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1.
Bactericidal properties of aprotinin, a proteinase inhibitor and possibly a defence molecule in bovine species, and of chicken egg white lysozyme, known as muramidase, were investigated. Incubation of various bacteria in the presence of either aprotinin or lysozyme showed that both proteins killed Gram-positive as well as Gram-negative bacteria without addition of complement or EDTA. Denaturation of the two proteins by dithiothreitol did not lead to loss of their bactericidal potency. Electron microscopic examination of Escherichia coli incubated either with lysozyme or aprotinin revealed that the bacterial cytoplasms gradually disintegrated. Both aprotinin and lysozyme were demonstrated within the affected cytoplasm by immunogold labelling. The results suggest that the bactericidal potency of lysozyme is not only due to muramidase activity but also to its cationic and hydrophobic properties. The bactericidal activity of aprotinin is probably also related to both these properties rather than to its activity as proteinase inhibitor.  相似文献   

2.
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.  相似文献   

3.
A. PELLEGRINI, U. THOMAS, R. VON FELLENBERG AND P. WILD. 1992. Bactericidal properties of aprotinin, a proteinase inhibitor and possibly a defence molecule in bovine species, and of chicken egg white lysozyme, known as muramidase, were investigated. Incubation of various bacteria in the presence of either aprotinin or lysozyme showed that both proteins killed Gram-positive as well as Gram-negative bacteria without addition of complement or EDTA. Denaturation of the two proteins by dithiothreitol did not lead to loss of their bactericidal potency. Electron microscopic examination of Escherichia coli incubated either with lysozyme or aprotinin revealed that the bacterial cytoplasms gradually disintegrated. Both aprotinin and lysozyme were demonstrated within the affected cytoplasm by immunogold labelling. The results suggest that the bactericidal potency of lysozyme is not only due to muramidase activity but also to its cationic and hydrophobic properties. The bactericidal activity of aprotinin is probably also related to both these properties rather than to its activity as proteinase inhibitor.  相似文献   

4.
Recently, we have found that partially unfolded lysozyme exerts broad spectrum antimicrobial action in vitro against Gram-negative and Gram-positive bacteria independent of its catalytic activity. In parallel, an internal peptide (residues 98-112) of hen egg white lysozyme, obtained after digestion with clostripain, possessed broad spectrum antimicrobial action in vitro. This internal peptide is part of a helix-loop-helix domain (87-114 sequence of hen lysozyme) located at the upper lip of the active site cleft of lysozyme. The helix-loop-helix (HLH) structures are known motifs commonly found in membrane-active and DNA-binding proteins. To evaluate the contribution of the HLH peptide to the antimicrobial properties of lysozyme, the HLH sequence and its secondary structure derivatives of chicken and human lysozyme were synthesized and tested for antimicrobial activity against several bacterial strains. We found that the full HLH peptide of both chicken and human lysozymes was potently microbicidal against both Gram-positive and Gram-negative bacteria and the fungus Candida albicans. The N-terminal helix of HLH was specifically bactericidal to Gram-positive bacteria, whereas the C-terminal helix was bactericidal to all tested strains. Outer and inner membrane permeabilization studies, as well as measurements of transmembrane electrochemical potentials, provided evidence that HLH peptide and its C-terminal helix domain kill Gram-negative bacteria by crossing the outer membrane via self-promoted uptake and causing damage to the inner membrane through channel formation. The results are discussed in terms of proposed mechanisms for the catalytically independent antimicrobial activity of lysozyme that offer a new strategy for the design of potential antimicrobial drugs in the treatment of infectious diseases.  相似文献   

5.
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.  相似文献   

6.
The goose-type lysozyme inhibitor PliG enhances the survival of Escherichia coli in goose but not in chicken egg white, which contains goose- and chicken-type lysozymes, respectively. These results indicate that both the type of host lysozyme and the type of bacterial lysozyme inhibitor may affect bacterium-host interactions.  相似文献   

