Inactivation of Gram-Negative Bacteria by Lysozyme, Denatured Lysozyme, and Lysozyme-Derived Peptides under High Hydrostatic Pressure

We have studied the inactivation of six gram-negative bacteria (Escherichia coli, Pseudomonas fluorescens, Salmonella enterica serovar Typhimurium, Salmonella enteritidis, Shigella sonnei, and Shigella flexneri) by high hydrostatic pressure treatment in the presence of hen egg-white lysozyme, partially or completely denatured lysozyme, or a synthetic cationic peptide derived from either hen egg white or coliphage T4 lysozyme. None of these compounds had a bactericidal or bacteriostatic effect on any of the tested bacteria at atmospheric pressure. Under high pressure, all bacteria except both Salmonella species showed higher inactivation in the presence of 100 μg of lysozyme/ml than without this additive, indicating that pressure sensitized the bacteria to lysozyme. This extra inactivation by lysozyme was accompanied by the formation of spheroplasts. Complete knockout of the muramidase enzymatic activity of lysozyme by heat treatment fully eliminated its bactericidal effect under pressure, but partially denatured lysozyme was still active against some bacteria. Contrary to some recent reports, these results indicate that enzymatic activity is indispensable for the antimicrobial activity of lysozyme. However, partial heat denaturation extended the activity spectrum of lysozyme under pressure to serovar Typhimurium, suggesting enhanced uptake of partially denatured lysozyme through the serovar Typhimurium outer membrane. All test bacteria were sensitized by high pressure to a peptide corresponding to amino acid residues 96 to 116 of hen egg white, and all except E. coli and P. fluorescens were sensitized by high pressure to a peptide corresponding to amino acid residues 143 to 155 of T4 lysozyme. Since they are not enzymatically active, these peptides probably have a different mechanism of action than all lysozyme polypeptides.     

Identification and isolation of a bactericidal domain in chicken egg white lysozyme

Chicken egg white lysozyme exhibits antimicrobial activity against both Gram-positive and Gram-negative bacteria. Fractionation of clostripain-digested lysozyme yielded a pentadecapeptide with antimicrobial activity but without muramidase activity. The peptide was isolated and its sequence found to be I-V-S-D-G-N-G-M-N-A-W-V-A-W-R (amino acids 98-112 of chicken egg white lysozyme). A synthesized peptide of identical sequence had the same bactericidal activity as the natural peptide. Replacement of Trp 108 with tyrosine significantly reduced the antibacterial capacity of the peptide. By replacement of Trp 111 with tyrosine the antibacterial activity was lost. Replacement of Asn 106 with the positively charged arginine strongly increased the antibacterial capacity of I-V-S-D-G-N-G-M-N-A-W-V-A-W-R. The peptide I-V-S-D-G-N-G-M consisting of the eight amino acids of the N-terminal side had no bactericidal properties, whereas the peptide N-A-W-V-A-W-R of the C-terminal side retained some bactericidal activity. Replacement of asparagine 106 by arginine (R-A-W-V-A-W-R) increased the bactericidal activity considerably. The D enantiomer of R-A-W-V-A-W-R was as active as the L form against five of the tested bacteria, but substantially less active against Serratia marcescens, Micrococcus luteus, Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus lentus. For these bacterial species some stereospecific complementarity between receptor structures of the bacteria and the peptide can be assumed.     

Inactivation of gram-negative bacteria by lysozyme, denatured lysozyme, and lysozyme-derived peptides under high hydrostatic pressure

We have studied the inactivation of six gram-negative bacteria (Escherichia coli, Pseudomonas fluorescens, Salmonella enterica serovar Typhimurium, Salmonella enteritidis, Shigella sonnei, and Shigella flexneri) by high hydrostatic pressure treatment in the presence of hen egg-white lysozyme, partially or completely denatured lysozyme, or a synthetic cationic peptide derived from either hen egg white or coliphage T4 lysozyme. None of these compounds had a bactericidal or bacteriostatic effect on any of the tested bacteria at atmospheric pressure. Under high pressure, all bacteria except both Salmonella species showed higher inactivation in the presence of 100 microg of lysozyme/ml than without this additive, indicating that pressure sensitized the bacteria to lysozyme. This extra inactivation by lysozyme was accompanied by the formation of spheroplasts. Complete knockout of the muramidase enzymatic activity of lysozyme by heat treatment fully eliminated its bactericidal effect under pressure, but partially denatured lysozyme was still active against some bacteria. Contrary to some recent reports, these results indicate that enzymatic activity is indispensable for the antimicrobial activity of lysozyme. However, partial heat denaturation extended the activity spectrum of lysozyme under pressure to serovar Typhimurium, suggesting enhanced uptake of partially denatured lysozyme through the serovar Typhimurium outer membrane. All test bacteria were sensitized by high pressure to a peptide corresponding to amino acid residues 96 to 116 of hen egg white, and all except E. coli and P. fluorescens were sensitized by high pressure to a peptide corresponding to amino acid residues 143 to 155 of T4 lysozyme. Since they are not enzymatically active, these peptides probably have a different mechanism of action than all lysozyme polypeptides.     

