| The use and abuse of antibiotics and the strong pressure of antibiotic screening in nature make drug-resistant strains gradually become dominant epidemic strains,resulting in a large number of infections caused by drug-resistant bacteria that cannot be treated with antibiotics.Therefore,phage therapy has received renewed attention,but the application of phages is limited because some phages carry virulence genes or a large number of genes with unknown functions,as well as the strict species specificity of phages.The bacteriophage derived lysin is not easy to produce drug resistance because its recognition site is the conservative structure of bacterial cell wall,and usually has a broader cleavage spectrum than the host bacteria.Therefore,phage therapy is considered to be one of the potential alternatives to antibiotics.Enterococci are bacteria that widely exist in nature,human and animal intestines.They are members of normal flora.Enterococcus itself has a thick cell wall and can obtain drug resistance through a variety of ways.Once it causes infection,it is difficult to treat.In the list of bacteria with high priority in looking for antibiotic substitutes published by the World Health Organization in 2017,drug-resistant enterococcus was ranked first.In this study,we cloned and expressed the lyase of enterococcal phage LY0322 with 6 x His tag,tested its biological characteristics and cleavage activity,and named it as Lys22.In order to purify the lysin,we designed to add 6 x His tag to the tail(C-end)of the modified lyase.Through the online protein database Swiss Server,the three-dimensional structure of Lys22 carrying 6 x His tag was predicted.It was found that the zinc ion binding domain composed of two histidines(52 and 160)and one aspartic acid(173)could still be retained at the N end of the protein.The C-terminal of lysin showed similar structure containing two antiparallel ?-Folds and one ?-Spiral.The purified Lys22 was obtained by prokaryotic expression of BL21,two rounds of ultrafiltration centrifugation and Ni purification.Then,the relevant biological characteristics of lysin were determined,mainly including cleavage spectrum,bactericidal activity and its activity in different p H,temperature,EDTA,Na Cl and other environments.The results showed that Lys22 broadened the cleavage spectrum of phage ly0322.Moreover,Lys22 showed cross-genus lysis to Staphylococcus.The sensitive species included S.aureus,S.epidermidis,S.haemolyticus,S.cohnii,S.hominis,and S.wameri.Some sensitive S.aureus are methicillin and vancomycin resistant strains.The bactericidal efficiency of Lys22 is directly proportional to the concentration of Lys22,and at p H 4-10,EDTA < 50?M,0-100?M of Na Cl,Lys22 showed relatively high lytic activity to E faecalis.It is particularly worthy that Lys22 maintained high activity after treatment at 55?for 30 min.Lys22 showed good resistance to high temperature because it still remained a part of lytic activity after treatment at 75?for 30 minute.Our study also aimed at effects of Lys22 to biofilm because biofilm is one of the most important reasons of resistant E.faecalis formation.Firstly,the formation matrix curve of E.faecalis biofilm was detected.Then,the effects of Lys22 was tested by crystal violet quantitative method and laser confocal electron microscopy.The results proved that Lys22 could not only inhibit the formation of E.faecalis biofilm,but also could destroy the mature biofilm.Finally,based on the dentin specimen,the effect of Lys22 on biofilm was observed under scanning electron microscopy.Lys22 also showed inhibition to biofilm formation and destroy to mature biofilm.In conclusion,Lys22,a Lysin with high temperature resistant,high cross-genus lytic activity to Enterococcus and Staphylococcus was obtained in this study.Lys22 also showed strong inhibition to both biofilm formation and mature biofilm of E.faecalis. |
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