Spin-label assay for lysozyme.

A spin-label assay for lysozyme, which is based on the enzymatic hydrolysis of spin-labeled peptidoglycan, is described. Hydrolysis of this polymer by lysozyme results in sharpening of the esr spectrum. The rate of spectral sharpening is a function of enzyme concentration. When the activities of hen egg-white and human lysozymes are compared by this method, human lysozyme is 3.5 times as active as the hen enzyme. The pH optima for both enzymes are pH 5.0. At this pH, the maximal activity for the hen egg-white lysozyme is observed at an ionic strength of 0.09. This assay is suitable for measuring lysozyme levels in biological fluids. It is a sensitive, continuous assay that measures muramidase activity on a defined substrate.     

Distinct functions of polysaccharide deacetylases in cell shape,neutral polysaccharide synthesis and virulence of Bacillus anthracis

Peptidoglycan deacetylases (PGNG‐dacs) belong to the Carbohydrate Esterase Family 4 (CE4) and have been described as required for bacterial evasion to lysozyme and innate immune responses. Interestingly, there is an unusual occurrence of 10 putative polysaccharide deacetylases, including five PGNG‐dacs, in the Bacillus sp. genomes, especially B. cereus and B. anthracis. To elucidate the physiological role of these multiple deacetylases, we employed genetic analysis and protein localization studies of five putative PGNG‐dacs from B. anthracis as well as biochemical analysis of their corresponding homologues from B. cereus. Our data confirm that three enzymes are PGNG‐dacs. While BA1977, associated with lateral peptidoglycan synthesis, is a bona fide peptidoglycan deacetylase involved in resistance to host lysozyme and required for full virulence, BA1961 and BA3679 participate in the biogenesis of the peptidoglycan during both elongation and cell division. Furthermore, two enzymes are important for neutral polysaccharide attachment to PG and consequently anchoring of S‐layer proteins (BA5436) and for polysaccharide modification (BA2944). Our results provide novel and fundamental insights into the function of polysaccharide deacetylases in a major bioterrorism agent.     

Peptidoglycan N-acetylglucosamine deacetylases from Bacillus cereus, highly conserved proteins in Bacillus anthracis

The genomes of Bacillus cereus and its closest relative Bacillus anthracis contain 10 polysaccharide deacetylase homologues. Six of these homologues have been proposed to be peptidoglycan N-acetylglucosamine deacetylases. Two of these genes, namely bc1960 and bc3618, have been cloned and expressed in Escherichia coli, and the recombinant enzymes have been purified to homogeneity and further characterized. Both enzymes were effective in deacetylating cell wall peptidoglycan from the Gram(+) Bacillus cereus and Bacillus subtilis and the Gram(-) Helicobacter pylori as well as soluble chitin substrates and N-acetylchitooligomers. However, the enzymes were not active on acetylated xylan. These results provide insight into the substrate specificity of carbohydrate esterase family 4 enzymes. It was revealed that both enzymes deacetylated only the GlcNAc residue of the synthetic muropeptide N-acetyl-D-glucosamine-(beta-1,4)-N-acetylmuramyl-L-alanine-D-isoglutamine. Analysis of the constituent muropeptides of peptidoglycan from B. subtilis and H. pylori resulting from incubation of the enzymes BC1960 and BC3618 with these polymers and subsequent hydrolysis by Cellosyl and mutanolysin, respectively, similarly revealed that both enzymes deacetylate GlcNAc residues of peptidoglycan. Kinetic analysis toward GlcNAc(2-6) revealed that GlcNAc4 was the favorable substrate for both enzymes. Identification of the sequence of N-acetychitooligosaccharides (GlcNAc(2-4)) following enzymatic deacetylation by using 1H NMR revealed that both enzymes deacetylate all GlcNAc residues of the oligomers except the reducing end ones. Enzymatic deacetylation of chemically acetylated vegetative peptidoglycan from B. cereus by BC1960 and BC3618 resulted in increased resistance to lysozyme digestion. This is the first biochemical study of bacterial peptidoglycan N-acetylglucosamine deacetylases.     

