| T.pyogenes is an important pathogen that can cause suppurative infection in livestock.The issue that decreasing curative effect in infectious diseases caused by antibiotic-resistant T.pyogenes has gradually become a real problem in clinical treatment.The efflux of MefA and MsrA are the main mechanisms leading to drug resistance in macrolide antibiotics,it is crucial to study efflux pump inhibitors(EPI)to prevent/reverse drug resistant in T.pyogenes.However,currently known EPI,such as reserpine,are not suitable for clinical treatment due to their high toxicity and side effects.The development of low-toxicity and high-efficiency EPI has become a new strategy for the treatment of infectious diseases caused by resistant T.pyogenes and other bacteria.Luteolin,a flavonoid,possesses antibacterial,anti-inflammatory,antiallergic,antitumor and other pharmacological activities.As further studies on the antibacterial activity of luteolin,they show that luteolin has the function of inhibiting bacterial efflux pump and reversing bacterial drug resistance.In this study,the complete sequences of mefA and msrA efflux genes are obtained from PCR amplification in the efflux pump of T.pyogenes,which was previously identified in our group,and the homology of mefA and msrA gene sequences with other strains was analyzed by bioinformatics.Meanwhile the structures of the two proteins,as well as cellular localization,were analyzed.On this basis,according to the predicted protein tertiary structure,the molecular docking between luteolin and exoprotein was simulated by computer aid.The results showed that the homology of mefA gene sequence with NCBI sequence was ranged from 89% to 99%,with amino acid sequence identity from 74% to 97%,MefA protein is a non-secretory hydrophobic stable membrane integrin,and no similar tertiary structure of MefA protein was found in PDB;The homology between msrA gene sequence and NCBI sequence was about 99% to 100%.MsrA protein is a non-secretory hydrophilic stable protein,and a reliable MsrA tertiary structure model is predicted,and luteolin can be bound to MsrA protein molecules through hydrogen bonding force..Broth microdilution method was selected to measure the MIC of six macrolides,including erythromycin,roxithromycin,acetylspiramycin,tylosin,azithromycin and tilmicosin,against T.pyogenes,which were treated or untreated by luteolin,and MIC variation were compared.The optimal conditions for luteolin to reverse the drug resistance of T.pyogenes were found to be 1/2 MIC for 36 h.The results showed that the MIC values of six macrocolides to T.pyogenes carrying mefA and msrA genes decreased in different degrees,meanwhile their drug resistance was reversed.While that MIC of the strain not carrying the efflux gene did not change.It suggested that luteolin could reverse the resistance of T.pyogenes to macrolides by inhibiting the efflux pump.DNAMAN was used to compare and analyze the mefA and msrA gene sequences in T.pyogenes before and after luteolin treatment to detect the effect of luteolin on the sequences of mefA and msrA genes,and the effect of luteolin on mRNA expression of mefA and msrA was detected by fluorescence quantitative PCR.The results showed that luteolin had no effect on the nucleotide sequences of mefA and msrA genes;luteolin can considerably reduce mRNA expression of mefA and msrA efflux gene of T.pyogenes,and the mRNA expression level of mefA and msrA gene of most strains had the same downward trend as the MIC value of antibiotic,and is consistent with the EPI reserpine result.The results suggested that luteolin might influence drug resistance by inhibiting the expression of mefA and msrA efflux genes in T.pyogenes.In this study,macrolide efflux genes mefA and msrA were studied.The relationship between the efflux proteins and drug resistance mechanism of T.pyogenes was analyzed by predicting the structure and function of the efflux proteins encoded by mefA and msrA.Computer aided simulation of docking of luteolin with protein molecules.The reversal effect of luteolin and the molecular mechanism of its anti-drug resistance in T.pyogenes were analysed,which will provide a theoretical foundation for the screening and development of innovative antimicrobial agents in combating drug-resistant bacteria. |
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