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Vincent A Fischetti - One of the best experts on this subject based on the ideXlab platform.
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Phage Lysins: Novel Alternative to Antibiotics
Phage Therapy: A Practical Approach, 2019Co-Authors: Vincent A FischettiAbstract:Bacteriophage (or phage) endoLysins (or Lysins) are highly evolved enzymes produced to cleave essential bonds in the bacterial cell wall peptidoglycan for phage progeny release. Small quantities of purified recombinant Lysin added externally to gram-positive or gram-negative bacteria cause immediate lysis resulting in log-fold death of the target bacterium. Lysins have now been used successfully in a variety of animal models to control pathogenic antibiotic-resistant bacteria found on mucosal surfaces, in the blood, and in infected tissues. While several Lysins against gram-negative bacteria are effective topically, they show limited activity in serum. The advantages over antibiotics are their specificity for the pathogen, minimal effects on the normal flora, low chance of bacterial resistance, and their ability to kill colonizing pathogens on mucosal surfaces, a capability previously unavailable. Lysins, therefore, may be a much-needed anti-infective (or enzybiotic) in an age of mounting antibiotic resistance. This chapter will outline the characteristics and remarkable potency of these enzymes in killing bacteria both in vitro and in vivo.
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development of phage Lysins as novel therapeutics a historical perspective
Viruses, 2018Co-Authors: Vincent A FischettiAbstract:Bacteriophage Lysins and related bacteriolytic enzymes are now considered among the top antibiotic alternatives for solving the mounting resistance problem. Over the past 17 years, Lysins have been widely developed against Gram-positive and recently Gram-negative pathogens, and successfully tested in a variety of animal models to demonstrate their efficacy. A Lysin (CF-301) directed to methicillin resistant Staphylococcus aureus (MRSA) has effectively completed phase 1 human clinical trials, showing safety in this novel therapeutic class. To validate efficacy, CF-301 is currently the first Lysin to enter phase 2 human trials to treat hospitalized patients with MRSA bacteremia or endocarditis. If successful, it could be the defining moment leading to the acceptance of Lysins as an alternative to small molecule antibiotics. This article is a detailed account of events leading to the first therapeutic use and ultimate development of phage-encoded Lysins as novel anti-infectives.
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novel phage Lysin capable of killing the multidrug resistant gram negative bacterium acinetobacter baumannii in a mouse bacteremia model
Antimicrobial Agents and Chemotherapy, 2015Co-Authors: Rolf Lood, Raymond Schuch, Adam J. Pelzek, Benjamin Y Winer, Roberto Diezmartinez, Mya Thandar, Chad W Euler, Vincent A FischettiAbstract:Acinetobacter baumannii, a Gram-negative multidrug-resistant (MDR) bacterium, is now recognized as one of the more common nosocomial pathogens. Because most clinical isolates are found to be multidrug resistant, alternative therapies need to be developed to control this pathogen. We constructed a bacteriophage genomic library based on prophages induced from 13 A. baumannii strains and screened it for genes encoding bacteriolytic activity. Using this approach, we identified 21 distinct Lysins with different activities and sequence diversity that were capable of killing A. baumannii. The Lysin (PlyF307) displaying the greatest activity was further characterized and was shown to efficiently kill (>5-log-unit decrease) all tested A. baumannii clinical isolates. Treatment with PlyF307 was able to significantly reduce planktonic and biofilm A. baumannii both in vitro and in vivo. Finally, PlyF307 rescued mice from lethal A. baumannii bacteremia and as such represents the first highly active therapeutic Lysin specific for Gram-negative organisms in an array of native Lysins found in Acinetobacter phage.
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Lysin Therapy for Staphylococcus aureus and Other Bacterial Pathogens.
