In Vitro/Vivo Antibacterial Activity Of Recombination Protein AtlM And AtlG From Staphylococcal Aureus

Objective Staphylococcus aureus is one of the important pathogens of humansand animals infected.It can cause local suppurative infections, septicemia,sepsis andother systemic infections.At present,the frequent use of antibiotics lead to human floraand the growing phenomenon of resistant Staphylococcus aureus.Antibiotics have beennot enough to cure infections caused by multidrug-resistant Staphylococcusaureus.Searching for new antimicrobial agents to treat Staphylococcus aureus infectionsbecome a research hotspot.The autolysin of S.aureus is a bifunctional protein,which areseparated by proteolytic processing to generate two extracellular lytic enzymes,a51-kDgucosaminidase and a62-kD amidase.The aim of this study was to obtain theStaphylococcal aureus autolysin AtlM and AtlG by prokaryotic expression system andinvestigate its antibacterial activity in vitro and vivo,which lay the basis for theautolysin fragment as an antibacterial drug.Methods1.Prokaryotic expression and purification for atlM and atlG:The specificprimers were designed according to atlM and atlG of Staphylococcal aureus genesequence,and atlM and atlG was amplified by PCR from the Staphylococcal aureusgenome,respectively.The recombiant plasmid pET-32а(+)/atlM and pET-32а(+)/atlGwas constructed.After IPTG inducing,the recombinant protein was purified byelectroeluting of bag filter.The MIC of rAtlM/rAtlG for ATCC25923and resistantS.aureus were determined by the broth microdilution method.2.In vitro antibacterial test:add rAtlM or rAtlG (final concentration50μg/ml) tosuspension of S. aureus with0.01M PBS (final concentration of5×105CFU/ml),monitored colonies at1h,3h and5h.3.In vivo antibacterial test:gave Staphylococcus aureus0.5ml (107CFU/ml) toeach group of mouse by intraperitoneal inoculation,one hour later,gave each group of mouse rAtlM/rAtlG0.2ml(1mg/mice)by tail vein injection.After two hours and fourhours,collected the10μl eye blood of each mice. At last,the colonies were counted atdifferent times.Results1.The recombiant plasmid pET-32а(+)-atlM and pET-32а(+)-atlG wereconstructed successfully.rAtlM and rAtlG determined by SDS-PAGE were80kD and66kD,met the expected results.The concentration of rAtlM and rAtlG were1.25mg/mland1.63mg/ml,respectively.The MIC of ATCC25923and resistant S.aureus for rAtlMwere8μg/ml and64μg/ml,and the MIC of ATCC25923and resistant S.aureus for rAtlGwere16μg/ml and64μg/ml.2.The vitro antibacterial test showed that rAtlM and rAtlG had bactericidal effecton ATCC25923S.aureus and resistant S.aureus,and rAtlM is better than rAtlG forresistant S.aureus.（At3h, rAtlM took effect to ATCC25923S.aureus and resistantS.aureus,P=0.004/0.001；At3h, rAtlG took effect to ATCC25923S.aureus and resistantS.aureus,P=0.004/0.003）3.The vivo antibacterial test showed that rAtlM and rAtlG both had bactericidaleffect on ATCC25923S.aureus and resistant S.aureus,and rAtlM is better than rAtlGfor ATCC25923S.aureus and resistant S.aureus.(At2h, rAtlM took effect toATCC25923S.aureus and resistant S.aureus,P=0.008/0.014；At2h, rAtlG took effect toATCC25923S.aureus and resistant S.aureus,P=0.011/0.026)Conclution rAtlM and rAtlG indicates a certain antibactericidal effect onATCC25923S.aureus and resistant S.aureus,and may be new potent antimicrobialagents.