L-Alanine Specifically Potentiates Fluoroquinolone Efficacy Against Mycobacterium Persisters Via Increased Intracellular Reactive Oxygen Species

Tuberculosis(TB)caused by Mycobacterium tuberculosis(Mtb)is still a major chronic infectious disease of global concern,which is related to drug resistance and persistention after infection.Persisters are phenotypic variations in bacterial populations.Persisters can survive under starvation,which is the main model to study the persistence of M.tuberculosis in vitro.Compounds that revive persisters can improve the germicidal efficacy of antibiotics.Amino acids are the key to the growth of bacteria and play an important role in the antibiotic tolerance of persistence,and have been deeply studied as adjuvants of antibiotics in order to better eradicate persistent infection.In this study,Lrp/AsnC family transcriptional regulator Rv2779c(alanine dehydrogenase transcription factor aldR)was used as the research object to explore its role in the persistence of M.tuberculosis,and L-alanine was used as an adjuvant to explore the effect of exogenous alanine on the killing effect of antibiotics on M.tuberculosis.Through bioinformatics comparison,it was found that Rv2779c has the structural characteristics of typical Lrp/AsnC family transcriptional regulators and has high similarity with M.smegmatis gene MSMEG2660.In order to further explore the functional characteristics of Rv2779c,we used the technology of homologous recombination to knock out the homologous gene MSMEG2660 of Rv2779c in M.smegmatis.Through electrophoretic mobility shift assay,we found that MSMEG2660could inhibit the expression of its target gene MSMEG2659(alanine dehydrogenase),but this regulation changed in the presence of L-alanine.In the presence of L-alanine,MSMEG2660 is beneficial to the expression of its target gene MSMEG2659,and we also verified this regulation by qRT-PCR analysis.After knockout of MSMEG2660 gene,there was no difference in the growth of wild-type and knockout strains,but when we cultured the strains with L-alanine as the only nitrogen source,it was found that the knockout strains could not effectively use L-alanine as the nitrogen source for growth.In order to explore the effect of L-alanine on MSMEG2660 and the role of both in the formation of Mycobacterium persister,we used different kinds of antibiotics to sterilize wild-type and knockout strains.L-alanine could enhance the killing effect of fluoroquinolones against M.smegmatis in a dose-dependent manner,and the auxiliary effect was the best when the concentration of alanine was 5mM.At the same time,we also found that the killing effect of exogenous alanine on fluoroquinolones was more obvious for knockout strains.But when we combined L-alanine with rifampin/isoniazid and other antibiotics,we didn't find this effect.We also found this effect in the germicidal experiment in which hunger was used as the persistence model.At the same time,we also found that knockout strains were more likely to recover from the persistence state,this may be the reason why knockout strains are more likely to be killed by the combination of L-Alanine and fluoroquinolones.In addition,we also detected the ATP content,proton-motive force and NAD~+/NADH ratio related to the tricarboxylic acid cycle,and found that the addition of L-alanine promoted the tricarboxylic acid cycle.The results of transcriptome data analysis showed that L-alanine may promote the whole central metabolism of Mycobacterium,while the increase of reactive oxygen species was the ultimate cause of mycobacterium death.To sum up,we found for the first time that L-alanine can promote the production of intracellular active oxygen and promote the killing of fluoroquinolones against mycobacteria by promoting tricarboxylic acid cycle.Knockout of MSMEG2660 is beneficial to the resuscitation of mycobacteria.This will provide new targets and approaches for the elimination of mycobacteria and better ideas and methods for the treatment of tuberculosis.