The Mechanism Of Drug Tolerance And Compensation For Rifampicin Resistance In Mycobacterium Tuberculosis

Purpose:Mycobacterium tuberculosis（MTB）remains one of the serious infectious pathogens in the world,posing a catastrophic threat to human health.According to the World Health Organization,there were approximately 10 million new tuberculosis cases in 2019,including 1.4 million deaths.At the same time,due to the imperfect treatment plan,poor patient compliance and the spread of drug-resistant strains,the drug resistance of M.tuberculosis has become a major challenge for global tuberculosis control.At present,although the resistance mechanisms of most antibiotics have been studied clearly,while the new "auxiliary" mechanisms of drug resistance were produced in the long-term killing of multiple antibiotics,which allowing bacteria to evade the killing of antibiotics or increase the spread of drugresistant strains.The compensatory mutations and drug tolerance are two important aspects that have gradually attracted attention,but more new mechanisms still need to be revealed.In the treatment of tuberculosis,due to the long-term combination of antibiotics,M.tuberculosis can not only directly develop resistance."Drug tolerance" refers to that bacteria may prolong survival time in antibiotics without changing the MIC,thereby increasing their survival under high concentrations（>MIC）.Studies have shown that since most antibiotics have a highly bactericidal effect against active bacteria,M.tuberculosis can slow down metabolism by regulating multiple interrelated pathways to survival.Studies have shown that drug tolerance could contribute to the development of drug resistance.Therefore,understanding the phenomenon of drug tolerance and its mechanism is important for predicting drug efficacy and designing targeted treatment strategies.Recently,a number of wholegenome sequencing data of clinical isolated MDR/XDR strains or a few sensitive strains have identified point or deletion mutations in the rv2752c gene（encoding M.tuberculosis RNase J）.Existing studies have shown that RNase J has nothing to do with antibiotic resistance.And the rv2752c gene mutation has also been identified in drug-sensitive strains,suggesting that the rv2752c gene mutation may be related to the drug tolerance in M.tuberculosis.RNase J is one of the components of the "RNA degradosome" of M.tuberculosis,it participates in the degradation of mRNA.Studies have shown that bacteria can regulate the expression of genes related to efflux pumps,metabolic enzymes,and transporters through mRNA regulation and metabolic processes to participate in antibiotic stress regulation.However,the phenotype and mechanisms of RNase J mediated drug tolerance in M.tuberculosis remain to be revealed.Since drug targets usually involve important physiological functions of bacteria,the drug-resistant mutations can not only result in the resistance,but also cause growth defects（fitness cost）.Mathematical models have predicted that because of the fitness cost associated with drug-resistant mutations that impair the reproduction or virulence of resistant strains,it is difficult for the bacteria to maintain large transmission networks or concentrated outbreaks.Many studies have proved that drug-resistant M.tuberculosis can compensate or even restore its fitness through compensatory evolution,the corresponding mutation is called compensatory mutations.Studies have shown that the acquisition of compensatory mutations may increase the competitive advantage of bacteria in the population and promoting their spread.Rifampicin（RIF）is one of the most important first-line anti-tuberculosis drugs,it acts as a bactericidal agent by binding to the β subunit of RNA polymerase（RNAP）and preventing the extension of the primary RNA product,which ultimately inhibits transcription.M.tuberculosis gains rifampicin resistance primarily through rpoB（encoding β subunit of RNA polymerase）mutations,and more than 95%of these mutations are present within an 81-bp rifampicin resistance-determining region（RRDR）.A number of studies have shown that rifampicin-resistant mutations can bring about fitness cost.In recent years,studies have also shown that mutations in rpoA or rpoC（encoding α andβ’ subunits of the polymerase）can alleviate the fitness cost of initial rpoB mutations.Further,several genome sequencing-based studies demonstrated that many rifampicin-resistant M.tuberculosis clinical isolates carry multiple rpoB mutations,with nonsynonymous mutations outside the RRDR commonly co-occurring with RRDR mutations and being more likely to appear in strains without rpoA or rpoC mutations,which suggesting that these mutations may compensatory,but the functions and mechanisms remain unclear.In summary,this study aims to explore whether the rpoB non-RRDR mutation in clinically isolated M.tuberculosis is compensation mutations and the mechanism;And to explore whether RNase J mediates drug tolerance and the possible mechanism.Methods:The inactivated hygromycin resistance selection marker（hygS）was introduced into the