Isolation and characterization of the peptidoglycans from selected gram-positive and gram-negative periodontal pathogens

The peptidoglycans from several Gram-negative and Gram-positive periodontal pathogens were isolated, purified, and characterized both morphologically and chemically. In addition, the effects of the mureolytic enzymes, lysozyme, M-1 N-acetyl-muramidase, and the AM-3 endopeptidase, on the peptidoglycans were examined. These enzymes were found to be highly effective in the degradation of the purified peptidoglycans; however, a Bacteroides capillus peptidoglycan-protein complex exhibited a greater resistance to these enzymes. Morphologically, the peptidoglycans consisted of large saccular sheets which, when viewed by scanning electron microscopy, contained numerous holes and tears. Chemically, the peptidoglycans consisted of muramic acid, glucosamine, alanine, glutamic acid, and meso-diaminopimelic acid (DAP). One Bacteroides species, Bacteroides gingivalis strain W, contained glycine and LL-DAP, suggestive of an indirectly cross-linked A3 gamma peptidoglycan.     

Peptidoglycan Loss During Hen Egg White Lysozyme-Inorganic Salt Lysis of Streptococcus mutans

Streptococcus mutans BHT was grown in Todd-Hewitt dialysate medium containing N-acetyl[¹⁴C]glucosamine for 6 to 11 generations. After treatment with cold and hot trichloroacetic acid and trypsin, 52 to 65% of the radioactivity remained present in insoluble peptidoglycan-containing residues. Hen egg white lysozyme or mutanolysin treatment of the peptidoglycan residues resulted in the release of 80 and 97%, respectively, of the ¹⁴C label to the supernatant fraction. Hydrochloric acid hydrolysates of such supernatants showed that essentially all of the radioactivity present in insoluble peptidoglycan fractions was present in compounds that comigrated on paper chromatography with glucosamine (~60%) or muramic acid (~30%). Treatment of whole cells with low and high concentrations of lysozyme alone resulted in losses of 45 and 70% of the insoluble peptidoglycan, respectively, yet release of deoxyribonucleic acid from cells was not detected. Sequential addition of appropriate concentrations of selected inorganic salts after lysozyme treatment did result in the liberation of deoxyribonucleic acid. Deoxyribonucleic acid release was correlated with a further release of peptidoglycan from the insoluble fraction. However, the total amount of peptidoglycan lost effected by the low concentration of lysozyme and NaSCN (lysis) was significantly less than the amount of peptidoglycan hydrolyzed by high concentrations of lysozyme alone (no lysis), suggesting that the overall amount of peptidoglycan lost did not correlate well with cellular lysis. The total amount of insoluble peptidoglycan lost at the highest salt concentrations tested was found to be greater than could be accounted for by lysozyme-sensitive linkages of the peptidoglycan, possibly implicating autolysins. The results obtained suggested that hydrolysis of peptidoglycan bonds in topologically localized, but strategically important, sites was a more significant factor in the sequence that results in loss of cellular integrity (lysis).     

Relationship of prostaglandin secretion by rabbit alveolar macrophages to phagocytosis and lysosomal enzyme release

The phospholipids of rabbit alveolar macrophages were pulse-labelled with [(14)C]-arachidonic acid, and the subsequent release of labelled prostaglandins was measured. Resting macrophages released measurable amounts of arachidonic acid, the prostaglandins E(2), D(2) and F(2alpha) and 6-oxoprostaglandin F(1alpha). Phagocytosis of zymosan increased the release of arachidonic acid and prostaglandins to 2.5 times the control value. In contrast, phagocytosis of inert latex particles had no effect on prostaglandin release. Indomethacin inhibited the release of prostaglandin, and, at high doses (20mug/ml), increased arachidonic acid release. Analysis of the cellular lipids showed that after zymosan stimulation the proportion of label was decreased in phosphatidylcholine, but not in other phospholipids or neutral lipids. Cytochalasin B, at a dose of 2mug/ml, inhibited the phagocytosis induced by zymosan but increased prostaglandin synthesis to 3.4 times the control. These data suggest that the stimulation of prostaglandin synthesis by zymosan is not dependent on phagocytosis. Exposure to zymosan also resulted in the release of the lysosomal enzyme, acid phosphatase. Furthermore, cytochalasin B augmented the zymosan-stimulated release of acid phosphatase at the same dose that stimulated prostaglandin synthesis. However, indomethacin, at a dose that completely inhibited prostaglandin synthesis, failed to block the lysosomal enzyme release. Thus despite some parallels between the release of prostaglandins and lysosomal enzymes, endogenous prostaglandins do not appear to mediate the release of lysosomal enzymes. The prostaglandins released from the macrophages may function as humoral substances affecting other cells.     

