The Mechanisms Underlying Mycobacterium Tuberculosis Rv1634 Mediated Fluoroquinolones Resistance

Mycobacterium tuberculosis(Mtb)is pathogen of tuberculosis(TB)and one of the most successful intracellular pathogens.With the emergence and spread of multidrugresistant and Extensive-resistant Mycobacterium tuberculosis(MDR-TB,XDR-TB),the treatment of tuberculosis is facing great challenges.Therefore,the research on the mechanism of bacillus resistance is particularly urgent.Increased efflux capacity,protection of antibiotic targets and mutations,degradation of drugs and decreased cell wall permeability are involved in M.tuberculosis resistance.Among them,the way to reduce intracellular drug content by efflux drugs is mainly mediated by efflux pumps.According to the structure of the efflux pump,the transport substrate and the energy source,the efflux pump can be divided into five families: ATP binding cassette(ABC),resistance nodulation cell division(RND),multidrug and toxic compound extrusion family(MATE),major facilitator superfamily(MFS),and small multidrug resistance family(SMR).Among them,the MFS family belongs to the secondary transporter family,which contains 74 members.It is an ancient and ubiquitous transport protein family that can rely on proton dynamics to drive substrate transport.In addition,MFS family proteins have a wide range of transport substrates,including ions,sugars,amino acids,peptides,drugs,and other small molecules.In this study,the MFS family transporter Rv1634 was used as a research object to explore its role in drug tolerance.By bioinformatics prediction of Rv1634 revealed that it has the structural characteristics of a typical MFS family of proteins.In order to further investigate the function of Rv1634,we successfully constructed the overexpressing strain Ms_Rv1634 and the control strain Ms_pALACE using molecular cloning technology with M.smegmatis as a model strain.Through the determination of MIC,antibiotic stress and intracellular ethidium bromide accumulation,it was found that overexpression of Rv1634 can significantly enhance the tolerance of fluoroquinolone to M.smegmatis and reduce intracellular ethidium bromide accumulation.In addition,under the treatment of the efflux pump inhibitors-carbonyl cyanide,reserpine and verapamil,the drug tolerance of Ms_Rv1634 was weakened,and the accumulation of intracellular ethidium bromide(EtBr)also appeared a certain degree of recovery under the treatment of carbonyl cyanide.The above results indicate that Rv1634 encode a functional efflux pump protein,which is consistent with the predicted results.In order to explore the mechanism of fluoroquinolone resistance regulated by the Rv1634 in M.smegmatis,we have utilized the quantitative PCR and β-galactosidase experiments confirmed that the transcription level and promoter activity of MSMEG3815,the homologous gene of Rv1634,were both elevated under the antibiotics stress.Besides,we determined the changes of intracellular reactive oxygen species and membrane potential of Ms_Rv1634 and Ms_pALACE under antibiotic pressure.It was found that the intracellular reactive oxygen species of Ms_Rv1634 was significantly lower than that of Ms_pALACE,and the membrane potential was stable.Hydrogen peroxide stress experiments proved Rv1634 Overexpression does not affect the sensitivity of M.smegmatis to hydrogen peroxide.This result demonstrates that Rv1634 cannot modulate the tolerance of bacteria to fluoroquinolones by means of substances such as hydroxyl radicals.The results of biofilm experiments show that overexpression of Rv1634 can lead to an increase in biofilm folds.To sum up,we speculate that Rv1634 may reduce the intracellular antibiotic content of bacteria and reduce the production of reactive oxygen species by means of drug efflux and weakening the permeability of bacterial cell wall.Thereby maintain the intracellular homeostasis and enhance tolerance of bacterial to drugs.This paper revealed that the M.tuberculosis Rv1634 were involved in the tolerance of fluoroquinolones and the potential mechanism.The elementary results may prove novel ideas for dealing the drug resistance of Mycobacterium tuberculosis.