| Mycobacterium tuberculosis（MTB）is the pathogen that causes tuberculosis.Tuberculosis is still one of the important diseases that endangers human health worldwide.According to the report from the World Health Organization,there are still 10 million new cases and 1.2 million deaths worldwide in2019.In addition,the co-infection of tuberculosis and AIDS has caused 250,000 lives death,which has become one of the restrictive factors hindering the economic development of developing countries.In recent years,with the emergence of extensively drug-resistant and multi-drug-resistant tuberculosis,the treatment of tuberculosis has become more complicated.Therefore,it is urgent to clarify the pathogenic mechanism of tuberculosis and find effective methods to combat MTB.Mycobacterium tuberculosis can enter dormancy and survive in host’s alveolar macrophages for a long time without causing the host’s immune response when encountering external stimuli such as hypoxia,NO,CO,and low p H.This adaptive mechanism is inseparable from the synergy of internal genes of Mycobacterium tuberculosis.Under hypoxia,the expression of about 100 genes of Mycobacterium tuberculosis are significantly different.Among them,the Dos R regulator in the two-component regulatory system Dos RST can regulate the expression of～48 genes.These 48 genes together constitute the Dos R rugulon,which regulates various physiological processes including amino acid synthesis,lipid metabolism,DNA damage repairs and carbohydrate metabolism.Our previous research found that the addition of vitamin C in the process of culturing BCG can lead to the up-regulation of arginine synthesis-related genes,and arginine synthesis cluster are necessary for the growth of Mycobacterium tuberculosis.Therefore,we tried to analyze the potential role of Dos R protein in regulating arginine synthesis,and whether Dos R is another key transcriptional regulator in addition to Arg R that regulates arginine synthesis.Gel retardation experiment was used to determine that Dos R can specifically bind to the minor groove in arg C promoter in vitro.The phosphorylation of Dos R can promote the binding of P-Dos R to the arg C promoter.The footprint experiment confirmed that Dos R has two binding sites within the arg C promoter which form an inconsensus reverse complementary structure to facilitate the recognition of Dos R.Cofactors specifity test showed that the cofactors including amino acids,vitamins and metal ions had different effects on Dos R binding to the arg C promoter.Arginine,glutamine and lysine acid can inhibit the binding of Dos R and P-Dos R to the arg C promoter.Vitamin B6,vitamin C,choline,Fe3+and Cu2+can inhibit the binding of Dos R and arg C promoters in a different way.Molecular docking experiments confirmed that the Asn-167 and Val-181 amino acid residues of the Dos R protein were essential for the interaction between the protein and small molecules.Subsequently,we confirmed that Dos R can bind to the arg C promoter in vivo through Ch IP which confirmed that Dos R has a regulatory effect on arginine synthesis in vivo and in vitro.In order to verify whether the Dos R protein has other targets in the BCG strain,we constructed the dos R deletion strain by homologous recombination in BCG,transcriptomics and proteomics were conducted to analyze the genes and protein expression levels in BCGΔdos R.The deletion of dos R gene can bring about significant changes in the transcription of 104 genes,99 genes were down-regulated while 5 genes up-regulated.Sequence conservation analysis confirmed that C7 and G12 are very critical for interaction with Dos R.The lack of Dos R protein changes the expression of 179 proteins.Many proteins play important roles in the virulence and metal ion metabolism of Mycobacterium tuberculosis.Our research showed that the Dos R protein plays an important role in the synthesis of arginine in Mycobacterium tuberculosis,providing a reference for the comprehensive analysis of the adaptive mechanism of Mycobacterium tuberculosis. |
