| Enterococcus faecium is a Gram-positive opportunistic pathogen that can cause a variety of local and systemic infections in immune-compromised patients. Virulent bacteriophages are bacterial viruses that can specifically infect and lyse host bacteria.Lysin is encoded by the phage genome at the end of the phage lytic life cycle to lyse the host cell. Phage and lysin are highly efficient in lysing bacteria. Due to the worldwide prevalence of antibiotic resistant strains and the shortage of new effective antimicrobial agents, phage and lysin have become a popular research topic.In this study, the E. faecium phage and its lysin were studied. First, 23 strains have been isolated from sewage, including 9 vancomycin-resistant strains(VREF). Then,we isolated a novel phage, IME-EFm5, using vancomycin-resistant E. faecium 4P-SA.The structural characteristic of IME-EFm5 suggest that it is a member of the Caudovirales, Siphoviridae family, which has an isometric head of about 50 nm in diameter and a about 165 nm long tail. When the MOI was 0.01, the phage IME-EFm5 titer was the highest, reaching approximately >109 PFU/m L. The one-step growth curve revealed that the latent period was approximately 30 min, and the release period was approximately 70 min. IME-EFm5 was resistant to ether and chloroform. IME-EFm5 was relatively stable at 40℃ and 50℃. The infectivity ability of IME-EFm5 was relatvely stable at p H 6.0 ~ 9.0. Both the spot testing and the double-layer agar plate method indicated that phage IME-EFm5 only showed sensitivity to E. faecium 4P-SA. So, IME-EFm5 displayed extremely specific infectivity to E. faecium. The size of the most abundant major structural protein was approximately 36 k D.The sequencing indicated that IME-EFm5 has a double-stranded, terminally non-redundant genome of 42,265 bp with a low G+C content of 35.51 mol%. The complete genome of IME-EFm5 encodes 70 ORFs. The IME-EFm5 genome only exhibited high similarity at the whole genome level with phage IME-EFm1.Furthermore, Lys EFm5, the lysin of phage IME-EFm5, was expressed. Lys EFm5 displayed a broad lytic spectrum: in vitro treatment killed a number of E. faecium strains rapidly and completely, including VREF strains, and this ability may allow it to serve as an alternative treatment strategy for controlling multidrug-resistant E.faecium. Sequence analysis indicated that the N-terminus of Lys EFm5 is a member of the amidase family. The structure model of Lys EFm5 amidase was built and was homologous with that of the Lys GH15 amidase-2 domain(staphylococcal phage lysin)despite the low sequence similarity. The circular dichroism(CD) spectroscopy showed that the mutations didn’t affect the secondary structures of the Lys EFm5.Structure-guided mutagenesis of the amidase domain revealed that the zinc ion binding residues(H27, H132, and C140), E90, and T138 are required for catalysis.The activity of Lys EFm5 was not significantly different(P>0.05) with or without 100 m M EDTA. However, the lytic activity of Lys EFm5 is zinc ion independent, which is inconsistent with other amidase members, enabling us to propose a distinct catalytic mechanism. The characteristics of Lys EFm5 represent a pivotal step forward in understanding the amidase domain of phage lysin. |
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