Lysozyme‐associated bactericidal activity in the ejaculate of a wild passerine

Numerous antibacterial substances have been identified in the ejaculates of animals and are suggested to protect sperm from bacterial‐induced damage in both the male and female reproductive tracts. Lysozymes, enzymes that exhibit bactericidal activity through their ability to break down bacterial cell walls, are likely to be particularly important for sperm defence as they are part of the constitutive innate immune system and are thus immediately available to protect sperm from bacterial attack. Birds are an ideal model for studies of ejaculate antimicrobial defences because of the dual function of the avian cloaca (i.e. waste excretion and sperm transfer), yet the antibacterial activity of avian ejaculates remains largely unexplored, and data on ejaculate lysozyme levels are only available for the domestic turkey (Meleagris gallopavo). Moreover, ejaculate lysozyme levels have not been reported for any species in the wild; which many argue is necessary to gain a comprehensive understanding of the function and dynamics of immune responses. Here, we show that lysozyme is present in the ejaculate of a wild passerine, the superb fairy‐wren (Malurus cyaneus), and that the concentration of lysozyme in ejaculates varies substantially among males. This variation, however, is not associated with male condition, sperm quality or plumage coloration. Nevertheless, we suggest that lysozyme‐associated antibacterial activity in ejaculates may be the target of natural and sexual selection and that these enzymes are likely to function in defending avian sperm from bacterial‐induced damage. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 92–100.     

Über die Bildung von Bakterienhemmstoffen durch Cosmarium impressulum

1. An already published test method for detecting bactericidal substances in paper chromatograms was further improved. 2. In cultures of Cosmarium impressulum free from bacteria, two bactericidal substances were found in the ether extracts from the algae and two others in the extracts of the culture medium. The are active against some or all bacteria testes (Serratia marcescens, Pseudomonas fluorescens; Aerobacter aerogenes or Bacillus pumilus). 3. If the culture medium of Cosmarium or another desmidiale was inoculated with the test bacteria, a clear bactericidal effect was never observed. 4. Because the activity of the bactericidal substances of Cosmarium is only small and the formation is not constant, it is concluded that the water-soluble bactericidal substances of the alga are not the cause that epiphytic bacteria do not grow normally on Cosmarium.     

The Amino Acid Sequence of Wood Duck Lysozyme

The amino acid sequence of wood duck (Aix sponsa) lysozyme was analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had the highest similarity to duck III lysozyme with four amino acid substitutions, and had eighteen amino acid substitutions from chicken lysozyme. The valine at position 75 was newly detected in chicken-type lysozymes. In the active site, Tyr34 and Glu57 were found at subsites F and D, respectively, when compared with chicken lysozyme.     

Purification of Staphylocidal β-Lysin from Rabbit Serum

A cationic protein of rabbit serum bactericidal for Staphylococcus aureus was purified. The specific activity per unit of protein of the purified staphylocidal preparation was approximately 37,000 times greater than that of the serum from which it was isolated. Similar techniques were used to purify serum beta-lysin active against Bacillus subtilis approximately 24,000 times. The staphylocidal activity cannot be attributed to the same beta-lysin active against B. subtilis, lysozyme, or antibody-complement systems. The concentrations of staphylocidal beta-lysin in the sera of the five mammalian species studied did not correlate with their beta-lysin activities against B. subtilis. The two beta-lysins are similar in that both were heat-stable, sensitive to trypsin digestion, had molecular weights near 6,000, and were found in higher concentrations in serum than in plasma. Furthermore, similar techniques can be used to absorb and elute both substances in highly purified forms using cellulose asbestos filter pads and ion exchange chromatography on carboxymethyl cellulose. In contrast to the beta-lysin against B. subtilis, the staphylocidal beta-lysin was not released from blood platelets, and it was inactive in the presence of heparin, sodium citrate, sodium oxalate, ethylenediaminetetraacetic acid, acidic phospholipids, and acid pH values. A variety of proteins, including those of normal serum, preferentially inhibited the bactericidal activity of staphylocidal beta-lysin but not the beta-lysin against B. subtilis.     

