Identification And Functional Study Of Three New Drug-resistant Genes In Mycobacterium

Tuberculosis?TB?remains a major global public health threat,infecting and killing mainly in developing countries.The reason why TB is so difficult to treat is because of the TB bacterium's three“weapons”:persistence,drug resistance and virulence.Among them,the infection of drug-resistant Mycobacterium tuberculosis is a major difficulty in TB control.In 2018,the world health organization?WHO?estimates that at least 6.3million new cases of tuberculosis?TB?caused by pyrazinamide?PZA?and Fluoroquinolone?Fluoroquinolone?are reported,including 490,000 cases of MDR-TB.In addition,due to the increasing incidence of multidrug-resistant?MDR?and extensively drug-resistant?XDR?TB,there are limited antibiotic options available to treat TB.To cure TB patients more effectively,new drug targets must be found.Mycobacterium cell wall and cell membrane together provide a barrier for bacteria to resist external stress and cause permeability barrier to antibiotics and host immunity.Mycobacterium cell envelope is a complex multilayer structure,mainly composed of five important components:plasma membrane?PM?,peptidoglycan?PG?,arabinogalactan?AG?,outer membrane?OM?and mycolic acid?MA?,while the cell wall is mainly composed of peptidoglycan.Cell walls and membranes are critical in supporting cell growth,toxicity,and providing a barrier to cellular immunity.In particular,the basic properties of cell membrane synthesis and assembly determine that mycobacterium cell membrane is the most extensive target for the development of anti-tuberculosis drugs.Therefore,the discovery of new cell wall-or membrane-specific genes and the use of multiple antibiotics in combination to enhance the ability of antibiotics to destroy cell membranes should lead to new strategies for TB eradication.First,we constructed 10,000 mutant strains of Mycobacterium smegmatis using Tn7 transcripts.Through screening,we found that the mutant mycobacterium M492would lead to increased cell membrane permeability.Using the plasmid rescue method,we further identified that the mutant gene of M492 was MSMEG₃314?membrane transporter?.Genomic environment analysis of non-tuberculous mycobacterium?NTM?has found that it is conserved in M.marinum,M.leprae,M.smegmatis,M.bovis BCG,M.avium,and M.abscess.Their consistency was higher than 60%.Then,MSMEG₃314protein was replenished and expressed in M492,and the gene function was restored,which was verified by experiments.The loss of MSMEG₃314 changes the proton gradient?pH difference?of the cell membrane and enhances the bactericidal efficiency of fluoroquinolones.The bactericidal action of moxifloxacin can be weakened by the addition of reactive oxygen scavengers?thiourea and bipyridine?.The accumulation and outflow experiments of fluorescence staining with ethyl bromide showed that MSMEG₃314 mutation would lead to a significant increase in cell membrane permeability.In summary,these results indicate that M.smegmatismc²155MSMEG₃314 plays an important role in ROS scavenging and maintaining membrane stability,and affects M.smegmatis'resistance to PZA and fluoroquinolones.Then,bioinformatics analysis was applied to detect the function of genes affecting the biological function of bacterial cell membrane and cell wall,and it was found that serine protease Rv2224c had significantly different expressions in response to the pressure of various antibiotics,including isoniazid,moxifloxacin,vancomycin and rifampin.Homologous gene MSMEG₄296 was knocked out in M.smegmatis by homologous recombination and exchange.After knockout of this gene,it was found that MSMEG₄296 knockout bacteria were significantly sensitive to SDS.It was further verified that both the biofilm formation ability of MSMEG₄296 knockout bacteria and the sliding ability of bacteria were found to be defective,indicating that MSMEG₄296is an important gene affecting the formation of bacterial cell wall.This was also confirmed by scanning electron microscopy.It was found that MSMEG₄296 had a weaker ability to form ridges on the surface of the knockout bacteria than the wild strain.At the same time,we also found that MSMEG₄296 knockout bacteria were more sensitive to vancomycin.The Rv2224c protein expression experiment confirmed that d-ala-d-ala,the target of vancomycin on the cell wall,could be hydrolyzed by Rv2224c protein,thus affecting the sensitivity of M.smegmatis to vancomycin.Finally,we investigated the effect of cell wall on copper homeostasis.There are many kinds of metal ion transporters on the cell wall.We went on to screen the gene mutation library and found a mutant strain that was particularly sensitive to the content of copper ions,and identified that its mutant gene was MSMEG₄702,and its corresponding tuberculous homologous gene was Rv0102.By detecting the growth curve of M.smegmatis under the conditions of different copper ion concentration medium,we found that the knockout of MSMEG₄702 led to a significant decrease in the growth ability of M.smegmatis under the low copper environment,and enhanced the survival ability under the high copper environment.Western blot revealed that the over-expressed Rv0102 protein was located on the cell wall of the bacteria,and the Nile red staining experiment also confirmed that the cell wall synthesis of the knockout strain MSMEG₄702 was damaged.The intracellular copper ion concentration of MSMEG₄702 mutant strain was found to be much lower than that of the wild type.Based on the comprehensive findings,we confirmed that Rv0102 was a copper ion entraining protein located on the cell wall,which,together with the previously discovered MctB and CtpV copper ion efflux protein,formed the copper transport system of M.tuberculosis copper cell wall.In this study,the important role of cell wall in mycobacterium antibiotic resistance was determined from three aspects of cell membrane permeability,biofilm formation and cell wall functional proteins,which provided a theoretical basis for the development of new cell wall targeted antibiotics in the future.