7.
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.  相似文献   

8.
Peptide fragments possessing antimicrobial activity were obtained by protease digestion of goose egg white lysozyme. Digested peptide purified from RP-HPLC which showed no lysozyme activity exhibited bactericidal activity toward Gram-negative and Gram-positive bacteria. LC/MS–MS and automated Edman degradation revealed the amino acid sequence to be Thr-Ala-Lys-Pro-Glu-Gly-Leu-Ser-Tyr. This sequence corresponds to amino acid positions 20–28, located at the N-terminal outer part of goose lysozyme. The peptide acted on bacterial membrane as shown by scanning electron microscopy. The mechanism of action could be explained from a helical structure that may be formed by the centered Pro residue and the terminal Lys residue after the peptide attaches to a cell membrane. This is the first study to report that a peptide derived from the protease digests of G-type lysozyme possesses antimicrobial activity with broad spectrum activity. Our result is comparative to the previous reports of Chicken lysozyme and T4 phage lysozyme, which showed antimicrobial activity after digestion with protease. These results might contribute to the usage of antimicrobial peptides engineered by genetic or chemical synthesis.  相似文献   

9.
Lysozymes are ancient and important components of the innate immune system of animals that hydrolyze peptidoglycan, the major bacterial cell wall polymer. Bacteria engaging in commensal or pathogenic interactions with an animal host have evolved various strategies to evade this bactericidal enzyme, one recently proposed strategy being the production of lysozyme inhibitors. We here report the discovery of a novel family of bacterial lysozyme inhibitors with widespread homologs in gram-negative bacteria. First, a lysozyme inhibitor was isolated by affinity chromatography from a periplasmic extract of Salmonella Enteritidis, identified by mass spectrometry and correspondingly designated as PliC (periplasmic lysozyme inhibitor of c-type lysozyme). A pliC knock-out mutant no longer produced lysozyme inhibitory activity and showed increased lysozyme sensitivity in the presence of the outer membrane permeabilizing protein lactoferrin. PliC lacks similarity with the previously described Escherichia coli lysozyme inhibitor Ivy, but is related to a group of proteins with a common conserved COG3895 domain, some of them predicted to be lipoproteins. No function has yet been assigned to these proteins, although they are widely spread among the Proteobacteria. We demonstrate that at least two representatives of this group, MliC (membrane bound lysozyme inhibitor of c-type lysozyme) of E. coli and Pseudomonas aeruginosa, also possess lysozyme inhibitory activity and confer increased lysozyme tolerance upon expression in E. coli. Interestingly, mliC of Salmonella Typhi was picked up earlier in a screen for genes induced during residence in macrophages, and knockout of mliC was shown to reduce macrophage survival of S. Typhi. Based on these observations, we suggest that the COG3895 domain is a common feature of a novel and widespread family of bacterial lysozyme inhibitors in gram-negative bacteria that may function as colonization or virulence factors in bacteria interacting with an animal host.  相似文献   

10.
Ivy is a lysozyme inhibitor that protects Escherichia coli against lysozyme-mediated cell wall hydrolysis when the outer membrane is permeabilized by mutation or by chemical or physical stress. In the current work, we have investigated whether Ivy is necessary for the survival or growth of E. coli MG1655 and Pseudomonas aeruginosa PAO1 in hen egg white and in human saliva and breast milk, which are naturally rich in lysozyme and in membrane-permeabilizing components. Wild-type E. coli was able to grow in saliva and breast milk but showed partial inactivation in egg white. The knockout of Ivy did not affect growth in breast milk but slightly increased sensitivity to egg white and caused hypersensitivity to saliva, resulting in the complete inactivation of 10(4) CFU ml(-1) of bacteria within less than 5 hours. The depletion of lysozyme from saliva completely restored the ability of the ivy mutant to grow like the parental strain. P. aeruginosa, in contrast, showed growth in all three substrates, which was not affected by the knockout of Ivy production. These results indicate that lysozyme inhibitors like Ivy promote bacterial survival or growth in particular lysozyme-rich secretions and suggest that they may promote the bacterial colonization of specific niches in the animal host.  相似文献   

