Study On The Bacteriostasis Of Lysin TSPpgh From A Thermophilic Phage

As a new type of protein antibiotic that is being attracted much attention,M23 family proteases with endopeptidase and amidase activities can degrade peptidoglycan in bacterial cell walls.Several bacteriophage lysins belonging to M23 family proteases can efficiently lyse multidrug-resistant bacteria,making it possibly replace traditional antibiotic therapy.Based on sequenced genome of the bacteriophage TSP4 from Thermus thermosyphon in Tengchong,characteristics of the bacteriophage lysin are analyzed in this study,as well as we also cloned,and then expressed the lysin gene,to esTable lish a rapid purification method of recombinant lysin based on thermal decomposition of thallus,and assess activity of purified lysin through antibacterial activity evaluation and atomic force microscope observation.Furthermore,the lysin is adsorbed with soluble starch to prepare enzyme preparation that was regularly evaluated enzyme activity changes.To explore cleavage mechanism of lysin,the peptidoglycan of gram-negative Escherichia coli and positive Staphylococcus aureus was reacted by the lysin,and adsorption characteristics of lysates solution containing lytic peptidoglycan was detected.The site of peptidoglycan hydrolysis targeted by the lysin was demonstrated by liquid chromatography-mass spectrometry(LC-MS),and we further validated active site and characteristics of lysin via site-specific mutation of TSPpgh gene sequence.To explore antibacterial effect of the lysin,we evaluated its antibacterial effect on E.coli,Salmonella,S.aureus and multidrug-resistant Klebsiella pneumoniae through lethality analysis and scanning electron microscope(SEM)observation.Effect of the lysin on the treatment of multi-drug resistant S.aureus in traumatic infection was explored by skin wound treatment,drug-resistant S.aureus colonization,and local treatment with TSPpgh on mice.The results indicate TSPpgh,a lysin of TSP4,belonging to the M23 peptidase family.Motif HXXXD and HXH were predicted to be the active sites of TSPpgh,andmutation verification was implemented.By cloning and expressing the lysin gene TSPpgh,a recombinant protein with a molecular weight of 19 KDa was obtained.By raising temperature of fermentation generated by inducible expression for TSPpgh to55? for 20 minutes,cell can be ruptured and denatured,which was precipitated to remove protein expressed mostly in strains of E.coli,realizing rapid purification of the lysin.Combined with high-density fermentation technology,concentration of the lysin in fermentation broth can reach 2 mg/mL.TSPpgh showed the highest bactericidal efficiency against S.aureus ATCC 6538,up to 90%.AFM observation showed that TSPpgh cracked cell walls of E.coli BL21(including TSPpgh plasmid)and S.aureus,and TSPpgh preparation can store at 20? for one year and still maintain 75% enzyme activity.Thermophilic lysin TSPpgh can adsorb and hydrolyze peptidoglycans of gram-negative bacteria and positive bacteria,and can hydrolyze elastin.Analysis of the hydrolysis site of peptidoglycan by LC-MS shows that TSPpgh can also hydrolyze glycosidic bond Glcp-?-1'-6'-Glcp-?-1'-1',with glycosidase activity.Bacteriostasis experiments showed that the lethal rate of the lysin TSPpgh to S.aureus,Salmonella CMCC(B)50094 and E.coli CMCC(B)44102 strains was 90%,that of multidrug-resistant K.pneumoniae was more than 80%,and the partial lethal rate was up to 100% at 37 ? and 68 ?g/mL of enzyme concentration.SEM observation shows that TSPpgh realizes its bactericidal function through lysis.In this study,skin wounds were conducted in mice,drug-resistant S.aureus2612-1606BL1486 were colonized,and then TSPpgh was used in local treatment.During 9-day treatment,both TSPpgh and kanamycin rapidly promoted wound healing.The role of TSPpgh in the treatment of multi-drug resistant S.aureus in traumatic infection was verified.In summary,a high-efficiency expression and rapid purification system of thermophilic lysin has been esTable lished here.Combined with wider bacterial lysis spectrum,higher activity and thermal sTable ility of thermophilic lysin,it presents a better prospect on production and application.A good killing effect on multidrug-resistant bacteria of thermophilic lysin is helpful for its application in pharmaceutical research and development as a new type of enzyme antibiotic.