Highly active fractions of the medicinal leech recombinant destabilase-lysozyme

Fractionation of the highly purified but low active recombinant protein destabilase-lysozyme (Dest-Lys) by cation exchange chromatography on a TSK CM 3-SW chromatography the non-active fraction (IV) containing 90% of total protein has been separated. Fractions I, II, and III contained proteins with lysozyme and isopeptidase activities and their lysozyme activity correlated with the activity of native Dest-Lys. However, the ratio of lysozyme and isopeptidase activities differed in these fractions; maximal lysozyme activity was found in fraction III, while maximal isopeptidase activity was associated with fraction I. Possible regulation of different functions of Dest-Lys is discussed in the context of formation of its various complexes.     

Antibacterial activity of <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> phage ϕAB2 endolysin (LysAB2) against both Gram-positive and Gram-negative bacteria

To investigate the nature and origin of the antibacterial activity of the lytic phage ϕAB2 toward Acinetobacter baumannii, we successfully isolated and characterized a novel phage lysozyme (endolysin) from ϕAB2 and named it LysAB2. To analyze antibacterial activity of LysAB2, the complete LysAB2 and two deletion derivatives were constructed, purified and characterized. Zymographic assays showed that only the intact LysAB2 could lyse the peptidoglycan of A. baumannii and the Staphylococcus aureus cell wall. Antibacterial analysis also showed that only the intact LysAB2 retained the complete bactericidal activity. When applied exogenously, LysAB2 exhibited a broad bacteriolytic activity against a number of Gram-negative and Gram-positive bacteria. Thermostability assays indicated that LysAB2 was stable at 20∼40°C. Its optimal pH was 6.0, and it was active from pH 4 to 8. Scanning electron microscopy revealed that exposure to 500 μg ml⁻¹ LysAB2 for up to 60 min caused a remarkable modification of the cell shape of the bacteria. Treating bacteria with LysAB2 clearly enhanced permeation of the bacterial cytoplasmic membrane. These results indicate that LysAB2 is an effective lysozyme against bacteria, and they suggest that it is a good candidate for a therapeutic/disinfectant agent to control nosocomial infections caused by multiple drug-resistant bacteria.     

Expression of functional recombinant human lysozyme in transgenic rice cell culture

Using particle bombardment-mediated transformation, a codon-optimized synthetic gene for human lysozyme was introduced into the calli of rice (Oryza sativa) cultivar Taipei 309. The expression levels of recombinant human lysozyme in the transformed rice suspension cell culture approached approximately 4% of total soluble protein. Recombinant human lysozyme was purified to greater than 95% homogeneity using a two-step chromatography process. Amino acid sequencing verified that the N-terminus of the mature recombinant human lysozyme was identical to native human lysozyme. This indicates that the rice RAmy3D signal peptide was correctly cleaved off from the human lysozyme preprotein by endogenous rice signal peptidase. Recombinant human lysozyme was found to have the same molecular mass, isoelectric point and specific activity as native human lysozyme. The bactericidal activity of recombinant human lysozyme was determined by turbidimetric assay using Micrococcus lysodeikticus in 96-well microtiter plates. The bactericidal activity of lysozyme on Gram-negative bacteria was examined by adding purified lysozyme to mid-log phase cultures of E. coli strain JM109. In this study, significant bactericidal activity was observed after E.coli cells were exposed to recombinant human lysozyme for 60min. Both native and recombinant human lysozyme displayed the same thermostability and resistance to degradation by low pH. The potential for using rice-derived lysozyme as an antimicrobial food supplement, particularly for infant formula and baby foods, is discussed.     

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.     

Purification and identification of antibacterial phenolics from Tripodanthus acutifolius leaves

