Unexpected Role Of Mycobacterium Tuberculosis AdhA In Persistence Via Metabolism Reprogramming

Since bacterium persistence was firstly found in Staphylococcus aureus after penicillin treatment,persisters are increasingly clinical difficult problems.Persisters are transient non-genetic phenotypic tolerant to antibiotics and associated with recurrent and chronic infections.Tuberculosis,caused by Mycobacterium tuberculosis,remains a threatening global public concern largely due to M.tuberculosis capacity to persistence and antimicrobial resistance.As a common phenomenon,M.tuberculosis persistence is prominent largely due to the huge burden of tuberculosis occasioned by M.tuberculosis infection.Now,persisting genes have been intensively studied,such as dnaK,clpB,rpoS,pspF,tnaA,sucB,ssrA,smpB,recA,umuD,uvrA,hipA,hspX,hspR,hsp70,katG,mqsR,relA,relE,dinJ,icl,gcvB,narGHJI,narX,narK2,pcaA,treS,mprA,dosR,tcrY,trcS,kdpDE,sigF,sigH,sigE and whiB3.Most persisting genes play a role in metabolism.However,alcohol dehydrogenase was never reported as persistence factor.Enzymes catalyzing the inter-conversion of ketones,aldehydes and alcohols essential for both prokaryotes and eukaryotes can be divided into three categories:the NAD（P）-dependent alcohol dehydrogenases（ADHs）;NAD（P）-independent alcohol dehydrogenases,which use pyrroloquinoline quinone（PQQ）;FAD-dependent alcohol oxidases,which can catalyze irreversible oxidation of alcohols.Microbial ADHs can be further divided into three major subcategories,their broad-spectrum substrates including aliphatic and aromatic alcohols,as well as corresponding ketones and aldehydes.ADHs facilitate the conversion of alcohol to aldehyde with the reduction of NAD⁺to NADH.Bacteria ADHs studies focused largely on their roles in metabolism,ethanol tolerance,nitrosative stress and virulence.However,their role in M.tuberculosis persistence lacked reports.M.tuberculosis genome encodes at least twenty putative alcohol dehydrogenases（Rv0765c,Rv1530,Rv3086,Rv1862,Rv3726,Rv3045,Rv3548c,Rv2259,Rv1865c,Rv3559c,Rv3085,Rv3549c,Rv0761c,Rv0927c,Rv1144,Rv1928c,Rv1895,Rv0162c,Rv3485c,Rv1941）.Based on their transcription altered by environmental cues such as non-replicating persistence stage（NRP）,pH and starvation,we hypothesize that alcohol dehydrogenase might play a role in persistence.M.tuberculosis alcohol dehydrogenase encoding gene adhA（Rv1862）,putatively involved in M.tuberculosis unique cell wall components biosynthesis,unexpectedly plays a role in persistence.AdhA could promote the persistence of bacteria intracellular and upon exposure to stresses.AdhA homolog MSMEG₃615 deletion M.smegmatis mutant cell wall was significantly changed,produced more ATP,more sensitive to multiple stresses,increased ratio of NAD⁺/NADH and decreased membrane potential.Compensatory increase of transcription of three genes crucial for persistence and involved in distinct metabolic pathways,namely icl,relA,treS,was notable in the deletion mutants.The findings implicate that adhA is a persister gene.This will provide new candidate for antimicrobials against persisters.