11.
Lysozyme and antimicrobial peptides are key factors of the humoral immune response in insects. In the present work lysozyme and anionic defense peptide (GMAP2) were isolated from the hemolymph of the greater wax moth Galleria mellonella and their antibacterial activity was investigated. Adsorption of G. mellonella lysozyme on the cell surface of Gram-positive and Gram-negative bacteria was demonstrated using immunoblotting with anti-G. mellonella lysozyme antibodies. Lysozyme effectively inhibited the growth of selected Gram-positive bacteria, which was accompanied by serious alterations of the cell surface, as revealed by atomic force microscopy (AFM) imaging. G. mellonella lysozyme used in concentrations found in the hemolymph of naive and immunized larvae, perforated also the Escherichia coli cell membrane and the level of such perforation was considerably increased by GMAP2. GMAP2 used alone did not perforate E. coli cells nor influence lysozyme muramidase activity. However, the peptide induced a decrease in the turgor pressure of the bacterial cell. Moreover, in the samples of bacteria treated with a mixture of lysozyme and GMAP2 the sodium chloride crystals were found, suggesting disturbance of ion transport across the membrane leading to cell disruption. These results clearly indicated the synergistic action of G. mellonella lysozyme and anionic peptide 2 against Gram-negative bacteria. The reported results suggested that, thanks to immune factors constitutively present in hemolymph, G. mellonella larvae are to some extent protected against infection caused by Gram-negative bacteria.  相似文献   

12.
Cyclization of R- and W-rich hexapeptides has been found to enhance specifically the antimicrobial activity against Gram-negative Escherichia coli. To gain insight into the role of the bacterial outer membrane in mediating selectivity, we assayed the activity of cyclic hexapeptides derived from the parent sequence c-(RRWWRF) against several E. coli strains and Bacillus subtilis, L-form bacteria, and E. coli lipopolysaccharide (LPS) mutant strains, and we also investigated the peptide-induced permeabilization of the outer and inner membrane of E. coli. Wall-deficient L-form bacteria were distinctly less susceptible than the wild type strain. The patterns of peptide-induced permeabilization of the outer and inner E. coli membranes correlated well with the antimicrobial activity, confirming that membrane permeabilization is a detrimental effect of the peptides upon bacteria. Truncation of LPS had no influence on the activity of the cyclic parent peptide, but the highly active c-(RRWFWR), with three adjacent aromatic residues, required the complete LPS for maximal activity. Furthermore, differences in the activity of the parent peptide and its all-D sequence indicated stereospecific interactions with the LPS mutant strains. We suggest that, depending on the primary sequence of the peptides, either hydrophobic interactions with the fatty acid chains of lipid A, or electrostatic interactions disturbing the polar core region and interference with saccharide-saccharide interactions prevail in the barrier-disturbing effect upon the outer membrane and thereby provide peptide accessibility to the inner membrane. The results underline the importance of tryptophan and arginine residues and their relative location for a high antimicrobial effect, and the activity-modulating function of the outer membrane of E. coli. In addition to membrane permeabilization, the data provided evidence for the involvement of other mechanisms in growth inhibition and killing of bacteria.  相似文献   

13.
Lysozymes are an important component of the innate immune system of animals that hydrolyze peptidoglycan, the major bacterial cell wall constituent. Many bacteria have contrived various means of dealing with this bactericidal enzyme, one of which is to produce lysozyme inhibitors. Recently, a novel family of bacterial lysozyme inhibitors was identified in various Gram-negative bacteria, named MliC (membrane bound lysozyme inhibitor of C-type lysozyme). Here, we report the crystal structure of Pseudomonas aeruginosa MliC in complex with chicken egg white lysozyme. Combined with mutational study, the complex structure demonstrates that the invariant loop of MliC plays a crucial role in the inhibition of the lysozyme by its insertion to the active site cleft of the lysozyme, where the loop forms hydrogen and ionic bonds with the catalytic residues. Since MliC family members have been implicated as putative colonization or virulence factors, the structures and mechanism of action of MliC will be of relevance to the control of bacterial growth in animal hosts.  相似文献   