Aims: To perform an activity‐guided purification, identification and quantification of antibacterial compounds from Tripodanthus acutifolius infusion. To validate the antibacterial activity of purified substances. Methods and Results: Bioautographic methods were employed as screening assays for purifying bioactive substances. Purification procedures included sephadex LH‐20 column chromatography and reverse phase HPLC. Identification was achieved by spectroscopic methods (UV‐Vis, MS, NMR and polarimetry) and chromatographic assays (paper chromatography and HPLC). Antibacterial activity was studied by microdilution, colony count and photometric assays, Sytox green stain and transmission electron microscopy (TEM). Four glycoflavonoids (rutin, nicotiflorin, hyperoside and isoquercitrin) and an unusual phenylbutanoid glycoside (tripodantoside) were purified and identified. Tripodantoside was found at 6·59 ± 0·82 g per 100 g of dry leaves. The flavonoids showed bactericidal effect at a concentration of 4 mg ml^?1 against Staphylococcus aureus and Pseudomonas aeruginosa strains from American Type Culture Collection, while tripodantoside was almost four times more active than those compounds, with a minimum bactericidal concentration = 1·024 mg ml^?1 against these strains. Tripodantoside aglycone showed bacteriolytic effects on the assayed strains, causing evident damages on cell wall and membrane, while tripodantoside did not exhibit those effects. Conclusions: The antibacterial activity of T. acutifolius infusion would be partially attributed to the purified glycoflavonoids and mainly to tripodantoside. Significance and Impact: The high extraction yield and the antibacterial activity exhibited by tripodantoside makes this chemical structure of interest to support further studies dealing with chemical modifications to increase the antibacterial activity or to seek another activities.     

State of the nonspecific humoral factors of the body's natural resistance in patients with suppurative and septic infections having various ABO system blood groups]

The results of surveying 140 patients with severe purulent and septic infections of staphylococcal etiology, when compared with the distribution of the blood groups (as classified according to the ABO system) in 180 healthy donors, revealed that generalized purulent infections occurred most frequently in patients with blood groups A (II) and AB (IV), and more seldom in patients with blood groups O (I) and B (III). The average content of lysozyme, complement and normal antibodies to E. coli, as well as the average level of general bactericidal activity in the blood sera of the patients were considerably lower than in the blood sera of healthy donors; at the same time content of lysozyme, complement and normal antibodies in the blood sera of patients having different groups of blood did not reflect the degree of their predisposition or resistance to staphylococcal infections. The general bactericidal activity of the blood serum was found to correlate with the degree of predisposition or resistance to purulent septic infections of staphylococcal etiology to a greater extent than other characteristics.     

Structure based discovery of small molecule suppressors targeting bacterial lysozyme inhibitors

The production of lysozyme inhibitors, competitively binding to the lysozyme active site, is a bacterial strategy to prevent the lytic activity of host lysozymes. Therefore, suppression of the lysozyme–inhibitor interaction is an interesting new approach for drug development since restoration of the bacterial lysozyme sensitivity will support bacterial clearance from the infected sites. Using molecular modelling techniques the interaction of the Salmonella PliC inhibitor with c-type lysozyme was studied and a protein–protein interaction based pharmacophore model was created. This model was used as a query to identify molecules, with potential affinity for the target, and subsequently, these molecules were filtered using molecular docking. The retained molecules were validated as suppressors of lysozyme inhibitory proteins using in vitro experiments revealing four active molecules.     

Expression of natural antimicrobial human lysozyme in rice grains

In the present study, we explored the expression of human lysozyme in maturing rice grains. Particle bombardment-mediated transformation was utilized to deliver the codon-optimized structural gene for human lysozyme to the callus of rice cultivar Taipei 309. Lysozyme expression is controlled by the promoter and signal peptide sequence for rice storage protein Glutelin 1. A total of 33 fertile plants were regenerated from independent transformation events and 12 of them with significant expression levels of lysozyme were advanced to further generations. The transgenes were characterized by PCR and Southern blot analysis. Segregation analysis indicated a typical Mendelian 3: 1 inheritance, suggesting a single locus or closely linked loci of gene insertion. The expression levels of lysozyme reached 0.6% of the brown rice weight or 45% of soluble proteins. Seven transgenic breeding lines have been selected and followed over six generations. Lysozyme expression levels were maintained in all generations. Biochemical, biophysical and functional comparisons of native and recombinant human lysozyme revealed identical N-terminal sequence, molecular weight, pI and specific activity. Similar thermal and pH stability was observed for lysozyme from two sources. Furthermore, similar bactericidal activity was displayed towards a laboratory strain of E. coli. The possibility of improving medical and nutritional quality of infant formulas and baby foods with rice flour or rice extract containing recombinant human lysozyme is discussed.     

Killing and Lysis of Gram-negative Bacteria Through the Synergistic Effect of Hydrogen Peroxide, Ascorbic Acid, and Lysozyme

A mixture of hydrogen peroxide and ascorbic acid has been found to generate an antibacterial mechanism which is active against gram-negative bacteria. It results in bacterial death and renders the organism sensitive to lysis by lysozyme. Under the conditions used, horseradish peroxidase did not augment the antibacterial effect. It is suggested that the effector mechanism involves the generation of short-lived free radicals which disturb the integrity of the cell wall. This effect alone might kill bacteria by interfering with selective permeability, but in the presence of lysozyme a further bactericidal activity is accomplished by complete disruption of the cell. It is proposed that a transient antibacterial system such as that described could exist within phagocytic cells. Free radicals would be formed through the interaction of certain oxidizable substances and hydrogen peroxide, which is produced during the enhanced metabolic activity that accompanies ingestion of bacteria. Such a system would help to explain why macrophages, which are apparently devoid of preformed bactericidins, are nonetheless very efficient in killing most phagocytosed bacteria.     

