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Yves Briers - One of the best experts on this subject based on the ideXlab platform.
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Domain of Unknown Function 3380 (DUF3380) confers potent N-acetylmuramidase activity to a Salmonella phage Endolysin
2020Co-Authors: Lorena Rodríguez-rubio, Hans Gerstmans, Simon Thorpe, Stephane Mesnage, Rob Lavigne, Yves BriersAbstract:Bacteriophage-encoded Endolysins are highly diverse enzymes that cleave the bacterial peptidoglycan layer at the end of the lytic infection cycle to allow the viral progeny release. The specific activity and structure of these proteins have boosted their study as new antimicrobials against pathogens including multidrug resistant bacteria as well as the development of new biotechnological tools for bacterial diagnostics and detection, among others. Despite the wealth of applications relying on the use of Endolysin, little is known about the enzymatic properties of these enzymes, especially in case of Endolysins of bacteriophages infecting Gram-negative species. Only a limited number of studies have analyzed the peptidoglycan bond cleaved by Endolysins and most annotations of enzymatic specificity only rely on sequence similarity. As a result, available databases contain inaccurate descriptions of biochemical specificities. In this study, we report the functional and biochemical characterization of the modular Salmonella phage Endolysin Gp110 which comprises an uncharacterized Domain of Unknown Function (DUF3380; pfam11860) in its C-terminus and shows the highest specific activity (34,240 U/µM) compared to fourteen previously characterized Endolysins active against peptidoglycan from Gram-negative bacteria (corresponding to a 1.7- to 364-fold higher activity). This Endolysin showed an optimal enzymatic activity at pH 8 and an elevated thermal resistance. Reversed phase-HPLC analysis coupled to mass spectrometry showed that DUF3380 has N-acetylmuramidase (lysozyme) activity cleaving the β-(1,4) glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine residues. Gp110 is active against directly cross-linked peptidoglycan with various peptide stem compositions, making it an attractive enzyme to develop novel antimicrobial agents.
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Innolysins: A novel approach to engineer Endolysins to kill Gram-negative bacteria
bioRxiv, 2018Co-Authors: Athina Zampara, Yves Briers, Martine C. Holst Sørensen, Dennis Grimon, Fabio Antenucci, Lone BrøndstedAbstract:Bacteriophage-encoded Endolysins degrading the essential peptidoglycan of bacteria are promising alternative antimicrobials to handle the global threat of antibiotic resistant bacteria. However, Endolysins have limited use against Gram-negative bacteria, since their outer membrane prevents access to the peptidoglycan. Here we present Innolysins, a novel concept for engineering Endolysins that allows the enzymes to pass through the outer membrane, hydrolyse the peptidoglycan and kill the target bacterium. Innolysins combine the enzymatic activity of Endolysins with the binding capacity of phage receptor binding proteins (RBPs). As our proof of concept, we used phage T5 Endolysin and receptor binding protein Pb5, which binds irreversibly to the phage receptor FhuA involved in ferrichrome transport in Escherichia coli. In total, we constructed twelve Innolysins fusing Endolysin with Pb5 or the binding domain of Pb5 with or without flexible linkers in between. While the majority of the Innolysins maintained their muralytic activity, Innolysin#6 also showed bactericidal activity against E. coli reducing the number of bacteria by 1 log, thus overcoming the outer membrane barrier. Using an E. coli fhuA deletion mutant, we demonstrated that FhuA is required for bactericidal activity, supporting that the specific binding of Pb5 to its receptor on E. coli is needed for the Endolysin to access the peptidoglycan. Accordingly, Innolysin#6 was able to kill other bacterial species that carry conserved FhuA homologs such as Shigella sonnei and Pseudomonas aeruginosa. In summary, the Innolysin approach expands recent protein engineering strategies allowing customization of Endolysins by exploiting phage RBPs to specifically target Gram-negative bacteria.
