Preliminary Study On The Mechanism Of Tgs1 And MSMEG＿5242 In The Tolerance Of Mycobacterium Smegmatis To Isoniazid

Tuberculosis(TB)is a respiratory infectious disease caused by Mycobacterium tuberculosis(Mtb).This pathogen is mainly transmitted through droplets and enters the lungs or other parts outside the lungs through the respiratory tract,with pulmonary infection leading to pulmonary tuberculosis as the main clinical phenotype.The typical pathological feature of TB is the formation of granulomatous structures in the lungs,which are composed of multiple immune cells and play an important role in killing and inhibiting the spread of Mtb;however,granulomas also provide a suitable site for the growth of Mtb.About 1/4 of the world’s people are infected with Mtb.Although the widespread use of antibacterial drugs has effectively controlled the spread of TB,the treatment of TB still faces many challenges.First,the emergence of drug-resistant Mycobacterium tuberculosis has made common anti-tuberculosis drugs lose their killing effect on Mtb.Secondly,in recent years,it has been found that the difficulty in treating TB is not only related to the increase in drug resistant strains,but also related to the tolerance of Mtb to drugs.Drug tolerance refers to the fact that Mtb’s genes has not changed,but under various stress reactions,intracellular gene expression regulation changes bacterial metabolism,making it insensitive to high concentrations of antibiotics.Lipids are an important source of energy within Mtb,and when it enters a dormant state,they are stored in the form of triacylglycerol(TAG).The dormant TB bacteria exhibit tolerance to anti TB drugs,in which lipids play a key role.Numerous studies have shown that lipid metabolism related genes induce drug tolerance in mycobacterium.Mycobacterium smegmatis(Msm),a Gram positive saprophytic bacterium,has characteristics of rapid growth,weak pathogenicity,high homology with Mtb genes,and similar cellular structure.It is a relatively ideal model bacterium for studying Mtb and related immunology.In this study,Msm was used as a model bacterium to study the mechanism of related genes in the tolerance to Mtb isoniazid.Tgs1 is a key gene for the synthesis of triacylglycerol in Mtb,and one of its homologous genes in Msm is MSMEG＿5242.In this paper,we used p MV261 plasmid to construct overexpression vectors for Tgs1 and MSMEG＿5242,and then constructed Msm that overexpress Tgs1 and MSMEG＿5242.The results showed that overexpression of Tgs1 or MSMEG＿5242 resulted in slower growth of Msm without changing the minimum inhibitory concentration(MIC).The experiment of co incubation of the overexpression strain with isoniazid in PBS showed that Tgs1 and MSMEG＿5242 could induce the tolerance of mycobacterium to isoniazid.In order to explore the mechanism of isoniazid-tolerance of mycobacterium inducing by Tgs1 and MSMEG＿5242 genes,we conducted a transcriptome analysis.The results showed that there were 1664 differentially expressed genes common to the four strains of Msm,Ms-WT,Ms-p MV261,Ms-Tgs1,and Ms-5242,which were treated with isoniazid.Through Go and KEGG path analysis,it was found that the upregulated genes were mainly related to the SEC protein transport system α-Dextran biosynthesis,glycogen biosynthesis,amino acid betaine biosynthesis,amino acid betaine metabolism,and other related genes.The down-regulated genes are mainly related to amino acid and protein metabolism,transcription,and translation.Compared to Ms-p MV261,Ms-Tgs1 strain specifically expressed 310 up-regulated genes and 256 down-regulated genes under isoniazid pressure.According to KEGG path analysis,up-regulated genes were mainly related to the two-component system and fatty acid synthesis,while down-regulated genes were mainly related to fatty acid degradation and type I NADH dehydrogenase subunits.Similarly,compared to Ms-p MV261,Ms-5242 strain specifically expressed 298 upregulated genes and 249 down-regulated genes under isoniazid pressure.According to KEGG pathway analysis,up-regulated genes were mainly related to the two-component system and cholesterol transport,while down-regulated genes were mainly related to nitrogen uptake and cytochrome c subunits.In addition,we conducted functional annotation analysis on 50 genes differentially expressed under isoniazid pressure between Ms-Tgs1 and Ms-5242,and found that the expression level of the heme oxygenase synthesis gene MSMEG＿6086 increased,indicating that the strain maintains redox levels by increasing the expression of this gene;Increased synthesis of mycobacterium cyclopropaneum acid synthase I encoded by MSMEG＿0902 leads to cell wall thickening and drug tolerance.In summary,this study established an experimental model of isoniazid tolerance in starvation by constructing Msm overexpressing Tgs1 and MSMEG＿5242 genes.The mechanism of Tgs1 and MSMEG＿5242 mediated isoniazid-tolerance in Msm was analyzed by RNA-seq and we found that genes related to lipid metabolism,energy metabolism,and redox in Msm play an important role in the process of isoniazid tolerance.The above results can provide a theoretical basis for the discovery of new antibacterial targets for mycobacterium.