Extent of peptidoglycan O acetylation in the tribe Proteeae.

The degree of peptidoglycan O acetylation in 18 strains of the different genera of the tribe Proteeae (Proteus, Providencia, and Morganella) has been determined by high-performance liquid chromatography-based organic acid analysis of mild-base-released acetic acid and quantitation of peptidoglycan concentrations by simultaneous amino sugar-amino acid analysis using high-performance anion-exchange chromatography with pulsed amperometric detection. The N,O-diacetylmuramyl content of all isolated and purified peptidoglycans was greater than 29% and ranged up to 57% relative to total muramic acid concentration. Each of the O-acetylated peptidoglycans was found to be resistant to solubilization by hen egg white lysozyme.     

Peptidoglycan fragments elicit antibacterial protein synthesis in larvae of Manduca sexta

In several insect species, serum lysozyme and antibacterial peptide concentration increases after injection of bacteria and other foreign substances. The purpose of this study was to characterize the specificity of this induction in the tobacco hornworm, Manduca sexta. By 48 h after injection of killed bacteria, lysozyme activity was approximately tenfold greater than in untreated insects. This maximal response was observed after injection of every bacterial species tested and after injection of purified cell walls of Micrococcus luteus. A variety of acellular particles, soluble molecules, and bacterial cell wall components were either poor lysozyme inducers or elicited no change in lysozyme concentration. The polysaccharide zymosan from yeast cell walls was a moderate lysozyme inducer. Peptidoglycan from M. luteus cell walls was found to induce lysozyme to a level as great or greater than whole cell walls. Small fragments of peptidoglycan generated by hen egg white lysozyme digestion were isolated, partially characterized, and shown to be good inducers of lysozyme as well as other antibacterial peptides. It appears that peptidoglycan provides a signal that initiates antibacterial responses in the insect.     

Induction of spheroplasts in Capnocytophaga ochracea

A gentle technique for preparing spheroplasts of Capnocytophaga ochracea strain 25 is described. Cells in the exponential phase were washed with 1.0 M-NaCl, agitated in 1.0 M-NaCl for 2 h at 30 degrees C and exposed to lysozyme in a Tris/salts buffer, pH 7.0. This procedure resulted in 98% spheroplast formation with complete removal of the peptidoglycan layer as detected by both phase-contrast and electron microscopy in combination with chemical analysis.     

Regulation of B cell tolerance by macrophage-derived mediators: antagonistic effects of prostaglandin E2 and interleukin 1

A role for prostaglandins in the mechanism of B cell tolerance induction in normal adult mouse spleen cells was examined. Two inhibitors of the cyclooxygenase pathway of arachidonic acid metabolism, indomethacin and acetylsalicylic acid, abrogated hapten-specific B cell tolerance induction by trinitrophenyl-human gamma-globulin. Tolerance was fully restored by the addition of prostaglandin E2 (PGE2) at a concentration of greater than or equal to 6 nM. T cell-depleted spleen cells produced comparable amounts of PGE2 in culture, indicating that the tolerance promoting activity of PGE2 occurred with physiologically relevant concentrations. Depletion and reconstitution experiments indicated that macrophages in the spleen cell preparations completely accounted for both PGE2 production and the effects of indomethacin and acetylsalicylic acid on B cell tolerance induction. The macrophage product interleukin 1 (IL 1) was also found to alter B cell susceptibility to tolerance induction. Thus, human IL 1 containing monocyte supernatants and purified IL 1 were found to interfere with B cell tolerance induction when added to macrophage- and T cell-depleted splenic B cells. Tolerance was restored in such cultures by the addition of 10 nM PGE2. These experiments demonstrate that within mixed lymphoid populations macrophages through the release of mediators modulate B cell susceptibility to tolerance induction.     

