Bacteriophage φEf11 ORF28 Endolysin, a Multifunctional Lytic Enzyme with Properties Distinct from All Other Identified Enterococcus faecalis Phage Endolysins

Abstract

Ef11 is a temperate Siphoviridae bacteriophage that infects strains of Enterococcus faecalis. The Ef11 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, ORF28, was predicted to code for an N-acetylmuramoyl-Lalanine amidase endolysin of 46.133 kDa, composed of 421 amino acids. The PCRamplified ORF28 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 E. faecalis populations and was active against 73 of 103 (71%) E. faecalis strains tested. In addition, it caused substantial destruction of E. faecalis 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 2ϩ . Liquid chromatography-mass spectrometry analysis of E. faecalis cell wall digestion products produced by the ORF28 endolysin indicated that the lysin acted as an N-acetylmuramidase, an endo-␤-N-acetylglucosaminidase, and an endopeptidase, rather than an N-acetylmuramoyl-L-alanine amidase. The Ef11 ORF28 lysin shared 10% to 37% amino acid identity with the lytic enzymes of all other characterized E. faecalis bacteriophages. IMPORTANCE 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 Enterococcus faecalis. The lysin is broadly active against most of the tested E. faecalis strains and exhibits multifunctional enzymatic specificities that differ from all other characterized endolysins produced by E. faecalis bacteriophages.

The prevalence and impact of lysogeny among oral isolates ofEnterococcus faecalis

“…The external application of endolysins to strains of most (Gram positive) bacteria will also cause cell wall degradation and result in cell lysis from without. Previously, we isolated and characterized an endolysin produced by E. faecalis bacteriophage ɸEf11 [20]. Preparations of this endolysin (ORF28 endolysin) were used to test the sensitivity of the isogenic pair of lysogenic (TUSoD11) and cured [TUSoD11(ΔɸEf11)] E. faecalis strains.…”

“…The cells were resuspended in 5 ml of PBS, and 1.5 ml of this suspension was transferred into sterile, clear tubes. Each tube then received 30 μl of a filter sterilized preparation of the phage ɸEf11 ORF28 endolysin [20]. Control tubes received 30 μl of PBS instead of the endolysin.…”

Characterization of the Bacteriophage vB_EfaS-271 Infecting Enterococcus faecalis

“…Generally, reports from recent years indicated a high biodiversity of bacteriophages specific for E. faecalis strains. Such reports can be exemplified by articles describing isolation and characterization of previously unknown phages of different properties [ 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 ], genetic modification of known phages and their use in experimental phage therapy (including effects on biofilms) [ 46 , 47 ], assessment of phages in therapy using animal models [ 48 , 49 , 50 , 51 , 52 , 53 ], and cloning of phage genes coding for specific lysins and characterization of the gene products in the light of killing E. faecalis cells [ 54 , 55 , 56 , 57 , 58 , 59 , 60 ].…”

Comparison of Enterococcus faecalis Biofilm Removal Efficiency among Bacteriophage PBEF129, Its Endolysin, and Cefotaxime

“…There are several reports describing various phage endolysins’ efficacy against bacterial biofilms where only limited activities were reported [ 50 , 51 , 52 , 53 ]. One report described an endolysin against Staphylococcus aureus biofilm on the surface of a medical device which showed no apparent efficacy [ 54 ].…”