The Biological Characteristics Of Enterococcus Faecalis Phage EF-P10 And Its Lysin And Therapeutic Experiment Study Of The Lysin

The antibiotic resistance problem of bacteria is becoming more and more serious,which makes that there is no available drug to treat some bacterial infection.Therefore,the development of new antibacterial drugs has become a focus of attention.Bacteriophages and their lysins can specifically and efficiently lyse bacteria,which makes them become new antibacterial drugs to treat bacterial infection,particularly the multidrug-resistance bacterial,showing great potential in the treatment of bacterial infections.Enterococcus faecalis is an opportunistic pathogen which can cause a variety of local or systemic infectious diseases.Because of the abuse of antibiotics and the inherent resistance of Enterococcus faecalis to many antibiotics,it is very difficult to treat.Therefore,this study took vancomycin-resistant Enterococcus faecalis phage EF-P10 and its lyase LysEF-P10 as the research object,and systematically studied its potential as an antimicrobial preparation and its cleavage mechanism.In this study,first,40 strains of multi-drug resistant Enterococcus faecalis were obtained,and a virulent phage EF-P10 was isolated and purified from sewage using vancomycin-resistant Enterococcus faecalis?VREF?N10 as the host bacterium.Transmission electron microscopy showed that EF-P10 belonged to Siphoviridae,and genomic type result showed that EF-P10 was a double-stranded DNA virus.EF-P10 showed high bactericidal activity against its host bacteria,but the lysis spectrum was narrow.The whole genome of EF-P10 was sequenced,and found that the total length of EF-P10 was 57 408 bp,with 127 open reading frames.After comparison and analysis,the protein encoded by ORF60 was the lyase LysEF-P10.LysEF-P10 was prokaryotically expressed and purified.In vitro experiments showed that LysEF-P10 had high bactericidal activity and its lysis spectrum was much wider than that of its parent phage.Continuous interaction of Enterococcus faecalis with LysEF-P10 for multiple generations will not induce resistance of bacteria to LysEF-P10,indicating a broad application prospect.Then,we conducted an in-depth study on the structure and function of LysEF-P10.According to comparison and analysis of amino acid sequence,LysEF-P10 contained a N-terminal CHAP structure domain?LysEF-P10C?and a C-terminal presumed binding structure domain?LysEF-P10B?.To verify the functions of each domain,LysEF-P10C and LysEF-P10B were prokaryotically expressed and purified,respectively.In vitro bactericidal experiments showed that neither LysEF-P10C nor LysEF-P10B had bactericidal activity,and only complete LysEF-P10 were active.To see visually the structure domain combined with the host bacteria,LysEF-P10C and LysEF-P10B were fused with GFP,respectively,to obtain LysEF-P10C-GFP and LysEF-P10B-GFP fusion proteins.The ability of the two fusion proteins to bind to Enterococcus faecalis N10 was detected by confocal laser microscopy.The result showed that only LysEF-P10B-GFP could specifically bind to Enterococcus faecalis N10,indicating that LysEF-P10B was the structural domain where LysEF-P10 played a binding role.Taking the CHAP fragment of Staphylococcus aureus phage lyase LysGH15 as the template,the 3D structure of LysEF-P10C with 100%confidence was predicted,and the two structures had high homology.Through sequence alignment and the construction of 3D structural model,we predicted the possible key amino acid sites D20,D22,D31,C29,H90 and N110in LysEF-P10C,and mutated these sites into alanine one by one.The results of circular dichroism showed that the secondary structure of these proteins did not change before and after mutation.In vitro bactericidal activity showed that the activity of LysEF-P10 was completely lost after EDTA treatment,and the activity was recovered after Ca²⁺supplementation,indicating that Ca²⁺was necessary to maintain the bactericidal activity of LysEF-P10.The activities of D20A,D22A and D31A mutant proteins were all lost,and the results of ICP-AES showed that the content of Ca²⁺was significantly reduced after the mutation,while the activity of mutant proteins did not recover after Ca²⁺supplementation,indicating that D20,D22and D31 were the key sites for binding Ca²⁺.The activity of C29A,H90A and N110A mutant proteins were also all lost,but the results of ICP-AES showed that there was no significant change in the content of Ca²⁺after mutation,indicating that these three sites were the key sites that played a catalytic role.Next,we further evaluated the therapeutic effect,immunogenicity,effect on intestinal flora and safety of LysEF-P10 in vivo.Treatment experiments showed that intraperitoneal injection of 5?g LysEF-P10 at 1 h after challenge could 100%protect mice infected with lethal dose of VREF,and significantly reduce the number of bacteria in the blood of mice.Immunogenicity experiments showed that LysEF-P10 could induce specific antibody against LysEF-P10 in mice,and the titer of antibody at the third week after one-time injection of 5?g LysEF-P10 reached the highest?1:100?,and the main type of antibody was IgG.Neutralization test showed that anti-LysEF-P10 antibody did not affect the bactericidal activity of LysEF-P10 in vitro,and also did not affect the therapeutic effect of LysEF-P10 in vivo.To evaluate the effect of LysEF-P10 on intestinal flora in vivo,16S rRNA sequencing analysis was performed on the bacteria in mouse feces.The results showed that the abundance of Enterococcus bacteria in mouse feces was significantly increased at 24h after lethal dose of VREF was intraperitoneally injected into mice,and the balance of intestinal flora was disturbed.At 1 h after challenge,5?g LysEF-P10 was used for treatment.The abundance of Enterococcus bacteria in mouse feces was significantly reduced,and the imbalance of intestinal flora was restored to a certain extent.Only intraperitoneal injection of 100?g LysEF-P10 also significantly reduced the abundance of Enterococcus bacteria in mouse feces within 24 h.The safety test results showed that even a large dose of 5 mg LysEF-P10 injected intraperitoneally would not cause obvious side effects on mice,indicating that LysEF-P10 has good safety when applied in vivo.In summary,this study provides a solid theoretical and experimental basis for the comprehensive analysis of the biological characteristics of Enterococcus faecalis phage and its lysin and the application of LysEF-P10 in the treatment of multidrug-resistant Enterococcus faecalis infection.