Journal articles on the topic 'Endolysins'
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1
Gontijo, Marco Túlio Pardini, Genesy Perez Jorge, and Marcelo Brocchi. "Current Status of Endolysin-Based Treatments against Gram-Negative Bacteria." Antibiotics 10, no. 10 (September 22, 2021): 1143. http://dx.doi.org/10.3390/antibiotics10101143.
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The prevalence of multidrug-resistant Gram-negative bacteria is a public health concern. Bacteriophages and bacteriophage-derived lytic enzymes have been studied in response to the emergence of multidrug-resistant bacteria. The availability of tRNAs and endolysin toxicity during recombinant protein expression is circumvented by codon optimization and lower expression levels using inducible pET-type plasmids and controlled cultivation conditions, respectively. The use of polyhistidine tags facilitates endolysin purification and alters antimicrobial activity. Outer membrane permeabilizers, such as organic acids, act synergistically with endolysins, but some endolysins permeate the outer membrane of Gram-negative bacteria per se. However, the outer membrane permeation mechanisms of endolysins remain unclear. Other strategies, such as the co-administration of endolysins with polymyxins, silver nanoparticles, and liposomes confer additional outer membrane permeation. Engineered endolysins comprising domains for outer membrane permeation is also a strategy used to overcome the current challenges on the control of multidrug-resistant Gram-negative bacteria. Metagenomics is a new strategy for screening endolysins with interesting antimicrobial properties from uncultured phage genomes. Here, we review the current state of the art on the heterologous expression of endolysin, showing the potential of bacteriophage endolysins in controlling bacterial infections.
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Chang, Yoonjee. "Bacteriophage-Derived Endolysins Applied as Potent Biocontrol Agents to Enhance Food Safety." Microorganisms 8, no. 5 (May 13, 2020): 724. http://dx.doi.org/10.3390/microorganisms8050724.
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Endolysins, bacteriophage-encoded enzymes, have emerged as antibacterial agents that can be actively applied in food processing systems as food preservatives to control pathogens and ultimately enhance food safety. Endolysins break down bacterial peptidoglycan structures at the terminal step of the phage reproduction cycle to enable phage progeny release. In particular, endolysin treatment is a novel strategy for controlling antibiotic-resistant bacteria, which are a severe and increasingly frequent problem in the food industry. In addition, endolysins can eliminate biofilms on the surfaces of utensils. Furthermore, the cell wall-binding domain of endolysins can be used as a tool for rapidly detecting pathogens. Research to extend the use of endolysins toward Gram-negative bacteria is now being extensively conducted. This review summarizes the trends in endolysin research to date and discusses the future applications of these enzymes as novel food preservation tools in the field of food safety.
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Son, Bokyung, Minsuk Kong, Yoyeon Cha, Jaewoo Bai, and Sangryeol Ryu. "Simultaneous Control of Staphylococcus aureus and Bacillus cereus Using a Hybrid Endolysin LysB4EAD-LysSA11." Antibiotics 9, no. 12 (December 14, 2020): 906. http://dx.doi.org/10.3390/antibiotics9120906.
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Bacteriophage endolysins have attracted attention as promising alternatives to antibiotics, and their modular structure facilitates endolysin engineering to develop novel endolysins with enhanced versatility. Here, we constructed hybrid proteins consisting of two different endolysins for simultaneous control of two critical foodborne pathogens, Staphylococcus aureus and Bacillus cereus. The full-length or enzymatically active domain (EAD) of LysB4, an endolysin from the B. cereus-infecting phage B4, was fused to LysSA11, an endolysin of the S. aureus-infecting phage SA11, via a helical linker in both orientations. The hybrid proteins maintained the lytic activity of their parental endolysins against both S. aureus and B. cereus, but they showed an extended antimicrobial spectrum. Among them, the EAD of LysB4 fused with LysSA11 (LysB4EAD-LyaSA11) showed significantly increased thermal stability compared to its parental endolysins. LysB4EAD-LysSA11 exhibited high lytic activity at pH 8.0–9.0 against S. aureus and at pH 5.0–10.0 against B. cereus, but the lytic activity of the protein decreased in the presence of NaCl. In boiled rice, treatment with 3.0 µM of LysB4EAD-LysSA11 reduced the number of S. aureus and B. cereus to undetectable levels within 2 h and also showed superior antimicrobial activity to LyB4EAD and LysSA11 in combination. These results suggest that LysB4EAD-LysSA11 could be a potent antimicrobial agent for simultaneous control of S. aureus and B. cereus.
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Love, Michael J., David Coombes, Sarah H. Manners, Gayan S. Abeysekera, Craig Billington, and Renwick C. J. Dobson. "The Molecular Basis for Escherichia coli O157:H7 Phage FAHEc1 Endolysin Function and Protein Engineering to Increase Thermal Stability." Viruses 13, no. 6 (June 9, 2021): 1101. http://dx.doi.org/10.3390/v13061101.
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Bacteriophage-encoded endolysins have been identified as antibacterial candidates. However, the development of endolysins as mainstream antibacterial agents first requires a comprehensive biochemical understanding. This study defines the atomic structure and enzymatic function of Escherichia coli O157:H7 phage FAHEc1 endolysin, LysF1. Bioinformatic analysis suggests this endolysin belongs to the T4 Lysozyme (T4L)-like family of proteins and contains a highly conserved catalytic triad. We then solved the structure of LysF1 with x-ray crystallography to 1.71 Å. LysF1 was confirmed to exist as a monomer in solution by sedimentation velocity experiments. The protein architecture of LysF1 is conserved between T4L and related endolysins. Comparative analysis with related endolysins shows that the spatial orientation of the catalytic triad is conserved, suggesting the catalytic mechanism of peptidoglycan degradation is the same as that of T4L. Differences in the sequence illustrate the role coevolution may have in the evolution of this fold. We also demonstrate that by mutating a single residue within the hydrophobic core, the thermal stability of LysF1 can be increased by 9.4 °C without compromising enzymatic activity. Overall, the characterization of LysF1 provides further insight into the T4L-like class of endolysins. Our study will help advance the development of related endolysins as antibacterial agents, as rational engineering will rely on understanding mutable positions within this protein fold.
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Swift, Steven M., Kevin P. Reid, David M. Donovan, and Timothy G. Ramsay. "Thermophile Lytic Enzyme Fusion Proteins that Target Clostridium perfringens." Antibiotics 8, no. 4 (November 8, 2019): 214. http://dx.doi.org/10.3390/antibiotics8040214.
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Clostridium perfringens is a bacterial pathogen that causes necrotic enteritis in poultry and livestock, and is a source of food poisoning and gas gangrene in humans. As the agriculture industry eliminates the use of antibiotics in animal feed, alternatives to antibiotics will be needed. Bacteriophage endolysins are enzymes used by the virus to burst their bacterial host, releasing bacteriophage particles. This type of enzyme represents a potential replacement for antibiotics controlling C. perfringens. As animal feed is often heat-treated during production of feed pellets, thermostable enzymes would be preferred for use in feed. To create thermostable endolysins that target C. perfringens, thermophile endolysin catalytic domains were fused to cell wall binding domains from different C. perfringens prophage endolysins. Three thermostable catalytic domains were used, two from prophage endolysins from two Geobacillus strains, and a third endolysin from the deep-sea thermophilic bacteriophage Geobacillus virus E2 (GVE2). These domains harbor predicted L-alanine-amidase, glucosaminidase, and L-alanine-amidase activities, respectively and degrade the peptidoglycan of the bacterial cell wall. The cell wall binding domains were from C. perfringens prophage endolysins (Phage LYtic enzymes; Ply): PlyCP18, PlyCP10, PlyCP33, PlyCP41, and PlyCP26F. The resulting fifteen chimeric proteins were more thermostable than the native C. perfringens endolysins, and killed swine and poultry disease-associated strains of C. perfringens.
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Rodríguez-Rubio, Lorena, Hans Gerstmans, Simon Thorpe, Stéphane Mesnage, Rob Lavigne, and Yves Briers. "DUF3380 Domain from a Salmonella Phage Endolysin Shows PotentN-Acetylmuramidase Activity." Applied and Environmental Microbiology 82, no. 16 (June 10, 2016): 4975–81. http://dx.doi.org/10.1128/aem.00446-16.
