Identification And Characterization Of Mycobacterium Tuberculosis Rv1473

Bacterial drug-resistance has become a global problem in the clinical treatment,especially the emergence of multidrug resistance,some known antibiotics have become useless to cure diseases.Several antibiotic resistance mechanisms were reported in bacteria.Changes in antibiotic targets by mutation and drug efflux pumps were two import approaches.Efflux pump could efflux antibiotics out of the cells of bacteria and decrease the intracellular concentrations of antibiotics.Beside,their activities may potentiate the acquisition of chromosomal mutations that offer higher levels of resistance.Therefore,further investigation of efflux pump may lead to the development of countermeasures against bacterial multidrug resistance.Tuberculosis（TB）,caused by Mycobacterium tuberculosis（Mtb）,is still a major public problem especially in poor countries,accounting for 10.4 million new cases and1.4 million deaths in 2015.In the absence of a more effective vaccine,chemotherapy is one of the main anti-tuberculosis tools.But the emergence of multidrug-resistant TB（MDR-TB）,extensively drug-resistant TB（XDR-TB）and the co-infection of TB and HIV have hampered the effective TB treatment and control.Efflux pump,also known as transporter,adopts a diversity of structures in bacteria and can be classified into five different families.There are the major facilitator superfamily（MFS）,small multidrug resistance（SMR）,ATP binding cassette（ABC）transporters,resistance nodulation division（RND）and the multidrug and toxic compound extrusion（MATE）family.ABC transporters require the energy of ATP hydrolysis for active efflux of drug.In contrast,the MFS,SMR,RND,and MATE-type efflux pumps require an energized membrane and the proton motive force（PMF）for activity.Several efflux pumps have been reported that related to drug resistance inM.tuberculosis,and they were found involved in efflux out a variety of drugs,such as aminoglycosides,fluoroquinolones,tetracycline,isoniazid,rifampin,doxorubicin.Rv1473,an incomplete ABC transporter,predicted as a macrolides efflux pump,was characterized in this study.In this study,the protein encoded by Rv1473 presents typical motifs of NBDs by bioinformatics analyses,and might be involved in ATP binding and hydrolysis.To measure whether Rv1473 has ATPase activity,Rv1473 was heterologous expressed in E.coli BL21,and purified by Ni-NTA column.Then,the ATPase activity was determined by PMA-Malachite green spectrophotometry.The results showed that Rv1473 has a relative higher enzyme activity.The non-pathogenic,fast-growing M.smegmatis,a widely used facile surrogate model for pathogenic mycobacteria,was adopted in this study.To further analyze the physiological role of Rv1473,its M.smegmatis homolog MSMEG₃140was deleted（△MSMEG₃140）and complementarystrains（△MSMEG₃140::Rv1473）was constructed.The △MSMEG₃140 strain wasfound more sensitive to macrolide and accumulated more EB.The complementary strains reverts the growth defect of △MSMEG₃140 in the presence of macrolides.RT-PCR results showed the expression of MEMEG₃140 was upregulated when exposure on macrolides.The expression of Rv1473 in M.tuberculosis was also increased in the presence of roxithromycin,according to previous reports.The promoter activity of Rv1473 was also enhanced when subjected to macrolides.Our results suggested that the ABC transporter Rv1473 and its homolog gene in M.smegmatis MSMEG₃140,playsan important role in mycobacterial macrolides efflux.Biofilm formation was not affected by disruption of MSMEG₃140.The co-transcription of Rv1473 with CtpD（Rv1469）,two thioredixin genes trxA（Rv1470）,trxB（Rv1471）and enoyl-CoA hydratase（Rv1472）implicated a synergism with CtpD to efflux macrolides.This is the first report on the function of Mycobacterium tuberculosis Rv1473.It is important to seek efflux pump inhibitors to relieve the bacterial drug resistance.