Study On The Correlation Between Rv1453 And Clofazimine Resistance In Mycobacterium Tuberculosis

Background:China has a large number of tuberculosis(TB)patients and ranks third in the number of new TB cases in 2019,after India and Indonesia.China is also one of the countries with a high burden of drug-resistant TB,accounting for 14%of the world's cases of drug-resistant TB.Because of the long treatment time,high cost and low treatment success rate of drug-resistant TB,extensive research has been carried out on drug development,treatment regimens optimization and mechanisms of drug-resistant tuberculosis.Clofazimine was initially used to treat leprosy,and in 2018 the World Health Organization designated it a as group B drug for the treatment of drug-resistant tuberculosis.In order to slow down the occurrence of drug resistance to clofazimine,it is necessary to study the mechanism of drug resistance.In our previous study,clofazimine resistant isolates were selected from 100 clinical strains that isolated from multi-drug resistant tuberculosis patients,and the resistance rate was 7%.The Rv1453 gene,which might be associated with clofazimine resistance,was screened out by whole genome sequencing.The Rv1453 gene encodes a transcriptional regulatory protein that may affect the sensitivity of the strain to clofazimine by regulating the transcription of neighboring genes.In this study,Rv1453 gene was studied mainly at the DNA level,RNA level and protein level to explore the ways through which Rv1453 protein affected the sensitivity of the strain to clofazimine.Methods:1.To clarify the correlation between Rv1453 and the susceptibility of the strain to clofazimine:Rv1453 gene knockout strain,Rv1453 gene complement strain and Rv1453 gene overexpression strain were constructed,and the minimum inhibitory concentration(MIC)of clofazimine against the above strains were detected by Microplate Alamar Blue Assay(MABA).2.Analysis of Rv1453 gene regulation at the transcriptional level:The transcription levels of Rv1453 gene and its adjacent genes were detected by reverse transcriptionpolymerase chain reaction(RT-PCR),and the effects of Rv1453 gene on the transcription levels of its adjacent genes were observed with or without clofazimine.3.The interaction between Rv1453 protein and target DNA was analyzed at protein level:The recombinant E.coli+Rv1453 was constructed.The purified Rv1453 protein was used for gel migration electrophoresis to detect the interaction between Rv1453 protein and target DNA,and further identify the binding site of Rv1453 protein.And rabbit polyclonal antibody against Rv1453 was prepared to detect the expression level of Rv1453 protein in Mycobacterium tuberculosis.Results:1.We successfully obtained Rv1453 gene knockout strain,Rv1453 gene complement strain and Rv1453 gene overexpression strain.The results of clofazimine against the recombinant strain showed that the MIC value increased about 4-fold for the Rv1453 knockout strain,decreased about 4-fold for the Rv1453 overexpression strain,and returned to the same level as H37Rv for the Rv1453 gene complement strain.2.RT-PCR results showed that the transcription levels of qor gene and Rv1455 gene were significantly up-regulated after gene knockout,and the transcript levels of qor and Rv1455 were slightly reduced by overexpression of Rv1453 gene.After adding clofazimine,the transcription levels of Rv1455 gene and qor gene of Rv1453 gene knockout strain increased with the increase of drug treatment time.However,the transcriptional levels of Rv1455 gene and qor gene of Rv1453 gene complement strain and Rv1453 gene overexpression strain were up-regulated at first and then down regulated.3.The results of gel electrophoresis showed that the purified Rv1453 protein could bind to the DNA fragments of the promoter region of qor gene and the promoter region of Rv1455 gene.And it was found that Rv1453 protein could bind to both double-stranded and single-stranded probes,but the binding force was weak.Further study found that RV1453 protein may bind to the RNA polymerase binding site of the qor gene,thus preventing the transcription of qor gene.Conclusions:1.Rv1453 gene is related to the resistance of clofazimine.2.Rv1453 gene can regulate the transcription level of Rv1455 gene and qor gene,and the regulation mechanism is that Rv1453 protein binds to the promoter region of target gene and hinders the transcription process.3.Mechanism of clofazimine resistance in Mycobacterium tuberculosis caused by loss of Rv1453 protein:As a regulatory protein,Rv1453 protein binds to the RNA polymerase binding site of qor gene and blocks the transcription process.When the Rv1453 protein was absent,the transcription of the qor gene was no longer inhibited,which resulted in the increase of quinone reductase and reduction of reactive oxygen species and oxidative stress,thus making the strain resistant to clofazimine.