| The disease caused by Streptococcus spp is a serious zoonosis which distributes widely in the world.Antibiotics includingβ-lactam(represented by penicillin),macrolide and quinolone were effective drug for the therapy for streptococcal infection.However,there have been reports of Streptococcus resistant phenotype and genotype(mef A,erm B,Tet M,etc.)in China and worldwide.In addition,the reduction of new candidate antibiotics has forced people to develop new antibacterial agents.Endolysins are hydrolytic enzymes which are encoded by phage,specifically cleave the specific bonds on peptidoglycan of bacterial cell wall,destroy the integrity of cell wall,and make bacteria disintegrate under the osmotic pressure.It can be expressed in vitro and has various features,such as easily accessible,facilely modified,quick effect,safety,and low resistance to bacteria.In addition,there are the similar cross-bridge in Streptococcal peptidoglycan which allows Streptococcal endolysins have a wider host spectrum than other species endolysins in nature,and can lyse a variety of Streptococci species.Therefore,endolysin is expected to become a new antimicrobial alternatives against Streptococcal infections.In this study,the clinical isolate 3518 of Streptococcus equi was used as the host bacteria,and a prophage endolysin with good antimicrobial activity was found in its whole genome sequence,which was named Lys LF1.Uploaded the amino acid sequence of Lys LF1 to the bioinformatics online analysis website NCBI for comparing the homology.The results of analysis indicated that Lys LF1 had the highest homology of21.88%with other Streptococcal endolysins that had been studied.Lys LF1 is a new Streptococcus endolysin.The results of biological characteristics indicated that Lys LF1had a wide host range and could lyse Streptococcus equi,Streptococcus suis,and Streptococcus gallolyticus in the tested strains,with a bacterial reduction rate of 30%to 99%.The p H stability test results showed that Lys LF1 has strong acid and alkali resistance,and maintained good antimicrobial activity within the p H range of 4～8,which can cleave about 70%to 99%of live bacteria;The temperature stability results indicated that Lys LF1 was more sensitive to temperature,and its activity is completely lost when the temperature was above 40°C;The stability results of Na Cl showed that high concentrations of Na Cl would affect the activity of Lys LF1,and the use of 200m M Na Cl would reduce its antimicrobial activity by 1.24×10~8 CFU/m L;The stability results of EDTA showed that metal ions were not necessary for the enzyme to exert its activity.The results of in vitro lysis experiments further indicated that Lys LF1 had good antimicrobial activity.Subsequently,the 3D structure and key catalytic sites of each domain of Lys LF1were predicted using the bioinformatics online analysis tool Phyre~2,and the predicted key catalytic sites were mutated into alanine using a one-step method.By comparing the antimicrobial activity of various mutant proteins(N11A,C26A,H91A,G108A,G158A,D241A,D276A,D337A)with Native Lys LF1,their key catalytic sites were determined.The experimental results showed that the antimicrobial activity of C26A and G108A decreased by 81.99%and 61.61%compared to Native Lys LF1,respectively.Thus,it was determined that C26 and G108 are key amino acid action sites for Lys LF1to exert proteolytic activity;By constructing truncated proteins(Lys LF1A(1～170 aa),Lys LF1B(1～360 aa),and Lys LF1C(330～445 aa)),the proteolytic activity of each domain was determined through in vitro antimicrobial experiments;The results showed that Lys LF1A(1～170 aa)and Lys LF1B(1～360 aa)exhibited proteolytic activity,while Lys LF1C(330～445 aa)did not exhibit proteolytic activity;By constructing fusion proteins(Lys LF1A-EGFP,Lys LF1B-EGFP,EGFP-Lys LF1C),the binding activity of each structural domain was determined using LSCM.The results showed that Lys LF1B-EGFP and EGFP-Lys LF1C had binding activity,while Lys LF1A-EGFP did not.Thus,the structure type of Lys LF1 was determined to be a dual-enzymatically active domain(EAD)and a cell wall-binding domain(CBD).The domain that exerts binding activity was similar to SH3,and the GH25 domain was also related to binding.The key domain that exerted catalytic activity was similar to CHAP.The above results have laid the foundation for the artificial design and transformation of Lys LF1 in the future.Finally,a mice pneumonia infection model was established using the host Streptococcus equi clinical isolate 3518.Different concentrations of Lys LF1 were used to treat mice and evaluated the in vivo antibacterial effect of Lys LF1.The results showed that 200μg/mice and 400μg/mice Lys LF1 had the effect of improving mice survival rate and slowing their death.200μg/mice Lys LF1 could protect 37.5%of mice from death,400μg/mice Lys LF1 could increase the survival rate of mice to 75%.The bacterial load results in the mice lungs showed that 400μg/mice Lys LF1 had a scavenging effect on isolate 3518.The results of lung histopathological showed that compared to the untreated group,400μg/mice Lys LF1 could alleviate pathological and histological damage to the lungs.The pulmonary interstitial thickness,alveolar hemorrhage,and inflammatory cell infiltration were reduced at 12 and 48 hours.In summary,this study found and successfully obtained a novel Streptococcal endolysin with good antimicrobial activity in vitro and vivo.Lys LF1 has good application potential in the treatment of Streptococcal infections,laying the foundation for enriching the Streptococcal endolysin library and developing new antibacterial agents. |