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ABSTRACTBacteriophage-encoded endolysins are highly diverse enzymes that cleave the bacterial peptidoglycan layer. Current research focuses on their potential applications in medicine, in food conservation, and as biotechnological tools. Despite the wealth of applications relying on the use of endolysin, little is known about the enzymatic properties of these enzymes, especially in the case of endolysins of bacteriophages infecting Gram-negative species. Automated genome annotations therefore remain to be confirmed. Here, we report the biochemical analysis and cleavage site determination of a novelSalmonellabacteriophage endolysin, Gp110, which comprises an uncharacterizeddomain ofunknownfunction (DUF3380; pfam11860) in its C terminus and shows a higher specific activity (34,240 U/μM) than that of 14 previously characterized endolysins active against peptidoglycan from Gram-negative bacteria (corresponding to 1.7- to 364-fold higher activity). Gp110 is a modular endolysin with an optimal pH of enzymatic activity of pH 8 and elevated thermal resistance. Reverse-phase high-performance liquid chromatography (RP-HPLC) analysis coupled to mass spectrometry showed that DUF3380 hasN-acetylmuramidase (lysozyme) activity cleaving the β-(1,4) glycosidic bond betweenN-acetylmuramic acid andN-acetylglucosamine residues. Gp110 is active against directly cross-linked peptidoglycans with various peptide stem compositions, making it an attractive enzyme for developing novel antimicrobial agents.IMPORTANCEWe report the functional and biochemical characterization of theSalmonellaphage endolysin Gp110. This endolysin has a modular structure with an enzymatically active domain and a cell wall binding domain. The enzymatic activity of this endolysin exceeds that of all other endolysins previously characterized using the same methods. A domain of unknown function (DUF3380) is responsible for this high enzymatic activity. We report that DUF3380 hasN-acetylmuramidase activity against directly cross-linked peptidoglycans with various peptide stem compositions. This experimentally verified activity allows better classification and understanding of the enzymatic activities of endolysins, which mostly are inferred by sequence similarities. Three-dimensional structure predictions for Gp110 suggest a fold that is completely different from that of known structures of enzymes with the same peptidoglycan cleavage specificity, making this endolysin quite unique. All of these features, combined with increased thermal resistance, make Gp110 an attractive candidate for engineering novel endolysin-based antibacterials.
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Kuty, Gabriel F., Min Xu, Douglas K. Struck, Elizabeth J. Summer, and Ry Young. "Regulation of a Phage Endolysin by Disulfide Caging." Journal of Bacteriology 192, no. 21 (September 10, 2010): 5682–87. http://dx.doi.org/10.1128/jb.00674-10.
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ABSTRACT In contrast to canonical phage endolysins, which require holin-mediated disruption of the membrane to gain access to attack the cell wall, signal anchor release (SAR) endolysins are secreted by the host sec system, where they accumulate in an inactive form tethered to the membrane by their N-terminal SAR domains. SAR endolysins become activated by various mechanisms upon release from the membrane. In its inactive form, the prototype SAR endolysin, LyzP1, of coliphage P1, has an active-site Cys covalently blocked by a disulfide bond; activation involves a disulfide bond isomerization driven by a thiol in the newly released SAR domain, unblocking the active-site Cys. Here, we report that Lyz103, the endolysin of Erwinia phage ERA103, is also a SAR endolysin. Although Lyz103 does not have a catalytic Cys, genetic evidence suggests that it also is activated by a thiol-disulfide isomerization triggered by a thiol in the SAR domain. In this case, the inhibitory disulfide in nascent Lyz103 is formed between cysteine residues flanking a catalytic glutamate, caging the active site. Thus, LyzP1 and Lyz103 define subclasses of SAR endolysins that differ in the nature of their inhibitory disulfide, and Lyz103 is the first enzyme found to be regulated by disulfide bond caging of its active site.
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Murray, Ellen, Lorraine A. Draper, R. Paul Ross, and Colin Hill. "The Advantages and Challenges of Using Endolysins in a Clinical Setting." Viruses 13, no. 4 (April 15, 2021): 680. http://dx.doi.org/10.3390/v13040680.
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Antibiotic-resistant pathogens are increasingly more prevalent and problematic. Traditional antibiotics are no longer a viable option for dealing with these multidrug-resistant microbes and so new approaches are needed. Bacteriophage-derived proteins such as endolysins could offer one effective solution. Endolysins are bacteriophage-encoded peptidoglycan hydrolases that act to lyse bacterial cells by targeting their cell’s wall, particularly in Gram-positive bacteria due to their naturally exposed peptidoglycan layer. These lytic enzymes have received much interest from the scientific community in recent years for their specificity, mode of action, potential for engineering, and lack of resistance mechanisms. Over the past decade, a renewed interest in endolysin therapy has led to a number of successful applications. Recombinant endolysins have been shown to be effective against prominent pathogens such as MRSA, Listeria monocytogenes, Staphylococcus strains in biofilm formation, and Pseudomonas aeruginosa. Endolysins have also been studied in combination with other antimicrobials, giving a synergistic effect. Although endolysin therapy comes with some regulatory and logistical hurdles, the future looks promising, with the emergence of engineered “next-generation” lysins. This review will focus on the likelihood that endolysins will become a viable new antimicrobial therapy and the challenges that may have to be overcome along the way.
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Gerstmans, Hans, Lorena Rodríguez-Rubio, Rob Lavigne, and Yves Briers. "From endolysins to Artilysin®s: novel enzyme-based approaches to kill drug-resistant bacteria." Biochemical Society Transactions 44, no. 1 (February 9, 2016): 123–28. http://dx.doi.org/10.1042/bst20150192.
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One of the last untapped reservoirs in nature for the identification of new anti-microbials is bacteriophages, the natural killers of bacteria. Lytic bacteriophages encode peptidoglycan (PG) lytic enzymes able to degrade the PG layer in different steps of their infection cycle. Endolysins degrade the bacterial cell wall at the end of the infection cycle, causing lysis of the host to release the viral progeny. Recombinant endolysins have been successfully applied as anti-bacterial agent against antibiotic-resistant Gram-positive pathogens. This has boosted the study of these enzymes as new anti-microbials in different fields (e.g. medical, food technology). A key example is the recent development of endolysin-based anti-bacterials against Gram-negative pathogens in which the exogenous application of endolysins is hindered by the outer membrane (OM). These novel anti-microbials, termed Artilysin®s, are able to pass through the OM and reach the PG where they exert their action. In addition, mycobacteria whose cell wall is structurally different from both Gram-positive and Gram-negative bacteria have also been reported to be inhibited by mycobacteriophage-encoded endolysins. Endolysins and endolysin-based anti-microbials can be considered as ideal candidates for an alternative to antibiotics for several reasons: (1) their unique mode of action and activity against bacterial persisters (independent of an active host metabolism), (2) their selective activity against both Gram-positive and Gram-negative pathogens (including antibiotic resistant strains) and mycobacteria, (3) the limited resistance development reported so far. The present review summarizes and discusses the potential applications of endolysins as new anti-microbials.
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Wu, Zhifeng, Yang Zhang, Xinyang Xu, Temoor Ahmed, Yong Yang, Belinda Loh, Sebastian Leptihn, Chenqi Yan, Jianping Chen, and Bin Li. "The Holin-Endolysin Lysis System of the OP2-Like Phage X2 Infecting Xanthomonas oryzae pv. oryzae." Viruses 13, no. 10 (September 28, 2021): 1949. http://dx.doi.org/10.3390/v13101949.
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Most endolysins of dsDNA phages are exported by a holin-dependent mechanism, while in some cases endolysins are exported via a holin-independent mechanism. However, it is still unclear whether the same endolysins can be exported by both holin-dependent and holin-independent mechanisms. This study investigated the lysis system of OP2-like phage X2 infecting Xanthomonas oryzae pv. oryzae, causing devastating bacterial leaf blight disease in rice. Based on bioinformatics and protein biochemistry methods, we show that phage X2 employs the classic "holin-endolysin" lysis system. The endolysin acts on the cell envelope and exhibits antibacterial effects in vitro, while the holin facilitates the release of the protein into the periplasm. We also characterized the role of the transmembrane domain (TMD) in the translocation of the endolysin across the inner membrane. We found that the TMD facilitated the translocation of the endolysin via the Sec secretion system. The holin increases the efficiency of protein release, leading to faster and more efficient lysis. Interestingly, in E. coli, the expression of either holin or endolysin with TMDs resulted in the formation of long rod shaped cells. We conclude that the TMD of X2-Lys plays a dual role: One is the transmembrane transport while the other is the inhibition of cell division, resulting in larger cells and thus in a higher number of released viruses per cell.
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Love, Michael J., David Coombes, Salim Ismail, Craig Billington, and Renwick C. J. Dobson. "The structure and function of modular Escherichia coli O157:H7 bacteriophage FTBEc1 endolysin, LysT84: defining a new endolysin catalytic subfamily." Biochemical Journal 479, no. 2 (January 28, 2022): 207–23. http://dx.doi.org/10.1042/bcj20210701.
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Bacteriophage endolysins degrade peptidoglycan and have been identified as antibacterial candidates to combat antimicrobial resistance. Considering the catalytic and structural diversity of endolysins, there is a paucity of structural data to inform how these enzymes work at the molecular level — key data that is needed to realize the potential of endolysin-based antibacterial agents. Here, we determine the atomic structure and define the enzymatic function of Escherichia coli O157:H7 phage FTEBc1 endolysin, LysT84. Bioinformatic analysis reveals that LysT84 is a modular endolysin, which is unusual for Gram-negative endolysins, comprising a peptidoglycan binding domain and an enzymatic domain. The crystal structure of LysT84 (2.99 Å) revealed a mostly α-helical protein with two domains connected by a linker region but packed together. LysT84 was determined to be a monomer in solution using analytical ultracentrifugation. Small-angle X-ray scattering data revealed that LysT84 is a flexible protein but does not have the expected bimodal P(r) function of a multidomain protein, suggesting that the domains of LysT84 pack closely creating a globular protein as seen in the crystal structure. Structural analysis reveals two key glutamate residues positioned on either side of the active site cavity; mutagenesis demonstrating these residues are critical for peptidoglycan degradation. Molecular dynamic simulations suggest that the enzymatically active domain is dynamic, allowing the appropriate positioning of these catalytic residues for hydrolysis of the β(1–4) bond. Overall, our study defines the structural basis for peptidoglycan degradation by LysT84 which supports rational engineering of related endolysins into effective antibacterial agents.
