The Identification Of Mycobacterium Tuberculosis Resistant Gene And Resistance Mechanism

Tuberculosis,caused by Mycobacterium tuberculosis（M.tuberculosis）,an ancient and important infection disease,remains a leading cause of mortality and morbidity worldwide.Nearly one-third of the global population was infected by latent tuberculosis.Multidrug-resistant tuberculosis（MDR-TB）and extensively drug-resistant tuberculosis（XDR-TB）have worsened this scenario and co-infection with HIV,make tuberculosis worse.Compared with other pathogens,M.tuberculosis exist nature resistance to multiple antibiotics because of specific cell wall.The cell wall of mycobacterium is impermeable to most drugs because of lipid-rich layer containing mycolic acids in the inner and long-chain fatty acids in the outer leaflet.Defects of lipid synthesis would damage the function of cell wall and increase the sensitive to various antituberculosis drugs.Antibiotics resistance poses catastrophic threat to global public health.Novel insights into the underlying mechanisms of action will inspire better measures to control drug resistance.At present,it is difficult to developed of new antibiotics.New inhibitors target resistant genes combined with old antibiotics is a better choice.First,we must understand the mechanism of resistance for developing new synergist.Fluoroquinolones are potent and widely prescribed broad-spectrum antibiotics.Bacterial protein degradation pathways represent novel druggable target for the development of new classes of antibiotics.Mycobacteria proteasome accessory factor C（pafC）,a component of bacterial proteasome,is involved in fluoroquinolones resistance.PafC deletion mutants are hypersensitive to fluoroquinolones,including moxifloxacin,norfloxacin,ofloxacin,ciprofloxacin,but not to other antibiotics such as isoniazid,rifampicin,spectinomycin,chloramphenicol,capreomycin.This phenotype can be restored by complementation.The pafC mutant is hypersensitive to H₂O₂ exposure.The iron chelator（bipyridyl）and a hydroxyl radical scavenger（thiourea）can abolish the diff erence.The fnding that pafC is a novel intrinsic selective resistance gene provided new evidence for the bacterial protein degradation pathway as druggable target for the development of new class of antibiotics.Transcriptional factors are essential for bacteria to adapt diverse environmental stresses,especially upon exposure to antibiotics.Mycobacterium tuberculosis,the causative agent of tuberculosis,which inflicts around one third of the global population,contains three IclR family transcriptional factors,namely Rv1719,Rv1773c and Rv2989.In this study,MSMEG₂386,the homolog of Rv2989 in Mycobacterium smegmatis,was deleted by homologous recombination and complemented.The gene did not affect the growth in conventional culture.However,the growth of deletion mutants upon isoniazid（INH）exposure was severely delayed.This growth defect is specific to INH and not observed in other antibiotics tested,including rifampicin,capreomycin,norfloxacin and ethionamide.The transcription of katG and enzymatic activity of its encoding product catalase were elevated in the MSMEG₂386 deleted M.smegmatis mutants.The survival of MSMEG₂386 deletion mutant within U937 macrophages was markedly reduced compared with the wild type strain.These results improve our understanding of the role of IclR family transcriptional factors in INH resistance in mycobacteria.Bacteriophages,as the natural enemies of bacteria,have an especially natural advantage in antimicrobial activity.Mycobacteriophage SWU1 is a newly isolated phage from soil sample collected in Sichuan province,China,which is highly similar to mycobacteriophage L5,but no homolog of SWU1gp39 in the L5 genome.No homolog was found in NCBI using BLAST until mycobacteriophage EagleEye and Serenity were isolated.SWU1gp39 is a novel gene from mycobacteriophage SWU1 with unknown function.To defne the function of SWU1gp39,it was amplifed from SWU1 genome and expressed a His-tagged gp39 protein using a recombinant pALACE plasmid.SWU1gp39 expressed in M.smegmatis conferred the host cell increased susceptibility to multiple antibiotics,including isoniazid,erythromycin,norfloxacin,ampicillin,ciprofloxacin,ofloxacin,rifampicin and vancomycin,and multiple environment stresses such as H₂O₂,heat shock,low pH and SDS.By using EtBr/Nile red uptake assays,WT-pAL-gp39 strain showed higher cell wall permeability than control strain WT-pAL.Moreover,the WT-pAL-gp39 strain produced more reactive oxygen species and reduced NAD+/NADH ratio.RNA-Seq transcriptomes of the WT-pAL-gp39 and WT-pAL revealed that the transcription of 867 genes was diff erentially regulated,including genes associated with lipid metabolism.Taken together,our results implicated that SWU1gp39,a novel gene from mycobacteriophage,disrupted the lipid metabolism of host and increased cell wall permeability,ultimately potentiated the efficacy of multiple antibiotics and stresses against mycobacteria.Antimicrobial killing is generally measured by incubating bacterial cells with antimicrobial at various concentrations and for various times followed by plating serially diluted samples on drug-free agar for colony formation unit（viable count）determination.While this method is widely adopted and considered a gold standard for bacterial survival assays,it would not be able to distinguish bacterial cells are still alive at the end of antimicrobial treatment but become dead on drug-free agar during recovery growth（termed post-stress programmed cell death）from those directly killed by antimicrobial before plating for viable count.The present work used a recently developed method that can specifically detect reactive oxygen species（ROS）-mediated bacterial cell death during post-antimicrobial treatment recovery growth by including ROS scavengers in antimicrobial-free agar to evaluate the dependence on ROS of quinolone-mediated killing.The classical killing assay has revealed two lethal pathways with various types of quinolones:one depends on ROS and ongoing protein synthesis and the other does not.Exponentially growing E.coli BW25113 cells were treated with moxifloxacin in the presence of chloramphenicol.Then the survival were calculated on the various LB plates in the presence of antioxidant,no killing was found.DNA repair is necessary but insufficient to rescue cells died during post-stress PCD because a deficiency in recA or recB suppressed the protective effect of ROS scavenger on PCD.These data indicate that all quinolones kill largely by the ROS-dependent pathway and DNA repair systems are necessary for bacteria survival under the treatment of quinolones.In short,we identified two genes（pafC and Rv2989）incolved in antibiotic resistance and the mechanisms are also discussed.Then we explored the nes antimicrobial strategy from the perspective of bacteriophage.Fially,we discuss the specific action mechanism of fluoroquinolones by new mechods.