14.
Mammalian peptidoglycan recognition proteins (PGRPs), similar to antimicrobial lectins, bind the bacterial cell wall and kill bacteria through an unknown mechanism. We show that PGRPs enter the Gram-positive cell wall at the site of daughter cell separation during cell division. In Bacillus subtilis, PGRPs activate the CssR-CssS two-component system that detects and disposes of misfolded proteins that are usually exported out of bacterial cells. This activation results in membrane depolarization, cessation of intracellular peptidoglycan, protein, RNA and DNA synthesis, and production of hydroxyl radicals, which are responsible for bacterial death. PGRPs also bind the outer membrane of Escherichia coli and activate the functionally homologous CpxA-CpxR two-component system, which kills the bacteria. We exclude other potential bactericidal mechanisms, including inhibition of extracellular peptidoglycan synthesis, hydrolysis of peptidoglycan and membrane permeabilization. Thus, we reveal a previously unknown mechanism by which innate immunity proteins that bind the cell wall or outer membrane exploit the bacterial stress defense response to kill bacteria.  相似文献   

15.
A novel toad lysozyme (named BA-lysozyme) was purified from skin secretions of Bufo andrewsi by a three-step chromatography procedure. BA-lysozyme is a single chain protein and the apparent molecular weight is about 15 kDa as judged by SDS-PAGE. The specific lytic activity against Micrococcus lysodeikticus of BA-lysozyme is 2.7 x 10(5) units/mg, indicating that it is a potent lysozyme. It displayed potent bactericidal activity against Staphylococcus aureus and Escherichia coli with minimal inhibitory concentrations (MIC) of 1 and 8 microM, respectively. The deduced primary structure of BA-lysozyme from cloned cDNA was confirmed by N-terminal sequencing and peptide mass fingerprinting. Its amino acid sequence shares 56.5% identity with that of chicken egg-white lysozyme. Phylogenetic analysis indicates that B. andrewsi lysozyme is closely related to that of turtle. This is the first report on the isolation and primary structure determination of amphibian lysozyme.  相似文献   

16.
Two peptides, RAWVAWR-NH2 and IVSDGNGMNAWVAWR-NH2, derived from human and chicken lysozyme, respectively, exhibit antimicrobial activity. A comparison between the L-RAWVAWR, D-RAWVAWR, and the longer peptide has been carried out in membrane mimetic conditions to better understand how their interaction with lipid and detergent systems relates to the reported higher activity for the all L-peptide. Using CD and 2D 1H NMR spectroscopy, the structures were studied with DPC and SDS micelles. Fluorescence spectroscopy was used to study peptide interactions with POPC and POPG vesicles and DOPC, DOPE, and DOPG mixed vesicle systems. Membrane-peptide interactions were also probed by ITC and DSC. The ability of fluorescein-labeled RAWVAWR to rapidly enter both E. coli and Staphylococcus aureus was visualized using confocal microscopy. Reflecting the bactericidal activity, the long peptide interacted very weakly with the lipids. The RAWVAWR-NH2 peptides preferred lipids with negatively charged headgroups and interacted predominantly in the solvent-lipid interface, causing significant perturbation of membrane mimetics containing PG headgroups. Peptide structures determined by 1H NMR indicated a well-ordered coiled structure for the short peptides and the C-terminus of the longer peptide. Using each technique, the two enantiomers of RAWVAWR-NH2 interacted in an identical fashion with the lipids, indicating that any difference in activity in vivo is limited to interactions not involving the membrane lipids.  相似文献   

17.
Lysozyme is an abundant, cationic antimicrobial protein that plays an important role in pulmonary host defense. Increased concentration of lysozyme in the airspaces of transgenic mice enhanced bacterial killing whereas lysozyme deficiency resulted in increased bacterial burden and morbidity. Lysozyme degrades peptidoglycan in the bacterial cell wall leading to rapid killing of Gram-positive organisms; however, this mechanism cannot account for the protective effect of lysozyme against Gram-negative bacteria. The current study was therefore designed to test the hypothesis that the catalytic activity (muramidase activity) of lysozyme is not required for bacterial killing in vivo. Substitution of serine for aspartic acid at position 53 (D53S) in mouse lysozyme M completely ablated muramidase activity. Muramidase-deficient recombinant lysozyme (LysM(D53S)) killed both Gram-positive and Gram-negative bacteria in vitro. Targeted expression of LysM(D53S) in the respiratory epithelium of wild-type (LysM(+/+)/LysM(D53S)) or lysozyme M(null) mice (LysM(-/-)/LysM(D53S)) resulted in significantly elevated lysozyme protein in the airspaces without any increase in muramidase activity. Intratracheal challenge of transgenic mice with Gram-positive or Gram-negative bacteria resulted in a significant increase in bacterial burden in LysM(-/-) mice that was completely reversed by targeted expression of LysM(D53S). These results indicate that the muramidase activity of lysozyme is not required for bacterial killing in vitro or in vivo.  相似文献   

