muramidase and endolysin

Peptidoglycan is the major structural component of bacterial cell walls. In this era of increasingly antibiotic resistant pathogens, peptidoglycan hydrolases that degrade this important cell wall structure have emerged as a potential novel source of new antimicrobials. Included in this class are bacteriocins (lysostaphin), lysozyme, and bacteriophage endolysins. Bacteriophage are viruses that infect and utilize bacteria as their host. They can reside in the bacterial genome as a prophage, or enter the lytic phase, take over the bacterial gene expression machinery, synthesize new phage particles, lyse the host, and release up to hundreds of phage progeny. Lysis occurs during the late phase of the lytic cycle when the phage endolysin and a holin molecule are produced. The holin creates holes in the cells lipid bilayer allowing the phage endolysin (peptidoglycan hydrolase) to escape and degrade the structural portion of the cell wall. These (and other phage encoded proteins) have been shown to inhibit bacterial growth. The ability to inhibit growth or kill bacteria make both the bacteriophage and their gene products a rich source of potential antimicrobials. This review summarizes the recent resurgence of these potential antimicrobials as both diagnostic and therapeutic agents and identifies recent patents that describe these technologies.

Topics: Anti-Infective Agents; Bacteria; Bacterial Infections; Bacteriophages; Cell Wall; Drug Resistance, Bacterial; Endopeptidases; Humans; Muramidase; Patents as Topic; Peptidoglycan; Viral Proteins

Endolysins are bacteriophage-encoded peptidoglycan-degrading enzymes with potential applications for treatment of multidrug-resistant bacterial infections. Hafnia phage Enc34 encodes an unusual endolysin with an N-terminal enzymatically active domain and a C-terminal transmembrane domain. The catalytic domain of the endolysin belongs to the conserved protein family PHA02564 which has no recognizable sequence similarity to other known endolysin types. Turbidity reduction assays indicate that the Enc34 enzyme is active against peptidoglycan from a variety of Gram-negative bacteria including the opportunistic pathogen Pseudomonas aeruginosa PAO1. The crystal structure of the catalytic domain of the Enc34 endolysin shows a distinctive all-helical architecture that distantly resembles the α-lobe of the lysozyme fold. Conserved catalytically important residues suggest a shared evolutionary history between the Enc34 endolysin and GH73 and GH23 family glycoside hydrolases and propose a molecular signature for substrate cleavage for a large group of peptidoglycan-degrading enzymes.

Topics: Bacteriophages; Catalytic Domain; Endopeptidases; Muramidase; Peptidoglycan

Organisms specialized to thrive in cold environments (so-called psychrophiles) produce enzymes with the remarkable ability to catalyze chemical reactions at low temperature. Cold activity relies on adaptive changes in the proteins' sequence and structural organization that result in high conformational flexibility. As a consequence of flexibility, several such enzymes are inherently heat sensitive. Cold-active enzymes are of interest for application in a number of bioprocesses, where cold activity coupled with easy thermal inactivation can be of advantage. We describe the biochemical and functional properties of two glycosyl hydrolases (named LYS177 and LYS188) of family 19 (GH19), identified in the genome of an Antarctic marine

Topics: Antarctic Regions; Bacterial Proteins; Cold Temperature; Endopeptidases; Enzyme Stability; Evolution, Molecular; Half-Life; Muramidase; Pseudomonas; Substrate Specificity

Two commonly encountered bottlenecks in the structure determination of a protein by X-ray crystallography are screening for conditions that give high-quality crystals and, in the case of novel structures, finding derivatization conditions for experimental phasing. In this study, the phasing molecule 5-amino-2,4,6-triiodoisophthalic acid (I3C) was added to a random microseed matrix screen to generate high-quality crystals derivatized with I3C in a single optimization experiment. I3C, often referred to as the magic triangle, contains an aromatic ring scaffold with three bound I atoms. This approach was applied to efficiently phase the structures of hen egg-white lysozyme and the N-terminal domain of the Orf11 protein from Staphylococcus phage P68 (Orf11 NTD) using SAD phasing. The structure of Orf11 NTD suggests that it may play a role as a virion-associated lysin or endolysin.

