membrane blebs. Paradoxically, ExoS and other P. aeruginosa T3SS effectors can also have antiphagocytic and cytotoxic activities. Here, we sought to reconcile these apparently contradictory activities of ExoS by studying the relationships between intracellular persistence and host epithelial cell death. Methods involved quantitative imaging and the use of antibiotics that vary in host cell membrane permeability to selectively kill intracellular and extracellular populations after invasion. Results showed that intracellular P. aeruginosa mutants lacking T3SS effector toxins could kill (permeabilize) cells when extracellular bacteria were eliminated. Surprisingly, wild-type strain PAO1 (encoding ExoS, ExoT and ExoY) caused cell death more slowly, the time extended from 5.2 to 9.5 h for corneal epithelial cells and from 10.2 to 13.0 h for HeLa cells. Use of specific mutants/complementation and controls for initial invasion showed that ExoS ADPr activity delayed cell death. Triggering T3SS expression only after bacteria invaded cells using rhamnose-induction in T3SS mutants rescued the ExoS-dependent intracellular phenotype, showing that injected effectors from extracellular bacteria were not required. The ADPr activity of ExoS was further found to support internalization by countering the antiphagocytic activity of both the ExoS and ExoT RhoGAP domains. Together, these results show two additional roles for ExoS ADPr activity in supporting the intracellular lifestyle of P. aeruginosa; suppression of host cell death to preserve a replicative niche and inhibition of T3SS effector antiphagocytic activities to allow invasion. These findings add to the growing body of evidence that ExoS-encoding (invasive) P. aeruginosa strains can be facultative intracellular pathogens, and that intracellularly secreted T3SS effectors contribute to pathogenesis.     

Lytic ability, in relation to gram-positive microorganisms, of a preparation isolated from a Pseudomonas lytica culture

A lytic enzyme isolated from P. lytica was studied with respect to its effect on pathogenic grampositive and gramnegative organisms. All the grampositive organisms were lyzed by the enzyme to this or that extent. The cells of staphylococci were the most sensitive. The gramnegative organisms were resistant. It was suggested that the lytic enzyme could be used in preparing drugs for treating certain skin diseases caused by pathogenic staphylococci.     

The cell walls of Pseudomonas aeruginosa. General composition

1. Cell walls of Pseudomonas aeruginosa were prepared and analysed. 2. Separate preparations were found to be reproducible, e.g. the phosphorus contents of all batches lay in the range 2.0-2.1%. 3. Ninhydrin-positive compounds in hydrolysates accounted for 43-46% of the cell wall and 79-87% of the nitrogen of the cell wall. Examination of the results for individual ninhydrin-positive compounds showed that 5-15% of the cell wall was murein and about 30% protein. 4. Trypsin treatment of crude cell-wall preparations preferentially liberated arginine, lysine and leucine, but it was not clear whether these arose from cell-wall or cytoplasmic proteins.     

Intracellular peptide hydrolysis by Pseudomonas putida and Pseudomonas maltophilia.

Amino acids liberated by peptidase hydrolysis of di- and oligopeptides by Pseudomonas putida were measured by trinitrobenzenesulphonate assay and high voltage electrophoresis or paper chromatography followed by ninhydrin spray. Intact bacteria or periplasmic contents released by lysozyme treatment did not hydrolyse peptides. Subcellular fractionation showed that glycylmethionine peptidase activity was cytoplasmic. This enzyme had a Km of 2 mM, and was stimulated fivefold by I mM-Co2+. Crude peptidase extract did not cleave peptides with D-residues, acylated N-terminal amino groups or N-methylated peptide bonds but otherwise showed a wide specificity. Di- or tripeptides with blocked C-terminus were hydrolysed. Leucylleucine (12 mM) and leucylglycylglycine (10 mM) did not compete with glycylmethionine (1-2 mM) and glycylmethionylglycine (1-0 mM), respectively, for hydrolysis. Pseudomonas maltophilia also contained peptidase activity (0-84 mumol amino acid released from glycylmethionylglycine/min/mg protein). Peptidases of both P. putida and P. maltophilia were constitutive.     