Identification of the namH gene, encoding the hydroxylase responsible for the N-glycolylation of the mycobacterial peptidoglycan

The peptidoglycan of most bacteria consists of a repeating disaccharide unit of beta-1,4-linked N-acetylmuramic acid and N-acetylglucosamine. However, the muramic acid moieties of the mycobacterial peptidoglycan are N-glycolylated, not N-acetylated. This is a rare modification seen only in the peptidoglycan of mycobacteria and five other closely related genera of bacteria. The N-glycolylation of sialic acids is a unique carbohydrate modification that has been studied extensively in eukaryotes. However, the significance of the N-glycolylation of bacterial peptidoglycan is unknown. The goal of this project was to identify the gene encoding the hydroxylase responsible for the N-glycolylation of the mycobacterial peptidoglycan. We developed a novel assay for the mycobacterial UDP-N-acetylmuramic acid hydroxylation reaction and demonstrated that Mycobacterium smegmatis has an enzyme activity that can convert UDP-N-acetylmuramic acid to UDP-N-glycolylmuramic acid. We identified the gene namH encoding the mycobacterial UDP-N-acetylmuramic acid hydroxylase by computer data base searching and motif comparisons with the eukaryotic enzymes responsible for the N-glycolyation of sialic acids. The namH gene is not essential for in vitro growth as we were successful in deleting the gene in M. smegmatis. The M. smegmatis mutant is devoid of UDP-N-acetylmuramic acid hydroxylase activity and synthesizes only N-acetylated muropeptide precursors. Furthermore, the mutant exhibits increased susceptibility to beta-lactam antibiotics and lysozyme. Our studies suggest that the N-glycolylation of mycobacterial peptidoglycan may play a role in lysozyme resistance or may contribute to the structural stability of the cell wall architecture.     

Detection of a gonococcal endo-beta-N-acetyl-D-glucosaminidase and its peptidoglycan cleavage site

Neisseria gonorrhoeae contains several hydrolases which may be responsible for gonococcal cell lysis. One of these enzymes, an endo-beta-N-acetyl-D-glucosaminidase, has been extracted from supernatants of sonicated gonococci and partially purified by ammonium sulfate precipitation and affinity and ion-exchange chromatography. This enzyme has a different specificity than egg white lysozyme and cleaves the beta 1 leads to 4 glycosidic linkage between N-acetylglucosamine and N-acetylmuramic acid in gonococcal peptidoglycan.     

Structural variation in the glycan strands of bacterial peptidoglycan

The normal, unmodified glycan strands of bacterial peptidoglycan consist of alternating residues of beta-1,4-linked N-acetylmuramic acid and N-acetylglucosamine. In many species the glycan strands become modified after their insertion into the cell wall. This review describes the structure of secondary modifications and of attachment sites of surface polymers in the glycan strands of peptidoglycan. It also provides an overview of the occurrence of these modifications in various bacterial species. Recently, enzymes responsible for the N-deacetylation, N-glycolylation and O-acetylation of the glycan strands were identified. The presence of these modifications affects the hydrolysis of peptidoglycan and its enlargement during cell growth. Glycan strands are frequently deacetylated and/or O-acetylated in pathogenic species. These alterations affect the recognition of bacteria by host factors, and contribute to the resistance of bacteria to host defence factors such as lysozyme.     

Structural Analysis of a Specialized Type III Secretion System Peptidoglycan-cleaving Enzyme

The Gram-negative bacterium enteropathogenic Escherichia coli uses a syringe-like type III secretion system (T3SS) to inject virulence or “effector” proteins into the cytoplasm of host intestinal epithelial cells. To assemble, the T3SS must traverse both bacterial membranes, as well as the peptidoglycan layer. Peptidoglycan is made of repeating N-acetylmuramic acid and N-acetylglucosamine disaccharides cross-linked by pentapeptides to form a tight mesh barrier. Assembly of many macromolecular machines requires a dedicated peptidoglycan lytic enzyme (PG-lytic enzyme) to locally clear peptidoglycan. Here we have solved the first structure of a T3SS-associated PG-lytic enzyme, EtgA from enteropathogenic E. coli. Unexpectedly, the active site of EtgA has features in common with both lytic transglycosylases and hen egg white lysozyme. Most notably, the β-hairpin region resembles that of lysozyme and contains an aspartate that aligns with lysozyme Asp-52 (a residue critical for catalysis), a conservation not observed in other previously characterized lytic transglycosylase families to which the conserved T3SS enzymes had been presumed to belong. Mutation of the EtgA catalytic glutamate, Glu-42, conserved across lytic transglycosylases and hen egg white lysozyme, and this differentiating aspartate diminishes type III secretion in vivo, supporting its essential role in clearing the peptidoglycan for T3SS assembly. Finally, we show that EtgA forms a 1:1 complex with the building block of the polymerized T3SS inner rod component, EscI, and that this interaction enhances PG-lytic activity of EtgA in vitro, collectively providing the necessary strict localization and regulation of the lytic activity to prevent overall cell lysis.     