Current topics in microbiology and immunology, 2015Co-Authors: Vincent A FischettiAbstract:Lysins are a new and novel class of anti-infectives derived from bacteriophage (or phage ). They represent highly evolved enzymes produced to cleave essential bonds in the bacterial cell wall peptidoglycan for phage progeny release. Small quantities of purified recombinant Lysin added externally to gram-positive bacteria results in immediate lysis causing log-fold death of the target bacterium. Lysins can eliminate bacteria both systemically and topically, from mucosal surfaces and biofilms, as evidenced by experimental models of sepsis, pneumonia, meningitis, endocarditis, and mucosal decolonization. Furthermore, Lysins can act synergistically with antibiotics by resensitizing bacteria to non-susceptible antibiotics. The advantages over antibiotics are their specificity for the pathogen without disturbing the normal flora, the low chance of bacterial resistance, and their ability to kill colonizing pathogens on mucosal surfaces, a capacity previously unavailable. Lysins, therefore, may be a much-needed anti-infective in an age of mounting antibiotic resistance.
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combination therapy with Lysin cf 301 and antibiotic is superior to antibiotic alone for treating methicillin resistant staphylococcus aureus induced murine bacteremia
The Journal of Infectious Diseases, 2014Co-Authors: Raymond Schuch, Brent Schneider, Karen Sauve, Babar Khan, Jimmy Rotolo, Yuki Horiuchi, Daniel E Couto, David B Huang, Vincent A Fischetti, Robert C NowinskiAbstract:Methicillin-resistant Staphylococcus aureus (MRSA) infections occur in both hospital and community settings, and in the United States approximately 100 000 patients are hospitalized annually with invasive MRSA infections resulting in >18 000 deaths [1]. Among invasive infections, the annual incidence rate of S. aureus bacteremia varies from 3.6 to 6.0 per 100 000 person-years [2]. Of further concern, MRSA strains are now evolving additional resistances against standard-of-care (SOC) antibiotics [3]. This high unmet clinical need and associated healthcare costs [4] underscore the need for new therapeutic alternatives to treat MRSA infections. Emerging options for treating gram-positive bacterial infections are Lysins [5]. In the natural setting, these bacteriophage-encoded hydrolytic enzymes liberate progeny phage from infected bacteria by degrading peptidoglycan from inside the cell, causing lysis of the host bacterium. Therapeutically, Lysins are being developed as antimicrobial agents to lyse pathogenic bacteria by attacking peptidoglycan from outside the cell [6, 7]. The efficacy of Lysin therapy has been demonstrated in rodents with experimental pharyngitis [7], pneumonia [8], otitis media [9], abscesses [10], bacteremia [11], endocarditis [12], and meningitis [13]. In addition, Lysins are generally highly specific for bacterial species and rarely lyse nontarget organisms, including commensal gut bacteria, which may be beneficial in maintaining gastrointestinal homeostasis [14, 15]. CF-301, also referred to as PlySs2, was identified as an antistaphylococcal Lysin encoded within a prophage of the Streptococcus suis genome [16]. It has a domain arrangement characteristic of most bacteriophage Lysins, defined by a catalytic N-terminal domain linked to a cell wall–binding C-terminal domain [5] (Figure (Figure11A). The N-terminal domain belongs to the cysteine-histidine–dependent amidohydrolases/peptidases (CHAP) family [20], common among Lysins and other bacterial cell wall–modifying enzymes. The C-terminal domain belongs to the SH3b family [21] that often forms the cell wall–binding element of Lysins. Unlike most recombinant Lysins that digest S. aureus, which are often poorly expressed and display low solubilities [22–24], recombinant CF-301 is highly expressed in E. coli within the soluble fraction [16]. Figure 1. General features of CF-301. A, Protein sequence and diagrammatic representation of CF-301 depicted with the N-terminal cysteine-histidine–dependent amidohydrolases/peptidases (CHAP) domain in blue and the C-terminal SH3b domain in green. The CHAP ... In the present study we show that CF-301 differs from SOC antibiotics by its potency, speed, specificity, and activity against antibiotic-resistant strains. We also demonstrate a low resistance profile, anti-biofilm activity, and synergy with antibiotics in vitro. Furthermore, we show that CF-301 significantly enhances SOC antibiotic activities against staphylococcal-induced murine bacteremia under challenging conditions where high doses of single antibiotics fail, underscoring the effectiveness of the combination strategy for treating bacteremia.