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Oechslin, Frank, Xiaojun Zhu, Moira B. Dion, Rong Shi, and Sylvain Moineau. "Phage endolysins are adapted to specific hosts and are evolutionarily dynamic." PLOS Biology 20, no. 8 (August 1, 2022): e3001740. http://dx.doi.org/10.1371/journal.pbio.3001740.
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Endolysins are produced by (bacterio)phages to rapidly degrade the bacterial cell wall and release new viral particles. Despite sharing a common function, endolysins present in phages that infect a specific bacterial species can be highly diverse and vary in types, number, and organization of their catalytic and cell wall binding domains. While much is now known about the biochemistry of phage endolysins, far less is known about the implication of their diversity on phage–host adaptation and evolution. Using CRISPR-Cas9 genome editing, we could genetically exchange a subset of different endolysin genes into distinct lactococcal phage genomes. Regardless of the type and biochemical properties of these endolysins, fitness costs associated to their genetic exchange were marginal if both recipient and donor phages were infecting the same bacterial strain, but gradually increased when taking place between phage that infect different strains or bacterial species. From an evolutionary perspective, we observed that endolysins could be naturally exchanged by homologous recombination between phages coinfecting a same bacterial strain. Furthermore, phage endolysins could adapt to their new phage/host environment by acquiring adaptative mutations. These observations highlight the remarkable ability of phage lytic systems to recombine and adapt and, therefore, explain their large diversity and mosaicism. It also indicates that evolution should be considered to act on functional modules rather than on bacteriophages themselves. Furthermore, the extensive degree of evolvability observed for phage endolysins offers new perspectives for their engineering as antimicrobial agents.
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Maliničová, Lenka, Peter Pristaš, and Peter Javorský. "Bioinformatic Analysis of Prophage Endolysins and Endolysin-Like Genes from the Order Lactobacillales." Nova Biotechnologica et Chimica 11, no. 1 (June 1, 2012): 1–10. http://dx.doi.org/10.2478/v10296-012-0001-4.
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Bioinformatic Analysis of Prophage Endolysins and Endolysin-Like Genes from the Order Lactobacillales Endolysins belonging to the group of peptigoglycan hydrolases, which are able to cleave peptidoglycan in bacterial cell walls, become an extensively studied group of enzymes. Thanks to their narrow target specificity and low probability of resistance they are considered to be an appropriate alternative to conventional antibiotics. The present paper concerns the occurrence of endolysin and endolysin-like genes in genomes of bacteria belonging to the order Lactobacillales. Using bioinformatic programmes we compared and analysed protein sequences of catalytic and cell wall binding (CWB) domains of these enzymes, their preferred combinations, their phylogenetic relationship and potential occurence of natural "domain shuffling". The existence of this phenomenon in selected group of enzymes was confirmed only in limited range, so we assume that the natural trend is the distribution of "well-tried" combinations of catalytic and CWB domains of endolysin genes as a whole.
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King, Harley, Sowmya Ajay Castro, Amol Arunrao Pohane, Cynthia M. Scholte, Vincent A. Fischetti, Natalia Korotkova, Daniel C. Nelson, and Helge C. Dorfmueller. "Molecular basis for recognition of the Group A Carbohydrate backbone by the PlyC streptococcal bacteriophage endolysin." Biochemical Journal 478, no. 12 (June 25, 2021): 2385–97. http://dx.doi.org/10.1042/bcj20210158.
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Endolysins are peptidoglycan (PG) hydrolases that function as part of the bacteriophage (phage) lytic system to release progeny phage at the end of a replication cycle. Notably, endolysins alone can produce lysis without phage infection, which offers an attractive alternative to traditional antibiotics. Endolysins from phage that infect Gram-positive bacterial hosts contain at least one enzymatically active domain (EAD) responsible for hydrolysis of PG bonds and a cell wall binding domain (CBD) that binds a cell wall epitope, such as a surface carbohydrate, providing some degree of specificity for the endolysin. Whilst the EADs typically cluster into conserved mechanistic classes with well-defined active sites, relatively little is known about the nature of the CBDs and only a few binding epitopes for CBDs have been elucidated. The major cell wall components of many streptococci are the polysaccharides that contain the polyrhamnose (pRha) backbone modified with species-specific and serotype-specific glycosyl side chains. In this report, using molecular genetics, microscopy, flow cytometry and lytic activity assays, we demonstrate the interaction of PlyCB, the CBD subunit of the streptococcal PlyC endolysin, with the pRha backbone of the cell wall polysaccharides, Group A Carbohydrate (GAC) and serotype c-specific carbohydrate (SCC) expressed by the Group A Streptococcus and Streptococcus mutans, respectively.
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Rodríguez-Rubio, Lorena, Dolores Gutiérrez, Beatriz Martínez, Ana Rodríguez, and Pilar García. "Lytic Activity of LysH5 Endolysin Secreted by Lactococcus lactis Using the Secretion Signal Sequence of Bacteriocin Lcn972." Applied and Environmental Microbiology 78, no. 9 (February 17, 2012): 3469–72. http://dx.doi.org/10.1128/aem.00018-12.
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ABSTRACTBacteriophage endolysins have an interesting potential as antimicrobials. The endolysin LysH5, encoded byStaphylococcus aureusphage vB_SauS-phi-IPLA88, was expressed and secreted inLactococcus lactisusing the signal peptide of bacteriocin lactococcin 972 and lactococcal constitutive and inducible promoters. Up to 80 U/mg of extracellular active endolysin was detected in culture supernatants, but most of the protein (up to 323 U/mg) remained in the cell extracts.
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Harhala, Marek, Daniel Nelson, Paulina Miernikiewicz, Ryan Heselpoth, Beata Brzezicka, Joanna Majewska, Sara Linden, et al. "Safety Studies of Pneumococcal Endolysins Cpl-1 and Pal." Viruses 10, no. 11 (November 15, 2018): 638. http://dx.doi.org/10.3390/v10110638.
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Bacteriophage-derived endolysins have gained increasing attention as potent antimicrobial agents and numerous publications document the in vivo efficacy of these enzymes in various rodent models. However, little has been documented about their safety and toxicity profiles. Here, we present preclinical safety and toxicity data for two pneumococcal endolysins, Pal and Cpl-1. Microarray, and gene profiling was performed on human macrophages and pharyngeal cells exposed to 0.5 µM of each endolysin for six hours and no change in gene expression was noted. Likewise, in mice injected with 15 mg/kg of each endolysin, no physical or behavioral changes were noted, pro-inflammatory cytokine levels remained constant, and there were no significant changes in the fecal microbiome. Neither endolysin caused complement activation via the classic pathway, the alternative pathway, or the mannose-binding lectin pathway. In cellular response assays, IgG levels in mice exposed to Pal or Cpl-1 gradually increased for the first 30 days post exposure, but IgE levels never rose above baseline, suggesting that hypersensitivity or allergic reaction is unlikely. Collectively, the safety and toxicity profiles of Pal and Cpl-1 support further preclinical studies.
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Dentovskaya, Svetlana V., Anastasia S. Vagaiskaya, Mikhail E. Platonov, Alexandra S. Trunyakova, Sergei A. Kotov, Ekaterina A. Krasil’nikova, Galina M. Titareva, et al. "Peptidoglycan-Free Bacterial Ghosts Confer Enhanced Protection against Yersinia pestis Infection." Vaccines 10, no. 1 (December 30, 2021): 51. http://dx.doi.org/10.3390/vaccines10010051.
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To develop a modern plague vaccine, we used hypo-endotoxic Yersinia pestis bacterial ghosts (BGs) with combinations of genes encoding the bacteriophage ɸX174 lysis-mediating protein E and/or holin-endolysin systems from λ or L-413C phages. Expression of the protein E gene resulted in the BGs that retained the shape of the original bacterium. Co-expression of this gene with genes coding for holin-endolysin system of the phage L-413C caused formation of structures resembling collapsed sacs. Such structures, which have lost their rigidity, were also formed as a result of the expression of only the L-413C holin-endolysin genes. A similar holin-endolysin system from phage λ containing mutated holin gene S and intact genes R-Rz coding for the endolysins caused generation of mixtures of BGs that had (i) practically preserved and (ii) completely lost their original rigidity. The addition of protein E to the work of this system shifted the equilibrium in the mixture towards the collapsed sacs. The collapse of the structure of BGs can be explained by endolysis of peptidoglycan sacculi. Immunizations of laboratory animals with the variants of BGs followed by infection with a wild-type Y. pestis strain showed that bacterial envelopes protected only cavies. BGs with maximally hydrolyzed peptidoglycan had a greater protectivity compared to BGs with a preserved peptidoglycan skeleton.
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Park, Taehyun, Douglas K. Struck, Chelsey A. Dankenbring, and Ry Young. "The Pinholin of Lambdoid Phage 21: Control of Lysis by Membrane Depolarization." Journal of Bacteriology 189, no. 24 (September 7, 2007): 9135–39. http://dx.doi.org/10.1128/jb.00847-07.
