Study On The Inhibition Mechanism Of Luteolin On The MATE Gene In T.Pyogenes

Trueperella pyogenes is a kind of pathogenic bacteria which causes pyogenic infection in animal.Due to the rapid development of antibiotic resistance,Trueperella pyogenes gained extensive attention.The active efflux system is one of the most important factors which causes bacterial drug resistance.Although the efflux pump inhibitor(EPIs)could reduce the ejectment of the antibiotic,but the EPIs were under restrictions for the high doses use and the major side effect.The study analyzed the correlation between the efflux gene in Trueperella pyogenes with its antibiotic resistance,and explored the effects of luteolin on efflux gene,so as to provide a basic foundation of luteolin as a new type antibiotic development,it is of great significance to reduce the occurrence of bacterial drug resistance.Using molecular biological technique and bioinformatics technique to screen MATE gene of Trueperella pyogenes,and projective parsing the second and tertiary structure of the coding protein.The results showed that the carrying rate of the MATE gene is 94.12%(32/34)in Trueperella pyogenes,and coding 436 amino acids with the relative molecular weight 44.94232 kD,the theoretical isoelectric point is 10.05,the grand average hydrophilia index(GRAVY)is 0.873;the most likely distribution location of the transporter is cytomembrane;it contains no signal peptide sequence,no N-glycosylation site,contains 12 transmembrane domains 25 phosphorylation sites.The dominating secondary structure is ?-helix,containing 2 conserved domain databases;None reliable three dimensional structure was projected.The minimum inhibitory concentrations(MICs)of luteolin and gentamicin against 34 strains of Trueperella pyogenes isolates were tested by micro-broth dilution method.Using chessboard method to test the co-antibacterial effect of gentamicin and luteolin against isolates which resistant gentamicin.Analysis the resistance level changes in gentamicin before and after luteolin treatment in sub-inhibition concentration.The results showed that the MIC of luteolin against Trueperella pyogenes was 39-156 p,g/mL,presented no co-antibacterial effect with gentamicin.The MICs of these resistant isolates become 1/2 to 1/32 after treating 36 h with a sub-inhibitory concentration(1/4MIC)of luteolin,the MICs decreased significantly as the antibiotic resistant levels become higher.The results show that luteolin has a good antibacterial activity in vitro and it can reverse the drug resistance of Trueperella pyogenes.8 gentamicin resistance isolates with MATE genes were chosen to study the effective of luteolin on MATE gene sequence and the expression quantity of mRNA.The results showed that after treating with a sub-inhibitory concentration(1/4MIC)of luteolin,there was no effect on MATE gene sequence,but the gene's mRNA expression quantity was declined.And with the decline of mRNA expression the MIC presented on a declining curve.But those changes un-conspicuous after passage.Suggesting luteolin impact the MIC of Trueperella pyogenes through the suppression the expression of mRNA of MATE gene.This study based on efflux gene,project and analysis the dependency between the MATE gene of Trueperella pyogenes and drug resistance.To inquiry the bacteriostatic activity and the influence of efflux gene of luteolin on Trueperella pyogenes,this study presents significant meaning on resistance mechanism and the prevention of drug resistance,meanwhile providing a basic foundation of luteolin as a new type antibiotic development.