Mechanism Of ArsR Family Transcription Factor Rv2640c Regulating Resistance To Copper And Isoniazid In Mycobacterium

Tuberculosis(TB),as an ancient disease,has always threatened human health.One of the reasons for its long-term existence is that its pathogenic bacterium,Mycobacterium tuberculosis(Mtb),is constantly evolving and can adapt to various environmental pressures,including the toxicity of metal ions in host macrophages.In order to maintain the homeostasis of metal ions in vivo,Mtb has evolved a coping mechanism,in which metal sensing proteins play a major regulatory role.Exploring the regulatory patterns of metal sensing proteins will help to understand the interaction between mycobacteria,metal ions and hosts.The ArsR(arsenical resistance operon repressor)family of transcription factors can relieve the inhibition of target genes by binding metal ions,thereby regulating metal ion homeostasis.This study investigated the role of the ArsR family transcription factor Rv2640c in maintaining the homeostasis of metal ions in bacteria and responding to antibiotic stress.Through bioinformatics analysis of Rv2640c,it was found that it has a highly conserved helix-turn-helix(HTH)DNA binding domain,and has a high similarity to M.smegmatis MSMEG＿1175.To explore the regulatory mechanism of Rv2640c,we first heterologously expressed and purified the Rv2640c protein in Escherichia coli.The protein structure detection experiment showed that Rv2640c mainly exists in the form of dimers in vitro,which is consistent with the modeling results.Electrophoretic mobility shift assay(EMSA)verification found that Rv2640c can bind to the target gene Rv2641promoter sequence,but when high concentrations of copper ions exist,Rv2640c loses its binding to the target gene promoter.To explore the mechanism behind this phenomenon,we treated the Rv2640c protein and the target gene Rv2641 with copper sulfate,respectively.The results showed that the Rv2640c protein was able to bind to copper,and at the same time,the activity of the Rv2641 promoter was further enhanced after copper sulfate treatment,suggesting that the target gene may play an important role under copper sulfate pressure.Point mutation experiments found that R44,S76,and H78 are the three key amino acid sites for binding DNA to Rv2640c protein.In order to further explore the regulatory mechanism of Rv2640c,we used nonpathogenic Mycolicibacterium smegmatis mc~2 155 as a model strain to knock out the homologous gene MSMEG＿1175 of Rv2640c,and electrically transferred the recombinant plasmid Rv2640c-p ALACE into the knockout strain as a complementary strain.Through RT-q PCR experiments,it was found that MSMEG＿1175 negatively regulates the transcription of the Rv2641 homologous gene MSMEG＿1174.At the same time,by comparing the genetic locus of Rv2640c and MSMEG＿1175 in Mtb and M.smegmatis,it was found that the upstream gene of these two genes had a similar arrangement pattern in the two bacteria,and corresponded to homology in sequence.Furthermore,it was further verified that Rv2641,Rv2642,and Rv2643 were co-transcribed.Combined with RT-q PCR results,Rv2640c negatively regulates the transcription of Rv2641-Rv2642-Rv2643 gene clusters.By treating wild type strains,knockout strains,and complementary strains with copper sulfate,we found that knockout strains exhibited copper sulfate resistance.Further testing of the content of reactive oxygen species(ROS)in the three strains treated with copper sulfate showed that the content of ROS in the knockout strains was significantly lower than that in the wild type strains,indicating that the knockout strains could significantly reduce the ROS content produced by copper sulfate in vivo.We also found that knockout strains respond to isoniazid pressure by changing the expression of Kat G and intracellular NAD~+content.In summary,we found for the first time that Rv2640c can not only regulate the copper resistance of Mycobacterium,but also play a role in isoniazid resistance.It is worth noting that the knockout strain produces resistance to copper and isoniazid simultaneously.This will provide new ideas for exploring the interaction between metal ions and antibiotics,as well as between Mycobacterium tuberculosis and its host.