Cloning,expression And Antibacterial Activity Of Endolysin And Holin Against Staphylococcus Aureus Phage

OBJECTIVEStaphylococcus aureus is a common human opportunistic pathogen.People infected with S.aureus often have severe illness and high mortality.In recent years,the drug resistance rate of S.aureus has been increasing,especially the emergence of methicillin resistant S.aureus（MRSA）and vancomycin resistant S.aureus（VRSA）,which leads to more and more difficulties in clinical treatment.In addition to the scientific and rational use of existing antibiotics,seeking new supplement and substitution strategies is one of the clinical problems to be solved urgently.Since the 1980 s,in order to deal with the severe situation of antibiotic resistance all over the world,the ancient phage therapy has attracted researchers’ attention again.A number of basic and clinical studies have shown good application prospects,providing new ideas and strategies for the treatment of drug-resistant bacteria.In addition to the traditional phage therapy,the research of phage derivatives such as endolysin has also attracted extensive attention,which expands the technical methods of phage therapy and improves the feasibility of clinical application.Based on the previous study of Acinetobacter baumannii phages,Klebsiella pneumoniae phages and their depolymerase,a clinically isolated multidrug-resistant S.aureus 35 was used as the host,and a lytic phage IME522 was isolated from the untreated sewage of the South Hospital of the Fifth Medical Center of the PLA General Hospital.The protein of endolysin and holin were predicted,cloned,expressed,and the antibacterial activity was studied,so as to provide theoretical basis for phage relative therapy of drug-resistant S.aureus infection.METHODThe S.aureus 35 phage IME522 was used as the research object.The bioinformatics analysis and functional prediction of ORF4 were carried out by Pfam database,NCBI conserved domain database（CDD）,signal P 4.0 server,CLC genomics workbench 3.6.1 and TMHMM server v.2.0 software.The endolysin（ORF4）gene of phage IME522 was amplified by PCR,and the recombinant plasmid was constructed with p ET-28 a expression vector by homologous recombination seamless cloning technology.The molecular size of endolysin was verified by SDS-PAGE and Western blot,the activity of endolysin was verified by spot plate method,and the protein purified by affinity chromatography.Antibacterial activity in vitro was carried out to compare the lytic ability of endolysin to logarithmic and stationary phase bacteria.The endolysin activity at different temperature and p H value and lytic spectrum were measured.The ability of endolysin to remove S.aureus biofilm was observed by crystal violet staining and scanning electron microscope.The minimum inhibitory concentration of the LysP4 combined with vancomycin was measured by checkerboard assay.The time-kill test was used to determine the bactericidal rate of the LysP4 combined with vancomycin.And to evaluate the hemolytic effect of endolysin on red blood cells and its toxicity to human cells.Double stranded DNA phages usually lysed the bacteria by the holin-endolysin lysis system.The ORF3 gene was predicted to encode holin.Then,its bioinformatics characteristics were analyzed,and the holin was cloned and identified.The effect of holin on the growth rate and activity of Escherichia coli BL21（DE3）during induction was measured.The molecular size of holin was verified by Western blot.The antibacterial effect of holin was determined by dot plate method,bacteria counting and resazurin staining,and the lytic spectrum of holin and endolysin were compared.RESULTProtein structure and function analysis that endolysin had two catalytic domains,a CHAP domain and an amidase-2 domain,as well as a C-terminal SH3 b cell-binding domain.The recombinant ORF4 was verified by SDS-PAGE and Western blot,and it could form transparent halo on the double-layer agar plate.It had good antibacterial activity in vitro,and named LysP4.LysP4 had a strong bactericidal ability on S.aureus,which can reduce logarithmic phase bacteria by 3 log CFU/m L and stationary phase bacteria about 2 log CFU/m L.The endolysin LysP4 activity was stable at the temperature between 16-40°C and p H5.0-10.0.S.aureus 35 had a strong biofilm forming ability.Crystal violet staining and scanning electron microscope showed that LysP4 can remove the biofilm.LysP4 will not cause hemolysis of red blood cells,nor will it affect the growth of embryonic kidney cells and lung cancer cells.After the synergistic action of LysP4 and vancomycin,the minimum inhibitory concentration from 64μg/m L and 2μg/m L decreased to 4μg/m L and0.5μg/m L,respectively.The time-kill curve showed that the number of bacteria is reduced by 4 log CFU/m L,while LysP4 alone（4μg/m L）only reduced the number of bacteria by 1 log CFU / ml,vancomycin alone（0.5μg/m L）has no bactericidal effect.Protein structure analysis of that holin and phage＿holin＿1 family protein had high homology.It belonged to class II holin,had two hydrophobic transmembrane structure regions.When holin is induced to express,it will lead to the growth arrest or even cleavage of host BL21（DE3）.Spot plate method,resazurin staining and counting verified that holin had lytic activity against S.aureus 35,and named HolP3.The lytic spectrum HolP3 and LysP4 were determined against seven different MLST（1,22,25,59,239,398 and 509）of S.aureus.Only 239 ST type could not be lysed,and the other 6 types all could be lysed.Staphylococcus pseudointermedius could be lysed about 5/10 strains by holin and 10/10 strains by LysP4.Thirteen strains of Staphylococcus epidermidis,8/13 strains by holin and 7/13 strains by LysP4.Holin can cleave Escherichia coli of serotype O16: H48,while LysP4 can not.CONCLUSION1.In this study,holin and endolysin LysP4 were successfully cloned and expressed in the lysis system of S.aureus phage IME522.2.LysP4 has a strong bactericidal ability on S.aureus,and has good stability at p H5.0-10.0 and temperature 16-40℃.It can effectively remove the biofilm on the surface of bacteria,cooperate with vancomycin reduced the dosage of antibiotics,have no hemolytic effect on human red blood cells,and have no toxic effect on embryonic kidney cells and lung cancer cells.3.When IPTG induced holin expression will lead to the cleavage of Escherichia coli BL21（DE3）,and it had lytic activity against S.aureus 35.Holin is a small molecule membrane protein with two hydrophobic transmembrane structural regions.4.HolP3 and LysP4 had a wide range of lysis to Staphylococcus.Holin can lyse E.coli of serotype O16: H48 treated with EDTA.,while Lys P4 have no lytic ability to Gram-negative bacteria.