Function Characterization Of Mycobacterium Tuberculosis Lrp/AsnC Family Transcriptional Factor Rv3050c

Tuberculosis(TB)is a fatal infectious disease caused by Mycobacterium tuberculosis(Mtb).At present,TB is still a public health problem closely related to human health.According to the World Health Organization(WHO),the whole world has 10 million people suffering from TB in 2019.With the emergence of multidrug-resistant tuberculosis(MDR-TB)and extremely drug-resistant tuberculosis(XDR-TB),the co-infection of Mtb and human immunodeficiency virus(HIV)and the unstable efficacy of bacillus calmette-guerin(BCG)make the treatment of TB complicated.Streptomycin is an aminoglycoside antibiotic,mainly used in the treatment of TB.With the clinical use of streptomycin,the drug resistance of streptomycin has gradually emerged.The 16S r RNA(rrs)and ribosomal protein S12(rps L)mutations in Mtb lead to high levels of streptomycin resistance,while gid B mutation leads to low level of streptomycin resistance.The hydroxyl or amino groups of streptomycin are catalysed by the enzymatic reaction of aminoglycoside modifying enzyme,resulting in the loss of bactericidal effect of streptomycin.It is worth noting that the uptake of streptomycin by Mtb needs to consume the proton-motive force(PMF)generated by the electron-transport chain(ETC).When the PMF is insufficient,streptomycin will be blocked out of the cell,and it is difficult to combine with the drug target,resulting in streptomycin resistance phenotype of Mtb.The Lrp/AsnC family of Mtb are global(Lrp)/specific(AsnC)transcriptional regulators,whose transcription regulatory factors can regulate cell metabolism globally or specifically,such as amino acid metabolism,central metabolism,drug resistance and persistence.In this study,the transcription factor Rv3050c of Lrp/AsnC family of Mtb was used to explore the transcriptional regulation and antibiotic resistance mechanism.Bioinformatics analysis showed that Rv3050c was highly conserved in mycobacterium.In order to further study the function of Rv3050c,we used Mycolicibacterium smegmatis as the model strain,and constructed the overexpression strain Ms＿Rv3050c and control strain Ms＿Vec by molecular cloning techniques.Through antibiotic stress and q RT-PCR experiments,we found that the overexpression strain Ms＿Rv3050c could significantly enhance the tolerance of M.smegmatis to streptomycin.To further explore the mechanism of streptomycin tolerance mediated by Rv3050c,we knocked out the Rv3050c homologous gene Ms2309 in M.smegmatis and obtained the knockout strain(ΔMs＿2309),which was supplemented by the recombinant plasmid p ALACE-Rv3050c.We found thatΔMs＿2309 was more sensitive to streptomycin and the complementation strain could restore phenotype.Moreover,the uptake mechanism of streptomycin involves energy consumption,so we measured the PMF and NAD~+/NADH ratio ofΔMs＿2309 under antibiotic stress,and found that the PMF and intracellular NADH content ofΔMs＿2309were higher than wild-type strain.Mtb has a complex transcriptional regulatory network,we predicted that the target genes regulated by Rv3050c might be Rv0886 and Rv3049c.The results of electrophoretic mobility shift assay(EMSA)and promoter activity assay showed that Rv3050c could regulate Rv0886 and Rv3049c,and the promoter activity of Rv3049c was stronger than Rv0886.Finally,we found that Rv3050c was a positive regulator of Rv3049c,and specifically regulated the 23 bp upstream fragment of Rv3049c promoter.All in all,we revealed the regulatory mechanism of the transcription factor Rv3050c and its streptomycin resistance mechanism,which provided a new reference for the treatment of drug-resistant TB.