Effect of lysozyme or modified lysozyme fragments on DNA and RNA synthesis and membrane permeability of Escherichia coli

Previously we have shown that chicken egg white lysozyme, an efficient bactericidal agent, affects both gram-positive and gram-negative bacteria independently of its muramidase activity. More recently we reported that the digestion of lysozyme by clostripain yielded a pentadecapeptide, IVSDGNGMNAWVAWR (amino acid 98-112 of chicken egg white lysozyme), with moderate bactericidal activity but without muramidase activity. On the basis of this amino acid sequence three polypeptides, in which asparagine 106 was replaced by arginine (IVSDGNGMRAWVAWR, RAWVAWR, RWVAWR), were synthesized which showed to be strongly bactericidal. To elucidate the mechanisms of action of lysozyme and of the modified antimicrobial polypeptides Escherichia coli strain ML-35p was used. It is an ideal organism to study the outer and the inner membrane permeabilization since it is cryptic for periplasmic beta-lactamase and cytoplasmic beta-galactosidase unless the outer or inner membrane becomes damaged. For the first time we present evidence that lysozyme inhibits DNA and RNA synthesis and in contrast to the present view is able to damage the outer membrane of Escherichia coli. Blockage of macromolecular synthesis, outer membrane damage and inner membrane permeabilization bring about bacterial death. Ultrastructural studies indicate that lysozyme does not affect bacterial morphology but impairs stability of the organism. The bactericidal polypeptides derived from lysozyme block at first the synthesis of DNA and RNA which is followed by an increase of the outer membrane permeabilization causing the bacterial death. Inner membrane permeabilization, caused by RAWVAWR and RWVAWR, follows after the blockage of macromolecular synthesis and outer membrane damage, indicating that inner membrane permeabilization is not the deadly event. Escherichia coli bacteria killed by the substituted bactericidal polypeptides appeared, by electron microscopy, with a condensed cytoplasm and undulated bacterial membrane. So the action of lysozyme and its derived peptides is not identical.     

Electrophoretic Forms of Chitinolytic and Lysozyme Activities in Ruminal Protozoa

Homogenates from a mixed ruminal protozoal population and a ruminal protozoon Entodinium caudatum were analyzed for chitinolytic and lysozyme activities by sodium dodecyl sulfate polyacrylamide gel electrophoresis. For chitinase activity, up to eight bands in mixed protozoa and seven bands in E. caudatum were detected. Estimated molecular mass ranged from 70 to 110 kDa. These enzymes did not display lysozyme activity. N-Acetyl-β-glucosaminidase activity was also detected in both samples with an estimated molecular mass of 37 kDa. Lysozyme activity in mixed protozoa was present in two major and three minor bands, where one major band displayed the same motility as chicken egg white (CEW) lysozyme, and the other had an approximate molecular mass of 17.5 kDa. The latter remained active even when denatured in the presence of dithiothreitol and renatured under anaerobic conditions. Entodinium caudatum presented one major band coincident with that of CEW lysozyme and a minor band at the 17.5-kDa point. This study showed that protozoal chitinase and lysozyme activities are originated from several enzymes and that none of these enzymes exhibited both activities.     

Recombinant Bacterial Expression of the Lysozyme from the Tobacco-Hornworm <Emphasis Type="Italic">Manduca sexta</Emphasis> with Activity at Low Temperatures

A gene coding for lysozyme from the insect Manduca sexta (Ms-lyz) was expressed in Escherichia coli. The protein was produced as an insoluble cytoplasmic inclusion body which was denatured in 8 M guanidine-HCl, renatured and purified by affinity and ion-exchange chromatography. The N-terminal sequence and the activity of the recombinant protein against Micrococcus luteus confirmed that correct expression had occurred. When Ms-lyz activity was compared to hen egg white lysozyme, the insect lysozyme was active at lower temperatures. These results demonstrate the feasibility of producing a disulfide-bonded lysozyme enzyme in bacteria and suggest that the insect Ms-lyz is an interesting system for further development of an antibacterial functional at low temperatures.