Peptidoglycan degrading activity of the broad-range Salmonella bacteriophage S-394 recombinant endolysin

Legotsky, S.A.; Vlasova, Kseniya Yu.; Priyma, A.D.; Shneider, Mikhail M.; Pugachev, V. G.; Totmenina, Olga Dmitrievna; Kabanov, Alexander V.; Miroshnikov, Konstantin A.; Klyachko, Natalia L.

doi:10.1016/j.biochi.2014.09.017

Cited by 31 publications

(17 citation statements)

References 22 publications

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“…1), in contrast to that of other previously described endolysins with a Gram-negative background, which have optimal activities at neutral pH (21,22,23,35). It is noteworthy that three other endolysins from phages in- fecting S. Typhimurium have been reported to show optimal activities at pH 9.5 (phage SPN1S endolysin) and pH 8.5 (phage SPN9CC endolysin and Lys394), respectively (36)(37)(38), whereas endolysins from phages infecting Salmonella enterica serovar Enteritidis (PVP-SE1gp146, Lys68, and PsP3gp10) have optimal activities at pH 7 (21,22,35). In terms of temperature resistance, Gp110 remains active after treatment at temperatures across the range tested (20 to 90°C), showing no significant activity loss with up to 10-min heat treatments at 60°C and only gradually decreasing activity at higher temperatures (Fig.…”

Section: Discussionmentioning

confidence: 84%

“…2). Whereas most endolysins irreversibly lose their enzymatic activities upon exposure to temperatures around 50°C (37,(39)(40)(41)(42)(43), some thermoresistant endolysins have also been described, including Lys68 from Salmonella phage phi68 (35), gp146 from Salmonella phage PVP-SE1 (21), the endolysins from bacteriophages Ph2119 and vB_Tsc2631 infecting the thermophile Thermus scotoductus (44,45), and the lysin from deep-sea thermophilic bacteriophage GVE2 (46). Gp110 shows an intermediate profile with elevated temperature resistance compared to that of most mesophilic endolysins, which may correlate to increased shelf life of Gp110.…”

Section: Discussionmentioning

confidence: 99%

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DUF3380 Domain from a Salmonella Phage Endolysin Shows Potent N -Acetylmuramidase Activity

Rodríguez-Rubio

Gerstmans

Thorpe

et al. 2016

Appl Environ Microbiol

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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/M) 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 ␤-(1,4) glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine residues. Gp110 is active against directly crosslinked peptidoglycans with various peptide stem compositions, making it an attractive enzyme for developing novel antimicrobial agents. IMPORTANCEWe 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 crosslinked 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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Section: Discussionmentioning

confidence: 84%

Section: Discussionmentioning

confidence: 99%

DUF3380 Domain from a Salmonella Phage Endolysin Shows Potent N -Acetylmuramidase Activity

Rodríguez-Rubio

Gerstmans

Thorpe

et al. 2016

Appl Environ Microbiol

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“…The possibility to modify endolysins to enable them attack Gram-negative bacteria from outside is a long term research interest in the context of therapeutic antibacterial application of such proteins. One of the strategies to overcome the outer membrane of the bacteria is the use of transmembrane peptides—either as a component of the reaction mixture [ 40 ] or as a fused part of the chimeric protein, “artilysin” [ 9 , 10 , 11 ]. One of the problems in artilysin design is the structural positioning of the engineered domain in the protein globule.…”

Section: Discussionmentioning

confidence: 99%

Structure of an Acinetobacter Broad-Range Prophage Endolysin Reveals a C-Terminal α-Helix with the Proposed Role in Activity against Live Bacterial Cells

Сыкилинда

Shneider

Mishkin

et al. 2018

Viruses

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Proteins that include enzymatic domain degrading the bacterial cell wall and a domain providing transport through the bacterial outer membrane are considered as prospective compounds to combat pathogenic Gram-negative bacteria. This paper presents an isolation and study of an enzyme of this class naturally encoded in the prophage region of Acinetobacter baumannii AB 5075 genome. Recombinant protein expressed in E. coli exhibits an antimicrobial activity with respect to live cultures of Gram-negative bacteria reducing the population of viable bacteria by 1.5–2 log colony forming units (CFU)/mL. However the protein becomes rapidly inactivated and enables the bacteria to restore the population. AcLys structure determined by X-ray crystallography reveals a predominantly α—helical fold similar to bacteriophage P22 lysozyme. The С-terminal part of AcLys polypeptide chains forms an α—helix enriched by Lys and Arg residues exposed outside of the protein globule. Presumably this type of structure of the C-terminal α—helix has evolved evolutionally enabling the endolysin to pass the inner membrane during the host lysis or, potentially, to penetrate the outer membrane of the Gram-negative bacteria.

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“…A multitude of chemical OM permeabilizers have been investigated. The majority of these compounds either destabilize the OM by chelation of the stabilizing divalent cations (Ca 2+ , Mg 2+ ) that bridge adjacent LPS molecules, like EDTA [25,26], or competitively displace divalent cations by the cationic nature of the compound (cationic antibiotics such as colistin and polymyxin B [19 ,27,28 ], cationic peptides such as PGLa, poly-L-Arg, e-poly-L-lysine [27,29] and poly(propyleneimine) dendrimers [30]). Organic acids such as citric acid and malic acid also have a weak chelating effect, but this effect seems to be overwhelmed by OM disruption based on acidification [31].…”

Section: Weakening the Om By Physical Or Chemical Methodsmentioning

confidence: 99%

Lysins breaking down the walls of Gram-negative bacteria, no longer a no-go

Gutiérrez

Briers

2021

Current Opinion in Biotechnology

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Peptidoglycan degrading activity of the broad-range Salmonella bacteriophage S-394 recombinant endolysin

Cited by 31 publications

References 22 publications

DUF3380 Domain from a Salmonella Phage Endolysin Shows Potent N -Acetylmuramidase Activity

DUF3380 Domain from a Salmonella Phage Endolysin Shows Potent N -Acetylmuramidase Activity

Structure of an Acinetobacter Broad-Range Prophage Endolysin Reveals a C-Terminal α-Helix with the Proposed Role in Activity against Live Bacterial Cells

Lysins breaking down the walls of Gram-negative bacteria, no longer a no-go

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