Characterization of the bacteriophage φEf11 ORF 28 endolysin: A multi-functional lytic enzyme with properties distinct from all other identified <i>Enterococcus. faecalis</i> phage endolysins.

Characterization of the bacteriophage φEf11 ORF 28 endolysin: A multi-functional lytic enzyme with properties distinct from all other identified Enterococcus. faecalis phage endolysins.

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Authors: Zhang H, Buttaro BA, Fouts DE, Sanjari S, Evans B, Stevens RH
Title: Characterization of the bacteriophage φEf11 ORF 28 endolysin: A multi-functional lytic enzyme with properties distinct from all other identified Enterococcus. faecalis phage endolysins.
Citation: Applied and environmental microbiology. 2019-04-12; .: .
Abstract:
φEf11 is a temperate, bacteriophage that infects strains of The φEf11 genome, encompassing 65 open reading frames (ORFs), is contained within 42,822 bp of DNA. Within this genome, a module of six lysis-related genes was identified. Based upon sequence homology, one of these six genes, ORF 28, was predicted to code for an N- acetylmuramoyl-L-alanine amidase endolysin of 46.133 kDa, composed of 421 amino acids. The PCR-amplified ORF 28 was cloned and expressed, and the resulting gene product was affinity purified to homogeneity. The purified protein was obtained from a fusion protein that exhibited a molecular mass of 72.5 kDa, consistent with a 46.1 kDa protein combined with a fused 26.5 kDa glutathione S-transferase tag. It produced rapid, profound lysis in populations, and was active against 73 of 103 (71%) strains tested. In addition, it caused substantial destruction of biofilms. The lysin was quite stable, retaining its activity for three years in refrigerated storage, was stable over a wide range of pHs, and was unaffected by the presence of a reducing agent, however, it was inhibited by increasing concentrations of Ca LC-MS analysis of cell wall digestion products produced by the ORF 28 endolysin indicated that the lysin acted as an N-acetylmuramidase, an endo-β-N-acetylglucosaminidase, and an endopeptidase, rather than an N-acetylmuramoyl-L-alanine amidase. The φEf11 ORF 28 lysin shared 10-37% amino acid identity with the lytic enzymes of all other characterized bacteriophages.The emergence of multidrug-resistant pathogenic microorganisms has brought increasing attention to the urgent need for the development of alternative antimicrobial strategies. One such alternative to conventional antibiotics employs lytic enzymes (endolysins) that are produced by bacteriophages in the course of lytic infection. During lytic infection by a bacteriophage, these enzymes hydrolyze the cell wall peptidoglycan, resulting in the lysis of the host cell. However, external endolysin application can result in lysis from without. In this study, we have cloned, expressed, purified and characterized an endolysin produced by a bacteriophage infecting strains of The lysin is broadly active against most of the tested strains, and exhibits multifunctional enzymatic specificities that differ from all other characterized endolysins produced by bacteriophages.
PMID: 30979842