Mechanisms Of Short Chain Dehydrogenase MSMEG＿5885 Regulates Bedaquiline Resistance Of Mycobacterium

Tuberculosis(TB)is an infectious disease caused by Mycobacterium tuberculosis(MTB)infection.In 2022,the number of newly diagnosed tuberculosis in the world exceed 10 million,and the number of deaths due to tuberculosis was about 1.6 million,with a mortality rate of 15.1%.About one quarter of the people in the world are MTB carriers,and 5-10% of them will develop into active tuberculosis.People with impaired immune system have a higher incidence rate to suffer this desease.The widespread MTB makes it difficult to end tuberculosis globally.In addition,the emergence of drug-resistant tuberculosis has increased the difficulty of overall cure of tuberculosis.Multidrug resistant tuberculosis(MDR-TB)refers to tuberculosis that is resistant to over two firstline anti tuberculosis drugs at the same time.The clinical treatment of MDR-TB is extremely challenging due to its long treatment time,poor treatment effect,and high cost.Moreover,MTB produces specific point mutation to resist environmental pressures such as antibiotics and rapidly evolves.With the accumulation of mutations,the situation of drug resistance becomes more complex,and there are fewer alternative anti-tuberculosis drugs and drug combination schemes available.Therefore,it is urgent to find more novel and effective treatment plans.Bedaquiline(BDQ)is a diarylquinoline compound that can achieve bactericidal effects by inhibiting ATP synthase.In 2012,the US Food and Drug Administration(FDA)approved BDQ for the treatment of MDR-TB and extensively drug-resistant tuberculosis(XDR-TB).Although BDQ is a new type of anti-tuberculosis drug that has not been clinically used for a long time,there have been strains that are resistant to the drug.This study screened the transposon mutation library of Mycobacterium smegmatis(M.smegmatis)and obtained strains with BDQ tolerant phenotype.The transposon insertion gene MSMEG＿5885 was identified by Thermal Asymmetric Interlaced PCR(TAIL-PCR).MSMEG＿5885 is annotated as "encoding a possible short chain dehydrogenase",with a high similarity of 65.82% homologous gene Rv3485 c base sequence in MTB.Bioinformatics analysis shows that the purification selection coefficient of Rv3485 c in the evolutionary process of MTB is 0.07(a coefficient less than 0.1 indicates evolutionary conservatism),indicating that it is crucial for the survival of MTB.In addition,transcriptome data showed that transcriptional level of Rv3485 c increased after BDQ treatment,suggesting that this gene may play an important role in responding to BDQ stress.To delve deeper into the relation of MSMEG＿5885 to BDQ,we constructed the gene deletion strain ΔMSMEG＿5885,and it was found that this strain increased its tolerance to BDQ.Based on the bactericidal mechanism of BDQ,we investigated the indicators related to ATP synthesis of ΔMSMEG＿5885,and found that,compared to the wild-type,The transcriptional level of ATP synthase subunit in ΔMSMEG＿5885 increased,and the intracellular ATP content increased with the enhance of trans membrane potential.These results indicate that knocking out MSMEG＿5885 is beneficial for Mycobacterium to increase intracellular ATP content,which is consistent with the bactericidal mechanism of BDQ.In addition,we also observed that the single colony morphology ofΔMSMEG＿5885 changed,which leads us to speculate that knocking out MSMEG＿5885 may affect the cell wall function of Mycobacterium.Experimental proof,MSMEG＿5885 deletion leads to an increasement of the lipid composition in bacterial cell walls,and a reduce the cell wall permeability.To find genes affected with MSMEG＿5885,we utilized the phage phi Myco Mar T7 once again to establish double mutated gene library based on strain ΔMSMEG＿5885.After screening by BDQ,four transposon insertion mutant strains were obtained,named A5,A7,A12,and G6,respectively.Blast analysis revealed that these mutated genes are associated with fatty acid metabolism in Mycobacterium.By analyzing the genes related to β Oxidation of fatty acids MSMEG＿2227,MSMEG＿4874 and MSMEG＿4876,we detected that the transcription level of those genes in ΔMSMEG＿5885 was significantly upregulated.This indicates that the absence of MSMEG＿5885 affects the fatty acid metabolism in Mycobacterium,resulting in cell wall thickening and hindering the entry of BDQ,so that the strain exhibits BDQ tolerance.We further found that drugs related to energy metabolism,such as ATP synthase inhibitors dicyclohexyl carbodiimide(DCC)or INH,can alleviate BDQ tolerance caused by MSMEG＿5885 mutation.However,treated with streptomycin(SM),which targeted bacterial protein synthesis alone,the drug resistance phenotype of ΔMSMEG＿5885 still exists.It is worth noting that combining BDQ with DCS can inhibits cell wall synthesis of Mycobacterium,which can greatly enhance the bactericidal ability of BDQ.The results above indicate that cell wall thickening can cause widespread resistance of Mycobacterium,but it is not the only reason why ΔMSMEG＿5885 is resistant to BDQ.The main reason for tolerance of ΔMSMEG＿5885 to BDQ is the increase of intracellular ATP levels caused by MSMEG＿5885 deletion in Mycobacterium.In summary,MSMEG＿5885 mutation can lead to multiple effects,thereby enhancing bacterial resistance to BDQ.The impact of this gene on bacterial energy metabolism and cell wall synthesis indicate that the factors leading to BDQ resistance in clinical are also diverse.Therefore,only using a single drug to treat drug-resistant tuberculosis caused by cell wall thickening and enhanced bacterial metabolism may not be well.In response to this complex drug resistance situation,combining conventional drugs with new drugs can solve this problem better.This study provides new research ideas for solving the BDQ resistance and guiding clinical medication.