John Baker - One of the best experts on this subject based on the ideXlab platform.
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LambdaSa1 and LambdaSa2 prophage Lysins of Streptococcus agalactiae.
Applied and environmental microbiology, 2007Co-Authors: David G. Pritchard, Shengli Dong, Marion Kirk, Robert T. Cartee, John BakerAbstract:Putative N-acetylmuramyl-l-alanine amidase genes from LambdaSa1 and LambdaSa2 prophages of Streptococcus agalactiae were cloned and expressed in Escherichia coli. The purified enzymes lysed the cell walls of Streptococcus agalactiae, Streptococcus pneumoniae, and Staphylococcus aureus. The peptidoglycan digestion products in the cell wall lysates were not consistent with amidase activity. Instead, the structure of the muropeptide digestion fragments indicated that both the LambdaSa1 and LambdaSa2 Lysins exhibited γ-d-glutaminyl-l-Lysine endopeptidase activity. The endopeptidase cleavage specificity of the Lysins was confirmed using a synthetic peptide substrate corresponding to a portion of the stem peptide and cross bridge of Streptococcus agalactiae peptidoglycan. The LambdaSa2 Lysin also displayed β-d-N-acetylglucosaminidase activity.
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LambdaSa1 and LambdaSa2 prophage Lysins of Streptococcus agalactiae.
Applied and environmental microbiology, 2007Co-Authors: David G. Pritchard, Shengli Dong, Marion Kirk, Robert T. Cartee, John BakerAbstract:Putative N-acetylmuramyl-l-alanine amidase genes from LambdaSa1 and LambdaSa2 prophages of Streptococcus agalactiae were cloned and expressed in Escherichia coli. The purified enzymes lysed the cell walls of Streptococcus agalactiae, Streptococcus pneumoniae, and Staphylococcus aureus. The peptidoglycan digestion products in the cell wall lysates were not consistent with amidase activity. Instead, the structure of the muropeptide digestion fragments indicated that both the LambdaSa1 and LambdaSa2 Lysins exhibited gamma-d-glutaminyl-l-Lysine endopeptidase activity. The endopeptidase cleavage specificity of the Lysins was confirmed using a synthetic peptide substrate corresponding to a portion of the stem peptide and cross bridge of Streptococcus agalactiae peptidoglycan. The LambdaSa2 Lysin also displayed beta-d-N-acetylglucosaminidase activity.
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the bifunctional peptidoglycan Lysin of streptococcus agalactiae bacteriophage b30
Microbiology, 2004Co-Authors: David G. Pritchard, Shengli Dong, John Baker, Jeffrey A EnglerAbstract:A group B streptococcal (GBS) bacteriophage Lysin gene was cloned and expressed in Escherichia coli. The purified recombinant enzyme, calculated to have a molecular mass of 49 677 Da, lysed GBS cells. The susceptibility of GBS cells to lysis by the enzyme depended upon the growth stage at which they were harvested, with early exponential phase cells most sensitive. Calcium ions enhanced the activity of the enzyme. The enzyme also lysed other β-haemolytic streptococci, including groups A, C, E and G streptococci, but not common oral streptococci, including Streptococcus mutans. The generation of both reducing activity and N-terminal alanine residues during lysis indicated that the Lysin is a bifunctional enzyme, possessing both glycosidase and endopeptidase activities. This is consistent with the presence of two conserved sequence domains, an Acm (acetylmuramidase) domain associated with lysozyme activity, and a CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) domain associated with endopeptidase activity. Site-directed mutagenesis of conserved cysteine and histidine residues in the CHAP domain and conserved aspartate and glutamate residues in the Acm domain confirmed their importance for lysozyme and endopeptidase activity respectively.