Lysis of Streptococcus mutans by hen egg white lysozyme and inorganic sodium salts.

Streptococcus mutans BHT was grown in a synthetic medium containing radioactive thymidine to monitor deoxyribonucleic acid release. Kinetic experiments demonstrated that although lysozyme alone could not liberate deoxyribonucleic acid, cellular deoxyribonucleic acid was liberated from lysozyme-treated cells by addition of low concentrations of inorganic sodium salts. When the salts were tested for their ability to dislodge cell-bound tritiated lysozyme, the extent of the initial release of enzyme by individual anions correlated with the anion potency for deoxyribonucleic acid liberation (SCN- greater than ClO4- greater than I- greater than Br- greater than NO3- greater than Cl- greater than F-), although the total amount of lysozyme dislodged did not correspond directly with cell lysis. Differences in the effectiveness of anions (SCN-, HCO3-, Cl- and F-) in potentiating cell lysis could be enhanced or minimized by varying the lysozyme, anion, and bacterial cell concentrations. As the anion concentration was increased for each enzyme concentration and cell concentration, the lysis increased, in some cases markedly, until maximum levels of released deoxyribonucleic acid were attained. The maximum levels of lysis of SCN- and HCO3- were similar and were greater than those for Cl- and F-. In addition, the maximum levels were observed to increase for each of the anions as the concentration of lysozyme increased.     

Characteristics of the active and inactive variants of Actinomyces sp. 26-115, a producer of actinomycin C

Two natural variants, i.e. No. 1 and No. 2, not producing actinomycin were isolated from cultures of the actinomycin C-producing organism Actinomyces sp. 26-115. Variant No. 1 differed from the active variant by the growth dynamics and colony morphology. Variant No. 2 was close to the active variant by the growth dynamics. It was shown with electron microscopy that the cells of variant No. 1 differed from those of the active variant in the number and form of the mycelial septa, more even and compact structure of the cell walls and higher sensitivity to actinomycin. Still, they were more stable to the effect of lysozyme and ultrasound. The cell walls of the inactive variant No. 1 gradually lost teichoic acid during development, while the loss of peptidoglycan was observed only on transfer to the stationary phase. The cell walls of the active variant lost teichoic acid and peptidoglycan at the same time on transfer to the stationary phase. Peptidoglycans of both variants contained diaminopimelic acid (the configuration of which was not determined) and glycine (1:1) as differentiating amino acids. The two adjacent tetrapeptides were joined with one glycine radical. The peptidoglycan peptide chains of both variants contained muramic, glutamic and diaminopimelic acids and alanine (1:1:1:2). The peptidoglycans of the inactive variant No. 1 contained in addition valine and isoleucine. However, it is hardly probable that they are contained by the peptidoglycan peptide chains.     

Intraperiplasmic growth of Bdellovibrio bacteriovorus 109J: attachment of long-chain fatty acids to escherichia coli peptidoglycan.

