Molecular Mechanism Of The Synergistic Activity Of Ethambutol And Isoniazid Against Mycobacterium Tuberculosis

For more than half a century,isoniazid(INH)and ethambutol(EMB)have been the two major first-line drugs for managing a serious infectious disease-tuberculosis(TB).In particular,the combination of these two drugs has inhibited the spread of tuberculosis effectively.Although co-use of these two drugs is very common in clinical practice,the mechanism for the potential synergistic interplay between them remains unclear.Previous studies found that in the presence of non-lethal dose EMB,the expression level of etbR significantly affected the bactericidal activity of INH against Mycobacterium tuberculosis.Based on this finding,the current study further persued the molecular mechanism of synergistic interplay between two drugs mediated by the transcription factorEtbR,and the following results were obtained:(1)In the presence of a non-lethal dose of EMB,etbR overexpression strain becomes more sensitive to INH and the minimum inhibitory concentration(MIC)of INH for the recombinant M.tuberculosis is significantly reduced.EtbR belongs to the TetR/AcrR family of transcription factors.In this study,the MICs of INH for M.tuberculosis H37Ra wild type strain and etbR overexpression strain were determined by Resazurin Microtiter Assay,the results showed that the MIC of INH for etbR overexpression strain was reduced by one times compared with the wild-type strain;when a non-lethal concentration of EMB was added,the MIC of etbR overexpression strain was reduced by three times if compared with the wild-type strain.(2)EtbR recognizes a conserved motif in the upstream region of both inhA gene and its own,and inhibits gene expression.EMB can directly bind to EtbR and enhance its DNA binding activity.Using ChIP,EMSA and DNase I footprinting experiments,we found that EtbR recognizes a 20 bp conserved motif in the upstream sequence of its own and inhA gene;Real-time quantitative PCR and?-galactosidase activity experiments showed that EtbR inhibits its own and inhA promoters'activities,indicating that EtbR,as a transcriptional suppressor,negatively regulates gene expression in M.bovis BCG.Using isothermal titration calorimetry(ITC)and surface plasmon resonance(SPR)assays,we observed that EMB binds EtbR in a 1:1ratio;EMSA and SPR experiments demonstrated that EMB can specifically stimulates its DNA-binding activity;?-galactosidase activity experiments and Real-time quantitative PCR showed that EMB enhances the transcriptional inhibition of EtbR on inhA.(3)In the presence of a non-lethal doses of EMB,etbR overexpression enhanced the bactericidal ability of INH against M.tuberculosis H37Ra and M.bovis BCG as well as M.smegmatis MC~2155,and etbR-overexpression reduced the viability of mycobacteria in cells treated with EMB+INH.By constructing etbR recombinant strain and detecting the growth curve of the single drug or the combination of the two drugs,the results showed that under the stress of 0.02?g/mL INH,the growth of three etbR overexpression strains were slightly inhibited compared with wildtype strains in M.tuberculosis H37Ra,M.bovis BCG and M.smegmatis MC~2155.However,under the condition of co-stress of EMB(0.2?g/mL)with INH(0.025?g/mL),the growth of three etbR overexpression strains were significantly inhibited.In addition,by determining the intracellular survival rate of the recombinant strain of M.tuberculosis H37Ra under drug stress,we found that the survival rate of the wild type strain decreased slightly under the combined treatment of EMB+INH compared with INH alone(P<0.05);while the etbR overexpression strain infected cells,the number of intracellular survival rate decreased significantly under the same treatment conditions(P<0.01),indicating that etbR overexpression strain reduced the viability of mycobacterium under drug stress.In summary,we present the first evidence that a transcription factor encoded by the Rv0273c gene in mycobacteria,which can inhibit the expression of inhA gene by binding EMB,and enhance the INH sensitivity of mycobacteria under the synergy of EMB.These results reveal that the mycobacterial transcription factor-mediated molecular mechanism for the combination of two first-line anti-TB drugs,INH and EMB,and also enhance our understanding of the mechanism on drug-resistant formation in bacteria,providing theoretical reference for using transcription factors as drug targets and developing new antimicrobial strategy.