Structure And Key Amino Acid Analysis Of Streptococcus Suis Lysin PlyARI

Streptococcus suis is an important type of gram-positive zoonotic cocci with high isolation rate in pig breeding.Among the widely pathogenic Streptococcus suis,serotype 2has the most serious prevalence and virulence.In addition,in recent years,new mutant strains such as Chz serotype have emerged continuously and been identified in many countries,indicating that it has the possibility of continuous transmission in the evolutionary process.Therefore,it is of great public health significance to pay attention to the prevention and control of pathogenic Streptococcus suis infection.In this study,lysogenic phage encoding lyase Plyari was found by analyzing the genome of a Streptococcus suis strain AH681.Further bioinformatics analysis revealed that Plyari belonged to cysteine-histidine dependent amidase,and sequence alignment showed that Ply30c was highly homologous to the known lyase of Streptococcus suis.Therefore,the study was carried out from two directions.The first part mainly studied the in vitroin vitro lytic activity,biological characteristics and in vivo therapeutic effect of prokaryotic lyase PLYARI.In the second part,the relationship between the binding and the structure and function of the catalytic domain was studied by analyzing the crystallization and the three-dimensional crystal structure of lyase.1 Study on biological characteristics and lytic activity of recombinant Streptococcus suis lysin PlyARIPhage lyases are a class of peptidoglycan hydrolyases derived from the genome coding of phages.Due to its strong cleavage activity in vitro and in vivo,low drug resistance rate and easy availability,it has attracted wide attention in recent years in the context of global restrictions on the use of antibiotics.In this study,a phage lyase Plyari was found in Streptococcus suis AH681,and it was found to have a high homology（90%）with lyase Ply30in the subsequent evolutionary relationship analysis with known Gram-positive coccal lyase Plyari.Liquid pyrolysis experiments in vitro,however,we found that the cracking activity of the significant differences of the two,so this experiment measured the including slab cracking,acid and alkali tolerance,such as the biological characteristics,the results show that except one without milk streptococcus,PlyARI outside a MRSA strains of 35（40）strains bacteria produced obvious bacteriostatic circle,and PlyARI can pyrolysis pig streptococcus streptococcus animal disease with horse from different host host specificity of subspecies is not clear.Lyase indicator bacteria were added into buffer solutions with different p H values for lytic experiments,the results showed that it had good lytic activity in the range of p H 4-10,which gives Plyari more possibilities in vivo drug delivery,adjuvant packaging,transportation and preservation,etc.The results showed that Plyari was a new strain of Streptococcus suis lyase with broad spectrum and wide p H activity range in vitro.2 Antibacterial activity of recombinant Streptococcus suis lyase PlyARI in vivoIn order to study the anti-infective effect of lyase Plyari in vivo and the side effects of continuous administration,an intraperitoneal infective treatment model of Streptococcus suis ICR mice was established in this study based on the in vitro lytic activity.In the abdominal cavity infection-treatment model with 1.5 times the minimum lethal dose of type 2 pig streptococcus HA9801 attack on the ICR mice poison,the minimum bacteriostasis concentration of HA9801 PlyARI is 16 mu g/Ml,4 weeks of ICR mice celiac injection of 7.5×10⁸ CFU HA9801 interval of 2 h after injection PlyARI respectively（0.5 mg）and PBS treatment,72 h after PlyARI to protect mice force reached 100%,whereas the control mice all died,Mice treated with 24h continuous injection of PlyARI（4 times in total 2mg）showed no abnormal clinical symptoms.The anti-infection results showed that low concentration of lyase PlyARI could quickly and effectively inhibit the infection of Streptococcus suis in mice,showing excellent protection.3 Crystallization and analysis of recombinant PlyARI proteinIn the body are showed excellent activity in vitro of lyase PlyARI in structure belongs to the CHAP class cysteine-histidine dependent amidase,experiments in order to clarify the PlyARI its structure and mechanism of action of hydrolysis of PG,will the Ply of molecular sieve purification enrichment ARI protein with A drop type gas phase diffusion method for protein crystallization,in 1.8 A successful diffraction quality of high quality crystal.The structure was analyzed by Phaser in the CCP4 software group,and then modified by manual modeling with REFMAC5 and COOT.Finally,further refinement was performed by Phenix software.The analysis results showed that the PlyARI exhibited amylase activity,consisting of N-terminal CHAP catalytic domain and C-terminal binding domain SH3b.4 Functional domain of recombinant Streptococcus suis lysin PlyARIOn the basis of success for protein crystallization parsing,in order to study the combination of functional domain SH3b combining ability,build the GFP respectively-ARI chimeric protein and CHAPk-ARI chimeric protein,the results showed that the GFP-ARI of Streptococcus suis（serotype Chz,2,4,9,16）streptococcus,staphylococcus aureus（MRSA）,horse with high combining ability,and chimeric lyase Lys CHAPk-AR activity for Streptococcus suis and horse streptococcus cracking Lys K were improved.In addition,the purpose of this study was to investigate why two lyases PLY ARI and PLY 30 with similar homology had such great activity differences.In this study,key amino acid point mutations around Ca²⁺ion binding sites in CHAP catalytic domain were designed based on crystal structure data.Results show that the alanine（Ala）mutant strains for almost all the host bacterium（Streptococcus suis,animal disease subspecies horse streptococcus,staphylococcus aureus,non-dairy streptococcus）cracking ability are promoted,which it has no animal disease and horse milk streptococcus streptococcus subspecies of cracking,significantly improve the capacity,on the contrary lysine（Lys29）mutant strains of cracking activity results fell.In conclusion,this paper studied the lytic activity of Plyari in vitro and in vivo and analyzed the holoenzyme crystal structure of Plyari.Based on this,it was found that the key amino acid Asn29 in Ca²⁺ion binding zone affected the lytic function,and the binding ability and effect of Plyari-SH3b as chimeric lyase activity functional domain were also studied.It provides a theoretical basis for the subsequent modification of lyase functional domain and key active sites.