During the initial stages of intraperiplasmic growth of Bdellovibrio bacteriovorus on Escherichia coli, the peptidoglycan of the E. coli becomes acylated with long-chain fatty acids, primarily palmitic acid (60%) and oleic acid (20%). The attachment of the fatty acids to the peptidoglycan involves a carboxylic-ester bond, i.e., they were removed by treatment with alkaline hydroxylamine. Their linkage to the peptidoglycan does not involve a protein molecule. When the bdelloplast peptidoglycan was digested with lysozyme, the fatty acid-containing split products behaved as lipopeptidoglycan, i.e., they were extracted into the organic phase of 1-butanol:acetic acid:water (4:15) two-phase system; all of the lysozyme split products generated from normal E. coli peptidoglycan were extracted into the water phase. It is suggested that the function of the acylation reaction is to help stabilize the bdelloplast outer membrane against osmotic forces. In addition, a model is presented to explain how a bdellovibrio penetrates, stabilizes, and lyses a substrate cell.     

Regulation of prostaglandin synthesis and of the selective release of lysosomal hydrolases by mouse peritoneal macrophages.

Macrophages isolated from the peritoneal cavity of untreated mice and maintained in tissue culture synthesize and release prostaglandins when challenged with zymosan. These cells also selectively release lysosomal acid hydrolases under the same conditions. The major prostaglandins released into the media are found to be prostaglandins E1, E2 and 6-oxoprostaglandin F1a, whereas prostaglandin F2a is not detected. Macrophages isolated from mice that have received an intraperitoneal injection of thioglycollate broth are far less responsive to zymosan challenge. These cells require 300 microgram of zymosan to synthesize and release one-third the amount of prostaglandins released from non-stimulated macrophages exposed to 50 microgram of zymosan. In addition, thioglycollate-stimulated macrophages release less than 10% of their lysosomal acid hydrolases when exposed to 300 microgram of zymosan whereas non-stimulated cells release approximately 50% of these enzymes after treatment with 50 microgram of zymosan. The zymosan-stimulated synthesis and release of prostaglandins are completely inhibited by indomethacin, whereas the increased selective release of lysosomal acid hydrolases is not affected. Macrophages, unlike fibroblasts, do not synthesize and release prostaglandins when exposed to serum or to bradykinin.     

Structure of Bordetella pertussis peptidoglycan.

Bordetella pertussis Tohama phases I and III were grown to the late-exponential phase in liquid medium containing [3H]diaminopimelic acid and treated by a hot (96 degrees C) sodium dodecyl sulfate extraction procedure. Washed sodium dodecyl sulfate-insoluble residue from phases I and III consisted of complexes containing protein (ca. 40%) and peptidoglycan (60%). Subsequent treatment with proteinase K yielded purified peptidoglycan which contained N-acetylglucosamine, N-acetylmuramic acid, alanine, glutamic acid, and diaminopimelic acid in molar ratios of 1:1:2:1:1 and less than 2% protein. Radiochemical analyses indicated that 3H added in diaminopimelic acid was present in peptidoglycan-protein complexes and purified peptidoglycan as diaminopimelic acid exclusively and that pertussis peptidoglycan was not O acetylated, consistent with it being degraded completely by hen egg white lysozyme. Muramidase-derived disaccharide peptide monomers and peptide-cross-linked dimers and higher oligomers were isolated by molecular-sieve chromatography; from the distribution of these peptidoglycan fragments, the extent of peptide cross-linking of both phase I and III peptidoglycan was calculated to be ca. 48%. Unambiguous determination of the structure of muramidase-derived peptidoglycan fragments by fast atom bombardment-mass spectrometry and tandem mass spectrometry indicated that the pertussis peptidoglycan monomer fraction was surprisingly homogeneous, consisting of greater than 95% N-acetylglucosaminyl-N-acetylmuramyl-alanyl-glutamyl-diaminopimelyl++ +-alanine.     

Structure of the rigid-layer of rhizobium cell wall. II. Evidence for a covalent bond between peptidoglycan and cellodextrins

Covalent linkages between peptidoglycan and cellodextrins in the cell walls of Rhizobium were defined by the analysis of lysozyme split products. Digestion of peptidoglycan with lysozyme resulted in the liberation, beside disaccharide tetrapeptide fragments composed of glucosamine, muramic acid, alanine, glutamic acid and diaminopimelic acid in a molar ratio 1:1:2:1:1, also significant amounts of glucose and its polymers. The neutral carbohydrates composed of glucose, were further purified and determined as cellobiose, cellotriose and cellotetrose. Peptidoglycans pretreated with cellulase, which librated glucose and cellobiose, still contains glucose linked by lysozyme sensitive but cellulase insensitive bond.     

