Localization And Function Analysis Of Mycobacterium Tuberculosis Cell Wall Synthesis Gene Network Modules

Mycobacterium tuberculosis is considered to be the most successful pathogen in the world.Tuberculosis caused by tuberculosis has been one of the global health challenges since ancient times.Since the 1950 s,the continuous discovery of first-line anti-tuberculosis drugs such as isoniazid,rifampicin and ethambutol have effectively improved the cure rate and survival rate of tuberculosis patients.But the emergence of various forms of resistant strains,including isoniazid(INH)rifampin(XDR)resistant strains of a single,multi-drug resistant(XDR)strains and extensive drug-resistant strains,tuberculosis has become the world’s mainly cause of death for 1.5 million deaths each year.At the same time,the co-infection of HIV virus and tuberculosis has increased the burden of curing tuberculosis.Nowadays,it is imminent to overcome the targets of new and effective anti-tuberculosis drugs.The important reason why tuberculosis is difficult to overcome is that the cell wall of Mycobacterium tuberculosis is very different from other bacteria.The cell wall structure of tuberculosis bacteria is very important for the invasion,survival and reproduction of tuberculosis bacteria in the host.The main reason why tuberculosis drugs are difficult to develop is that tuberculosis bacteria have a hard cell wall and extremely low permeability.The resistance of tuberculosis bacteria is also related to its cell wall.Khosravi et al.found that compared with multidrug-resistant strains and tuberculosis susceptible strains,Drra and Drrb,which are related to cell wall synthesis,play an important role in the process of antibiotic resistance.Howard et al.found that tuberculosis bacteria carrying rifampicin-resistant can re-induce macrophage metabolism through changes in cell wall lipids.It can be seen that the cell wall of tuberculosis bacteria is an important target for the development of new anti-tuberculosis drugs.In this study,we first screened out the key gene groups in the cell wall based on bioinformatics methods.We used Biocarta,the National Cancer Institute Pathway Interaction Database(NCI-PID),Human Cyc and Reactome to perform high-throughput genome-wide screening of Mycobacterium tuberculosis.Then,we use the database established in the laboratory(DOOR)to classify the operons of the M.tuberculosis cell wall synthesis genes.We use GO,KEGG to perform functional analysis of bacterial cell wall genes,and screen out key subnets through protein interaction networks.Finally,we use Cytoscape to visualize the results.In this part of the study,we identified 893 Mycobacterium tuberculosis H37 Rv cell wall synthesis genes,which are distributed in 20 pathways and involve 46 different functions related to cell wall synthesis.We have identified important key genes and operons in the cell wall synthesis pathway.For example,the genes contained in operon ID6951 are all cell wall synthesis genes.In addition,we discovered five co-expression networks of molecular complexes involved in peptidoglycan biosynthesis,membrane transporter synthesis and other cell wall processes,and selected the key cell wall gene ftsW for follow-up research.Using the ftsW gene of Mycobacterium smegmatis as a template,we cloned the ftsW gene,ligated it with the p MV361-Rtet R plasmid,and transformed it into Mycobacterium smegmatis to construct an overexpression strain,and then constructed a homologous exchange site for the target gene.It is integrated into the mycobacterial phage genome to obtain a phagemid.Subsequently,The phagemid was transfected into the overexpression strain of Mycobacterium smegmatis,and the transfected Mycobacterium smegmatis was spread on the hygromycin and kanamycin resistant solid medium,and the bacteria were grown to In the logarithmic phase,single strains were picked and verified by fluorescent quantitative PCR.After the ftsW knockdown strain was successfully constructed,PCR verification found that when anhydrous tetracycline was not added,the strain showed ftsW overexpression.When 3ug/ml anhydrous tetracycline was added,the strain showed a decrease in Fsw expression.Subsequently,we tested the growth rate,colony observation,cell wall morphology observation under electron microscope,and bacterial survival rate under external pressure on the wild strain,overexpression strain and knockdown strain.The results showed that when the expression of ftsW gene changes,the growth rate of bacteria slows down.In addition,the reduction of ftsW gene expression will cause barriers to bacterial biofilm formation,changes in cell wall morphology,and changes in colony morphology,suggesting that the gene ftsW may be related to bacterial cell wall components.When faced with extreme environmental pressures,ftsW knockdown plants showed obvious growth advantages.Through this research,we first target and screen key genes based on big data through bioinformatics methods,and select ftsW genes for follow-up research.Through experimental verification,we found that the ftsW gene may be related to the cell wall components of Mycobacterium tuberculosis and play a key role in the growth and survival of Mycobacterium tuberculosis.