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duf3380 domain from a salmonella phage Endolysin shows potent n acetylmuramidase activity
Applied and Environmental Microbiology, 2016Co-Authors: Lorena Rodriguezrubio, Hans Gerstmans, Simon Thorpe, Stephane Mesnage, Rob Lavigne, Yves BriersAbstract:ABSTRACT Bacteriophage-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 novel Salmonella bacteriophage Endolysin, Gp110, which comprises an uncharacterized domain of unknown function (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 has N-acetylmuramidase (lysozyme) activity cleaving the β-(1,4) glycosidic bond between N-acetylmuramic acid and N-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. IMPORTANCE We report the functional and biochemical characterization of the Salmonella phage 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 has N-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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engineered Endolysin based artilysins to combat multidrug resistant gram negative pathogens
Mbio, 2014Co-Authors: Yves Briers, Maarten Walmagh, Anneleen Cornelissen, William Cenens, Gunther Verween, Victor Van Puyenbroeck, Hugo Oliveira, Joana Azeredo, Abram Aertsen, Jean Paul PirnayAbstract:The global threat to public health posed by emerging multidrug-resistant bacteria in the past few years necessitates the development of novel approaches to combat bacterial infections. Endolysins encoded by bacterial viruses (or phages) repre- sent one promising avenue of investigation. These enzyme-based antibacterials efficiently kill Gram-positive bacteria upon con- tact by specific cell wall hydrolysis. However, a major hurdle in their exploitation as antibacterials against Gram-negative patho- gens is the impermeable lipopolysaccharide layer surrounding their cell wall. Therefore, we developed and optimized an approach to engineer these enzymes as outer membrane-penetrating Endolysins (Artilysins), rendering them highly bactericidal against Gram-negative pathogens, including Pseudomonas aeruginosa and Acinetobacter baumannii. Artilysins combining a polycationic nonapeptide and a modular Endolysin are able to kill these (multidrug-resistant) strains in vitro with a4t o 5l og reduction within 30 min. We show that the activity of Artilysins can be further enhanced by the presence of a linker of increasing length between the peptide and Endolysin or by a combination of both polycationic and hydrophobic/amphipathic peptides. Time-lapse microscopy confirmed the mode of action of polycationic Artilysins, showing that they pass the outer membrane to degrade the peptidoglycan with subsequent cell lysis. Artilysins are effective in vitro (human keratinocytes) and in vivo (Caeno- rhabditis elegans). IMPORTANCE Bacterial resistance to most commonly used antibiotics is a major challenge of the 21st century. Infections that can- not be treated byfirst-line antibiotics lead to increasing morbidity and mortality, while millions of dollars are spent each year by health care systems in trying to control antibiotic-resistant bacteria and to prevent cross-transmission of resistance. Endoly- sins—enzymes derived from bacterial viruses—represent a completely novel, promising class of antibacterials based on cell wall hydrolysis. Specifically, they are active against Gram-positive species, which lack a protective outer membrane and which have a low probability of resistance development. We modified Endolysins by protein engineering to create Artilysins that are able to pass the outer membrane and become active against Pseudomonas aeruginosa and Acinetobacter baumannii, two of the most hazardous drug-resistant Gram-negative pathogens.
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Engineered Endolysin-based “Artilysins” to combat multidrug-resistant gram-negative pathogens
mBio, 2014Co-Authors: Yves Briers, Maarten Walmagh, Victor Van Puyenbroeck, Anneleen Cornelissen, William Cenens, Gunther Verween, Hugo Oliveira, Joana Azeredo, Abram Aertsen, Jean Paul PirnayAbstract:The global threat to public health posed by emerging multidrug-resistant bacteria in the past few years necessitates the development of novel approaches to combat bacterial infections. Endolysins encoded by bacterial viruses (or phages) represent one promising avenue of investigation. These enzyme-based antibacterials efficiently kill Gram-positive bacteria upon contact by specific cell wall hydrolysis. However, a major hurdle in their exploitation as antibacterials against Gram-negative pathogens is the impermeable lipopolysaccharide layer surrounding their cell wall. Therefore, we developed and optimized an approach to engineer these enzymes as outer membrane-penetrating Endolysins (Artilysins), rendering them highly bactericidal against Gram-negative pathogens, including Pseudomonas aeruginosa and Acinetobacter baumannii. Artilysins combining a polycationic nonapeptide and a modular Endolysin are able to kill these (multidrug-resistant) strains in vitro with a 4 to 5 log reduction within 30 min. We show that the activity of Artilysins can be further enhanced by the presence of a linker of increasing length between the peptide and Endolysin or by a combination of both polycationic and hydrophobic/amphipathic peptides. Time-lapse microscopy confirmed the mode of action of polycationic Artilysins, showing that they pass the outer membrane to degrade the peptidoglycan with subsequent cell lysis. Artilysins are effective in vitro (human keratinocytes) and in vivo (Caenorhabditis elegans). Importance: Bacterial resistance to most commonly used antibiotics is a major challenge of the 21st century. Infections that cannot be treated by first-line antibiotics lead to increasing morbidity and mortality, while millions of dollars are spent each year by health care systems in trying to control antibiotic-resistant bacteria and to prevent cross-transmission of resistance. Endolysins--enzymes derived from bacterial viruses--represent a completely novel, promising class of antibacterials based on cell wall hydrolysis. Specifically, they are active against Gram-positive species, which lack a protective outer membrane and which have a low probability of resistance development. We modified Endolysins by protein engineering to create Artilysins that are able to pass the outer membrane and become active against Pseudomonas aeruginosa and Acinetobacter baumannii, two of the most hazardous drug-resistant Gram-negative pathogens.
Hugo Oliveira - One of the best experts on this subject based on the ideXlab platform.
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Staphylococci phages display vast genomic diversity and evolutionary relationships
BMC Genomics, 2019Co-Authors: Hugo Oliveira, Welkin H. Pope, Graham F. Hatfull, Marta Sampaio, Oscar Dias, Luís D. R. Melo, Joana AzeredoAbstract:BackgroundBacteriophages are the most abundant and diverse entities in the biosphere, and this diversity is driven by constant predator–prey evolutionary dynamics and horizontal gene transfer. Phage genome sequences are under-sampled and therefore present an untapped and uncharacterized source of genetic diversity, typically characterized by highly mosaic genomes and no universal genes. To better understand the diversity and relationships among phages infecting human pathogens, we have analysed the complete genome sequences of 205 phages of Staphylococcus sp.ResultsThese are predicted to encode 20,579 proteins, which can be sorted into 2139 phamilies (phams) of related sequences; 745 of these are orphams and possess only a single gene. Based on shared gene content, these phages were grouped into four clusters (A, B, C and D), 27 subclusters (A1-A2, B1-B17, C1-C6 and D1-D2) and one singleton. However, the genomes have mosaic architectures and individual genes with common ancestors are positioned in distinct genomic contexts in different clusters. The staphylococcal Cluster B siphoviridae are predicted to be temperate, and the integration cassettes are often closely-linked to genes implicated in bacterial virulence determinants. There are four unusual Endolysin organization strategies found in Staphylococcus phage genomes, with Endolysins predicted to be encoded as single genes, two genes spliced, two genes adjacent and as a single gene with inter-lytic-domain secondary translational start site. Comparison of the Endolysins reveals multi-domain modularity, with conservation of the SH3 cell wall binding domain.ConclusionsThis study provides a high-resolution view of staphylococcal viral genetic diversity, and insights into their gene flux patterns within and across different phage groups (cluster and subclusters) providing insights into their evolution.