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ABSTRACT The phage 21 holin, S21, forms small membrane holes that depolarize the membrane and is designated as a pinholin, as opposed to large-hole-forming holins, like Sλ. Pinholins require secreted SAR endolysins, a pairing that may represent an intermediate in the evolution of canonical holin-endolysin systems.
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Wang, Luokai, Xiaochen Ju, Yu Cong, Hong Lin, and Jingxue Wang. "A Single Catalytic Endolysin Domain Plychap001: Characterization and Application to Control Vibrio parahaemolyticus and Its Biofilm Directly." Foods 11, no. 11 (May 27, 2022): 1578. http://dx.doi.org/10.3390/foods11111578.
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Endolysins are enzymes used by bacteriophages to cleave the host cell wall in the final stages of the lytic cycle. As such, they are considered promising antibacterial agents for controlling and combating multidrug-resistant (MDR) bacteria. However, the application of endolysins targeting Gram-negative bacteria is greatly hindered by the outer membrane on these bacteria. Lysqdvp001, an endolysin with modular structure, has been reported as one of the most efficient endolysins against the Gram-negative bacterium Vibrio parahaemolyticus. In this study, Plychap001, the truncated recombinant catalytic domain of Lysqdvp001, was demonstrated to exhibit a direct and efficient bactericidal activity against broad spectrum of V. parahaemolyticus strains. Plychap001 was shown to be highly stable and retain high bactericidal activity at high temperatures, over a wide pH range, and at high NaCl concentrations. Plychap001 also exhibited a synergistic lytic effect with EDTA. Additionally, Plychap001 was found to efficiently degrade and eliminate V. parahaemolyticus biofilms on polystyrene surfaces. Our study establishes Plychap001 as a promising method for controlling V. parahaemolyticus in the food industry.
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Antonova, Nataliia P., Daria V. Vasina, Evgeny O. Rubalsky, Mikhail V. Fursov, Alina S. Savinova, Igor V. Grigoriev, Evgeny V. Usachev, et al. "Modulation of Endolysin LysECD7 Bactericidal Activity by Different Peptide Tag Fusion." Biomolecules 10, no. 3 (March 12, 2020): 440. http://dx.doi.org/10.3390/biom10030440.
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The use of recombinant endolysins is a promising approach for antimicrobial therapy capable of counteracting the spread of antibiotic-resistant strains. To obtain the necessary biotechnological product, diverse peptide tags are often fused to the endolysin sequence to simplify enzyme purification, improve its ability to permeabilize the bacterial outer membrane, etc. We compared the effects of two different types of protein modifications on endolysin LysECD7 bactericidal activity in vitro and demonstrated that it is significantly modulated by specific permeabilizing antimicrobial peptides, as well as by widely used histidine tags. Thus, the tags selected for the study of endolysins and during the development of biotechnological preparations should be used with the appropriate precautions to minimize false conclusions about endolysin properties. Further, modifications of LysECD7 allowed us to obtain a lytic enzyme that was largely devoid of the disadvantages of the native protein and was active over the spectra of conditions, with high in vitro bactericidal activity not only against Gram-negative, but also against Gram-positive, bacteria. This opens up the possibility of developing effective antimicrobials based on N-terminus sheep myeloid peptide of 29 amino acids (SMAP)-modified LysECD7 that can be highly active not only during topical treatment but also for systemic applications in the bloodstream and tissues.
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Orlando, Marco, Patrick C. F. Buchholz, Marina Lotti, and Jürgen Pleiss. "The GH19 Engineering Database: Sequence diversity, substrate scope, and evolution in glycoside hydrolase family 19." PLOS ONE 16, no. 10 (October 26, 2021): e0256817. http://dx.doi.org/10.1371/journal.pone.0256817.
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The glycoside hydrolase 19 (GH19) is a bifunctional family of chitinases and endolysins, which have been studied for the control of plant fungal pests, the recycle of chitin biomass, and the treatment of multi-drug resistant bacteria. The GH19 domain-containing sequences (22,461) were divided into a chitinase and an endolysin subfamily by analyzing sequence networks, guided by taxonomy and the substrate specificity of characterized enzymes. The chitinase subfamily was split into seventeen groups, thus extending the previous classification. The endolysin subfamily is more diverse and consists of thirty-four groups. Despite their sequence diversity, twenty-six residues are conserved in chitinases and endolysins, which can be distinguished by two specific sequence patterns at six and four positions, respectively. Their location outside the catalytic cleft suggests a possible mechanism for substrate specificity that goes beyond the direct interaction with the substrate. The evolution of the GH19 catalytic domain was investigated by large-scale phylogeny. The inferred evolutionary history and putative horizontal gene transfer events differ from previous works. While no clear patterns were detected in endolysins, chitinases varied in sequence length by up to four loop insertions, causing at least eight distinct presence/absence loop combinations. The annotated GH19 sequences and structures are accessible via the GH19 Engineering Database (GH19ED, https://gh19ed.biocatnet.de). The GH19ED has been developed to support the prediction of substrate specificity and the search for novel GH19 enzymes from neglected taxonomic groups or in regions of the sequence space where few sequences have been described yet.
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Żebrowska, Joanna, Olga Żołnierkiewicz, Małgorzata Ponikowska, Michał Puchalski, Natalia Krawczun, Joanna Makowska, and Piotr Skowron. "Cloning and Characterization of a Thermostable Endolysin of Bacteriophage TP-84 as a Potential Disinfectant and Biofilm-Removing Biological Agent." International Journal of Molecular Sciences 23, no. 14 (July 9, 2022): 7612. http://dx.doi.org/10.3390/ijms23147612.
Full textAbstract:
The obligatory step in the life cycle of a lytic bacteriophage is the release of its progeny particles from infected bacterial cells. The main barrier to overcome is the cell wall, composed of crosslinked peptidoglycan, which counteracts the pressure prevailing in the cytoplasm and protects the cell against osmotic lysis and mechanical damage. Bacteriophages have developed two strategies leading to the release of progeny particles: the inhibition of peptidoglycan synthesis and enzymatic cleavage by a bacteriophage-coded endolysin. In this study, we cloned and investigated the TP84_28 endolysin of the bacteriophage TP-84, which infects thermophilic Geobacillus stearothermophilus, determined the enzymatic characteristics, and initially evaluated the endolysin application as a non-invasive agent for disinfecting surfaces, including those exposed to high temperatures. Both the native and recombinant TP84_28 endolysins, obtained through the Escherichia coli T7-lac expression system, are highly thermostable and retain trace activity after incubation at 100 °C for 30 min. The proteins exhibit strong bacterial wall digestion activity up to 77.6 °C, decreasing to marginal activity at ambient temperatures. We assayed the lysis of various types of bacteria using TP84_28 endolysins: Gram-positive, Gram-negative, encapsulated, and pathogenic. Significant lytic activity was observed on the thermophilic and mesophilic Gram-positive bacteria and, to a lesser extent, on the thermophilic and mesophilic Gram-negative bacteria. The thermostable TP84_28 endolysin seems to be a promising mild agent for disinfecting surfaces exposed to high temperatures.
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Tham, Hong Y., Adelene A.-L. Song, Khatijah Yusoff, and Geok H. Tan. "Effect of different cloning strategies in pET-28a on solubility and functionality of a staphylococcal phage endolysin." BioTechniques 69, no. 3 (September 2020): 161–70. http://dx.doi.org/10.2144/btn-2020-0034.
Full textAbstract:
Endolysins have been studied intensively as an alternative to antibiotics. In this study, endolysin derived from a phage which infects methicillin-resistant Staphylococcus aureus (MRSA) was cloned and expressed in Escherichia coli pET28a. Initially, the endolysin was cloned using BamHI/ XhoI, resulting in expression of a recombinant endolysin which was expressed in inclusion bodies. While solubilization was successful, the protein remained nonfunctional. Recloning the endolysin using NcoI/ XhoI resulted in expression of soluble and functional proteins at 18°C. The endolysin was able to form halo zones on MRSA plates and showed a reduction in turbidity of MRSA growth. Therefore, cloning strategies should be chosen carefully even in an established expression system as they could greatly affect the functionality of the expressed protein.
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Muharram, Magdy Mohamed, Ashraf Tawfik Abulhamd, Mohammed F. Aldawsari, Mohamed Hamed Alqarni, and Nikolaos E. Labrou. "Development of Staphylococcus Enzybiotics: The Ph28 Gene of Staphylococcus epidermidis Phage PH15 Is a Two-Domain Endolysin." Antibiotics 9, no. 4 (March 30, 2020): 148. http://dx.doi.org/10.3390/antibiotics9040148.
Full textAbstract:
Given the worldwide increase in antibiotic resistant bacteria, bacteriophage derived endolysins represent a very promising new alternative class of antibacterials in the fight against infectious diseases. Endolysins are able to degrade the prokaryotic cell wall, and therefore have potential to be exploited for biotechnological and medical purposes. Staphylococcus epidermidis is a Gram-positive multidrug-resistant (MDR) bacterium of human skin. It is a health concern as it is involved in nosocomial infections. Genome-based screening approach of the complete genome of Staphylococcus virus PH15 allowed the identification of an endolysin gene (Ph28; NCBI accession number: YP_950690). Bioinformatics analysis of the Ph28 protein predicted that it is a two-domain enzyme composed by a CHAP (22-112) and MurNAc-LAA (171-349) domain. Phylogenetic analysis and molecular modelling studies revealed the structural and evolutionary features of both domains. The MurNAc-LAA domain was cloned, and expressed in E. coli BL21 (DE3). In turbidity reduction assays, the recombinant enzyme can lyse more efficiently untreated S. epidermidis cells, compared to other Staphylococcus strains, suggesting enhanced specificity for S. epidermidis. These results suggest that the MurNAc-LAA domain from Ph28 endolysin may represent a promising new enzybiotic.