Differentiation of bacterial autolysins by zymogram analysis

The use of zymograms in which the bacterial cell wall heteropolymer peptidoglycan is incorporated into the resolving gel of SDS-PAGE has led to the identification of various SDS stable peptidoglycan hydrolases (autolysins). To examine the specificity of autolysins with respect to O-acetylated peptidoglycan, a discontinuous SDS-PAGE system has been developed that operates under neutral conditions. [Bis(2-hydroxyethyl)imino]tris(hydroxymethyl)methane (Bis-Tris) buffers are employed with pH 6.8 and 6.3 for the separating and stacking gels, respectively, while the anode buffer N-2-acetamido-2-hydroxyethanesulfonic acid (Aces)-HCl and the Bis-Tris cathode buffer both had a pH of 6.8. These conditions resulted in a relative trailing ion mobility of 0.349 and 0.137 in the resolving and staking gel, respectively, under room temperature conditions. Peptides and proteins were resolved in the 3-100 kDa range with a 10% acrylamide resolving gel. Comparison of zymograms that incorporated unacetylated or chemically O-acetylated peptidoglycan revealed the specificity of hen egg-white lysozyme for the unacetylated material. A preliminary analysis of the autolysins produced by the urinary tract pathogen Proteus mirabilis indicated that some enzymes were specific for either O-acetylated or non-O-acetylated peptidoglycan while others displayed no clear preference toward either of the two substrates.     

A column chromatographic method for determination of plasma and erythrocyte levels of inosine and hypoxanthine

A column chromatographic separation of inosine and hypoxanthine in plasma and erythrocytic samples after deproteination by ultrafiltration, adsorption of the compounds onto charcoal, elution in pyridine/ethanol solution, and filtration by celite gel is described. Sephadex G-10 was used to separate the compounds with small but different molecular weights. Inosine and hypoxanthine values were determined by enzymatic spectrophotometric assay. In arterial, coronary venous, and erythrocyte samples the mean ± SD values for inosine were calculated as 1365 ± 560 nmol/liter of plasma, 915 ± 310 nmol/liter of plasma, and 8925 ± 6720 nmol/liter of blood, respectively. The corresponding values for hypoxanthine were 2525 ± 1950 nmol/liter, 1835 ± 1315 nmol/liter, and 11090 ± 7600 nmol/liter, respectively.     

Stimulation of PgdA‐dependent peptidoglycan N‐deacetylation by GpsB‐PBP A1 in Listeria monocytogenes

Listeria monocytogenes and other pathogenic bacteria modify their peptidoglycan to protect it against enzymatic attack through the host innate immune system, such as the cell wall hydrolase lysozyme. During our studies on GpsB, a late cell division protein that controls activity of the bi‐functional penicillin binding protein PBP A1, we discovered that GpsB influences lysozyme resistance of L. monocytogenes as mutant strains lacking gpsB showed an increased lysozyme resistance. Deletion of pbpA1 corrected this effect, demonstrating that PBP A1 is also involved in this. Susceptibility to lysozyme mainly depends on two peptidoglycan modifying enzymes: The peptidoglycan N‐deacetylase PgdA and the peptidoglycan O‐acetyltransferase OatA. Genetic and biochemical experiments consistently demonstrated that the increased lysozyme resistance of the ΔgpsB mutant was PgdA‐dependent and OatA‐independent. Protein‐protein interaction studies supported the idea that GpsB, PBP A1 and PgdA form a complex in L. monocytogenes and identified the regions in PBP A1 and PgdA required for complex formation. These results establish a physiological connection between GpsB, PBP A1 and the peptidoglycan modifying enzyme PgdA. To our knowledge, this is the first reported link between a GpsB‐like cell division protein and factors important for escape from the host immune system.     

Lytic transglycosylases: bacterial space-making autolysins

Lytic transglycosylases are an important class of bacterial enzymes that act on peptidoglycan with the same substrate specificity as lysozyme. Unlike the latter enzymes, however, the lytic transglycosylases are not hydrolases but instead cleave the glycosidic linkage between N-actetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anydromuramoyl product. They are ubiquitous in bacteria which produce a compliment of different forms that are responsible for creating space within the peptidoglycan sacculus for its biosynthesis and recycling, cell division, and the insertion of cell-envelope spanning structures, such as flagella and secretion systems. As well, the lytic transglyosylases may have a role in pathogenesis of some bacterial species. Given their important role in bacterial cell wall metabolism, the lytic transglycosylases may present an attractive new target for the development of broad-spectrum antibiotics.     