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engineered Endolysin based artilysins to combat multidrug resistant gram negative pathogens
Mbio, 2014Co-Authors: Yves Briers, Maarten Walmagh, Anneleen Cornelissen, William Cenens, Gunther Verween, Victor Van Puyenbroeck, Hugo Oliveira, Joana Azeredo, Abram Aertsen, Jean Paul PirnayAbstract:The global threat to public health posed by emerging multidrug-resistant bacteria in the past few years necessitates the development of novel approaches to combat bacterial infections. Endolysins encoded by bacterial viruses (or phages) repre- sent one promising avenue of investigation. These enzyme-based antibacterials efficiently kill Gram-positive bacteria upon con- tact by specific cell wall hydrolysis. However, a major hurdle in their exploitation as antibacterials against Gram-negative patho- gens is the impermeable lipopolysaccharide layer surrounding their cell wall. Therefore, we developed and optimized an approach to engineer these enzymes as outer membrane-penetrating Endolysins (Artilysins), rendering them highly bactericidal against Gram-negative pathogens, including Pseudomonas aeruginosa and Acinetobacter baumannii. Artilysins combining a polycationic nonapeptide and a modular Endolysin are able to kill these (multidrug-resistant) strains in vitro with a4t o 5l og reduction within 30 min. We show that the activity of Artilysins can be further enhanced by the presence of a linker of increasing length between the peptide and Endolysin or by a combination of both polycationic and hydrophobic/amphipathic peptides. Time-lapse microscopy confirmed the mode of action of polycationic Artilysins, showing that they pass the outer membrane to degrade the peptidoglycan with subsequent cell lysis. Artilysins are effective in vitro (human keratinocytes) and in vivo (Caeno- rhabditis elegans). IMPORTANCE Bacterial resistance to most commonly used antibiotics is a major challenge of the 21st century. Infections that can- not be treated byfirst-line antibiotics lead to increasing morbidity and mortality, while millions of dollars are spent each year by health care systems in trying to control antibiotic-resistant bacteria and to prevent cross-transmission of resistance. Endoly- sins—enzymes derived from bacterial viruses—represent a completely novel, promising class of antibacterials based on cell wall hydrolysis. Specifically, they are active against Gram-positive species, which lack a protective outer membrane and which have a low probability of resistance development. We modified Endolysins by protein engineering to create Artilysins that are able to pass the outer membrane and become active against Pseudomonas aeruginosa and Acinetobacter baumannii, two of the most hazardous drug-resistant Gram-negative pathogens.
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Engineered Endolysin-based “Artilysins” to combat multidrug-resistant gram-negative pathogens
mBio, 2014Co-Authors: Yves Briers, Maarten Walmagh, Victor Van Puyenbroeck, Anneleen Cornelissen, William Cenens, Gunther Verween, Hugo Oliveira, Joana Azeredo, Abram Aertsen, Jean Paul PirnayAbstract:The global threat to public health posed by emerging multidrug-resistant bacteria in the past few years necessitates the development of novel approaches to combat bacterial infections. Endolysins encoded by bacterial viruses (or phages) represent one promising avenue of investigation. These enzyme-based antibacterials efficiently kill Gram-positive bacteria upon contact by specific cell wall hydrolysis. However, a major hurdle in their exploitation as antibacterials against Gram-negative pathogens is the impermeable lipopolysaccharide layer surrounding their cell wall. Therefore, we developed and optimized an approach to engineer these enzymes as outer membrane-penetrating Endolysins (Artilysins), rendering them highly bactericidal against Gram-negative pathogens, including Pseudomonas aeruginosa and Acinetobacter baumannii. Artilysins combining a polycationic nonapeptide and a modular Endolysin are able to kill these (multidrug-resistant) strains in vitro with a 4 to 5 log reduction within 30 min. We show that the activity of Artilysins can be further enhanced by the presence of a linker of increasing length between the peptide and Endolysin or by a combination of both polycationic and hydrophobic/amphipathic peptides. Time-lapse microscopy confirmed the mode of action of polycationic Artilysins, showing that they pass the outer membrane to degrade the peptidoglycan with subsequent cell lysis. Artilysins are effective in vitro (human keratinocytes) and in vivo (Caenorhabditis elegans). Importance: Bacterial resistance to most commonly used antibiotics is a major challenge of the 21st century. Infections that cannot be treated by first-line antibiotics lead to increasing morbidity and mortality, while millions of dollars are spent each year by health care systems in trying to control antibiotic-resistant bacteria and to prevent cross-transmission of resistance. Endolysins--enzymes derived from bacterial viruses--represent a completely novel, promising class of antibacterials based on cell wall hydrolysis. Specifically, they are active against Gram-positive species, which lack a protective outer membrane and which have a low probability of resistance development. We modified Endolysins by protein engineering to create Artilysins that are able to pass the outer membrane and become active against Pseudomonas aeruginosa and Acinetobacter baumannii, two of the most hazardous drug-resistant Gram-negative pathogens.