Raymond Schuch - One of the best experts on this subject based on the ideXlab platform.
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novel phage Lysin capable of killing the multidrug resistant gram negative bacterium acinetobacter baumannii in a mouse bacteremia model
Antimicrobial Agents and Chemotherapy, 2015Co-Authors: Rolf Lood, Raymond Schuch, Adam J. Pelzek, Benjamin Y Winer, Roberto Diezmartinez, Mya Thandar, Chad W Euler, Vincent A FischettiAbstract:Acinetobacter baumannii, a Gram-negative multidrug-resistant (MDR) bacterium, is now recognized as one of the more common nosocomial pathogens. Because most clinical isolates are found to be multidrug resistant, alternative therapies need to be developed to control this pathogen. We constructed a bacteriophage genomic library based on prophages induced from 13 A. baumannii strains and screened it for genes encoding bacteriolytic activity. Using this approach, we identified 21 distinct Lysins with different activities and sequence diversity that were capable of killing A. baumannii. The Lysin (PlyF307) displaying the greatest activity was further characterized and was shown to efficiently kill (>5-log-unit decrease) all tested A. baumannii clinical isolates. Treatment with PlyF307 was able to significantly reduce planktonic and biofilm A. baumannii both in vitro and in vivo. Finally, PlyF307 rescued mice from lethal A. baumannii bacteremia and as such represents the first highly active therapeutic Lysin specific for Gram-negative organisms in an array of native Lysins found in Acinetobacter phage.
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combination therapy with Lysin cf 301 and antibiotic is superior to antibiotic alone for treating methicillin resistant staphylococcus aureus induced murine bacteremia
The Journal of Infectious Diseases, 2014Co-Authors: Raymond Schuch, Brent Schneider, Karen Sauve, Babar Khan, Jimmy Rotolo, Yuki Horiuchi, Daniel E Couto, David B Huang, Vincent A Fischetti, Robert C NowinskiAbstract:Methicillin-resistant Staphylococcus aureus (MRSA) infections occur in both hospital and community settings, and in the United States approximately 100 000 patients are hospitalized annually with invasive MRSA infections resulting in >18 000 deaths [1]. Among invasive infections, the annual incidence rate of S. aureus bacteremia varies from 3.6 to 6.0 per 100 000 person-years [2]. Of further concern, MRSA strains are now evolving additional resistances against standard-of-care (SOC) antibiotics [3]. This high unmet clinical need and associated healthcare costs [4] underscore the need for new therapeutic alternatives to treat MRSA infections. Emerging options for treating gram-positive bacterial infections are Lysins [5]. In the natural setting, these bacteriophage-encoded hydrolytic enzymes liberate progeny phage from infected bacteria by degrading peptidoglycan from inside the cell, causing lysis of the host bacterium. Therapeutically, Lysins are being developed as antimicrobial agents to lyse pathogenic bacteria by attacking peptidoglycan from outside the cell [6, 7]. The efficacy of Lysin therapy has been demonstrated in rodents with experimental pharyngitis [7], pneumonia [8], otitis media [9], abscesses [10], bacteremia [11], endocarditis [12], and meningitis [13]. In addition, Lysins are generally highly specific for bacterial species and rarely lyse nontarget organisms, including commensal gut bacteria, which may be beneficial in maintaining gastrointestinal homeostasis [14, 15]. CF-301, also referred to as PlySs2, was identified as an antistaphylococcal Lysin encoded within a prophage of the Streptococcus suis genome [16]. It has a domain arrangement characteristic of most bacteriophage Lysins, defined by a catalytic N-terminal domain linked to a cell wall–binding C-terminal domain [5] (Figure (Figure11A). The N-terminal domain belongs to the cysteine-histidine–dependent amidohydrolases/peptidases (CHAP) family [20], common among Lysins and other bacterial cell wall–modifying enzymes. The C-terminal domain belongs to the SH3b family [21] that often forms the cell wall–binding element of Lysins. Unlike most recombinant Lysins that digest S. aureus, which are often poorly expressed and display low solubilities [22–24], recombinant CF-301 is highly expressed in E. coli within the soluble fraction [16]. Figure 1. General features of CF-301. A, Protein sequence and diagrammatic representation of CF-301 depicted with the N-terminal cysteine-histidine–dependent amidohydrolases/peptidases (CHAP) domain in blue and the C-terminal SH3b domain in green. The CHAP ... In the present study we show that CF-301 differs from SOC antibiotics by its potency, speed, specificity, and activity against antibiotic-resistant strains. We also demonstrate a low resistance profile, anti-biofilm activity, and synergy with antibiotics in vitro. Furthermore, we show that CF-301 significantly enhances SOC antibiotic activities against staphylococcal-induced murine bacteremia under challenging conditions where high doses of single antibiotics fail, underscoring the effectiveness of the combination strategy for treating bacteremia.