Chemical Composition of the Cell Walls of Clostridium botulinum Type A

Cell walls were isolated by sonic disruption of log-phase cells of Clostridium botulinum type A strain 190L and purified by treatment with sodium dodecyl sulfate (SDS) followed by digestion with proteases. Electron microscopy revealed that the cell walls thus obtained were free of both cytoplasmic membrane and cytoplasmic fragments. The purified cell wall contained 8.7% total nitrogen, 15.0% total hexosamines, 22.4% reducing groups, 8.3% carbohydrate, and 3.1% glucose. The content of total phosphorus was very low (0.02%), and therefore it was expected that teichoic acid might be absent in the cell wall. The wall peptidoglycan contained glutamic acid, alanine, diaminopimelic acid, glucosamine and muramic acid in the molar ratios of 1.00:1.85:0:85:1.06:0.67. A low amount of galactosamine was also present, but no other amino acids were found in significant quantities. The SDS-treated cell walls were not attacked by lysozyme, but after extraction with hot formamide they were completely dissolved by the enzyme and released reducing groups. The lysozyme digest was separated into two constituents, the saccharide moiety and the peptide moiety on Sephadex G-50.     

Lysozyme in the pericardial complex of Manduca sexta

The pericardial cell-heart complex (pericardial complex) of fifth instar Manduca sexta larvae has been shown to contain, to synthesize and to release lysozyme. Lysozyme activity was present in homogenates of pericardial complex. Immunocytochemical analysis demonstrated that lysozyme in the pericardial complex was located in pericardial cells. Injection of peptidoglycan elicitors, which markedly increase levels of hemolymph lysozyme, also elevated lysozyme activity in homogenates of pericardial complex, but only moderately. Lysozyme synthesis in the pericardial complex was demonstrated in vitro by the incorporation of [³H]leucine into immunoprecipitable lysozyme. This tissue did exhibit an increase in the release of a variety of newly synthesized proteins but not a selective increase in the synthesis and release of lysozyme after peptidoglycan stimulation.In similar experiments, cultured fat body from naive larvae incorporated [³H]leucine into secreted, immunoprecipitable lysozyme at a rate 100-fold greater than that observed for pericardial complex and exhibited a selective increase in lysozyme synthesis and release to 6.5 times its basal level when stimulated with peptidoglycan.We conclude that, of these two tissues, fat body is the primary source of hemolymph lysozyme. On the other hand, pericardial cell lysozyme may function in the intracellular, lysosomal degradation of pinocytosed fragments of bacterial invaders.     

The influence of phenelzine and tranylcypromine on the release of prostaglandins from the rat mesenteric vascular bed

We investigated the effects of phenelzine and tranylcypromine on the release of prostacyclin, thromboxane A2, prostaglandin E2, and prostaglandin E1 from the isolated perfused rat mesenteric vascular bed. Perfusion of the preparation with phenelzine in concentrations of 15, 45, and 135 microM for 150 min led to attenuated release of all four prostaglandins measured. Inhibition generally occurred with the lowest dose used and was most prominent with the highest concentration. Tranylcypromine also decreased prostaglandin formation. However, low doses were not effective in the suppression of prostacyclin release. Both drugs had an inhibitory effect on production of prostaglandin E1, which is a metabolite of dihomo-gamma-linolenic acid, the precursor of arachidonic acid, but this was only shown to be significant with phenelzine. In this work we demonstrate that phenelzine and tranylcypromine have an inhibitory effect on the production of 2-series prostaglandins derived from arachidonic acid, and possibly a similar effect on prostaglandins of the 1-series derived from dihomo-gamma-linolenic acid.     