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Engineered Endolysin Based “ Artilysins ” To Combat Multidrug Resistant GramNegative pathogens
mBio, 2014Co-Authors: Yves Briers, Maarten Walmagh, Anneleen Cornelissen, William Cenens, Victor Van Puyenbroeck, Abram Aertsen, Hugo OliveiraAbstract:The global threat to public health posed by emerging multidrug-resistant bacteria in the past few years necessitates the development of novel approaches to combat bacterial infections. Endolysins encoded by bacterial viruses (or phages) repre- sent one promising avenue of investigation. These enzyme-based antibacterials efficiently kill Gram-positive bacteria upon con- tact by specific cell wall hydrolysis. However, a major hurdle in their exploitation as antibacterials against Gram-negative patho- gens is the impermeable lipopolysaccharide layer surrounding their cell wall. Therefore, we developed and optimized an approach to engineer these enzymes as outer membrane-penetrating Endolysins (Artilysins), rendering them highly bactericidal against Gram-negative pathogens, including Pseudomonas aeruginosa and Acinetobacter baumannii. Artilysins combining a polycationic nonapeptide and a modular Endolysin are able to kill these (multidrug-resistant) strains in vitrowitha4to5log reduction within 30 min.Weshow that the activity of Artilysins can be further enhanced by the presence of a linker of increasing length between the peptide and Endolysin or by a combination of both polycationic and hydrophobic/amphipathic peptides. Time-lapse microscopy confirmed the mode of action of polycationic Artilysins, showing that they pass the outer membrane to degrade the peptidoglycan with subsequent cell lysis. Artilysins are effective in vitro (human keratinocytes) and in vivo (Caeno- rhabditis elegans).
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Molecular aspects and comparative genomics of bacteriophage Endolysins.
Journal of virology, 2013Co-Authors: Hugo Oliveira, Sílvio B. Santos, Luís D. R. Melo, Nuno Cerca, Eugénio C. Ferreira, Franklin Luzia Nóbrega, Joana Azeredo, Leon D KluskensAbstract:Phages are recognized as the most abundant and diverse entities on the planet. Their diversity is determined predominantly by their dynamic adaptation capacities when confronted with different selective pressures in an endless cycle of coevolution with a widespread group of bacterial hosts. At the end of the infection cycle, progeny virions are confronted with a rigid cell wall that hinders their release into the environment and the opportunity to start a new infection cycle. Consequently, phages encode hydrolytic enzymes, called Endolysins, to digest the peptidoglycan. In this work, we bring to light all phage Endolysins found in completely sequenced double-stranded nucleic acid phage genomes and uncover clues that explain the phage-Endolysin-host ecology that led phages to recruit unique and specialized Endolysins.
David M. Donovan - One of the best experts on this subject based on the ideXlab platform.
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Endolysins as Antimicrobials
Advances in Virus Research, 2020Co-Authors: Daniel C. Nelson, Mathias Schmelcher, Lorena Rodríguez-rubio, Jochen Klumpp, David G. Pritchard, Shengli Dong, David M. DonovanAbstract:Peptidoglycan (PG) is the major structural component of the bacterial cell wall. Bacteria have autolytic PG hydrolases that allow the cell to grow and divide. A well-studied group of PG hydrolase enzymes are the bacteriophage Endolysins. Endolysins are PG-degrading proteins that allow the phage to escape from the bacterial cell during the phage lytic cycle. The Endolysins, when purified and exposed to PG externally, can cause “lysis from without.” Numerous publications have described how this phenomenon can be used therapeutically as an effective antimicrobial against certain pathogens. Endolysins have a characteristic modular structure, often with multiple lytic and/or cell wall-binding domains (CBDs). They degrade the PG with glycosidase, amidase, endopeptidase, or lytic transglycosylase activities and have been shown to be synergistic with fellow PG hydrolases or a range of other antimicrobials. Due to the coevolution of phage and host, it is thought they are much less likely to invoke resistance. Endolysin engineering has opened a range of new applications for these proteins from food safety to environmental decontamination to more effective antimicrobials that are believed refractory to resistance development. To put phage Endolysin work in a broader context, this chapter includes relevant studies of other well-characterized PG hydrolase antimicrobials.
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The Endolysin from the Enterococcus faecalis bacteriophage VD13 and conditions stimulating its lytic activity
Fems Microbiology Letters, 2016Co-Authors: Steven M. Swift, D. Treva Rowley, Carly Young, Ashley Franks, Paul Hyman, David M. DonovanAbstract:Bacteriophage produce Endolysins (peptidoglycan hydrolases) to lyse the host cell from within and release nascent bacteriophage particles. Recombinant Endolysins can lyse Gram-positive bacteria when added exogenously. As a potential alternative antimicrobial, we cloned and expressed the enterococcal VD13 bacteriophage Endolysin. VD13 Endolysin has a CHAP catalytic domain with 92% identity with the bacteriophage IME-EF1 Endolysin. The predicted size of VD13 Endolysin is ∼27 kDa as verified by SDS-PAGE. The VD13 Endolysin lyses Enterococcus faecalis strains, but not Enterococcus faecium or other non-enterococci. VD13 Endolysin has activity from pH 4 to pH 8, with peak activity at pH 5, and exhibits greater activity in the presence of calcium. Optimum activity at pH 5 occurs in the absence of NaCl. VD13 Endolysin, in ammonium acetate (C2H3O2NH4) calcium chloride (CaCl2) buffer pH 5, is stimulated to higher activity upon heating at temperatures up to 65°C for 30 minutes, whereas activity is lost upon heating to 42°C, in pH 7 buffer.