Specificity of hydrolysis of bovine kappa-casein by cell envelope-associated proteinases from Lactococcus lactis strains.

The cell envelope-associated proteinases from Lactococcus lactis subsp. cremoris H2 (a PI-type proteinase-producing strain) and SK11 (a PIII-type proteinase-producing strain) both actively hydrolyze the kappa-casein component of bovine milk but with significant differences in the specificity of peptide bond hydrolysis. The peptide bonds Ala-23-Lys-24, Leu-32-Ser-33, Ala-71-Gln-72, Leu-79-Ser-80, Met-95-Ala-96, and Met-106-Ala-107 were cleaved by both proteinase types, although the relative rates of hydrolysis at some of these sites were quite different for the two proteinases. Small histidine-rich peptides were formed as early products of the action of the cell envelope-associated proteinases on kappa-casein, implicating this casein as a possible significant source of histidine, which is essential for starter growth. The major difference between the two proteinase types in their action on kappa-casein was in their ability to cleave bonds near the C-terminal end of the molecule. The bond Asn-160-Thr-161 and, to a lesser extent, the bond Glu-151-Val-152 were very rapidly cleaved by the PIII-type proteinase, whereas hydrolysis of these bonds by the PI-type proteinase was barely detectable (even after 24 h of digestion). Differential hydrolysis of kappa-casein at these sites by the two different proteinase types resulted in the formation of distinctive, high-M(r) products detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a lytic enzyme of Clostridium acetobutylicum that degrades choline-containing pneumococcal cell walls

Abstract A protein that degrades pneumococcal walls containing choline, but not ethanolamine, in the teichoic acids has been isolated and purified from supernatants obtained from cultures of Clostridium acetobutylicum . The analyses of the degradation products of [³H]choline-labeled cell walls treated with this enzyme indicated that the purified protein, showing an apparent M _r of 115 000, is an N-acetylmuramyl- l -alanine amidase. Our results also suggest that C. acetobutylicum contains choline in its cell wall.     

Isolation of atypical lipopolysaccharides from purified cell walls of Pseudomonas cepacia.

Wall fragments were prepared from two strains of Pseudomonas cepacia and from P. aeruginosa, and their contents of readily extractable lipid, pronase-digestible protein and lipopolysaccharide were measured. Lipopolysaccharide extracted from P. cepacia, although biologically active, contained no detectable 2-keto-3-deoxyoctonic acid, but contained phosphate, rhamnose, glucose, heptose and hexosamine in concentrations comparable to those found in P. aeruginosa.     

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.     

The preparation of immunogenic cell walls from a highly protective strain of Clostridium chauvoei.

Conventional methods for the preparation of cell walls of a highly protective strain of Clostridium chauvoei destroy the protective antigen. Bacteria were therefore lysed by the enzyme pronase instead of by the mechanical disintegration methods commonly employed. Final purification and separation of cell walls and membranes was achieved by equilibrium density-gradient centrifugation with sodium iodide in a zonal rotor. The resultant cell walls had a two-layered structure when seen in ultra-thin section and were highly immunogenic when used to immunize mice against challenge with C. chauvoei. Rabbit antisera raised against the cell walls provided passive protection against challenge in mice and the level of protection was not diminished by the absorption of all agglutinins from the sera. These results confirm previous observations that the protective antigen is a heatlabile cell wall antigen which stimulates the production of non-agglutinating protective antibody.     

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.     

Chemical composition of a preparation of the cell walls of Corynebacterium diphtheriae

The chemical characteristics of 6 batches of the preparation, obtained from the cell wall of C. diphtheriae grown in liquid and solid culture media, with respect to their content of nitrogen, hexoses, pentoses, total amino sugars, lipids and to the possible admixture of nucleic acids are presented. From the results of the chemical analysis of these batches their standardization according to the ratio of total amino sugars and pentoses to total nitrogen in C. diphtheriae cell-wall preparation is proposed.