GlnR And RegX3-mediated Regulation Of Cholesterol Metabolism In Mycobacterium.smegmatis

Mycobacterium tuberculosis continues to be a huge public health threat.Cholesterol,as a characteristic carbon source of M.tuberculosis,is essential for its survival in its host macrophages.GlnR,as a global regulator of nitrogen metabolism,can regulate the metabolism of carbon and phosphorus while regulating nitrogen metabolism in response to environmental nitrogen source signals.In addition,SenX3/RegX3 as a phosphorus source sensing two-component control system can coordinately regulate carbon source metabolism while regulating phosphorus metabolism.Therefore,in addition to maintaining the normal physiological functions of the cell and providing energy,nitrogen,carbon and phosphorus have a complex and delicate synergistic regulation effect between them.Cholesterol is a key carbon source for M.tuberculosis to survive and maintain virulence in macrophages,and the cholesterol metabolic mechanism in mycobacteria is still not fully understood.Here,based on the exploration and research of our group,GlnR and RegX3 are selected as media to further analyze the cooperative regulation mechanism of carbon,nitrogen,phosphorus in mycobacteria.The detailed research contents are as follows:(1)GlnR responding environmental nitrogen signal regulates cholesterol metabolism in M.smegmatis.In this study,we found a typical GlnR binding motif(CCGAC-AACAGT-GACAC)in the promoter region of kstR via bioinformatic analysis,and then EMSA assay was performed to determine the binding in vitro.Real-Time Quantitative Polymerase Chain Reaction(RT-qPCR)was conducted to analyze the differential expression of genes involved in cholesterol degradation from wild type(WT),glnR-deleted mutant,and glnR supplementary strains in nitrogen-limited condition.ATP test of macrophage demonstrated that the coordinated regulation of GlnR-KstR significantly reduced the invasion of M.smegmatis.The results indicated that many genes involved in the cholesterol catabolism were affected by the regulation of GlnR via KstR responding the environmental nitrogen level,and further influenced the invasion of M.smegmatis.(2)RegX3 regulates cholesterol transport in M.smegmatis in response to phosphorus source signals.First,bioinformatics technology is used to predict and analyze the structure,function,and binding motif of RegX3 protein,and then electrophoretic mobility shift assay(EMSA)initially confirmed that.Then RT-qPCR experiment was conducted to analyze the differential expression of mce4 gene cluster involved in cholesterol transport from wild type(WT),regX3-knock down mutant,and regX3 overexpression strain in phosphorus-limited condition.The results indicated that many genes of mce4 cluster involved in the cholesterol transport were affected by the regulation of RegX3 responding the environmental phosphorus level,and further influenced the growth and survival of M.smegmatis.Through the above research,we have revealed the regulation mechanism of M.smegmatis on cholesterol transport and metabolism in response to environmental nitrogen and phosphorus signals,which provides a new strategy for the survival control of mycobacteria in host cells.