The Key Sites Of Staphylococcus Aureus Phage Lysin LysGH15 And The Combination Therapy Of Lys GH15 And Baicalin For Treating Pneumonia Of Mice

Staphylococcus aureus is one of the most common community and hospital-associated pathogens.Prevention and treatment of the disease caused by S.aureus has become more challenging due to the emergence of resistant strains.It is imperative to seek new treatment strategies.Phage lytic enzymes are bacterial cell wall hydrolases that are produced during the infection cycle of double-stranded DNA phages.Phage was capable of releasing progeny virus particles by cleaving the essential bonds in the cell wall peptidoglycan,resulting in hypotonic lysis.Phage lytic enzymes are thought to be novel agents for controlling bacterial pathogens.Baicalin can target against S.aureus virulence factor ?-hemolysin and effectively treat S.aureus infection.In the previous period,our laboratory obtained the phage lysin Lys GH15 of S.aureus,which showed broad spectrum and high bactericidal activity against S.aureus.In order to further explore the molecular mechanism of Lys GH15 and its synergistic mechanism with baicalin,this study further analyzed the key location of the interaction between the Lys GH15 catalytic zone and the substrate,and combined Lys GH15 with baicalin to evaluate the efficacy in treating pneumoniae infection.Molecular mechanism of Lys GH15 actionThe CHAP fragment of Lys GH15 was docked with the peptide DGln NH2-LLys-DAla-5Gly by Auto Dock 4.2 software.It predicted that Asp33,Ser35,Phe36,Tyr50,Arg71,Tyr72,Gln53 and Asn75 should play a role in the process of CHAP bond to its substrate.These seven sites were all mutated to alanine.The mutations of Asp33 and Asn75 could not express proteins after mutated to alanine,but Arg71,Tyr72,Ser35,Phe36 and Tyr50 mutants successfully obtained expression.The CHAP fragment and five mutants of Arg71,Tyr72,Ser35,Phe36 and Tyr50 were analyzed by circular dichroism.The secondary structure of the native and mutants were similar,indicating that mutants did not affect the secondary structure of CHAP,and did not affect the tertiary structure of CHAP fragment.The result of zymogram showed that only F36 A mutant could not form transparent band,the other four mutants could form.Indicating that the F36 A mutant lost its cleavage activity against S.aureus.Native CHAP and S35 A,Y50A,R71 A,T72A mutants can decrease the OD600 value from 0.6 to almost undetectable in 60 min under the amount of 50 ?g/ml;the control group added equal amount of buffer,only the OD600 value of F36 A mutant and the control group were similar and the concentration of the bacteria did not decline.But also in the colony count test we found only mutant F36 A activity was completely lost,while the antimicrobial activity of the four mutants S35 A,Y50A,R71 A,T72A was not significantly different from that of native CHAP.The results showed that F36 plays a key role in the interaction between CHAP protein and substrate.Combined Lys GH15 with baicalinWe then investigated the interaction of Lys GH15 and baicalin in vitro.USA300 treated with Lys GH15(dose 50 ?g/ml)alone(The OD600 value of USA300 suspension decreased from 0.785 to 0.194 within 10 min)and co-treated with Lys GH15(dose 50?g/ml)and baicalin(dose 16 ?g/ml)(The OD600 of the combination treatment suspension decreased from 0.745 to 0.124 in 10 min).The cleavage activity was similar(P>0.05).In hemolysis experiments,hemolysis was not detected when rabbit erythrocytes were incubated simultaneously with baicalin(16 ?g/ml)and Lys GH15(?50 ?g/ml).In contrast,the hemolysis rate was 13.1% when treated with baicalin alone;and when rabbit erythrocytes were incubated with 25,50 or 100 ?g/ml Lys GH15,hemolysis rate was 34.5%,22.74% and 20.7%.Western blot results showed that adding 16 ?g/ml baicalin and 100 ?g/ml Lys GH15 lead to almost no ?-hemolysin production.In vitro experiments,the combined threpy effect of Lys GH15 and baicalin was determined,we further study of Lys GH15 combined with baicalin in the treatment of S.aureus pneumonia.The results showed that the combined treatment group in improving the health status of mice,reducing lung W / T ratio and BALF cellnumber,protein content and cytokine levels was effective.In the case of Lys GH15 alone or in combination therapy group,the bacterial colonies of the lung were reduced to 8.0×102CFU/mg and 5.0 ×101CFU/mg,respectively,and the bacterial load was1.0×104CFU/mg under the treatment of baicalin alone.As a control,the number of bacteria loaded in the lung increased to 3.3×1010CFU/mg in the lungs after 24 h in the absence of treatment,indicating that Lys GH15 combined with baicalin can produce effective treatment in Staphylococcus aureus pneumonia.