title: Characterization of lytic enzyme open reading frame 9 (ORF9) derived from Enterococcus faecalis bacteriophage phiEF24C.
abstract: In bacteriophage (phage) therapy against Gram-positive bacteria, such as Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis, members of a genus of SPO1-like viruses are typically employed because of their extreme virulence and broad host spectrum. Phage φEF24C, which is a SPO1-like virus infecting E. faecalis, has previously been characterized as a therapeutic phage candidate. In addition to the phage itself, phage endolysin is also recognized as an effective antimicrobial agent. In this study, a putative endolysin gene (orf9) of E. faecalis phage φEF24C was analyzed in silico, and its activity was characterized using the recombinant form. First, bioinformatics analysis predicted that the open reading frame 9 (ORF9) protein is N-acetylmuramoyl-l-alanine amidase. Second, bacteriolytic and bactericidal activities of ORF9 against E. faecalis were confirmed by zymography, decrease of peptidoglycan turbidity, decrease of the viable count, and morphological analysis of ORF9-treated cells. Third, ORF9 did not appear to require Zn(2+) ions for its activity, contrary to the bioinformatics prediction of a Zn(2+) ion requirement. Fourth, the lytic spectrum was from 97.1% (34 out of 35 strains, including vancomycin-resistant strains) of E. faecalis strains to 60% (6 out of 10 strains) of Enterococcus faecium strains. Fifth, N-acetylmuramoyl-l-alanine amidase activity of ORF9 was confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and the subsequent MALDI-postsource decay (PSD) analyses. Finally, functional analysis using N- or C-terminally deleted ORF9 mutants suggested that a complete ORF9 molecule is essential for its activity. These results suggested that ORF9 is an endolysin of phage φEF24C and can be a therapeutic alternative to antibiotics.
authors.affiliation: Department of Microbiology and Infection, Faculty of Medicine, Kochi University, Nankoku City, Kochi, Japan.
authors.forename: Jumpei
authors.initials: J
authors.lastname: Uchiyama
authors.forename: Iyo
authors.initials: I
authors.lastname: Takemura
authors.forename: Ikue
authors.initials: I
authors.lastname: Hayashi
authors.forename: Shigenobu
authors.initials: S
authors.lastname: Matsuzaki
authors.forename: Miho
authors.initials: M
authors.lastname: Satoh
authors.forename: Takako
authors.initials: T
authors.lastname: Ujihara
authors.forename: Masanao
authors.initials: M
authors.lastname: Murakami
authors.forename: Masayuki
authors.initials: M
authors.lastname: Imajoh
authors.forename: Motoyuki
authors.initials: M
authors.lastname: Sugai
authors.forename: Masanori
authors.initials: M
authors.lastname: Daibata
mesh_terms: D000595:Amino Acid Sequence; D001433:Bacteriolysis; D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics; D003067:Coenzymes / Q000378:metabolism; D015169:Colony Count, Microbial; D019295:Computational Biology; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D013293:Enterococcus faecalis / Q000821:virology*; D050296:Microbial Viability / Q000187:drug effects; D008969:Molecular Sequence Data; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics* / Q000378:metabolism*; D009391:Nephelometry and Turbidimetry; D016366:Open Reading Frames; D015032:Zinc / Q000378:metabolism
chemical_list: D003067:Coenzymes; D010450:Endopeptidases; C001143:endolysin; D009238:N-Acetylmuramoyl-L-alanine Amidase; D015032:Zinc