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A genetic screen to identify bacteriophage Lysins.
Methods in molecular biology (Clifton N.J.), 2009Co-Authors: Raymond Schuch, Vincent A Fischetti, Daniel C NelsonAbstract:Lysins are phage-encoded, peptidoglycan (cell wall) hydrolases that accumulate in the bacterial cytoplasm during a lytic infection cycle. Late during infection, the Lysins undergo holin-mediated translocation across the inner membrane into the peptidoglycan matrix where they cleave cell wall covalent bonds required for wall stability and allow bacterial lysis and progeny phage release. This potent hydrolytic activity is now the foundation of a powerful genetic-based screening process for the identification and analysis of phage Lysin proteins. Here, we describe a method for identifying a Lysin, PlyG, from a bacteriophage that specifically infects the Gram-positive organism Bacillus anthracis; however, the techniques described can be adapted to clone, express, and analyze Lysins from any phage infecting Gram-positive bacteria or possibly even Gram-negative bacteria.
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Current Protocols Essential Laboratory Techniques - Isolation of Bacteriophages from Environmental Sources, and Creation and Functional Screening of Phage DNA Libraries
Current Protocols Essential Laboratory Techniques, 2008Co-Authors: Adam J. Pelzek, Raymond Schuch, Jonathan E. Schmitz, Vincent A FischettiAbstract:Bacteriophages (phages) have evolved specific classes of proteins such as adhesins and Lysins to mediate specific bacterial host recognition and rapid and efficient lysis following infection. However, because many bacterial species, and thus phages, cannot be cultured in the laboratory, techniques for the efficient cloning and screening of phage gene libraries are required to aid in the discovery of novel phage proteins for use in diagnostic and therapeutic applications. This article contains protocols for the isolation of phages from environmental samples, enrichment of phages targeting host bacteria of interest, and induction of phages from lysogenized host strains. We also provide an optimized protocol for the creation and functional screening of phage DNA libraries derived from environmental samples. Curr. Protoc. Essential Lab. Tech. 7:13.3.1-13.3.35. © 2013 by John Wiley & Sons, Inc. Keywords: bacteriophage; phage; phage isolation; phage culture; metagenomics; phage titer; phage characterization; phage DNA; single-step growth curve; DNA library; Lysin screen
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PlyC: a multimeric bacteriophage Lysin.