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a bacteriophage Endolysin that eliminates intracellular streptococci
eLife, 2016Co-Authors: Yang Shen, Marilia Barros, Tarek Vennemann, Sara B Linden, Ryan D Heselpoth, Dennis J Spencer, David M. Donovan, Travis D Gallagher, John MoultAbstract:Streptococcus pyogenes is the bacterium that causes throat infections and other serious infections in humans. Antibiotics such as penicillin are used to treat active infections, but so-called “strep throat infections” often return after treatment. This is because S. pyogenes can enter the cells that line the throat and hide from the antibiotics, which cannot enter the throat cells. Endolysins are enzymes produced by viruses that attack bacteria, and these enzymes target and destroy the bacterial cell wall. A previous study revealed that an Endolysin known as PlyC could destroy S. pyogenes bacteria on contact. PlyC and other Endolysins have the potential to act as alternatives to common antibiotics, but before these enzymes can be developed as therapeutics, it is important to understand how they interact with human host cells. Like antibiotics, the PlyC Endolysin was not expected to enter throat cells. However, Shen, Barros et al. have now discovered that not only can PlyC enter throat cells, it can essentially chase down and kill S. pyogenes that are hiding inside. Other similar enzymes could not act in this way, and further studies confirmed that PlyC could move around inside a throat cell without causing it damage. Shen, Barros et al. also determined that PlyC has a pocket on its surface that binds with a specific component of the throat cell membrane, a molecule called phosphatidylserine. This interaction – which is a bit like a lock and key – grants PlyC access into the cell. While it is clear that PlyC eventually kills S. pyogenes hiding inside throat cells, future experiments will aim to determine how PlyC moves around once inside an infected throat cell. Together, an understanding of how an Endolysin enters cells and destroys hiding S. pyogenes will contribute to the development of Endolysins with broader activity, which can be used as alternatives to common antibiotics.
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A bacteriophage Endolysin that eliminates intracellular streptococci
eLife, 2016Co-Authors: Yang Shen, Marilia Barros, Tarek Vennemann, D Travis Gallagher, Yizhou Yin, Sara B Linden, Ryan D Heselpoth, Dennis J Spencer, David M. Donovan, John MoultAbstract:PlyC, a bacteriophage-encoded Endolysin, lyses Streptococcus pyogenes (Spy) on contact. Here, we demonstrate that PlyC is a potent agent for controlling intracellular Spy that often underlies refractory infections. We show that the PlyC holoenzyme, mediated by its PlyCB subunit, crosses epithelial cell membranes and clears intracellular Spy in a dose-dependent manner. Quantitative studies using model membranes establish that PlyCB interacts strongly with phosphatidylserine (PS), whereas its interaction with other lipids is weak, suggesting specificity for PS as its cellular receptor. Neutron reflection further substantiates that PlyC penetrates bilayers above a PS threshold concentration. Crystallography and docking studies identify key residues that mediate PlyCB–PS interactions, which are validated by site-directed mutagenesis. This is the first report that a native Endolysin can traverse epithelial membranes, thus substantiating the potential of PlyC as an antimicrobial for Spy in the extracellular and intracellular milieu and as a scaffold for engineering other functionalities.Streptococcus pyogenes is the bacterium that causes throat infections and other serious infections in humans. Antibiotics such as penicillin are used to treat active infections, but so-called “strep throat infections” often return after treatment. This is because S. pyogenes can enter the cells that line the throat and hide from the antibiotics, which cannot enter the throat cells.Endolysins are enzymes produced by viruses that attack bacteria, and these enzymes target and destroy the bacterial cell wall. A previous study revealed that an Endolysin known as PlyC could destroy S. pyogenes bacteria on contact. PlyC and other Endolysins have the potential to act as alternatives to common antibiotics, but before these enzymes can be developed as therapeutics, it is important to understand how they interact with human host cells.Like antibiotics, the PlyC Endolysin was not expected to enter throat cells. However, Shen, Barros et al. have now discovered that not only can PlyC enter throat cells, it can essentially chase down and kill S. pyogenes that are hiding inside. Other similar enzymes could not act in this way, and further studies confirmed that PlyC could move around inside a throat cell without causing it damage. Shen, Barros et al. also determined that PlyC has a pocket on its surface that binds with a specific component of the throat cell membrane, a molecule called phosphatidylserine. This interaction – which is a bit like a lock and key – grants PlyC access into the cell.While it is clear that PlyC eventually kills S. pyogenes hiding inside throat cells, future experiments will aim to determine how PlyC moves around once inside an infected throat cell. Together, an understanding of how an Endolysin enters cells and destroys hiding S. pyogenes will contribute to the development of Endolysins with broader activity, which can be used as alternatives to common antibiotics.