Topics: Crystallization; Crystallography, X-Ray; Endopeptidases; Models, Molecular; Muramidase; Staphylococcus Phages; Triiodobenzoic Acids; Viral Proteins

Most bacteriophages rapidly infect and kill bacteria and, therefore, qualify as the next generation therapeutics for rapidly emerging drug-resistant bacteria such as Mycobacterium tuberculosis. We have previously characterized the mycobacteriophage D29-generated endolysin, Lysin A, for its activity against mycobacteria. Here, we present a detailed characterization of the lysozyme domain (LD) of D29 Lysin A that hydrolyzes peptidoglycan of both gram-positive and gram-negative bacteria with high potency. By characterizing an exhaustive LD protein variant library, we have identified critical residues important for LD activity and stability. We further complement our in vitro experiments with detailed in silico investigations. We present LD as a potent candidate for developing phage-based broad-spectrum therapeutics.

Topics: Amino Acid Sequence; Base Sequence; Catalytic Domain; Cloning, Molecular; Endopeptidases; Escherichia coli; Gene Expression; Kinetics; Ligands; Lysogeny; Molecular Dynamics Simulation; Muramidase; Mutation; Mycobacteriophages; Mycobacterium tuberculosis; N-Acetylmuramoyl-L-alanine Amidase; Peptide Library; Protein Binding; Protein Conformation, alpha-Helical; Protein Domains; Protein Interaction Domains and Motifs; Protein Stability; Recombinant Proteins; Structure-Activity Relationship; Substrate Specificity; Thermodynamics; Viral Proteins

Endolysins are peptidoglycan-degrading enzymes utilized by bacteriophages to release the progeny from bacterial cells. The lytic properties of phage endolysins make them potential antibacterial agents for medical and industrial applications. Here, we present a comprehensive characterization of phage AP3 modular endolysin (AP3gp15) containing cell wall binding domain and an enzymatic domain (DUF3380 by BLASTP), both widespread and conservative. Our structural analysis demonstrates the low similarity of an enzymatic domain to known lysozymes and an unusual catalytic centre characterized by only a single glutamic acid residue and no aspartic acid. Thus, our findings suggest distinguishing a novel class of muralytic enzymes having the activity and catalytic centre organization of DUF3380. The lack of amino acid sequence homology between AP3gp15 and other known muralytic enzymes may reflect the evolutionary convergence of analogous glycosidases. Moreover, the broad antibacterial spectrum, lack of cytotoxic effect on human cells and the stability characteristics of AP3 endolysin advocate for its future application development.

Topics: Amino Acid Sequence; Bacteriophages; Burkholderia; Catalytic Domain; Cell Line, Tumor; Computer Simulation; Endopeptidases; Escherichia coli; Humans; Models, Molecular; Muramidase; Mutagenesis, Site-Directed; Protein Conformation; Recombinant Proteins

Analysis of autolysis of derivatives of Lactococcus lactis subsp. cremoris MG1363 and subsp. lactis IL1403, both lacking the major autolysin AcmA, showed that L. lactis IL1403 still lysed during growth while L. lactis MG1363 did not. Zymographic analysis revealed that a peptidoglycan hydrolase activity of around 30 kDa is present in cell extracts of L. lactis IL1403 that could not be detected in strain MG1363. A comparison of all genes encoding putative peptidoglycan hydrolases of IL1403 and MG1363 led to the assumption that one or more of the 99 % homologous 27.9-kDa endolysins encoded by the prophages bIL285, bIL286 and bIL309 could account for the autolysis phenotype of IL1403. Induced expression of the endolysins from bIL285, bIL286 or bIL309 in L. lactis MG1363 resulted in detectable lysis or lytic activity. Prophage deletion and insertion derivatives of L. lactis IL1403 had a reduced cell lysis phenotype. RT-qPCR and zymogram analysis showed that each of these strains still expressed one or more of the three phage lysins. A homologous gene and an endolysin activity were also identified in the natural starter culture L. lactis subsp. cremoris strains E8, Wg2 and HP, and the lytic activity could be detected under growth conditions that were identical as those used for IL1403. The results presented here show that these endolysins of L. lactis are expressed during normal growth and contribute to autolysis without production of (lytic) phages. Screening for natural strains expressing homologous endolysins could help in the selection of strains with enhanced autolysis and, thus, cheese ripening properties.