mycobacterial recombinant engineering vector pJV53 to achieve high-efficiency screening of genomic point mutations.In order to study the drug tolerance and the mechanism mediated by RNase J inactivation,the M.smegmatis was used as a model to construct RNase J inactivated strain;and the RNase J of M.tuberculosis was transformed into the inactivated strain to construct its complementing strain;The effect of RNase J on the growth of the strain was evaluated by the determination of the growth curve and the colony morphology;The MIC of the isoniazid,rifampicin and ofloxacin was determined to evaluate effect of RNase J on the resistance;The effect of RNase J on the tolerance of various antibiotics was evaluated by measuring the antibiotic time-kill（MDK）curve;The potential mechanism of RNase J inactivation-mediated drug tolerance was explored by transcriptome sequencing.In order to study the compensation effect of rpoB non-RRDR mutations in M.tuberculosis,phylogenetic and rpoB mutations analysis was performed on the published genome sequencing data of rifampicin-resistant clinical strains to identify rpoB non-RRDR convergent evolutionary mutations;Using M.smegmatis as an model,through DNA fragment homologous recombination experiments to study whether rpoB non-RRDR mutations cause rifampicin resistance;Using the modified pJV53 system to construct rpoB RRDR single mutant strains and rpoB RRDR,nonRRDR double mutant strains;through MIC assay evaluates the effect of rpoB nonRRDR mutations on the level of rifampicin resistance;Through growth curve and competitive growth test,evaluate the compensation effect of rpoB non-RRDR mutations on the fitness of resistant strain.Measure the transcription efficiency of RNA polymerase of single/double mutant strains by qPCR,evaluate whether nonRRDR mutations can compensate by increasing the transcription efficiency of RNA polymerase;Finally,analyze the protein structure to explore the potential mechanism of rpoB non-RRDR mutations affecting the transcription activity of RNA polymerase.Results:In the drug tolerance research,the MSMEG₂685 gene（encoding RNase J）was inactivated in M.smegmatis,the complemented strain MSmMSMEG₂685PTC::CP and vector complementing strain MSmMSMEG₂685PTC::Vec were constructed successfully;The growth curve and the colony morphology showed that the growth rate of MSm-MSMEG₂685PTC decreased significantly and the colony phenotype changed under the condition of insufficient carbon source,which proved that the inactivation of RNase J affects the growth of M smegmatis;The MIC ofMSm-MSMEG₂685PTC,MSm-MSMEG₂685PTC::CP,MSm-MSMEG₂685PTC::Vec and wild-type to isoniazid,rifampin and ofloxacin were no significant difference,which proved that RNase J has no effect on drug resistance;The drug time-kill curve results proved that RNase J inactivated strains are tolerance to the antibiotics under the insufficient carbon source;The transcriptome analysis showed that RNase J inactivation changes the expression of genes related to pathways such as carbon source metabolism and energy metabolism.In the rifampicin compensation study,the isolates that carry different rpoB mutations outside the RRDR were identified through mutation analyses from genome sequencing data of M.tuberculosis isolates worldwide.By mapping the mutations onto the ML phylogeny,we identified 33 convergent mutations that independently emerged at least twice;The screening of rifampicin resistance was proved that except for the V170F and 1491V mutations,the other mutations did not cause rifampicin resistance;The rpoB S450L mutant strain and S450L+P45L,S450L+P45S,S450L+I480V,S450L+V534M,S450L+R827L strains were constructed;MIC results proved that rpoB non-RRDR has no effect on the resistance level of the initial resistance mutation S450L;Growth curve and in vitro competitive growth showed that the S450L mutation causes the fitness cost of M.smegmatis and the double mutant strain has a significant growth advantage to the S450L strain（0.97-1.08 vs 0.87）,which proveed that rpoB non-RRDR can compensate for the growth defect caused by the S450L resistance mutation;The transcription efficiency measurement results showed that the rpoB non-RRDR mutations can increase the transcription efficiency of RNA polymerase;Protein structure analysis showed that rpoB non-RRDR mutations possible increase the transcription efficiency of RNA polymerase by modifying the active center of RpoB or changing the interaction between subunits.Conclusion:By optimizing the mycobacterial recombinant engineering plasmid pJV53,an efficient screening method for mycobacterial "no drug-resistance phenotype" point mutations was successfully constructed,and this method was used to construct MSMEG₂685 gene-inactivated M.smegmatis and rpoB single or double mutant strains;Using M.smegmatis as a model,we proved that RNase J inactivated can mediate drug tolerance,and the possible mechanism is to reduce the rate of carbon source metabolism;Using M.smegmatis as a model,we proved that 21 rpoB non-RRDR mutations are compensatory mutations,and revealed the underlying mechanisms.