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Jarábková, Veronika, Lenka Tišáková, and Andrej Godány. "Phage Endolysin: A Way To Understand A Binding Function Of C-Terminal Domains A Mini Review." Nova Biotechnologica et Chimica 14, no. 2 (December 1, 2015): 117–34. http://dx.doi.org/10.1515/nbec-2015-0021.
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AbstractEndolysins are bacteriophage-encoded peptidoglycan hydrolases, which are synthesized in the end of phage reproduction cycle, in an infected host cell. Usually, for endolysins from phages that infect Gram-positive bacteria, a modular structure is typical. Therefore, these are composed of at least two separate functional domains: an N-terminal catalytic domain (EAD) and a C-terminal cell wall binding domain (CBD). Specific ligand recognition of CBDs and following peptidoglycan (PG) binding mostly allows a rapid lytic activity of an EAD. Here we briefly characterize phage endolysin CBDs in conjuction with their domain architecture, (non)necessity for the following lytic activity and a high/low specificity of their ligands as well. Such an overall assessment of CBDs may help to find new ways to widen opportunities in their protein design to create ‛designer recombinant endolysins’ with diverse applications.
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Kaspar, Ursula, Nina Schleimer, Evgeny A. Idelevich, Sonja Molinaro, and Karsten Becker. "Exploration of Bacterial Re-Growth as In Vitro Phenomenon Affecting Methods for Analysis of the Antimicrobial Activity of Chimeric Bacteriophage Endolysins." Microorganisms 10, no. 2 (February 15, 2022): 445. http://dx.doi.org/10.3390/microorganisms10020445.
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Drug alternatives to combat methicillin-resistant Staphylococcus aureus (MRSA) in human and animal healthcare are urgently needed. Recently, the recombinant bacteriophage endolysins, PRF-119 and its successor substance HY-133, have proven to be highly active against various S. aureus clonal lineages and to exhibit a very rapid bactericidal effect when standard methods for susceptibility testing are applied. Along with subsequent growth curve experiments, a re-growth phenomenon was observed in vitro necessitating its clarification for the assessment of the agent’s stability and activity as well as for methodological aspects of endolysin testing in general. Distinct in vitro parameters were comparatively examined applying also scanning electron microscopy, fluorescence assays and SDS-PAGE analysis. The shape and material of the culture vessels as well as the shaking conditions were identified as factors influencing the in vitro stability and activity of HY-133. The highest function maintenance was observed in plain centrifuge tubes. Based on this, the conditions and parameters of assays for testing the antimicrobial activities of phage endolysins were determined and adjusted. In particular, shear forces should be kept to a minimum. Our results form the basis for both future test standardization and re-growth-independent experiments as prerequisites for exact determination of the antimicrobial activities of engineered endolysins.
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Leprince, Audrey, Manon Nuytten, Annika Gillis, and Jacques Mahillon. "Characterization of PlyB221 and PlyP32, Two Novel Endolysins Encoded by Phages Preying on the Bacillus cereus Group." Viruses 12, no. 9 (September 21, 2020): 1052. http://dx.doi.org/10.3390/v12091052.
Full textAbstract:
Endolysins are phage-encoded enzymes implicated in the breaching of the bacterial cell wall at the end of the viral cycle. This study focuses on the endolysins of Deep-Blue (PlyB221) and Deep-Purple (PlyP32), two phages preying on the Bacillus cereus group. Both enzymes exhibit a typical modular organization with an enzymatically active domain (EAD) located in the N-terminal and a cell wall binding domain (CBD) in the C-terminal part of the protein. In silico analysis indicated that the EAD domains of PlyB221 and PlyP32 are endowed with peptidase and muramidase activities, respectively, whereas in both proteins SH3 domains are involved in the CBD. To evaluate their antimicrobial properties and binding specificity, both endolysins were expressed and purified. PlyB221 and PlyP32 efficiently recognized and lysed all the tested strains from the B. cereus group. Biochemical characterization showed that PlyB221 activity was stable under a wide range of pHs (5–9), NaCl concentrations (up to 200 mM), and temperature treatments (up to 50 °C). Although PlyP32 activity was less stable than that of PlyB221, the endolysin displayed high activity at pH 6–7, NaCl concentration up to 100 mM and the temperature treatment up to 45 °C. Overall, PlyB221 and PlyP32 display suitable characteristics for the development of biocontrol and detection tools.
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Matamp, Nandita, and Sarita Bhat. "Phage Endolysins as Potential Antimicrobials against Multidrug Resistant Vibrio alginolyticus and Vibrio parahaemolyticus: Current Status of Research and Challenges Ahead." Microorganisms 7, no. 3 (March 18, 2019): 84. http://dx.doi.org/10.3390/microorganisms7030084.
Full textAbstract:
Vibrio alginolyticus and V. parahaemolyticus, the causative agents of Vibriosis in marine vertebrates and invertebrates, are also responsible for fatal illnesses such as gastroenteritis, septicemia, and necrotizing fasciitis in humans via the ingestion of contaminated seafood. Aquaculture farmers often rely on extensive prophylactic use of antibiotics in farmed fish to mitigate Vibrios and their biofilms. This has been postulated as being of serious concern in the escalation of antibiotic resistant Vibrios. For this reason, alternative strategies to combat aquaculture pathogens are in high demand. Bacteriophage-derived lytic enzymes and proteins are of interest to the scientific community as promising tools with which to diminish our dependency on antibiotics. Lysqdvp001 is the best-characterized endolysin with lytic activity against multiple species of Vibrios. Various homologues of Vibrio phage endolysins have also been studied for their antibacterial potential. These novel endolysins are the major focus of this mini review.
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Abdelrahman, Fatma, Maheswaran Easwaran, Oluwasegun I. Daramola, Samar Ragab, Stephanie Lynch, Tolulope J. Oduselu, Fazal Mehmood Khan, et al. "Phage-Encoded Endolysins." Antibiotics 10, no. 2 (January 28, 2021): 124. http://dx.doi.org/10.3390/antibiotics10020124.
Full textAbstract:
Due to the global emergence of antibiotic resistance, there has been an increase in research surrounding endolysins as an alternative therapeutic. Endolysins are phage-encoded enzymes, utilized by mature phage virions to hydrolyze the cell wall from within. There is significant evidence that proves the ability of endolysins to degrade the peptidoglycan externally without the assistance of phage. Thus, their incorporation in therapeutic strategies has opened new options for therapeutic application against bacterial infections in the human and veterinary sectors, as well as within the agricultural and biotechnology sectors. While endolysins show promising results within the laboratory, it is important to document their resistance, safety, and immunogenicity for in-vivo application. This review aims to provide new insights into the synergy between endolysins and antibiotics, as well as the formulation of endolysins. Thus, it provides crucial information for clinical trials involving endolysins.
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Bai, Jaewoo, Sangmi Lee, and Sangryeol Ryu. "Identification and in vitro Characterization of a Novel Phage Endolysin that Targets Gram-Negative Bacteria." Microorganisms 8, no. 3 (March 21, 2020): 447. http://dx.doi.org/10.3390/microorganisms8030447.
Full textAbstract:
Most double-stranded (ds) DNA phages utilize holin proteins to secrete endolysin for host peptidoglycan lysis. In contrast, several holin-independent endolysins with secretion sequences or signal-arrest-release (SAR) sequences are secreted via the Sec pathway. In this study, we characterized a novel lysis protein (M4Lys) encoded by the dsDNA phage BSPM4, whose lysis function is not dependent on either holin or the Sec pathway in vitro. In silico analysis of M4Lys revealed that it contains a putative virion protein domain and an unusual C-terminal transmembrane domain (TMD). Turbidity reduction assays and liquid chromatography-mass spectrometry using purified peptidoglycan showed that the virion protein domain of M4Lys has peptidoglycan lysis activity. In vitro overproduction of M4Lys in Escherichia coli revealed that M4Lys alone caused rapid cell lysis. Treatment of E. coli with a Sec inhibitor did not inhibit the lysis activity of M4Lys, indicating that the Sec pathway is not involved in M4Lys-mediated cell lysis. Truncation of the TMD eliminated the cell lysis phenomenon, while production of the TMD alone did not induce the cell lysis. All these findings demonstrate that M4Lys is a novel endolysin that has a unique mosaic structure distinct from other canonical endolysins and the TMD plays a critical role in M4Lys-mediated in vitro cell lysis.
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Escobedo, Campelo, Wegmann, García, Rodríguez, and Martínez. "Insight into the Lytic Functions of the Lactococcal Prophage TP712." Viruses 11, no. 10 (September 20, 2019): 881. http://dx.doi.org/10.3390/v11100881.