Cell wall changes in nisin-resistant variants of Listeria innocua grown in the presence of high nisin concentrations

Abstract Two nisin-resistant variants of a strain of Listeria innocua were isolated after growth in the presence of 500 and 4000 IU ml⁻¹ of nisin A showed increased cell wall hydrophobicity, resistance to phage attack and three different cell wall-acting antibiotics, as well as to the peptidoglycan hydrolytic enzymes lysozyme and mutanolysin, as compared to the parental strain. Transmission electron microscopy revealed marked thickening of the wall of nisin-resistant cells with an irregular surface. Differences in thickness were lost after cell wall purification and no significant difference in gross wall composition was observed between the parental and resistant variants. Cell wall changes in nisin-resistant listeriae are attributed to abnormal cell wall synthesis and autolysin inhibition, the latter possibly associated with subtle changes in cell wall structures and function.     

A fast screening method for surface layers on Gram-positive bacteria

A two-step screening method is described to identify regularly arranged surface layers (S layers) on Gram-positive bacterial strains. A non-destructive release of S-layer sheets is achieved by enzymatic hydrolysis of the underlying peptidoglycan using lysozyme. The existence of regular S layers is then directly confirmed by scanning force microscopy or transmission electron microscopy. This method requires a minimal amount of bacterial cells and may be used as a `quick test' for demonstrating the presence of S layers.     

First report of a bifunctional chitinase/lysozyme produced by Bacillus pumilus SG2

Bacillus pumilus SG2 isolated from high salinity ecosystem in Iran produces two chitinases (ChiS and ChiL) and secretes them into the medium. In this study, chiS and chiL genes were cloned in pQE-30 expression vector and were expressed in the cytoplasm of Escherichia coli strain M15. The recombinant proteins were purified using Ni-NTA column. The optimum pH and optimum temperature for enzyme activity of ChiS were pH 6, 50°C; those of ChiL were pH 6.5, 40°C. The purified chitinases showed antifungal activity against Fusarium graminearum, Rhizoctonia solani, Magnaporthe grisea, Sclerotinia sclerotiorum, Trichoderma reesei, Botrytis cinerea and Bipolaris sp. Moreover, purified ChiS was identified as chitinase/lysozyme, which are capable of degrading the chitin component of fungal cell walls and the peptidoglycan component of cell walls with many kinds of bacteria (Xanthomonas translucens pv. hordei, Xanthomonas axonopodis pv. citri, Bacillus licheniformis, E. coli C600, E. coli TOP10, Pseudomonas aeruginosa and Pseudomonas putida). Strong homology was found between the three-dimensional structures of ChiS and a chitinase/lysozyme from Bacillus circulans WL-12. This is the first report of a bifunctional chitinase/lysozyme from B. pumilus.     

Analytical subcellular fractionation of alveolar macrophages from normal and BCG-vaccinated rabbits with particular reference to heterogeneity of hydrolase-containing granules.

Macrophages were obtained by pulmonary lavage from normal rabbits or rabbits that had developed pulmonary granulomas after receiving intravenous BCG vaccine 2-3 weeks earlier. The cells were disrupted in iso-osmotic sucrose and a low-speed supernatant was fractionated by isopycnic centrifugation on a linear sucrose density gradient. Three populations of hydrolase-containing granules (putative lysosomes) were found in both normal and BCG-induced macrophages. They were distinguished by their different distributions in the gradient and different sensitivities to disruption by digitonin and were termed:type A, containing lysozyme; type B, containing N-acetyl-beta-glucosaminidase, beta-glactosidase, beta-glucuronidase and possibly some lysozyme; type C, containing cathepsin D. Acid phosphatase appeared to be about equally distributed between type B and C granules. Type A and B granules from BCG-induced macrophages showed markedly greater equilibrium density than did those from normal macrophages. Beta-glucuronidase and acid phosphatase had greater specific activity in the induced cells.