| Streptococcus suis is a serious zoonotic pathogen.The emergence of drugresistant and multi-drug resistant strains made the prevention and control of antibiotics more difficult.Under the background of "reducing resistance","restricting resistance" and "banning resistance",there is an urgent need to find a new type of antimicrobial agent that is not easy to develop resistance to Streptococcus suis.Phage lysin is a type of hydrolase that targeting bacterial cell wall peptidoglycan encoded by phage genome.Compared with traditional antibiotic drugs,phage lysin has the advantages of stable protein performance,high sterilization efficiency,less resistance,and ease of artificial design and modification.These advantages make lysin have great potential to be used in the treatment of clinical drug-resistant and multi-drug resistant bacterial infections.In this study,a prophage-encoded lysin was excavated from the whole genome of a strain of Streptococcus suis and named Ply1228.The comparison of the amino acid sequence of Ply1228 shows low homology with the reported Streptococcus suis lysin,indicating that Ply1228 is a new Streptococcus suis lysin.Ply1228 was constructed,prokaryotically expressed and purified to obtain a protein with a molecular weight of approximately 54 kDa.In vitro,the bactericidal of Ply1228 was determined,and the results showed that Ply1228 has broad and high-efficiency antibacterial activity against Streptococcus suis.The host spectrum of Ply1228 span 8 serotypes(type 2,3,7,9,10,12,14 and 27),but has no antibacterial activity against bacteria other than Streptococcus.Ply1228 has high-efficiency bactericidal activity and the action time is very short.It can reduce the number of viable host bacteria by about 4 Log within 10 min.The bacteria can not be developed resistance to Ply1228 when continuous passage in vitro.In addition,Ply1228 showed favorable thermal stability(temperature was 4 ?-50 ?)and acid-base stability(p H was 3-9)in vitro.The above results indicate that Ply1228 has great application potential.Subsequently,the 3D structure of Ply1228 was predicted,the key amino acid action sites were identified,and the antibacterial and binding activity of Ply1228 functional domains were verified.Bioinformatics analysis shows that Ply1228 contains an N-terminal CHAP catalytic domain,a C-terminal amidase-2 catalytic domain,and a binding domain(CWBD)composed of a CW-7 binding motif in the middle.The experimental results showed that the catalytic domain of CHAP exhibited bactericidal activity similar to that of the holoenzyme and the addition of EDTA did not affect the activity of the holoenzyme while the binding domain and amidase-2catalytic domain did not have bactericidal activity.This indicates that the CHAP catalytic domain is essential for the bactericidal function of lysin Ply1228 and lysin Ply1228 does not depend on the presence of Ca2+ for its bactericidal function which is different from other reported lysins with CHAP catalytic domain.The results of the binding activity experiment showed that both the CHAP catalytic domain and the cell wall binding domain(CWBD)have specific binding activity to Streptococcus suis and the CHAP catalytic domain has weak binding activity.This indicats that the cell wall binding domain of Ply1228 plays an important role in the specific binding activity of Ply1228.This study successfully predicted the 3D structure of three domains of Ply1228 with 100% confidence.The amino acid positions related to the activity were predicted to be C34,H99,E305 and E350 through sequence and structure alignment.The C34 and H99 of Ply1228 were mutated to alanine,and the activity was reduced by 88% and 63%,respectively.This indicats that C34 and H99 are the key active sites for the bactericidal activity of Ply1228.It also proves that the CHAP fragment plays a key role in the bactericidal activity of the holoenzyme.There is no significant change in bactericidal activity after E305 and E350 of Ply1228 were mutated to alanine,indicating that amidase-2 fragment has little effect on the cleavage activity of the holoenzyme.In order to evaluate the anti-infection effect of the lysin Ply1228 in vivo,a mouse bacteremia model caused by type 2 Streptococcus suis infection was established.The results show that intraperitoneal injection of 2×109/mouse of Streptococcus suis SC225 cause bacteremia in mice.After treatment of this model,it is found that 200?g/mouse of lysin Ply1228 can protect 100% of mice in a state of bacteremia mouse.The number of colonies in the blood of mice decreased by 3 Log,and the number of colonies in the liver,spleen,lung and kidney decreased by 2.3,2.3,2.6,and 2.4 Log,respectively,48 h after the treatment.The bacteria can not be developed resistance in vivo.Compared with the control group,the levels of inflammatory cytokines TNF-?and IL-6 in the treatment group were significantly reduced and the pathological changes of the liver,spleen,lung and kidney tissues were similar to the normal group,indicating that the lysin Ply1228 significantly reduced the inflammation and pathological changes of organs in vivo.In summary,this study systematically studied the bactericidal activity of Ply1228 in vivo and in vitro and found functional domains and key active sites that can be modified,providing a theoretical basis for further application of phage lysin in clinical research and drug development. |
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