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a thermophilic phage Endolysin fusion to a clostridium perfringens specific cell wall binding domain creates an anti clostridium antimicrobial with improved thermostability
Viruses, 2015Co-Authors: Steven M. Swift, Bruce S Seal, Johnna K Garrish, Brian B Oakley, Kelli L Hiett, Rebekah Woolsey, Kathleen M Schegg, J E Line, David M. DonovanAbstract:Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Many enzymes are added to animal feed during production and are subjected to high-heat stress during feed processing. To produce a thermostabile Endolysin for treating poultry, an E. coli codon-optimized gene was synthesized that fused the N-acetylmuramoyl-l-alanine amidase domain from the Endolysin of the thermophilic bacteriophage ΦGVE2 to the cell-wall binding domain (CWB) from the Endolysin of the C. perfringens-specific bacteriophage ΦCP26F. The resulting protein, PlyGVE2CpCWB, lysed C. perfringens in liquid and solid cultures. PlyGVE2CpCWB was most active at pH 8, had peak activity at 10 mM NaCl, 40% activity at 150 mM NaCl and was still 16% active at 600 mM NaCl. The protein was able to withstand temperatures up to 50 °C and still lyse C. perfringens. Herein, we report the construction and characterization of a thermostable chimeric Endolysin that could potentially be utilized as a feed additive to control the bacterium during poultry production.
Jean Paul Pirnay - One of the best experts on this subject based on the ideXlab platform.
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engineered Endolysin based artilysins to combat multidrug resistant gram negative pathogens
Mbio, 2014Co-Authors: Yves Briers, Maarten Walmagh, Anneleen Cornelissen, William Cenens, Gunther Verween, Victor Van Puyenbroeck, Hugo Oliveira, Joana Azeredo, Abram Aertsen, Jean Paul PirnayAbstract:The global threat to public health posed by emerging multidrug-resistant bacteria in the past few years necessitates the development of novel approaches to combat bacterial infections. Endolysins encoded by bacterial viruses (or phages) repre- sent one promising avenue of investigation. These enzyme-based antibacterials efficiently kill Gram-positive bacteria upon con- tact by specific cell wall hydrolysis. However, a major hurdle in their exploitation as antibacterials against Gram-negative patho- gens is the impermeable lipopolysaccharide layer surrounding their cell wall. Therefore, we developed and optimized an approach to engineer these enzymes as outer membrane-penetrating Endolysins (Artilysins), rendering them highly bactericidal against Gram-negative pathogens, including Pseudomonas aeruginosa and Acinetobacter baumannii. Artilysins combining a polycationic nonapeptide and a modular Endolysin are able to kill these (multidrug-resistant) strains in vitro with a4t o 5l og reduction within 30 min. We show that the activity of Artilysins can be further enhanced by the presence of a linker of increasing length between the peptide and Endolysin or by a combination of both polycationic and hydrophobic/amphipathic peptides. Time-lapse microscopy confirmed the mode of action of polycationic Artilysins, showing that they pass the outer membrane to degrade the peptidoglycan with subsequent cell lysis. Artilysins are effective in vitro (human keratinocytes) and in vivo (Caeno- rhabditis elegans). IMPORTANCE Bacterial resistance to most commonly used antibiotics is a major challenge of the 21st century. Infections that can- not be treated byfirst-line antibiotics lead to increasing morbidity and mortality, while millions of dollars are spent each year by health care systems in trying to control antibiotic-resistant bacteria and to prevent cross-transmission of resistance. Endoly- sins—enzymes derived from bacterial viruses—represent a completely novel, promising class of antibacterials based on cell wall hydrolysis. Specifically, they are active against Gram-positive species, which lack a protective outer membrane and which have a low probability of resistance development. We modified Endolysins by protein engineering to create Artilysins that are able to pass the outer membrane and become active against Pseudomonas aeruginosa and Acinetobacter baumannii, two of the most hazardous drug-resistant Gram-negative pathogens.
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Engineered Endolysin-based “Artilysins” to combat multidrug-resistant gram-negative pathogens
mBio, 2014Co-Authors: Yves Briers, Maarten Walmagh, Victor Van Puyenbroeck, Anneleen Cornelissen, William Cenens, Gunther Verween, Hugo Oliveira, Joana Azeredo, Abram Aertsen, Jean Paul PirnayAbstract:The global threat to public health posed by emerging multidrug-resistant bacteria in the past few years necessitates the development of novel approaches to combat bacterial infections. Endolysins encoded by bacterial viruses (or phages) represent one promising avenue of investigation. These enzyme-based antibacterials efficiently kill Gram-positive bacteria upon contact by specific cell wall hydrolysis. However, a major hurdle in their exploitation as antibacterials against Gram-negative pathogens is the impermeable lipopolysaccharide layer surrounding their cell wall. Therefore, we developed and optimized an approach to engineer these enzymes as outer membrane-penetrating Endolysins (Artilysins), rendering them highly bactericidal against Gram-negative pathogens, including Pseudomonas aeruginosa and Acinetobacter baumannii. Artilysins combining a polycationic nonapeptide and a modular Endolysin are able to kill these (multidrug-resistant) strains in vitro with a 4 to 5 log reduction within 30 min. We show that the activity of Artilysins can be further enhanced by the presence of a linker of increasing length between the peptide and Endolysin or by a combination of both polycationic and hydrophobic/amphipathic peptides. Time-lapse microscopy confirmed the mode of action of polycationic Artilysins, showing that they pass the outer membrane to degrade the peptidoglycan with subsequent cell lysis. Artilysins are effective in vitro (human keratinocytes) and in vivo (Caenorhabditis elegans). Importance: Bacterial resistance to most commonly used antibiotics is a major challenge of the 21st century. Infections that cannot be treated by first-line antibiotics lead to increasing morbidity and mortality, while millions of dollars are spent each year by health care systems in trying to control antibiotic-resistant bacteria and to prevent cross-transmission of resistance. Endolysins--enzymes derived from bacterial viruses--represent a completely novel, promising class of antibacterials based on cell wall hydrolysis. Specifically, they are active against Gram-positive species, which lack a protective outer membrane and which have a low probability of resistance development. We modified Endolysins by protein engineering to create Artilysins that are able to pass the outer membrane and become active against Pseudomonas aeruginosa and Acinetobacter baumannii, two of the most hazardous drug-resistant Gram-negative pathogens.
