Preliminary Study On The Effect And Mechanism Of Sigma E Gene On Drug Sensitivity Of Mycobacterium Smegmatis

Objective:Mycobacterium smegmatis(MS)overexpressing SigE is constructed,and its function is understood by stress response,indicating that SigE expression is successful and active.By observing the survival rate of MS(sigE overexpressing MS,sigE knockout MS,and anti-sigE expressing MS)to understand the drug sensitivity of sigE.RpfE was used to promote resuscitation of persistence phase bacteria.By detecting the resuscitaton index(RI),it is possible to clarify the possible mechanism which SigE promotes bacterial persistence and affects anti-tuberculosis drug susceptibility.Finally,high-throughput sequencing analyzes differential gene expression to further understand which genes may be associated with sigE,and lay the foundation for further research on Mycobacterial resistance.Methods:1.The sigE gene of MS was amplified by PCR,cloned into the multiple cloning site of plasmid pMV261,and the recombinant pMV261-SigE plasmid was constructed and verified by sequencing.The recombinant plasmid was electrotransformated into MS and the expression of SigE was detected by Western blot.2.The identification of stress function of SigE,understand the effect of SigE on sodium dodecyl sulfate,acid and hydrogen peroxide stress,and the effect of SigE on DNA damage response after DDP and MMC treatment.3.The MS containing the empty plasmid was designed as the control group.The survival rate of the bacteria to anti-tuberculosis drugs was detected by the microtiter plate method using resazurin as an indicator and the colony counting method.The effect of SigE on anti-tuberculosis drugs sensitivity is assessed by bacterial survival.4.To fully understand the function of sigE,we constructed sigE gene knockout variants and Mycobacterium smegmatis expressing MSMEG-5071(anti-sigE)and observed changes in their sensitivity to anti-tuberculosis drugs.5.The recombinant bacteria were induced into non-replication persistence phase treated with corresponding higher concentration antibiotic for 48 hours.Resuscitation-promoting factor E(RpfE)of Mycobacterium tuberculosis was cloned and expressed to enhance resuscitation and growth of persistence phase bacteria.The bacteria in persistence phase were incubated in 7H9 containing 20 ng/mL of RpfE for 2 weeks.The bacterial CFU and most probable number(MPN)were measured to count resuscitation index(RI).6.High-throughput sequencing analysis of differential gene expression,reference gene annotation,clustering,functional enrichment analysis.Results:1 The pMV261-SigE was successfully constructed,and the sequence was confirmed by sequencing.The expression of sigE in MS was detected by Western blot.2 After DDP treatment,the expression level of SigE was higher than that of the untreated control group.Compared with the no-load strain,the pMV261-SigE recombinant MS growth delayed into the log phase,the survival rate was higher under sodium dodecyl sulfate,acid and hydrogen peroxide stress(P<0.05),the survival rate of the SigE recombinant MS group treated with cisplatin or mitomycin c was also higher(P < 0.05).3.Compared with the control group,the SigE overexpressing of MS group had a higher percentage of fluorescence intensity(response survive rate)after anti-tuberculosis drug treatment.4.SigE gene knockout plasmid and pMV261(+)-MSMEG-5071/MS were successfully constructed.Recombinant MS expressing MSMEG-5071(anti-sigE)increased sensitivity to anti-tuberculosis drugs.However,the MS sigE gene mutant still needs further screening and identification.5.After the action of isoniazid and ethambutol,the resuscitation index of SigE overexpressing MS was significantly higher than that of the control group.In addition to streptomycin,the resuscitation index of MSMEG-5071 overexpressing bacteria was lower than that of the control group after the other five drugs treatment.6.High-throughput sequencing,differential gene expression analysis showed that MSMEG-5071 overexpressing bacteria mobilized stress and other mechanisms after drug treatment,but at the same time physiological metabolism was strong,reducing the expression of quorum sensing genes.Conclusion:1.Through these experiments we can infer that DNA damage agent(DDP)can increase the expression of SigE.The pMV261-SigE/MS is more resistant to DNA damage reagents than the pMV261/MS.2.SigE may influence the sensitivity of isoniazid and ethambutol by promoting Mycobacterium smegmatis into the non-replication persistence phase.3.MSMEG-5071 plays an important role in reducing the production of persistence bacteria.