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

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/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.

Bacteriophage-encoded virion-associated enzymes to overcome the carbohydrate barriers during the infection process

“…The cleavage specificity of phages lysins is only rarely experimentally examined and is often based on in silico homology and therefore biased by historic misannotations. However, in those few experimentally validated cases, liquid chromatography–mass spectrometry is the most common method (Caldentey and Bamford 1992; Sudiarta et al 2010; Stockdale et al 2013; Rodríguez-Rubio et al 2016; Maciejewska et al 2017). …”

Modular endolysin of Burkholderia AP3 phage has the largest lysozyme-like catalytic subunit discovered to date and no catalytic aspartate residue

“…Rare examples of modular endolysins are usually composed of a single N-terminal cell wall-binding domain (CBD) responsible for high-affinity tying to the cell wall receptor and a C-terminal enzymatically active domain (EAD) responsible for breaking specific bonds within the PG structure 4 . Modular architecture has been demonstrated for several endolysins produced by Gram-negatives infecting phages: KZ144, EL188, OBPgp279 and 201φ2-1gp229 encoded by Pseudomonas phages 12 , 13 and PVP-SE1gp146, SPN1S_0028, Lys68 and Gp110 encoded by Salmonella phages 10 , 13 – 15 .…”

Expression of a Peptidoglycan Hydrolase from Lytic Bacteriophages Atu_ph02 and Atu_ph03 Triggers Lysis of Agrobacterium tumefaciens

“…S3). A recent characterization of an endolysin (Gp110) from a Salmonella phage 10 containing DUF3380 revealed that this domain functions as an N-acetylmuramidase and cleaves the ␤1 to 4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan (55). The predicted PPH transmembrane topology according to a hidden Markov model (56) oriented the transmembrane domain with the EAD in the periplasm and the short C-terminal tail in the cytosol (Fig.…”