Structural And Functional Analysis Of Endolysin Ly7917 From Bacteriophage Of Streptococcus Suis

Streptoccocus suis serotype 2（S.suis 2）is an virulent zoonotic pathogen that makes it important to control the S.suis 2 infection for public health.Lysin Ly7917encoded by prophage phi7917 harbored in S.suis serotype 7 was found as a novel of high–efficiency antibiotic with significant catalytic effect on multiple serotypes of S.suis.Based on bioinformatics analysis,Ly7917 was predicted to have the N–terminal cysteine/histidine–dependent amidohydrolase/peptidase（CHAP）catalytic domain to lysis peptidoglycan（PG）and with the assistance of C–terminal SH3b binding domain in charge of binding to target PG site.To investigate the biological characteristics of Ly7917 and it’s catalytic mechanism and interaction mode with substrate,the antimicrobial effect against S.suis in vitro and in vivo were carried out systematiclly followed by the resolution of its three-dimentional structure.To verify the predicted ly7917 gene function in vitro,Ly7917 was expressed,purified and characterized.4.5 mg/mL Ni–NTA purified product showed obvious catalytic activity in plate lysis assay with enzyme activity units of 114 U/mg.Ly7917can lysis all provided S.suis strain in different serotypes and showed the highest efficiency in type 2 HA05729–2 with 40%decrease,meanwhile it lysis staphylococcus aureus with decrease of 10%,but with no lytic effect on examined gram-negative strains as expected.By addition of 1.25 mM Ca²⁺,the activity of Ly7917 on HA9801 is improved 20%while Mg²⁺,Zn²⁺and Co²⁺showed no obvious influence on the activity of Ly7917,and inhibited by Fe²⁺,Cu²⁺.Ly7917 prefered high pH value and inhibited with EDTA at concentration of over 50 mM.It can stay in 42℃for over 15 min.The stable efficiency of lytic activity Ly7917 on wide range of S.suis stains makes it possible for clinical therapy.To find the anti-microbial activity of Ly7917 in vivo,2 type of animal therapy model established.The optimal challenging dose of HA9801 to BALB/c mice was 1.4×10⁸ CFU/mouse,the minimal inhibitory concentrations（MIC）of Ly7917 in HA9801 strains is 64 U/mL,compared to 625 U/mL of penicillin G.The optimal treatment time of the Ly7917 was 3 h after challenge.In intraperitoneal infection-intraperitoneal therapy mice model,BALB/c mice challenged with HA9801at concentration of 1.4×10⁸ CFU/mouse,the bacteria concentration of buffer treated mice reached up to 2–18×10⁸ CFU/mL with 20%survival rate.Meanwhile,treatment with Ly7917 was 1–12×10⁴ CFU/mL resulted in 90%survival rate,by contrast a 50%survival rate with 1–5×10⁷ CFU/mL of HA9801 in blood was observedinthepenicillin-G-treatedgroup.Inmuscle incision-infection–intraperitoneal treatment mice model,there was no bacteria detected in the blood of BALB/c in Ly7917 treatment group within 2–2.25 h after the treatment while that of mice in buffer control group shows dynamic changes of normal distribution with the highest level of 10⁶ CFU/mL with no death of the mice,that improved excellent antimicrobial activity of Ly7917 with efficient catalytic activity against S.suis 2.In order to reveal the crystal structure of Ly7917 and interaction with ligand,Ly7917 was purified and condensed for crystallization screening with sitting-drop vapor diffusion method.Crystals of native Ly7917（PBD code:5D74）with high quality of 1.9?diffraction resolution obtained and its structural data revealed after SAD phase-analysis,phenix refinement and coot correction methods,which help to find that there are two Ly7917 molecules in one crystal unit and the CHAP domain is similar with chain A residues 331-462 of 4f88 while SH3b is similar to residues161-245 of 4lxc.Also,the cystal of complex of Ly7917 and hydrolysate of general ligand MDP called L-Alanyl-D-isoglutamine is suscessfully obtained and analysed（PBD code:5D76）with high diffraction resolution of 2.5?.The data reveals that Ly7917 with the typical catalytic CHAP domain acted as an amidase by hydrolyzing the amido bond between N-acetylmuramic and L-Alanine.The MDP hydrolysate L-Alanyl-D-isoglutamine bind with Ly7917 SH3b domain at 176A、180R.Combined with the results of the structure analysis and the active site prediction,serial truncated proteins and site mutative proteins were expressed and purified for activity analyzing.The results showed that the N–terminal truncated protein Ly7917_17–245 completely lost the catalytic activity as well as the point mutant proteins Q14A–Ly7917 and S17A–Ly7917.The activity of LyCHAP was almost the same as Ly7917.The N–terminal sequence is essential for Ly7917 and LyCHAP_1–100 was the smallest unit with detectable activity.With 1 mM Ca²⁺,LyCHAP_1–130 is compatable with Ly7917 as alternative.A176G-Ly7917 and R180A-Ly7917 are still remaining full catalytic activity of Ly7917 for SH3b binding domain is not necessary to endolysin.This study confirmed the catalytic activity of Ly7917 in vitro and in vivo,the resolution of crystal structure and the substrate catalytic sites,active domain and key amino acid site of CHAP,will provide useful data for the further clinical application and drug development.