Fluorescamine-labelled cortical fragments as a substrate for lysozyme and initiation protein (spore-lytic enzyme)

A fluorescamine assay for the detection of a spore-lytic enzyme from Clostridium perfringens is described. The substrate is prepared by treatment of cortical fragments with fluorescamine which reacts with amino terminal groups in the peptidoglycan which are not cross-linked, presumably diaminopimelic acid. Treatment of the labelled substrate with lytic enzymes results in the release of soluble fluorescent products which can be easily measured in a basic fluorometer. The assay is very sensitive, inexpensive and reproducible. As little as 1 μg of lysozyme can be detected by this assay.     

Luminescent immobilized enzyme test systems for inorganic pyrophosphate: assays using firefly luciferase and nicotinamide-mononucleotide adenylyl transferase or adenosine-5'-triphosphate sulfurylase.

Inorganic pyrophosphate was measured by luminescence produced by a pyrophosphatase (NAD adenylyl-transferase or ATP sulfurylase) coimmobilized with firefly luciferase on Sepharose beads, with continuous flow of saturating concentrations of substrates (NAD plus luciferin or adenylophosphosulfate plus luciferin, respectively) and intermittent injections of samples containing pyrophosphate. In this scheme, the limiting substrate (pyrophosphate) is regenerated, a situation that is well suited to a bioluminescent assay. The instrumentation allowed for automation with a through-put of approximately one sample every 4 min. With standard solutions or samples that do not contain ATP, the sensitivity of the assay permits detection of less than 1 pmol pyrophosphate in a volume of 20 microliters (50 nmol/liter) with a coefficient of variation approximately equal to 4%. To assay biological samples, it was shown that endogenous ATP can be inactivated by oxidation with sodium periodate. Periodate treatment and quenching engenders dilution that limits the sensitivity to approximately 600 nmol/liter pyrophosphate in the starting material. The assay has been applied to the determination of intracellular pyrophosphate in human lymphocytes and to the measurement of nucleoside-triphosphate pyrophosphohydrolase in human fibroblasts. The variability of the assay was greater with biological samples than with standard samples, with a coefficient of variation of 15.3% in a series of determinations of intracellular pyrophosphate in a series of replicate lymphocyte lysates. Bioluminescent systems of coupled coimmobilized enzymes offer great promise for sensitive, safe, automated assaying of metabolites.     

Listeria monocytogenes Is Resistant to Lysozyme through the Regulation,Not the Acquisition,of Cell Wall-Modifying Enzymes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is highly resistant to lysozyme, a ubiquitous enzyme of the innate immune system that degrades cell wall peptidoglycan. Two peptidoglycan-modifying enzymes, PgdA and OatA, confer lysozyme resistance on L. monocytogenes; however, these enzymes are also conserved among lysozyme-sensitive nonpathogens. We sought to identify additional factors responsible for lysozyme resistance in L. monocytogenes. A forward genetic screen for lysozyme-sensitive mutants led to the identification of 174 transposon insertion mutations that mapped to 13 individual genes. Four mutants were killed exclusively by lysozyme and not other cell wall-targeting molecules, including the peptidoglycan deacetylase encoded by pgdA, the putative carboxypeptidase encoded by pbpX, the orphan response regulator encoded by degU, and the highly abundant noncoding RNA encoded by rli31. Both degU and rli31 mutants had reduced expression of pbpX and pgdA, yet DegU and Rli31 did not regulate each other. Since pbpX and pgdA are also present in lysozyme-sensitive bacteria, this suggested that the acquisition of novel enzymes was not responsible for lysozyme resistance, but rather, the regulation of conserved enzymes by DegU and Rli31 conferred high lysozyme resistance. Each lysozyme-sensitive mutant exhibited attenuated virulence in mice, and a time course of infection revealed that the most lysozyme-sensitive strain was killed within 30 min of intravenous infection, a phenotype that was recapitulated in purified blood. Collectively, these data indicate that the genes required for lysozyme resistance are highly upregulated determinants of L. monocytogenes pathogenesis that are required for avoiding the enzymatic activity of lysozyme in the blood.     

Bacteriolytic enzymes produced by actinomycetes. II. Biosynthesis and areas of practical application

The data on physiological conditions of the bacteriolytic enzyme formulation of actinomycetes, the population structure of producing cultures, the search of producers of enzymes able to hydrolyze the peptidoglycan of cellular walls of bacteria are reviewed. The fields of application of lytic enzymes in fundamental and applied microbiological investigations are pointed out. These enzymes are of considerable interest as potentially useful chemotherapeutics and food preservatives. They may be successfully used in biochemical and genetic investigation, in the study of peptidoglycan structure. The ability of bacteriolytic enzymes to cause the lysis of microorganisms resistant to the lysozyme action is of special importance. The application of these enzymes allows to work out gentle methods of lysis of bacterial cells used in various fields of microbiology.     