Joana Azeredo - One of the best experts on this subject based on the ideXlab platform.
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Staphylococci phages display vast genomic diversity and evolutionary relationships
BMC Genomics, 2019Co-Authors: Hugo Oliveira, Welkin H. Pope, Graham F. Hatfull, Marta Sampaio, Oscar Dias, Luís D. R. Melo, Joana AzeredoAbstract:BackgroundBacteriophages are the most abundant and diverse entities in the biosphere, and this diversity is driven by constant predator–prey evolutionary dynamics and horizontal gene transfer. Phage genome sequences are under-sampled and therefore present an untapped and uncharacterized source of genetic diversity, typically characterized by highly mosaic genomes and no universal genes. To better understand the diversity and relationships among phages infecting human pathogens, we have analysed the complete genome sequences of 205 phages of Staphylococcus sp.ResultsThese are predicted to encode 20,579 proteins, which can be sorted into 2139 phamilies (phams) of related sequences; 745 of these are orphams and possess only a single gene. Based on shared gene content, these phages were grouped into four clusters (A, B, C and D), 27 subclusters (A1-A2, B1-B17, C1-C6 and D1-D2) and one singleton. However, the genomes have mosaic architectures and individual genes with common ancestors are positioned in distinct genomic contexts in different clusters. The staphylococcal Cluster B siphoviridae are predicted to be temperate, and the integration cassettes are often closely-linked to genes implicated in bacterial virulence determinants. There are four unusual Endolysin organization strategies found in Staphylococcus phage genomes, with Endolysins predicted to be encoded as single genes, two genes spliced, two genes adjacent and as a single gene with inter-lytic-domain secondary translational start site. Comparison of the Endolysins reveals multi-domain modularity, with conservation of the SH3 cell wall binding domain.ConclusionsThis study provides a high-resolution view of staphylococcal viral genetic diversity, and insights into their gene flux patterns within and across different phage groups (cluster and subclusters) providing insights into their evolution.
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the first paenibacillus larvae bacteriophage Endolysin plypl23 with high potential to control american foulbrood
PLOS ONE, 2015Co-Authors: A Oliveira, Leon D Kluskens, Luís D. R. Melo, Marta Leite, Silvio Roberto Branco Santos, Joana AzeredoAbstract:Endolysins, which are peptidoglycan-degrading enzymes expressed during the terminal stage of the reproduction cycle of bacteriophages, have great potential to control Gram-positive pathogens. This work describes the characterization of a novel Endolysin (PlyPl23) encoded on the genome of Paenibacillus larvae phage phiIBB_Pl23 with high potential to control American foulbrood. This bacterial disease, caused by P. larvae, is widespread in North America and Europe and causes important economic losses in apiculture. The restriction to antibiotic residues in honey imposed by the EU legislation hinders its therapeutic use to combat American foulbrood and enforces the development of alternative antimicrobial methods. The new Endolysin described herein has an N-acetylmuramoyl-L-alanine amidase catalytic domain and exhibits a broad-spectrum activity against common P. larvae genotypes. Moreover, the enzyme displays high antimicrobial activity in a range of pH that matches environmental conditions (pH between 5.0 and 7.0), showing its feasible application in the field. At pH 7.0, a concentration of 0.2 μM of enzyme was enough to lyse 104 CFU.mL-1 of P. larvae in no more than 2 h. The presence of sucrose and of the substances present in the larvae gut content did not affect the enzyme activity. Interestingly, an increase of activity was observed when PlyPl23 was previously incubated in royal jelly. Furthermore, in vivo safety evaluation assays demonstrated that this enzyme is not toxic to the bee larvae. The present work describes for the first time an Endolysin encoded in a P. larvae phage that presents high potential to integrate a commercial product to control the problematic American foulbrood.
