Molecular Mechanisms Of Drug-resistance Regulations Triggered By Two TetR Family Transcription Factors In Mycobacterium Smegmatis

Tuberculosis is an important human infectious disease caused by Mycobacterium tuberculosis,and the problem of drug resistance has caused widespread concern in the world.However,pathogenic M.tuberculosis grows extremely slow and is high infectious.The regulatory mechanism of its drug resistance remains largely unclear.In contrast,Mycobacterium smegmatis grows fast,the genetic manipulation is relatively mature.In particular,there are lots of highly homologous genes encoded by the genomes of both M.smegmatis and M.tuberculosis.Therefore,M.smegmatis has become an important model organism for studying the gene regulation and physiological metabolism of pathogenic mycobacteria.In this study,using M.smegmatis as a model,two novel TetR-family transcription regulators were found to positively modulate the mycobacterial resistance to Rifampin(RIF)and Ethambutol(EMB),respectively.The detailed results are as follows:(1)Ms4022,a function unknown transcription regulator in M.smegmatis,consists of 199 amino acids and the N-terminal HTH domain of TetR family.By constructing ms4022 knockout and overexpressing recombinantion strains and determining the minimal inhibitory concentration(MIC)to rifampicin,found that the MIC of ms4022 knockout strain(3.13 μg / ml)was two-fold lower than that of wild type strain(6.25 μl / ml),while the MIC of ms4022 overexpressing strain(25 μg / ml)was about four fold higher than that of wild-type strain.Therefore,the expression level of ms4022 observably affected the resistance of M.smegmatis to rifampicin.Further,EMSA and DNA footprint assays revealed two binding sites on Ms4022 promoter region.The first binding site named motif 1 containing 19 bp near the translation initiation site.The second binding site contained two motifs which were named motif 2 and motif 3.They were far from the translation initiation site.Sequence alignment analysis showed that all three motifs had high homology and obvious conservation at the 5’ end 9 bp.315 potential target genes were found to be broadly regulated by Ms4022 through using these three motifs to search the promoter region of M.smegmatis.Interestingly,these potential target genes included 31 transport-related genes(9.84%).Subsequently,EMSA and ChIP assays confirmed that Ms4022 specifically bound to the target promoter sequence of these transport-related genes in vitro and in vivo.Then,qRT-PCR analysis showed that the expressions of most transport-related genes were significantly down-regulated in ms4022 knockout strain compared to the wild type.On the contrary,the expressions level of these genes in ms4022 overexpressing strain were obviously up-regulated.Furthermore,β-galactosidase activity experiments clearly demonstrated that Ms4022 positively regulated the expressions of these genes.Finally,by constructing the overexpressing strains of these target genes and measuring the growth curves,we found that the overexpression of seven target genes could enhance the M.smegmatis resistance to rifampicin.In summary,Ms4022 is a broad transcriptional activator which could enhances M.smegmatis rifampicin resistance by positively regulating the expression of target genes associated transport.(2)LerR is another transcription factor in M.smegmatis,which contains a LuxR domain and belongs to the TetR family.LerR and kinase LerS form a two-component system that regulats M.smegmatis resistance to anti-tuberculosis drug ethambutol.By constructing lerR overexpressing strain and measuring the growth curve under different anti-tuberculosis drugs showed that the ethambutol tolerance of overexpressing strain was significantly higher than that of wild type strain.Subsequently,EMSA and Ch IP experiments confirmed that LerR was able to specifically bind the promoter sequence of ms6242 and ms6239(1,3-propanediol dehydrogenase,PDH)in vitro and in vivo.Further qRT-PCR analysis showed that the expressions level of PDH and operon ms6242,ms6241,ms6240 were significantly decreased in the lerR knockout strain compared with the wild-type strain.In contrast,the expressions level of these target genes were increase in the lerR overexpressing strain.Transcriptome analysis found that the expression level of lerR and its target gene PDH,ms6242,ms6241,ms6240 increased 2-4.5 fold in M.smegmatis treated with ethambutol compared with no drug treatment.This result indicated that these genes could respond to ethambutol stimulation.Finally,by constructing the overexpressing strains of the LerR target genes and measuring the growth curves found that only overexpressing PDH could increase the resistance to ethambutol in M.smegmatis.Therefore,LerR is also a positive regulator which can regulate the resistance of M.smegmatis to ethambutol by activating the expression of multiple target genes including PDH.