Mechanism Of TetR Family Transcription Factor Rv1353c In Energy Metabolism Regulation And Drug Tolerance Of Mycobacterium

Tuberculosis(TB)caused by Mycobacterium tuberculosis(Mtb)infection is a chronic infectious disease,which is still a major public health problem at present.The prevention and treatment of TB has been stagnated or even reversed due to the COVID-19 pandemic in recent years.Currently,first-line anti-tuberculosis drugs include rifampicin,isoniazid,pyrazinamide,streptomycin,and ethambutol.With the use of antibiotics,Mtb can avoid drug killing by changing its metabolism,resulting in drug resistance,making anti-tuberculosis treatment increasingly difficult.The main reason why TB is difficult to eradicate is that Mtb has complex cell wall structure,flexible metabolic pathways,and strong regulatory networks.The TetR family is a class of single component signal transduction systems with a large number and important functions.This family was originally discovered in Escherichia coli,and has a direct regulatory effect on the tet A gene encoding the tetracycline efflux pump,making E.coli resistant to tetracycline.The most common role of TetR family of regulators(TFRs)is to participate in the regulation of drug or toxic efflux proteins.In addition,they are also involved in the regulation of bacterial osmotic balance,cell division,virulence,and pathogenicity.As broad-spectrum transcription factors,TFRs are involved in regulating a variety of metabolism,but also interact with various types of ligand molecules,including amino acids,cofactors,and antibiotics.There are 49 transcription factors in Mtb and 137 transcription factors in M.smegmatis,indicating that the TetR family transcription factors have a very important regulatory role in the normal physiological functions of bacteria.In this study,we conducted experimental research around the transcription factor Rv1353 c of the TetR family of Mtb.First,using the pALACE shuttle plasmid as a vector,we constructed an Rv1353 c overexpression strain Ms＿Rv1353c and an control strain Ms＿Vec in M.smegmatis.Further experiments showed that overexpression of Rv1353 c would cause growth delays in M.smegmatis,but when the expression of Rv1353 c was not induced by an inducer,M.smegmatis could grow normally.This indicates that overexpression of Rv1353 c did affect the growth of Mycobacterium.We first speculated that this was related to cell division,and then detected the expression level of cell division related genes by RT-q PCR.The results showed that ftsB was significantly upregulated in Rv1353 c overexpression strain.However,when we overexpressed ftsB in wild type M.smegmatis,it was found that overexpression of ftsB would cause damage to the growth of M.smegmatis until the end of the stationary phase,ultimately not consistent with the growth of the control strain,and would not change the cell length of M.smegmatis,which is inconsistent with the growth delay pattern caused by overexpression of Rv1353 c.This suggests that Rv1353 c may have other regulatory pathways that affect the growth and division of Mycobacterium.Next,we deeply explored the response of Rv1353 c to different pressures.Through bioinformatics analysis of transcriptome data,we found that Rv1353 c was upregulated under treatment conditions related to electron transfer chains such as succinate,potassium cyanide,and thiolidazine,suggesting that Rv1353 c may participate in the regulation of the electron transfer chain.Using RT-q PCR to detect the expression of electron transfer chain related genes,it was found that overexpression of Rv1353 c downregulates the expression of ndh,nuo G,and upregulates the expression of genes such as sdh A,sdh C,and atp B.At the same time,EMSA experiments have demonstrated that Rv1353 c protein can directly bind to the promoter regions of these genes,indicating that Rv1353 c has a direct regulatory effect on these genes in the electron transfer chain.After detecting ATP and NADH levels and proton-motive force closely related to the electron transport chain,it was found that overexpression of Rv1353 c decreased intracellular NADH levels,but increased ATP content and proton-motive force in M.smegmatis.At the same time,the resistance to rifampicin,vancomycin,bedaquiline,sodium dodecyl sulfate(SDS),and lysozyme was stronger of Rv1353 c overexpression strain than the control strain,while the sensitivity to isoniazid and ethionimide increased.This is related to the decreased lipid synthesis and impaired mycolic acids synthesis caused by overexpression of Rv1353 c..In order to further study the regulatory mechanism of Rv1353 c,we knocked out the homologous gene of Rv1353 c in M.smegmatis MSMEG＿2225,but experimental results show that MSMGE＿2225 deletion did not affect the normal growth of M.smegmatis,nor did it affect the intracellular ATP level and proton-motive force.However,after supplementation with Rv1353 c,it still increases the proton-motive force of M.smegmatis.This indicates that there may be multiple pathways in Mycobacterium that regulate electron transfer,and when one pathway is inhibited,other regulatory pathways have a compensatory effect.In addition,After knock out MSMEG＿2225,M.smegmatis became more sensitive to antibiotics.In summary,our research indicates that Rv1353 c,as a transcription factor,participates in the regulation of Mycobacterium electron transfer chain genes,affecting the level changes of the core elements of intracellular energy metabolism,NADH and ATP,which may mediate changes in Mycobacterium sensitivity to antibiotics.