Topics: Bacteriolysis; Cheese; Endopeptidases; Lactococcus lactis; Muramidase; N-Acetylmuramoyl-L-alanine Amidase; Prophages; Real-Time Polymerase Chain Reaction; Sequence Deletion

Investigation of the chaperonin encoded by gene 146 of bacteriophage EL Pseudomonas aeruginosa that we characterized earlier has been continued. To reveal the mechanism of its functioning, new recombinant substrate proteins, fragments of gene product (gp) 183 containing the lysozyme domain were prepared. Their interaction with gp146 was studied. The influence of the phage chaperonin on the thermal aggregation of one of these gp183 fragments and endolysin (gp188) was investigated in both the presence and the absence of ATP by dynamic light scattering. In the absence of ATP, the phage chaperonin forms stable complexes with substrate proteins, thereby protecting them against thermal aggregation. Experimental data obtained for different substrate proteins are analyzed.

Topics: Chaperonins; Endopeptidases; Hot Temperature; Muramidase; Protein Aggregates; Pseudomonas aeruginosa; Pseudomonas Phages; Viral Proteins

Unlike other Salmonella, which can infect a broad range of hosts causing self-limiting infection, Salmonella Typhi is an exclusively human pathogen that causes typhoid fever, a life-threatening systemic disease. Typhoid toxin is a unique virulence factor of Salmonella Typhi, which is expressed when the bacteria are within mammalian cells. Here, we report that an N-acetyl-β-D-muramidase similar to phage endolysins encoded within the same pathogenicity islet as the toxin is required for typhoid toxin secretion. Genetic and functional analysis of TtsA revealed unique amino acids at its predicted peptidoglycan-binding domain that are essential for protein secretion and that distinguishes this protein from other homologues. We propose that TtsA defines a new protein secretion mechanism recently evolved from the machine that mediates phage release.

Topics: Amino Acid Sequence; Bacterial Proteins; Bacterial Toxins; Bacteriophages; Cell Line; Endopeptidases; Genomic Islands; Humans; Molecular Sequence Data; Muramidase; Salmonella typhi; Sequence Alignment; Sequence Homology, Amino Acid; Viral Proteins; Virulence

Lysozyme (Lyz) encoded by phage P1 is required for host cell lysis upon infection. Lyz has a N-terminal Signal Anchor Release (SAR) domain, responsible for its secretion into the periplasm and for its accumulation in a membrane tethered inactive form. Here, we report sequence-specific (1)H, (13)C and (15)N resonance assignments for secreted inactive form of Lyz at pH 4.5.

Topics: Bacteriophage P1; Endopeptidases; Hydrogen-Ion Concentration; Models, Molecular; Muramidase; Nuclear Magnetic Resonance, Biomolecular; Protein Structure, Secondary

Bacteriophage enzyme preparations exolysin and endolysin were studied. Exolysin (a phage-associated enzyme) was obtained from tail fraction and endolysin from phage-free cytoplasmic fraction of disintegrated Salmonella enteritidis cells. A new method for purification of these enzymes was developed, and their molecular masses were determined. The main catalytic properties of the studied enzymes (pH optimum and specificity to bacterial substrates) were found to be similar. Both enzymes lyse Escherichia coli cells like chicken egg lysozyme, but more efficiently lyse S. enteritidis cells and cannot lyse Micrococcus luteus, a good substrate for chicken egg lysozyme. Similar properties of exolysin and endolysin suggest that these enzymes are structurally similar or even identical.

Topics: Animals; Biocatalysis; Chickens; Endopeptidases; Escherichia coli; Muramidase; Salmonella enteritidis; Salmonella Phages; Substrate Specificity; Viral Proteins

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.

