Mechanism Study Of The Orphan Response Regulator Rv3143 Regulates Drug Sensitivity Of Mycobacterium Tuberculosis

Tuberculosis?TB?is a zoonotic infectious disease mainly caused by Mycobacterium tuberculosis.Previous studies have demonstrated that the expression of the Rv3143 gene in two-component signal transduction system was associated with the drug resistance of mycobacterium tuberculosis,but the function remains unknown.For the present study,we constructed the Rv3143 gene recombinant strains in Mycobacterium smegmatis and focused on the differences of recombinant strains under the drug stimulated.As the result,we determined the relationship between Rv3143 and drug-sensitivity.1.The Rv3143 inceases the drug sensitivity of Mycobacterium tuberculosisIn this study,we found that the inhibition ratio of drugs in H37Ra-Rv3143 were higher than the wild strains and the deletion mutant by measurement cell activity.Meanwhile,the mc2155-Rv3143 strain exhibited lower MICs than mc2155 and mc2155-p MV261 strains.The growth curve of mc2155-p MV261 and mc2155-Rv3143 had been determined by measured OD600 and CFUs,the results showed that there was no significant difference on the growth characteristic between mc2155-p MV261 and mc2155-Rv3143 in vitro.However,the mc2155-Rv3143 strain grown more solwer than the mc2155-p MV261 strain with the RIF.Our results showed that Rv3143 can make Mycobacterium tuberculosis more sensitively for the drug.2.The Rv3143 inceases survival ability and drug sensitivity of Mycobacterium smegmatis in intracellularThe intracellular CFUs of mc2155-p MV261 and mc2155-Rv3143 in the process of intracellular susceptibility test were compared.We found that mc2155-Rv3143 showed stronger ability in intracellular survival in the absence of RIF at different time post infection?48h,72h?.But in the presence of RIF,the growth of mc2155-Rv3143 was subdued in intracellular.It reveals that the Rv3143 can incease intracellular viability and drug sensitivity of Mycobacterium smegmatis in THP-1 cells.3.Analysis and screen of the differentially expressed genes on Rv3143 overexpressed strain of Mycobacterium smegmatis with or without RIFA total of 2356 differentially expressed genes have been screened by RNA-seq between mc2155-p MV261 and mc2155-Rv3143 with or without RIF.According to the functional annotation,several genes were differentially regulated which mainly classified into cell wall and cell processes,information pathways,lipid metabolism,intermediary metabolism and respiration and so on.We screened 15 genes including MSMEG₄740? MSMEG₃615?MSMEG₂130,which might be regulated by protein Rv3143.4.Rv3143 protein conbined MSMEG₄740 geneThe protein of Rv3143 was successfully prokaryotic expressed,then we used the SPR experiment to screen the probable genes that could be regulated by protein Rv3143.In order to make the results crediblely,we used the EMSA experiment to verify the results of the SPR.Finally,we found out one gene,MSMEG₄740,that is conbined and regulated specifically by Rv3143 protein.5.Rv3143 inceases the cell wall permeability of Mycobacterium smegmatisWe employed the use of accumulation rate of the ethidium bromide?EB?to determine the cell wall permeability of mc2155-p MV261 and mc2155-Rv3143 by fluorescence intensity.Contrast with mc2155-p MV261,the fluorescence intensity was found significantly enhanced in the mc2155-Rv3143 strain.It shows that Rv3143 can increase cell wall permeability.