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Daniel C Nelson, Raymond Schuch, Peter Chahales, Shiwei Zhu, Vincent A FischettiAbstract:Lysins are murein hydrolases produced by bacteriophage that act on the bacterial host cell wall to release progeny phage. When added extrinsically in their purified form, these enzymes produce total lysis of susceptible Gram-positive bacteria within seconds, suggesting a unique antimicrobial strategy. All known Gram-positive Lysins are produced as a single polypeptide containing a catalytic activity domain, which cleaves one of the four major peptidoglycan bonds, and a cell-wall-binding domain, which may bind a species-specific carbohydrate epitope in the cell wall. Here, we have cloned and expressed a unique Lysin from the streptococcal bacteriophage C1, termed PlyC. Molecular characterization of the plyC operon reveals that PlyC is, surprisingly, composed of two separate gene products, PlyCA and PlyCB. Based on biochemical and biophysical studies, the catalytically active PlyC holoenzyme is composed of eight PlyCB subunits for each PlyCA. Inhibitor studies predicted the presence of an active-site cysteine, and bioinformatic analysis revealed a cysteine, histidine-dependent amidohydrolase/peptidase domain within PlyCA. Point mutagenesis confirmed that PlyCA is responsible for the observed catalytic activity, and Cys-333 and His-420 are the active-site residues. PlyCB was found to self-assemble into an octamer, and this complex alone was able to direct streptococcal cell-wall-specific binding. Similar to no other proteins in sequence databases, PlyC defines a previously uncharacterized structural family of cell-wall hydrolases.
Victor D. Vacquier - One of the best experts on this subject based on the ideXlab platform.
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The abalone egg vitelline envelope receptor for sperm Lysin is a giant multivalent molecule
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Willie J. Swanson, Victor D. VacquierAbstract:Abalone sperm Lysin is a 16-kDa acrosomal protein, which nonenzymatically and species selectively creates a hole in the egg vitelline envelope (VE) through which the sperm passes to reach the egg cell membrane. The crystal structures of both monomeric and dimeric Lysins have been solved and the sequences of Lysins from 20 abalone species have been determined. As a first step in understanding the molecular mechanism by which Lysin creates a hole in the VE, its VE receptor was isolated. The VE receptor for Lysin (VERL) is an unbranched, rod-like molecule with an approximate relative molecular mass of 2 million; half the mass being carbohydrate. Fluorescence polarization studies showed positive cooperativity in the binding of Lysin to VERL (EC50 ≈9 nM) and were consistent with the species selectivity of Lysin in dissolving VEs. Each molecule of VERL bound between 126 and 142 molecules of monomeric Lysin (two independent assays), showing that VERL possesses repetitive Lysin-binding motifs.
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The species-specificity and structure of abalone sperm Lysin
Seminars in Developmental Biology, 1994Co-Authors: Andrew Shaw, Youn-ho Lee, C. David Stout, Victor D. VacquierAbstract:Abstract Abalone sperm Lysin is a 16 kDa protein that creates a hole in the egg vitelline envelope (VE) to allow the sperm to fuse with the egg. Purified Lysin exhibits quantitative species-specificity in the dissolution of isolated VE. The molecular basis for this specificity has been studied by sequencing Lysin cDNA and by solving the Lysin crystal structure. In the deduced amino acid sequences of Lysins of seven species of California abalones 50% of the positions are invariant. The most highly variable and strictly species-specific region is the amino-terminal domain of residues 2-12. The crystal structure of Lysin reveals a highly α-helical protein with a novel fold. Two tracks of basic amino acids run the length of the molecule. A hydrophobic patch of 11 residues lies on the opposite surface from the basic tracks. The species-specific domain of positions 2-12 extends away from the helical core. Mapping the species-variable positions onto the Lysin structure indicates regions which could be involved in species-specific molecular recognition.
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The Divergence of Species-Specific Abalone Sperm Lysins is Promoted by Positive Darwinian Selection.
The Biological bulletin, 1992Co-Authors: Youn-ho Lee, Victor D. VacquierAbstract:Recognition by sperm Lysin of the egg vitelline envelope may be one component in determining the species-specificity of fertilization in abalones. The amino acid sequences of Lysin proteins of seven California abalone species were deduced from the cDNA sequences. This is the first extensive comparison of a gamete recognition protein from congeneric species. Each preLysin has a highly conserved signal peptide of 18 amino acids, followed by a mature sequence of 136-138 residues. Of 136 aligned positions, 68 have the same amino acid in all seven sequences. The % identity relative to the red abalone Lysin sequence is: white 90%, flat 83%, pinto 82%, pink 78%, black 71%, and green 65%. Hydropathy plots and a distance tree of the seven Lysins show that red, white, and flat Lysins are more closely related to each other than to the Lysins of the other four species. A hypervariable, species-specific, domain exists in all sequences between positions 2-12. Amino acid replacements between any two Lysins are mostly no...