Topics: Anti-Bacterial Agents; Bacteriological Techniques; Bacteriophages; Cell Membrane Permeability; Colony Count, Microbial; Endopeptidases; Green Fluorescent Proteins; Hydrostatic Pressure; Microscopy, Fluorescence; Muramidase; Peptidoglycan; Pseudomonas aeruginosa; Recombinant Fusion Proteins

Screening of a genomic library with an antiserum raised against whole Lactobacillus fermentum BR11 cells identified a clone expressing an immunoreactive 37-kDa protein. Analysis of the 3010-bp DNA insert contained within the clone revealed four open reading frames (ORFs). One ORF encodes LysA, a 303 amino acid protein which has up to 35% identity with putative endolysins from prophages Lj928 and Lj965 from Lactobacillus johnsonii and Lp1 and Lp2 from Lactobacillus plantarum as well as with the endolysin of Lactobacillus gasseri bacteriophage Phiadh. The immunoreactive protein was shown to be encoded by a truncated ORF downstream of lysA which has similarity to glutamyl-tRNA synthetases. The N-terminus of LysA has sequence similarity with N-acetylmuramidase catalytic domains while the C-terminus has sequence similarity with putative cell envelope binding bacterial SH3b domains. C-terminal bacterial SH3b domains were identified in the majority of Lactobacillus bacteriophage endolysins. LysA was expressed in Escherichia coli and unusually was found to have a broad bacteriolytic activity range with activity against a number of different Lactobacillus species and against Lactococcus lactis, streptococci and Staphylococcus aureus. It was found that LysA is 2 and 8000 times more active against L. fermentum than L. lactis and Streptococcus pyogenes, respectively.

Topics: Amino Acid Sequence; Bacterial Proteins; Bacteriophages; Binding Sites; Catalytic Domain; Cell Wall; DNA, Bacterial; Endopeptidases; Escherichia coli; Gene Order; Glutamate-tRNA Ligase; Lactobacillus; Lactococcus lactis; Molecular Sequence Data; Muramidase; Open Reading Frames; Prophages; Recombinant Proteins; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Staphylococcus aureus; Streptococcus; Viral Proteins

Pneumococcal bacteriophage-encoded lysins are modular choline binding proteins that have been shown to act as enzymatic antimicrobial agents (enzybiotics) against streptococcal infections. Here we present the crystal structures of the free and choline bound states of the Cpl-1 lysin, encoded by the pneumococcal phage Cp-1. While the catalytic module displays an irregular (beta/alpha)(5)beta(3) barrel, the cell wall-anchoring module is formed by six similar choline binding repeats (ChBrs), arranged into two different structural regions: a left-handed superhelical domain configuring two choline binding sites, and a beta sheet domain that contributes in bringing together the whole structure. Crystallographic and site-directed mutagenesis studies allow us to propose a general catalytic mechanism for the whole glycoside hydrolase family 25. Our work provides the first complete structure of a member of the large family of choline binding proteins and reveals that ChBrs are versatile elements able to tune the evolution and specificity of the pneumococcal surface proteins.

Topics: Bacteriophages; Binding Sites; Cell Wall; Choline; Endopeptidases; Muramidase; N-Acetylmuramoyl-L-alanine Amidase; Peptidoglycan; Protein Structure, Tertiary; Streptococcus pneumoniae

Genetic and DNA heteroduplex analyses of lambda imm22 hybrid phages were used to compare the Salmonella bacteriophage P22 and coliphage lambda genes which control late gene regulation and lysis. Homologous DNA sequences were correlated with P22 gene 23 and lambda gene Q (late gene regulation) and with P22 gene 13 and lambda gene S (lysis control). Nonhomologous DNA sequences were correlated with P22 gene 19 and lambda gene R (lysozyme and endolysin) and with the region encoding the P22 alpha and lambda 6S transcripts.

Topics: Bacteriophage lambda; Chromosome Deletion; Chromosome Mapping; DNA, Viral; Endopeptidases; Gene Expression Regulation; Genes, Viral; Muramidase; Recombination, Genetic; Salmonella Phages; Sequence Homology, Nucleic Acid; Virus Replication