A rapid fluorimetric test for lysozyme with purified fluorescamine-labelled peptidoglycan

A sensitive and rapid fluorometric lysozyme assay is described. It is based on the hydrolysis of fluorescamine-labelled peptidoglycan from Micrococcus luteus cell walls. Lysozyme levels as low as 0.1 microgram can be detected.     

Evidence for the synthesis of soluble peptidoglycan fragments by protoplasts of Streptococcus faecalis.

Growing protoplasts of Streptococcus faecalis 9790 were found to synthesize and excrete soluble peptidoglycan fragments. The presence of soluble peptidoglycan derivatives in culture supernatants was determined by (i) incorporation of three different radioactively labeled precursors (L-lysine, D-alanine, and acetate) into products which, after hen egg-white lysozyme hydrolysis, had the same KD values on gel filtration as muramidase hydrolysis products of isolated walls; (ii) inhibition of net synthesis of these products by cycloserine and vancomycin; and (iii) identification of disaccharide-peptide monomer using the beta-elimination reaction, gel filtration, and high-voltage paper electrophoresis. Under the conditions of these experiments the presence of newly synthesized, acid-precipitable (macromolecular) peptidoglycan was not detected. The predominance of monomer (70 to 80%) in lysozyme digests of peptidoglycan synthesized by protoplasts was in sharp contrast to digest of walls from intact streptococci which contain mostly peptide cross-linked products. Biosynthesis and release of relatively uncross-linked, soluble peptidoglycan fragments by protoplasts was related to the absence of suitable, preexisting acceptor wall.     

Direct quantitation of the number of individual penicillin-binding proteins per cell in Escherichia coli.

The penicillin-binding proteins (PBPs) are a set of enzymes that participate in the terminal stages of bacterial peptidoglycan assembly. As their name implies, these proteins also covalently bind and are inhibited by beta-lactam antibiotics. Although many studies have examined the relative binding affinities of a number of beta-lactam antibiotics, a surprisingly small number of studies have addressed the absolute numbers of each of the PBPs present in the bacterial cell. In the present study, the PBP values initially reported in Escherichia coli almost 20 years ago by B. G. Spratt (Eur. J. Biochem. 72:341-352, 1977) were refined. The individual PBPs from a known number of bacteria radiolabeled with [3H]benzylpenicillin were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The radioactive bands were located, excised, and quantitatively extracted from the gel slices. The radioactivity was measured by scintillation counting, and the absolute disintegrations per minute were calculated. From the specific activity of the labeled penicillin, the absolute disintegrations per minute, and the CFU per milliliter, a determination of the number of each of the PBPs per cell was made. The measurements were performed on multiple samples to place statistical limits on the numbers obtained. The values for the individual PBPs found in E. coli deviated in several ways from the previously reported observations. Of particular significance is the higher number of molecules of PBP 2 and 3 observed, since these PBPs are known to participate in cell morphogenesis. The PBP content in both rich Luria broth medium and M9 minimal medium was determined, with the slower-growing cells in minimal medium possessing fewer of the individual PBPs per cell.     

Cell wall substrate specificity of six different lysozymes and lysozyme inhibitory activity of bacterial extracts

We have investigated the specificity of six different lysozymes for peptidoglycan substrates obtained by extraction of a number of gram-negative bacteria and Micrococcus lysodeikticus with chloroform/Tris-HCl buffer (chloroform/buffer). The lysozymes included two that are commercially available (hen egg white lysozyme or HEWL, and mutanolysin from Streptomyces globisporus or M1L), and four that were chromatographically purified (bacteriophage lambda lysozyme or LaL, bacteriophage T4 lysozyme or T4L, goose egg white lysozyme or GEWL, and cauliflower lysozyme or CFL). HEWL was much more effective on M. lysodeikticus than on any of the gram-negative cell walls, while the opposite was found for LaL. Also the gram-negative cell walls showed remarkable differences in susceptibility to the different lysozymes, even for closely related species like Escherichia coli and Salmonella Typhimurium. These differences could not be due to the presence of lysozyme inhibitors such as Ivy from E. coli in the cell wall substrates because we showed that chloroform extraction effectively removed this inhibitor. Interestingly, we found strong inhibitory activity to HEWL in the chloroform/buffer extracts of Salmonella Typhimurium, and to LaL in the extracts of Pseudomonas aeruginosa, suggesting that other lysozyme inhibitors than Ivy exist and are probably widespread in gram-negative bacteria.