Functional Study Of Mycobacterium Tuberculosis Rv0217c In Stress Response

Tuberculosis（TB）,caused by Mycobacterium tuberculsis（Mtb）,is a chronic and zoonotic disease.The emergence of MDR and XDR-resistant tuberculosis has exacerbated the prevalence of tuberculosis.It may be directly related to the unique cell wall composition of Mycobacterium tuberculosis.The cell wall of M.tuberculosis is rich in lipids.The main component is mycolic acid.In the case of latent infection,M.tuberculosis mainly performs lipid metabolism rather than carbohydrate metabolism.M.tuberculosis infects macrophages,and the accumulated lipids are stored in the form of triacylglycerol（TAG）.In the genome of M.tuberculosis,250 genes are involved in lipid metabolism.These 250 genes can be divided into five families:lip family,phospholipase family,PE/PPE family,β-lactam family and cutinase family.This paper focuses on the related functions of Lipw（Rv0217c）,a member of the lipid family of Mycobacterium tuberculosis.This subject mainly studies LipW（Rv0217c）,a member of the lip family of Mycobacterium tuberculosis,to explore the biological role of LipW protein in Mycobacterium tuberculosis.The strain of LipW is resistant to stress environment（SDS,acid and H₂O₂,etc.）.LipW have an immune response to macrophages and can be used as a new type of protection vaccine.The following test methods were used to explore the properties of the LipW protein.（1）The Lip family members and other branches wereidentifiedbytheNCBIdatabaseandESPript3.0（http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi）.The amino acid sequences encoded by the homologous genes in the bacilli were aligned to determine the LipW subfamily and its genetic stability;（2）The recombinant plasmid of pET-28a-Rv0217c was transferred to E.coli BL21 by gene cloning technique.It was expressed in the form of inclusion bodies,and the protein LipW was purified.The LipW protein with higher purity was obtained by renaturation,and the optimal substrate,optimal temperature and most of the protein were determined.Appropriate pH,including kinetic parameters.（3）Point mutations（Ser149,Asp239,His269）were performed on the predicted active sites,and the three mutant proteins were purified and renatured to determine the change in protein activity.（4）The LipW deletion strain（ΔMSMEG₀280）was constructed by homologousrecombinationinM.smegmatis.Thecomplementstrain（ΔMSMEG₀280::Rv0217c）was constructed,and then the knockout strain was determined by the paper method.After simulating M.tuberculosis infection of macrophages,knock out the growth of the strain in the intracellular environment（acid and hypoxia,etc.）may be encountered;（5）It was found through experiments that the LipW protein is localized on the cell wall of M.smegmatis,so we suspect that LipW may be involved in virulence as an antigen.The LipW protein was overexpressed in Mycobacterium smegmatis.After induction,the overexpressing strain was used to infect THP-1 macrophages,and the transcript levels of intracellular bacteria and cytokines were investigated over time.We obtained the following results:（1）Successfully constructed the recombinant plasmid pET-28a-Rv0217c,and three mutant plasmids（S149A,D239A,H269A）.（2）Protein experiments showed that pNP-C₂ was the optimal substrate for this protein.SDS,PMSF and Tween 80 effect the activity of LipW protein;（3）Compared with the wild-type strain,the knockout strain was more sensitive to SDS,H₂O₂,low acid and low oxygen pressure environment,and the supplemental strain can replenish the phenotype of its wild strain;（4）Compared with the empty bacteria,the expression strain was more viable.IL-6 and IL-12 were significantly up-regulated,indicating LipW may participate in the host immune response as an antigen.In summary,LipW protein prefers to hydrolyze short-chain carbon.It is affected by SDS,H₂O₂,hypoxia stress and acid,which indicates that LipW can directly participate in intracellular stress response.The LipW promote the release of inflammatory factors.LipW can act as a catalyst to catalyze the hydrolysis of intracellular unsaturated fatty acids and provide energy for bacterial growth.In addition,LipW may be immunogenic,providing a theoretical guide for the development of new drugs.