Recombination Of A Novel Chimeolysin And Its Antimicrobial Effects

Objectives:Bacteriophage lysins and their derivatives has long been viewed as promising novel class of antibacterials for the treatment of antibiotic-resistant pathogens.However,lysins with broad-spectrum of bactericidal activity are still not adequately addressed,which impedes the translational development of such molecules.Herein,we reported a novel chimeolysin,ClyE,which is consisted of the CD of PlyGBS(GBS180,N-terminal 180 amino acids)and the CBD of PlySs2(PlySb,C-terminal 85 amino acids).Methods:①E.coli BL21 and HisTrap FF columns were used to express and purify of ClyE.②The bactericidal activity of ClyE against Strecptococcus agalactiae S12 in PBS buffer and milk environment was quantified by dilution plate counting method.③Transmission electron microscopy(TEM)was used to visualize the effects of ClyE on bacterial cell wall.④The relative activities of ClyE in different NaCl,EDTA,and pH buffers were determined by bacteria turbidity decrease assay using Synergy H1 microplate reader.⑤Bacteria turbidity decrease assay was used to determine the lytic spectrum of ClyE.⑥The cytotoxicity of ClyE to A549 cells was evaluated by CCK-8 assay.⑦Furthermore,the antimicrobial efficacy of ClyE was confirmed in a S.agalactiae infected zebrafish model.Results:①Chimeolysin ClyE can be well expressed in E.coli BL21 and purified with HisTrap FF columns.②The lytic activity of ClyE against Strecptococcus agalactiae S12 was rapid,with reduction of over 2 log 10 after treatment for 60 min both in buffer and milk environments.③TEM showed that cells exposed to ClyE lost the intact of their cell walls.This observation is quite consistent with the typical phenomenon of osmosis-mediated cell lysis caused by lysins reported elsewhere.④The biochemical analysis showed that ClyE was highly active and stable under NaCl up to 500 mM,and displayed the optimum pH of 7.0-9.0.⑤ClyE demonstrated robust lytic activity against multiple species,including S.agalactiae,S.dysgalactiae,S.pyogenes,S.suis,S.mutans,S.pneumoniae,E.faecalis and S.aureus.⑥The CCK-8 assay showed that no obvious cytotoxicity was observed in A549 cell line under ClyE concentration of less than 1000μg/ml.⑦ The protective efficacy of ClyE was further confirmed in a S.agalactiae infected zebrafish model,showing that one single i.p.injection of ClyE(2 μg/zebrafish)greatly increased the survival rate of zebrafish in comparison with that of penicillin.Conclusions:The broad-spectrum and good safety of ClyE provides a new candidate for treating streptococcal and other infections.