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.

FIG. 1.

Bioinformatics analysis of the ORF9 amino acid sequence. The N-acetylmuramoyl-l-alanine amidase domain predicted by BLASTp is surrounded by a gray line. The amino acid residues for substrate binding (His-35, Asn-36, Ala-54, Ala-58, Val-71, Phe-78, His-79, Asn-95, His-145, Thr-149, Thr-151, Ala-152, and Cys-153), Zn²⁺ binding (His-34, His-145, and Cys-153), and amidase catalysis (His-34, Ala-58, His-145, Thr-151, and Cys-153) were also predicted in the N-acetylmuramoyl-l-alanine amidase domain predicted by BLASTp. According to the structure-based bioinformatics analysis by I-TASSER, ligand binding amino acid residues were predicted (shown in boldface and underlined).

FIG. 2.

Lytic activity of ORF9 against E. faecalis peptidoglycan and cells. (A) Zymography of ORF9-His-overexpressing E. coli. In the left and right lanes, E. coli BL21(pColdIII ORF9-His) without and with IPTG induction, respectively, is shown. Only E. coli BL21(pColdIII ORF9-His) with IPTG induction (right lane) showed a clear band, which was detected at the expected molecular size (arrow). (B) Dose-response curve of ORF9-His against E. faecalis peptidoglycan, summarizing the data at 10 min after the turbidity assay. Concentration-dependent ORF9 effects were observed. (C) Time course assay of ORF9-His treatment against E. faecalis peptidoglycan. Various concentrations of ORF9-His were treated with E. faecalis peptidoglycan; limited concentrations of ORF9-His treatment are presented. The ORF9-His concentrations at 0 (○), 10⁻⁴ (•), 10⁻² (▴), and 1 (⧫) mg/ml are shown. The turbidity decreased time dependently. (D) Dose-response bactericidal effect of ORF9 against E. faecalis cells. Three hours after ORF9 treatment of E. faecalis cells, the viable count was measured. The error bars indicate the SD.

FIG. 3.

Electron micrograph of PBS-treated (A) and ORF9-His-treated (B) E. faecalis. In both panels, the right and left images show ×13,000 and ×22,000 magnification, respectively. Scale bars are also shown.

FIG. 4.

Structural analysis of ORF9-His-treated E. faecalis peptidoglycan. (A) Reversed-phase HPLC separation of ORF9-His-treated E. faecalis peptidoglycan. The numbers from 1 to 6 above the peaks indicate the isolated fractions. (B) Structures and molecular masses of the isolated fractions. *, peak numbers refer to the HPLC chromatograms; **, 1r, Ala-iGln-Lys(ɛ)-Ala-Ala; 2r, Ala-iGln-Lys[(ɛ)-Ala-Ala]-Ala-Ala; iGln, isoglutamine. (C) Determined structure of the ORF9-His-treated E. faecalis peptidoglycan.

FIG. 5.

(A) Schematic overview of ORF9-His and eight deletion mutants of ORF9-His. ORF9ΔAmi1, ORF9ΔAmi2, ORF9ΔAmi3, ORF9ΔAmi4, and ORF9ΔAmi5 are N-terminal deletions of ORF9-His. ORF9ΔPGNB1, ORF9ΔPGNB2, and ORF9ΔPGNB3 are C-terminal deletions of ORF9-His. (B) Purified forms and lytic activities of ORF9-His and the deletion mutants. Lytic activity was tested against E. faecalis strain EF24. Only the soluble deletion mutant ORF9s and ORF9-His were subjected to lysis assay. N.A., not applicable; yes, lysed; no, not lysed.