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The lytic cassette of Lactococcus lactis prophage TP712 contains a putative membrane protein of unknown function (Orf54), a holin (Orf55), and a modular endolysin with a N-terminal glycoside hydrolase (GH_25) catalytic domain and two C-terminal LysM domains (Orf56, LysTP712). In this work, we aimed to study the mode of action of the endolysin LysTP712. Inducible expression of the holin-endolysin genes seriously impaired growth. The growth of lactococcal cells overproducing the endolysin LysTP712 alone was only inhibited upon the dissipation of the proton motive force by the pore-forming bacteriocin nisin. Processing of a 26-residues signal peptide is required for LysTP712 activation, since a truncated version without the signal peptide did not impair growth after membrane depolarization. Moreover, only the mature enzyme displayed lytic activity in zymograms, while no lytic bands were observed after treatment with the Sec inhibitor sodium azide. LysTP712 might belong to the growing family of multimeric endolysins. A C-terminal fragment was detected during the purification of LysTP712. It is likely to be synthesized from an alternative internal translational start site located upstream of the cell wall binding domain in the lysin gene. Fractions containing this fragment exhibited enhanced activity against lactococcal cells. However, under our experimental conditions, improved in vitro inhibitory activity of the enzyme was not observed upon the supplementation of additional cell wall binding domains in. Finally, our data pointed out that changes in the lactococcal cell wall, such as the degree of peptidoglycan O-acetylation, might hinder the activity of LysTP712. LysTP712 is the first secretory endolysin from a lactococcal phage described so far. The results also revealed how the activity of LysTP712 might be counteracted by modifications of the bacterial peptidoglycan, providing guidelines to exploit the biotechnological potential of phage endolysins within industrially relevant lactococci and, by extension, other bacteria.
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Vasina, Daria V., Nataliia P. Antonova, Aleksei M. Vorobev, Aleksei I. Laishevtsev, Andrei V. Kapustin, Eldar R. Zulkarneev, Svetlana S. Bochkareva, et al. "Efficacy of the Endolysin-Based Antibacterial Gel for Treatment of Anaerobic Infection Caused by Fusobacterium necrophorum." Antibiotics 10, no. 10 (October 16, 2021): 1260. http://dx.doi.org/10.3390/antibiotics10101260.
Full textAbstract:
Abscess formation is a common complication of severe life-threatening infections caused by obligate anaerobes. Fusobacterium necrophorum is among the frequently detected anaerobic pathogens from clinical specimens associated with liver abscesses, skin and soft tissue infections, or oral abscesses. The antimicrobial therapy for this kind of infection needs to be optimized. Here, we examined the possibility of treating F. necrophorum-induced abscess wound infections with candidate therapeutics based on three endolysins with activity against a broad spectrum of aerobe Gram-negative pathogens. Antibacterial gel containing three Gram-negative bacteria-targeting endolysins, LysAm24, LysAp22, and LysECD7, was formulated for topical use. Abscess formation was induced in rabbits with F. necrophorum and caused systemic infection. The survival and lifespan of the animals, general parameters, and biochemical and hematological blood tests were analyzed to assess the effectiveness of the gel treatment for the wound infection. The administration of the investigated gel twice per day for 5 days resulted in less acute inflammation, with decreased leukocytes and segmented neutrophils in the blood, retardation of infection progression, and an almost two-fold increase in the lifespan of the animals compared to the placebo group. The results indicate that endolysin-based therapy is an effective approach to treat anaerobic bacterial infections. The use of endolysins as independent pharmaceuticals, or their combination with antibiotics, could significantly reduce the development of complications in infectious diseases caused by sensitive bacterial species.
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Vander Elst, Niels, Sara B. Linden, Rob Lavigne, Evelyne Meyer, Yves Briers, and Daniel C. Nelson. "Characterization of the Bacteriophage-Derived Endolysins PlySs2 and PlySs9 with In Vitro Lytic Activity against Bovine Mastitis Streptococcus uberis." Antibiotics 9, no. 9 (September 19, 2020): 621. http://dx.doi.org/10.3390/antibiotics9090621.
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Bovine mastitis, an infection of the cow’s mammary gland, is frequently caused by Streptococcus uberis and causes major economic losses in the dairy industry. The intramammary administration of antibiotics currently remains the predominant preventive and therapeutic measure. These antimicrobial compounds, of which some are considered critical in human health care, are frequently applied as dry therapy resulting in their consistent overuse. Therefore, the use of antibiotics in the dairy sector is being questioned. We here identified two endolysins, i.e., PlySs2 and PlySs9, respectively derived from Streptococcus suis serotype-2 and -9 prophages, with lytic activity against S. uberis in an in vitro setting. Both endolysins gave clear lysis zones in spot-on-plate assays and caused a reduction of the optical density in a turbidity reduction assay. In depth characterization identified PlySs9 as the more potent endolysin over PlySs2 with a lower MIC value and about one additional log of killing. PlySs2 and PlySs9 were challenged to a panel of subclinical and clinical S. uberis milk isolates and were both able to lyse all strains tested. Molecular dissection of these endolysins in catalytic and cell wall binding subdomains resulted in major loss of killing and binding activity, respectively. Taken together, we here propose PlySs2 and PlySs9 as candidate compounds to the current antimicrobial arsenal known against bovine mastitis-causing S. uberis as future add-on or replacement strategy to the currently used intramammary antibiotics.
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Wittmann, Johannes, Rudolf Eichenlaub, and Brigitte Dreiseikelmann. "The endolysins of bacteriophages CMP1 and CN77 are specific for the lysis of Clavibacter michiganensis strains." Microbiology 156, no. 8 (August 1, 2010): 2366–73. http://dx.doi.org/10.1099/mic.0.037291-0.
Full textAbstract:
Putative endolysin genes of bacteriophages CMP1 and CN77, which infect Clavibacter michiganensis subsp. michiganensis and C. michiganensis subsp. nebraskensis, respectively, were cloned and expressed in Escherichia coli. The His-tagged endolysin of CMP1 consists of 306 amino acids and has a calculated molecular mass of 34.8 kDa, while the His-tagged endolysin of CN77 has 290 amino acids with a molecular mass of 31.9 kDa. The proteins were purified and their bacteriolytic activity was demonstrated. The bacteriolytic activity of both enzymes showed a host range which was limited to the respective C. michiganensis subspecies and did not affect other bacteria, even those closely related to Clavibacter. Due to the high specificity of the CMP1 and CN77 endolysins they may be useful tools for biocontrol of plant-pathogenic C. michiganensis without affecting other bacteria in the soil.
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Rahman, Mujeeb ur, Weixiao Wang, Qingqing Sun, Junaid Ali Shah, Chao Li, Yanmei Sun, Yuanrui Li, Bailing Zhang, Wei Chen, and Shiwei Wang. "Endolysin, a Promising Solution against Antimicrobial Resistance." Antibiotics 10, no. 11 (October 20, 2021): 1277. http://dx.doi.org/10.3390/antibiotics10111277.
Full textAbstract:
Antimicrobial resistance (AMR) is a global crisis for human public health which threatens the effective prevention and control of ever-increasing infectious diseases. The advent of pandrug-resistant bacteria makes most, if not all, available antibiotics invalid. Meanwhile, the pipeline of novel antibiotics development stagnates, which prompts scientists and pharmacists to develop unconventional antimicrobials. Bacteriophage-derived endolysins are cell wall hydrolases which could hydrolyze the peptidoglycan layer from within and outside of bacterial pathogens. With high specificity, rapid action, high efficiency, and low risk of resistance development, endolysins are believed to be among the best alternative therapeutic agents to treat multidrug resistant (MDR) bacteria. As of now, endolysins have been applied to diverse aspects. In this review, we comprehensively introduce the structures and activities of endolysins and summarize the latest application progress of recombinant endolysins in the fields of medical treatment, pathogen diagnosis, food safety, and agriculture.
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Imanishi, Ichiro, Jumpei Uchiyama, Toshihiro Tsukui, Junzo Hisatsune, Kaori Ide, Shigenobu Matsuzaki, Motoyuki Sugai, and Koji Nishifuji. "Therapeutic Potential of an Endolysin Derived from Kayvirus S25-3 for Staphylococcal Impetigo." Viruses 11, no. 9 (August 22, 2019): 769. http://dx.doi.org/10.3390/v11090769.
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Impetigo is a contagious skin infection predominantly caused by Staphylococcus aureus. Decontamination of S. aureus from the skin is becoming more difficult because of the emergence of antibiotic-resistant strains. Bacteriophage endolysins are less likely to invoke resistance and can eliminate the target bacteria without disturbance of the normal microflora. In this study, we investigated the therapeutic potential of a recombinant endolysin derived from kayvirus S25-3 against staphylococcal impetigo in an experimental setting. First, the recombinant S25-3 endolysin required an incubation period of over 15 minutes to exhibit efficient bactericidal effects against S. aureus. Second, topical application of the recombinant S25-3 endolysin decreased the number of intraepidermal staphylococci and the size of pustules in an experimental mouse model of impetigo. Third, treatment with the recombinant S25-3 endolysin increased the diversity of the skin microbiota in the same mice. Finally, we revealed the genus-specific bacteriolytic effect of recombinant S25-3 endolysin against staphylococci, particularly S. aureus, among human skin commensal bacteria. Therefore, topical treatment with recombinant S25-3 endolysin can be a promising disease management procedure for staphylococcal impetigo by efficient bacteriolysis of S. aureus while improving the cutaneous bacterial microflora.