18.
During apoptosis, an important pathway leading to caspase activation involves the release of cytochrome c from the intermembrane space of mitochondria. Using a cell-free system based on Xenopus egg extracts, we examined changes in the outer mitochondrial membrane accompanying cytochrome c efflux. The pro-apoptotic proteins, Bid and Bax, as well as factors present in Xenopus egg cytosol, each induced cytochrome c release when incubated with isolated mitochondria. These factors caused a permeabilization of the outer membrane that allowed the corelease of multiple intermembrane space proteins: cytochrome c, adenylate kinase and sulfite oxidase. The efflux process is thus nonspecific. None of the cytochrome c-releasing factors caused detectable mitochondrial swelling, arguing that matrix swelling is not required for outer membrane permeability in this system. Bid and Bax caused complete release of cytochrome c but only a limited permeabilization of the outer membrane, as measured by the accessibility of inner membrane-associated respiratory complexes III and IV to exogenously added cytochrome c. However, outer membrane permeability was strikingly increased by a macromolecular cytosolic factor, termed PEF (permeability enhancing factor). We hypothesize that PEF activity could help determine whether cells can recover from mitochondrial cytochrome c release.  相似文献   

19.
The parameters influencing outer membrane permeability of Pseudomonas aeruginosa PAO1 under high hydrostatic pressure were quantified and optimized, using fusion between a specific A1gamma peptidoglycan-binding domain and green fluorescent protein (PBD-GFP). Based on the obtained data, optimal conditions were defined to assess the synergistic bactericidal action between high hydrostatic pressure and peptidoglycan hydrolysis by bacteriophage-encoded endolysins KZ144 and EL188. Under high hydrostatic pressure, both endolysins show similar inactivation of P. aeruginosa as the commonly used hen egg white lysozyme or slightly higher inactivation in the case of EL188 at 150 and 200 MPa. The partial contribution of pressure to the bacterial inactivation increases with higher pressure, while the partial contribution of the enzymes is maximal at the onset pressure of outer membrane permeabilization for the PBD-GFP protein (175 MPa). This study's results demonstrate the usefulness of this approach to determine optimal synergy for hurdle technology applications.  相似文献   

20.
Increasing bacterial resistance towards antibiotics has stimulated research for novel antimicrobials. Proteins acting on bacterial membranes could be a solution. Lysozyme has been proven active against E. coli by disruption of both outer and cytoplasmic membranes, with dry‐heating increasing lysozyme activity. Dry‐heated lysozyme (DH‐L) is a mixture of isoforms (isoaspartyl, native‐like and succinimide lysozymes), giving rise to two questions: what effects does each form have, and which physicochemical properties are critical as regards the antibacterial activity? These issues were investigated by fractionating DH‐L, analyzing structural properties of each fraction, and testing each fraction in vivo on bacteria and in vitro on membrane models. Positive net charge, hydrophobicity and molecular flexibility of the isoforms seem key parameters for their interaction with E. coli membranes. The succinimide lysozyme fraction, the most positive, flexible and hydrophobic, shows the highest antimicrobial activity, induces the strongest bacterial membrane disruption and is the most surface active on model lipid monolayers. Moreover, each fraction appears less efficient than DH‐L against E. coli , indicating a synergetic cooperation between lysozyme isoforms. The bacterial membrane modifications induced by one isoform could facilitate the subsequent action of the other isoforms.  相似文献   

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