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engineered Endolysin based artilysins to combat multidrug resistant gram negative pathogens
Mbio, 2014Co-Authors: Yves Briers, Maarten Walmagh, Anneleen Cornelissen, William Cenens, Gunther Verween, Victor Van Puyenbroeck, Hugo Oliveira, Joana Azeredo, Abram Aertsen, Jean Paul PirnayAbstract:The global threat to public health posed by emerging multidrug-resistant bacteria in the past few years necessitates the development of novel approaches to combat bacterial infections. Endolysins encoded by bacterial viruses (or phages) repre- sent one promising avenue of investigation. These enzyme-based antibacterials efficiently kill Gram-positive bacteria upon con- tact by specific cell wall hydrolysis. However, a major hurdle in their exploitation as antibacterials against Gram-negative patho- gens is the impermeable lipopolysaccharide layer surrounding their cell wall. Therefore, we developed and optimized an approach to engineer these enzymes as outer membrane-penetrating Endolysins (Artilysins), rendering them highly bactericidal against Gram-negative pathogens, including Pseudomonas aeruginosa and Acinetobacter baumannii. Artilysins combining a polycationic nonapeptide and a modular Endolysin are able to kill these (multidrug-resistant) strains in vitro with a4t o 5l og reduction within 30 min. We show that the activity of Artilysins can be further enhanced by the presence of a linker of increasing length between the peptide and Endolysin or by a combination of both polycationic and hydrophobic/amphipathic peptides. Time-lapse microscopy confirmed the mode of action of polycationic Artilysins, showing that they pass the outer membrane to degrade the peptidoglycan with subsequent cell lysis. Artilysins are effective in vitro (human keratinocytes) and in vivo (Caeno- rhabditis elegans). IMPORTANCE Bacterial resistance to most commonly used antibiotics is a major challenge of the 21st century. Infections that can- not be treated byfirst-line antibiotics lead to increasing morbidity and mortality, while millions of dollars are spent each year by health care systems in trying to control antibiotic-resistant bacteria and to prevent cross-transmission of resistance. Endoly- sins—enzymes derived from bacterial viruses—represent a completely novel, promising class of antibacterials based on cell wall hydrolysis. Specifically, they are active against Gram-positive species, which lack a protective outer membrane and which have a low probability of resistance development. We modified Endolysins by protein engineering to create Artilysins that are able to pass the outer membrane and become active against Pseudomonas aeruginosa and Acinetobacter baumannii, two of the most hazardous drug-resistant Gram-negative pathogens.
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Engineered Endolysin-based “Artilysins” to combat multidrug-resistant gram-negative pathogens
mBio, 2014Co-Authors: Yves Briers, Maarten Walmagh, Victor Van Puyenbroeck, Anneleen Cornelissen, William Cenens, Gunther Verween, Hugo Oliveira, Joana Azeredo, Abram Aertsen, Jean Paul PirnayAbstract:The global threat to public health posed by emerging multidrug-resistant bacteria in the past few years necessitates the development of novel approaches to combat bacterial infections. Endolysins encoded by bacterial viruses (or phages) represent one promising avenue of investigation. These enzyme-based antibacterials efficiently kill Gram-positive bacteria upon contact by specific cell wall hydrolysis. However, a major hurdle in their exploitation as antibacterials against Gram-negative pathogens is the impermeable lipopolysaccharide layer surrounding their cell wall. Therefore, we developed and optimized an approach to engineer these enzymes as outer membrane-penetrating Endolysins (Artilysins), rendering them highly bactericidal against Gram-negative pathogens, including Pseudomonas aeruginosa and Acinetobacter baumannii. Artilysins combining a polycationic nonapeptide and a modular Endolysin are able to kill these (multidrug-resistant) strains in vitro with a 4 to 5 log reduction within 30 min. We show that the activity of Artilysins can be further enhanced by the presence of a linker of increasing length between the peptide and Endolysin or by a combination of both polycationic and hydrophobic/amphipathic peptides. Time-lapse microscopy confirmed the mode of action of polycationic Artilysins, showing that they pass the outer membrane to degrade the peptidoglycan with subsequent cell lysis. Artilysins are effective in vitro (human keratinocytes) and in vivo (Caenorhabditis elegans). Importance: Bacterial resistance to most commonly used antibiotics is a major challenge of the 21st century. Infections that cannot be treated by first-line antibiotics lead to increasing morbidity and mortality, while millions of dollars are spent each year by health care systems in trying to control antibiotic-resistant bacteria and to prevent cross-transmission of resistance. Endolysins--enzymes derived from bacterial viruses--represent a completely novel, promising class of antibacterials based on cell wall hydrolysis. Specifically, they are active against Gram-positive species, which lack a protective outer membrane and which have a low probability of resistance development. We modified Endolysins by protein engineering to create Artilysins that are able to pass the outer membrane and become active against Pseudomonas aeruginosa and Acinetobacter baumannii, two of the most hazardous drug-resistant Gram-negative pathogens.
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Molecular aspects and comparative genomics of bacteriophage Endolysins.
Journal of virology, 2013Co-Authors: Hugo Oliveira, Sílvio B. Santos, Luís D. R. Melo, Nuno Cerca, Eugénio C. Ferreira, Franklin Luzia Nóbrega, Joana Azeredo, Leon D KluskensAbstract:Phages are recognized as the most abundant and diverse entities on the planet. Their diversity is determined predominantly by their dynamic adaptation capacities when confronted with different selective pressures in an endless cycle of coevolution with a widespread group of bacterial hosts. At the end of the infection cycle, progeny virions are confronted with a rigid cell wall that hinders their release into the environment and the opportunity to start a new infection cycle. Consequently, phages encode hydrolytic enzymes, called Endolysins, to digest the peptidoglycan. In this work, we bring to light all phage Endolysins found in completely sequenced double-stranded nucleic acid phage genomes and uncover clues that explain the phage-Endolysin-host ecology that led phages to recruit unique and specialized Endolysins.