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Camacho-González, Carlos E., César S. Cardona-Félix, Victor Zamora-Gasga, Alejandro Pérez-Larios, and Jorge Alberto Sánchez-Burgos. "Biofunctionalization of Endolysins with Oligosacharides: Formulation of Therapeutic Agents to Combat Multi-Resistant Bacteria and Potential Strategies for Their Application." Polysaccharides 3, no. 2 (March 23, 2022): 306–25. http://dx.doi.org/10.3390/polysaccharides3020018.
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In the aquaculture sector, the biofunctionalization of biomaterials is discussed using materials from algae and analyzed as a possible potential strategy to overcome the challenges that hinder the future development of the application of endolysins in this field. Derived from years of analysis, endolysins have recently been considered as potential alternative therapeutic antibacterial agents, due to their attributes and ability to combat multi-resistant bacterial cells when applied externally. On the other hand, although the aquaculture sector has been characterized by its high production rates, serious infectious diseases have led to significant economic losses that persist to this day. Although there are currently interesting data from studies under in vitro conditions on the application of endolysins in this sector, there is little or no information on in vivo studies. This lack of analysis can be attributed to the relatively low stability of endolysins in marine conditions and to the complex gastrointestinal conditions of the organisms. This review provides updated information regarding the application of endolysins against multi-resistant bacteria of clinical and nutritional interest, previously addressing their important characteristics (structure, properties and stability). In addition, regarding the aquaculture sector, the biofunctionalization of biomaterials is discussed using materials from algae and analyzed as a possible potential strategy to overcome the challenges that hinder the future development of the application of endolysins in this field.
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Olsen, Nanna, Elowine Thiran, Tobias Hasler, Thomas Vanzieleghem, Georgios Belibasakis, Jacques Mahillon, Martin Loessner, and Mathias Schmelcher. "Synergistic Removal of Static and Dynamic Staphylococcus aureus Biofilms by Combined Treatment with a Bacteriophage Endolysin and a Polysaccharide Depolymerase." Viruses 10, no. 8 (August 18, 2018): 438. http://dx.doi.org/10.3390/v10080438.
Full textAbstract:
Staphylococcus aureus is an important pathogen and biofilm former. Biofilms cause problems in clinics and food production and are highly recalcitrant to antibiotics and sanitizers. Bacteriophage endolysins kill bacteria by degrading their cell wall and are therefore deemed promising antimicrobials and anti-biofilm agents. Depolymerases targeting polysaccharides in the extracellular matrix have been suggested as parts of a multi-enzyme approach to eradicate biofilms. The efficacy of endolysins and depolymerases against S. aureus biofilms in static models has been demonstrated. However, there is a lack of studies evaluating their activity against biofilms grown under more realistic conditions. Here, we investigated the efficacy of the endolysin LysK and the poly-N-acetylglucosamine depolymerase DA7 against staphylococcal biofilms in static and dynamic (flow cell-based) models. LysK showed activity against multiple S. aureus strains, and both LysK and DA7 removed static and dynamic biofilms from polystyrene and glass surfaces at low micromolar and nanomolar concentrations, respectively. When combined, the enzymes acted synergistically, as demonstrated by crystal violet staining of static biofilms, significantly reducing viable cell counts compared to individual enzyme treatment in the dynamic model, and confocal laser scanning microscopy. Overall, our results suggest that LysK and DA7 are potent anti-biofilm agents, alone and in combination.
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Defraine, Valerie, Joris Schuermans, Barbara Grymonprez, Sander K. Govers, Abram Aertsen, Maarten Fauvart, Jan Michiels, Rob Lavigne, and Yves Briers. "Efficacy of Artilysin Art-175 against Resistant and Persistent Acinetobacter baumannii." Antimicrobial Agents and Chemotherapy 60, no. 6 (March 28, 2016): 3480–88. http://dx.doi.org/10.1128/aac.00285-16.
Full textAbstract:
Bacteriophage-encoded endolysins have shown promise as a novel class of antibacterials with a unique mode of action, i.e., peptidoglycan degradation. However, Gram-negative pathogens are generally not susceptible due to their protective outer membrane. Artilysins overcome this barrier. Artilysins are optimized, engineered fusions of selected endolysins with specific outer membrane-destabilizing peptides. Artilysin Art-175 comprises a modified variant of endolysin KZ144 with an N-terminal fusion to SMAP-29. Previously, we have shown the high susceptibility ofPseudomonas aeruginosato Art-175. Here, we report that Art-175 is highly bactericidal against stationary-phase cells of multidrug-resistantAcinetobacter baumannii, even resulting in a complete elimination of large inocula (≥108CFU/ml). Besides actively dividing cells, Art-175 also kills persisters. Instantaneous killing ofA. baumanniiupon contact with Art-175 could be visualized after immobilization of the bacteria in a microfluidic flow cell. Effective killing of a cell takes place through osmotic lysis after peptidoglycan degradation. The killing rate is enhanced by the addition of 0.5 mM EDTA. No development of resistance to Art-175 under selection pressure and no cross-resistance with existing resistance mechanisms could be observed. In conclusion, Art-175 represents a highly active Artilysin against bothA. baumanniiandP. aeruginosa, two of the most life-threatening pathogens of the orderPseudomonadales.
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Herpers, Bjorn. "Endolysins: redefining antibacterial therapy." Future Microbiology 10, no. 3 (March 2015): 309–11. http://dx.doi.org/10.2217/fmb.14.142.
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Loc, Nguyen Tan, Bui Thanh Huyen, Hoang Anh Hoang, and Le Phi Nga. "Cloning and expression of the bacteriophage-derived endolysin against Aeromonas hydrophila." IOP Conference Series: Earth and Environmental Science 947, no. 1 (December 1, 2021): 012035. http://dx.doi.org/10.1088/1755-1315/947/1/012035.
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Abstract Hemorrhagic septicemia disease in striped catfish is caused by Aeromonas hydrophila bacterium. Antibiotics are commonly used to treat this disease, however, due to antibiotic resistance in A. hydrophila, it is necessary to have an alternative antibacterial agent to antibiotics. Endolysins are bacteriophage-encoded peptidoglycan hydrolases that are synthesized at the end of the lytic phage replication cycle, they lyse the host bacterial cell wall and release new bacteriophage virions. In this study, an endolysin (cell wall hydrolase) derived from A. hydrophila phage PVN02 was artificially synthesized, cloned into pET28a(+) and successfully expressed in E. coli BL21 (DE3). The recombinant endolysin, cell wall hydrolase strongly exhibited antimicrobial activity against A. hydrophila with a reduction of 3-log CFU/ml of A. hydrophila after 30 minutes of mixing and further 30 minutes of incubation, the bacterial cells were lysed completely. It should be emphasized that the lytic activity by the recombinant endolysin to A. hydrophila bacteria did not require a pretreatment with an outer-membrane permeabilizer. The results of our study showed a potential of use this recombinant endolysin as a novel antibacterial agent to replace antibiotics in the treatment of hemorrhagic septicemia diseases in striped catfish.
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Landlinger, Christine, Lenka Tisakova, Vera Oberbauer, Timo Schwebs, Abbas Muhammad, Agnieszka Latka, Leen Van Simaey, et al. "Engineered Phage Endolysin Eliminates Gardnerella Biofilm without Damaging Beneficial Bacteria in Bacterial Vaginosis Ex Vivo." Pathogens 10, no. 1 (January 8, 2021): 54. http://dx.doi.org/10.3390/pathogens10010054.
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Bacterial vaginosis is characterized by an imbalance of the vaginal microbiome and a characteristic biofilm formed on the vaginal epithelium, which is initiated and dominated by Gardnerella bacteria, and is frequently refractory to antibiotic treatment. We investigated endolysins of the type 1,4-beta-N-acetylmuramidase encoded on Gardnerella prophages as an alternative treatment. When recombinantly expressed, these proteins demonstrated strong bactericidal activity against four different Gardnerella species. By domain shuffling, we generated several engineered endolysins with 10-fold higher bactericidal activity than any wild-type enzyme. When tested against a panel of 20 Gardnerella strains, the most active endolysin, called PM-477, showed minimum inhibitory concentrations of 0.13–8 µg/mL. PM-477 had no effect on beneficial lactobacilli or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested by fluorescence in situ hybridization on vaginal samples of fifteen patients with either first time or recurring bacterial vaginosis. In thirteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome. The high selectivity and effectiveness in eliminating Gardnerella, both in cultures of isolated strains as well as in clinically derived samples of natural polymicrobial biofilms, makes PM-477 a promising alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.
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Landlinger, Christine, Lenka Tisakova, Vera Oberbauer, Timo Schwebs, Abbas Muhammad, Agnieszka Latka, Leen Van Van Simaey, et al. "Engineered Phage Endolysin Eliminates Gardnerella Biofilm without Damaging Beneficial Bacteria in Bacterial Vaginosis Ex Vivo." Pathogens 10, no. 1 (January 8, 2021): 54. http://dx.doi.org/10.3390/pathogens10010054.
