Construction And Characterization Of A Chimeric Lysin With Improved Bactericidal Activity Against Streptococcus Agalactiae

β-hemolytic streptococcus agalactiae,also known as Group B streptococci(GBS),is one of the most common symbiotic organisms in the lower digestive tract and vaginal flora of healthy adults.The pathogenicity and epidemiological investigation of Streptococcus agalactiae showed that GBS is the main cause of infant morbidity and mortality in developed and developing countries.Antibiotics are still the main way to deal with Streptococcus agalactiae infections.Penicillin is the first-line antibiotic for GBS infection.Macrolides are the recommended second-line drugs.However,in recent years,antibiotic sensitivity analysis showed that Streptococcus agalactiae isolates has reduced sensitivity even fully resistance to many antibiotics,including penicillin.Therefore,the emergence of GBS-resistant strains poses an increasing threat to global public health,necessitating the needs for options alternative to conversional antibiotics.Bacteriophage endolysins(lysins)is a new class of antimicrobial agents encoded by the phage genome.The bactericidal mechanism of lysins is the hydrolysis of cell wall peptidoglycan to cause bacterial lysis and death.Studies have shown that lysins have typical modular structure,that is,an N-terminal enzymatically active domain(EAD)and a C-terminal cell wall binding domain(CBD).CBD is used to target specific parts of the bacterial cell wall,and the EAD plays a bactericidal role by destroying the bonds of bacterial peptidoglycan,thereby causing bacterial cells to lyse and die.As an attempt to create a novel lysin with improved activity against S.agalactiae,a chimeric lysin,Cly V,was constructed by fusing the EAD from Ply GBS lysin(GBS180)and the CBD from Ply V12 lysin(V12CBD).Plate lysis assay combined with lytic kinetic analysis demonstrated that Cly V had improved activity than its parental enzymatic domain GBS180 against multiple streptococci.Biochemical characterization showed that Cly V was active from p H 7 to 10,with the optimum p H of 9,and was stable when it was exposed to Na Cl solution below 500 m M.In a S.agalactiae infection model,a single intraperitoneally administration of 0.1 mg/mouse of Cly V protected 100% mice,while it was observed that ～ 29% survive in group that received a single dose of 0.1 mg/mouse of GBS180.Moreover,a high dose of 0.8 mg/mouse Cly V did not show adverse effects to the health or survival rate of the mice.Although the pharmacokinetics and drugability of Cly V still need to be studied,the robust bactericidal activity and good safety profile showed in this study clearly suggest that Cly V may represents a potential candidate for the treatment of S.agalactiae infections.