Action Mechanism Of Berberine To Inhibit Mycobacteria And Screening Of M.Tuberculosis MurA Inhibitors

Tuberculosis(TB),an infectious disease caused by Mycobacterium tuberculosis,remains a global public health burden around the world.With the emergence of HIV-TB co-infection cases and multiple drug resistant tuberculosis(MDR-TB)cases,the existing anti-tuberculosis drugs have not been able to meet the requirements of TB treatment and the spread control.Therefore,developing new anti-tuberculosis drugs or exploring the anti-tuberculosis function of traditional drugs has been an urgent task.Currently,there were two main distinct methodologies in the search for new TB drugs:phenotypic screening and target based screens.M.smegmatis is a non-pathogenic and fast growing species which was homologous with M.tuberculosis.Furthermore,M.smegmatis had the same cell wall structure as M.tuberculosis.We had found that berberine had inhibitory activity against M.smegmatis.Berberine is a natural isoquinoline-type alkaloid which isolated from many kinds of medicinal plants including Menispermaceae,Ranunculaceae,Berberidaceae,etc.Berberine was reported to possess a variety of new potential pharmacological functions such as anti-microbial,anti-inflammatory,anti-cancer activities,etc.Studying the inhibitory mechanism of berberine to M.smegmatis will help us to develop new anti-tuberculosis drugs.M.tuberculosis has a unique cell wall structure which plays essential roles in controlling bacterial growth and its peptidoglycan is one of core structure.The peptidoglycan is a reticular molecule that is composed of N-acetylmuramate and N-acetylglucosamine as well as oligo peptides.MurA,UDP-N-acetylglucosamine enolpyruvyle transferase catalyzes the first reaction for the formation of UDP-N-acetylmuramate.M.tuberculosis murA was proved as an essential gene for the growth of cells.Furthermore,the metabolic pathway of UDP-N-acetylmuramate does not exist in mammalian cells.Therefore,in this study we used MurA as a target to screen anti-tuberculosis drugs.The objectives of this study:1.To utilize M.smegmatis as a mycobacterial model strain to screen mycobacterial inhibitors and investigate the effect of berberine on the growth,morphology,structure,proteins,drug-sensitivity and biofilm of mycobacteria.2.To establish a screening system for screening specific inhibitors to M.tuberculosis MurA enzyme.The results of this study:1.The action mechanism of berberine to inhibit mycobacteria was investigated.The growth curve and CFU affected by different concentrations of berberine indicated that berberine could inhibit M.smegmatis.After M.smegmatis was treated with different concentrations of berberine for 24 h,SEM results showed that M.smegmatis had a wrinkled cellular surface and an obvious enlarged shape.TEM results showed that M.smegmatis had a lower density of cell contents.These indicated that berberine was able to affect bacterial morphology and structure.The protein analysis results showed that the expression of a DNA topoisomerase Ⅱ(DNA gyrase)subunit B and a TetR family transcriptional regulator was up-regulated and a DNA-binding protein was obviously down-regulated.Berberine exhibited not only an inhibitory effect on the formation of M.smegmatis biofilm,but also had a capacity to reduce the 5 days-developed biofilm.In addition,M.smegmatis treated with berberine was more sensitive against INH,RMP and STR.2.Screening system for M.tuberculosis MurA inhibitors was established and specific inhibitors for M.tuberculosis MurA enzyme were found.The E.coli MurA was expressed,purified and characterized.MurA enzymes from Streptococcus mutans,E.coli,M.smegmatis and M.tuberculosis had different properties.Screening system for M tuberculosis MurA inhibitors was established and some specific inhibitors for M.tuberculosis MurA enzyme were found.Conclusions:1.Berberine was proved in this study as a potential medicine for the treatment of tuberculosis.2.Three compounds(#0025 and#0030)from the secondary metabolite library of marine actinomycetes were able to inhibit M.tuberculosis MurA enzyme activity specifically.Further studies:1.To optimize the approach in proteomic analysis to figure out the berberine action pathway.2.To expand the numbers and varieties of natural compound libraries to screen more MurA inhibitors and determine their types of inhibition.3.To clarify the mechanism of MurA inhibitors through determining the crystal structure of MurA-inhibitor complex or homology modeling.4.To establish virtual computer screening method for M tuberculosis MurA inhibitors to facilitate the discovery of M.tuberculosis MurA inhibitors.