Full textAbstract:
Bacterial vaginosis is characterized by an imbalance of the vaginal microbiome and a characteristic biofilm formed on the vaginal epithelium, which is initiated and dominated by Gardnerella bacteria, and is frequently refractory to antibiotic treatment. We investigated endolysins of the type 1,4-beta-N-acetylmuramidase encoded on Gardnerella prophages as an alternative treatment. When recombinantly expressed, these proteins demonstrated strong bactericidal activity against four different Gardnerella species. By domain shuffling, we generated several engineered endolysins with 10-fold higher bactericidal activity than any wild-type enzyme. When tested against a panel of 20 Gardnerella strains, the most active endolysin, called PM-477, showed minimum inhibitory concentrations of 0.13–8 µg/mL. PM-477 had no effect on beneficial lactobacilli or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested by fluorescence in situ hybridization on vaginal samples of fifteen patients with either first time or recurring bacterial vaginosis. In thirteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome. The high selectivity and effectiveness in eliminating Gardnerella, both in cultures of isolated strains as well as in clinically derived samples of natural polymicrobial biofilms, makes PM-477 a promising alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.
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Díez-Martínez, Roberto, Héctor de Paz, Noemí Bustamante, Ernesto García, Margarita Menéndez, and Pedro García. "Improving the Lethal Effect of Cpl-7, a Pneumococcal Phage Lysozyme with Broad Bactericidal Activity, by Inverting the Net Charge of Its Cell Wall-Binding Module." Antimicrobial Agents and Chemotherapy 57, no. 11 (August 19, 2013): 5355–65. http://dx.doi.org/10.1128/aac.01372-13.
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ABSTRACTPhage endolysins are murein hydrolases that break the bacterial cell wall to provoke lysis and release of phage progeny. Recently, these enzymes have also been recognized as powerful and specific antibacterial agents when added exogenously. In the pneumococcal system, most cell wall associated murein hydrolases reported so far depend on choline for activity, and Cpl-7 lysozyme constitutes a remarkable exception. Here, we report the improvement of the killing activity of the Cpl-7 endolysin by inversion of the sign of the charge of the cell wall-binding module (from −14.93 to +3.0 at neutral pH). The engineered variant, Cpl-7S, has 15 amino acid substitutions and an improved lytic activity againstStreptococcus pneumoniae(including multiresistant strains),Streptococcus pyogenes, and other pathogens. Moreover, we have demonstrated that a single 25-μg dose of Cpl-7S significantly increased the survival rate of zebrafish embryos infected withS. pneumoniaeorS. pyogenes, confirming the killing effect of Cpl-7Sin vivo. Interestingly, Cpl-7S, in combination with 0.01% carvacrol (an essential oil), was also found to efficiently kill Gram-negative bacteria such asEscherichia coliandPseudomonas putida, an effect not described previously. Our findings provide a strategy to improve the lytic activity of phage endolysins based on facilitating their pass through the negatively charged bacterial envelope, and thereby their interaction with the cell wall target, by modulating the net charge of the cell wall-binding modules.
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Jiang, Min, Zhongxing Wang, Fufang Xia, Zhe Wen, Rui Chen, Dongyu Zhu, Min Wang, Xiangkai Zhuge, and Jianjun Dai. "Reductions in bacterial viability stimulate the production of Extra-intestinal Pathogenic Escherichia coli (ExPEC) cytoplasm-carrying Extracellular Vesicles (EVs)." PLOS Pathogens 18, no. 10 (October 19, 2022): e1010908. http://dx.doi.org/10.1371/journal.ppat.1010908.
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Extra-intestinal Pathogenic Escherichia coli (ExPEC) is defined as an extra-intestinal foodborne pathogen, and several dominant sequence types (STs) ExPEC isolates are highly virulent, with zoonotic potential. Bacteria extracellular vesicles (EVs) carry specific subsets of molecular cargo, which affect various biological processes in bacteria and host. The mechanisms of EVs formation in ExPEC remains to be elucidated. Here, the purified EVs of ExPEC strains of different STs were isolated with ultracentrifugation processes. A comparative analysis of the strain proteomes showed that cytoplasmic proteins accounted for a relatively high proportion of the proteins among ExPEC EVs. The proportion of cytoplasm-carrying vesicles in ExPEC EVs was calculated with a simple green fluorescent protein (GFP) expression method. The RecA/LexA-dependent SOS response is a critical mediator of generation of cytoplasm-carrying EVs. The SOS response activates the expression of prophage-associated endolysins, Epel1, Epel2.1, and Epel2.2, which triggered cell lysis, increasing the production of ExPEC cytoplasm-carrying EVs. The repressor LexA controlled directly the expression of these endolysins by binding to the SOS boxes in the endolysin promoter regions. Reducing bacterial viability stimulated the production of ExPEC EVs, especially cytoplasm-carrying EVs. The imbalance in cell division caused by exposure to H2O2, the deletion of ftsK genes, or t6A synthesis defects activated the RecA/LexA-dependent SOS response, inducing the expression of endolysins, and thus increasing the proportion of cytoplasm-carrying EVs in the total ExPEC EVs. Antibiotics, which decreased bacterial viability, also increase the production of ExPEC cytoplasm-carrying EVs through the SOS response. Changes in the proportion of cytoplasm-carrying EVs affected the total DNA content of ExPEC EVs. When macrophages are exposed to a higher proportion of cytoplasm-carrying vesicles, ExPEC EVs were more cytotoxic to macrophages, accompanied with more-severe mitochondrial disruption and a higher level of induced intrinsic apoptosis. In summary, we offered comprehensive insight into the proteome analysis of ExPEC EVs. This study demonstrated the novel formation mechanisms of E. coli cytoplasm-carrying EVs.
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Ajuebor, Jude, Olivia McAuliffe, Jim O'Mahony, R. Paul Ross, Colin Hill, and Aidan Coffey. "Bacteriophage Endolysins and their Applications." Science Progress 99, no. 2 (June 2016): 183–99. http://dx.doi.org/10.3184/003685016x14627913637705.
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Schmelcher, Mathias, David M. Donovan, and Martin J. Loessner. "Bacteriophage endolysins as novel antimicrobials." Future Microbiology 7, no. 10 (October 2012): 1147–71. http://dx.doi.org/10.2217/fmb.12.97.
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Gerstmans, Hans, Bjorn Criel, and Yves Briers. "Synthetic biology of modular endolysins." Biotechnology Advances 36, no. 3 (May 2018): 624–40. http://dx.doi.org/10.1016/j.biotechadv.2017.12.009.
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Jun, Soo Youn, Gi Mo Jung, Seong Jun Yoon, Yun-Jaie Choi, Woo Suk Koh, Kyoung Sik Moon, and Sang Hyeon Kang. "Preclinical Safety Evaluation of Intravenously Administered SAL200 Containing the Recombinant Phage Endolysin SAL-1 as a Pharmaceutical Ingredient." Antimicrobial Agents and Chemotherapy 58, no. 4 (January 21, 2014): 2084–88. http://dx.doi.org/10.1128/aac.02232-13.
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ABSTRACTPhage endolysins have received increasing attention as potent antibacterial agents. However, although safety evaluation is a prerequisite for the drug development process, a good laboratory practice (GLP)-compliant safety evaluation has not been reported for phage endolysins. A safety evaluation of intravenously administered SAL200 (containing phage endolysin SAL-1) was conducted according to GLP standards. No animals died in any of the safety evaluation studies. In general toxicity studies, intravenously administered SAL200 showed no sign of toxicity in rodent single- and repeated-dose toxicity studies. In the dog repeated-dose toxicity test, there were no abnormal findings, with the exception of transient abnormal clinical signs that were observed in some dogs when daily injection of SAL200 was continued for more than 1 week. In safety pharmacology studies, there were also no signs of toxicity in the central nervous and respiratory system function tests. In the cardiovascular function test, there were no abnormal findings in all tested dogs after the first and second administrations, but transient abnormalities were observed after the third and fourth administrations (2 or 3 weeks after the initial administration). All abnormal findings observed in these safety evaluation studies were slight to mild, were apparent only transiently after injection, and resolved quickly. The safety evaluation results for SAL200 support the implementation of an exploratory phase I clinical trial and underscore the potential of SAL200 as a new drug. We have designed an appropriate phase I clinical trial based on the results of this study.
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Love, Michael J., Renwick C. J. Dobson, and Craig Billington. "Stemming the tide of antibiotic resistance by exploiting bacteriophages." Biochemist 42, no. 6 (November 11, 2020): 6–11. http://dx.doi.org/10.1042/bio20200074.
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The growing prevalence of antibiotic resistance is a global crisis. It is predicted that by 2050, antibiotic resistance-related deaths will exceed by 10 million per year. Thus, there is an urgent need for alternative strategies that can either replace or supplement antibiotic use. Bacteriophages and their encoded lytic proteins, called endolysins, have both shown promise as antibiotic alternatives. Bacteriophages were first investigated as therapeutics nearly a century ago, but the success of antibiotics led to phage therapy being largely abandoned in Western medicine until recently. While sporadic reports of life-saving successes in the ad hoc use of phage therapy have emerged, properly designed, robust clinical trials and clear regulatory guidelines are required before the true potential of phage therapy can be realized. In addition, despite endolysin research still being in its infancy, the early successes of endolysin-based therapeutics already entering clinical trials are an exciting glimpse into the future. No stone can be left unturned in the discovery and development of novel therapeutics if we are to ensure a future supply of effective treatments for bacterial infections.