David G. Pritchard - One of the best experts on this subject based on the ideXlab platform.
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LambdaSa1 and LambdaSa2 prophage Lysins of Streptococcus agalactiae.
Applied and environmental microbiology, 2007Co-Authors: David G. Pritchard, Shengli Dong, Marion Kirk, Robert T. Cartee, John BakerAbstract:Putative N-acetylmuramyl-l-alanine amidase genes from LambdaSa1 and LambdaSa2 prophages of Streptococcus agalactiae were cloned and expressed in Escherichia coli. The purified enzymes lysed the cell walls of Streptococcus agalactiae, Streptococcus pneumoniae, and Staphylococcus aureus. The peptidoglycan digestion products in the cell wall lysates were not consistent with amidase activity. Instead, the structure of the muropeptide digestion fragments indicated that both the LambdaSa1 and LambdaSa2 Lysins exhibited γ-d-glutaminyl-l-Lysine endopeptidase activity. The endopeptidase cleavage specificity of the Lysins was confirmed using a synthetic peptide substrate corresponding to a portion of the stem peptide and cross bridge of Streptococcus agalactiae peptidoglycan. The LambdaSa2 Lysin also displayed β-d-N-acetylglucosaminidase activity.
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LambdaSa1 and LambdaSa2 prophage Lysins of Streptococcus agalactiae.
Applied and environmental microbiology, 2007Co-Authors: David G. Pritchard, Shengli Dong, Marion Kirk, Robert T. Cartee, John BakerAbstract:Putative N-acetylmuramyl-l-alanine amidase genes from LambdaSa1 and LambdaSa2 prophages of Streptococcus agalactiae were cloned and expressed in Escherichia coli. The purified enzymes lysed the cell walls of Streptococcus agalactiae, Streptococcus pneumoniae, and Staphylococcus aureus. The peptidoglycan digestion products in the cell wall lysates were not consistent with amidase activity. Instead, the structure of the muropeptide digestion fragments indicated that both the LambdaSa1 and LambdaSa2 Lysins exhibited gamma-d-glutaminyl-l-Lysine endopeptidase activity. The endopeptidase cleavage specificity of the Lysins was confirmed using a synthetic peptide substrate corresponding to a portion of the stem peptide and cross bridge of Streptococcus agalactiae peptidoglycan. The LambdaSa2 Lysin also displayed beta-d-N-acetylglucosaminidase activity.
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the bifunctional peptidoglycan Lysin of streptococcus agalactiae bacteriophage b30
Microbiology, 2004Co-Authors: David G. Pritchard, Shengli Dong, John Baker, Jeffrey A EnglerAbstract:A group B streptococcal (GBS) bacteriophage Lysin gene was cloned and expressed in Escherichia coli. The purified recombinant enzyme, calculated to have a molecular mass of 49 677 Da, lysed GBS cells. The susceptibility of GBS cells to lysis by the enzyme depended upon the growth stage at which they were harvested, with early exponential phase cells most sensitive. Calcium ions enhanced the activity of the enzyme. The enzyme also lysed other β-haemolytic streptococci, including groups A, C, E and G streptococci, but not common oral streptococci, including Streptococcus mutans. The generation of both reducing activity and N-terminal alanine residues during lysis indicated that the Lysin is a bifunctional enzyme, possessing both glycosidase and endopeptidase activities. This is consistent with the presence of two conserved sequence domains, an Acm (acetylmuramidase) domain associated with lysozyme activity, and a CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) domain associated with endopeptidase activity. Site-directed mutagenesis of conserved cysteine and histidine residues in the CHAP domain and conserved aspartate and glutamate residues in the Acm domain confirmed their importance for lysozyme and endopeptidase activity respectively.
