Molecular Mechanism And Signaling Pathway On Antioxidant Adaptation Regulated By Transcriptional Factor CmtR In Mycobacterium Bovis BCG

During infection of intracellular pathogens,host macrophages usually produce a large number of reactive oxygen species(ROS)to inhibit the bacterial growth.Accordingly,pathogens also develop their unique antioxidant survival strategies in the long evolution.As two representatives of the most successful intracellular pathogens,Mycobacterium tuberculosis and Mycobacterium bovis possess extremely strongresistance to adverse stresses including oxidation.However,the signaling pathway and molecular mechanism on responding and withstanding the stress remain still unclear in these mycobacteria.Here,we used Mycobacterium bovis BCG as a model and identified a novel oxidation sensing transcription factor Cmt R,and further analyzed the signaling pathway and molecular mechanism on Cmt R-mediated antioxidant adaptation ofmycobacteria.The main results arrived in this study are listed below:(1)The regulator Cmt R enhances mycobacterial antioxidant survival ability and the Cys24 residue of Cmt R plays a unique role in responding and combating oxidation stress.By detecting effects of different expression levels of cmt R on thegrowth of recombinant strains in M.bovis BCG within H₂O₂ stress,we found overexpression of cmt R significantly increased the mycobacterial survival whileknockout of the gene obviously reduced the bacterial survival under the stress.RT-q PCR assays analyzed the expression of cmt R in mycobacteria under H₂O₂ stress.And wefound H₂O₂ strikingly induced the expression of cmt R in M.bovis BCG.The amino acid site-directed mutation,in combination with EMSA assays,revealed H₂O₂inhibited the DNA-binding activity of the wild-type Cmt R protein but didn't affect the activity of the mutant protein Cmt R(C24S).Furthermore,the growth curve determinations confirmed that the wild-type gene cmt R,but not the mutant gene cmt R(C24S),could complement H₂O₂resistance of the cmt R-deleted strain in M.bovis BCG.(2)Cmt R triggers the expression of the esx-3 operon by interacting with Zur,and it regulates mycobacterial antioxidant survival ability via the Cmt R-Zur-Esx H-Zn²⁺signaling pathway.Transcriptomic sequencings were performed to compare the gene expression difference between the wild-type and cmt R-deleted strains.We found that Cmt R positively regulated the expression of the esx-3 operon.However,EMSAassays confirmed that Cmt R could not bind directly with the operon promoter.Furthermore,the specific interaction between Cmt R and Zur was identified by the B2H and SPR assays.Cmt R could specifically inhibit the DNA-binding of Zur with the esx-3operon promoter,and H₂O₂promoted the inhibition of Cmt R.Then,the growth curve and ICP-OES assays showed that knockout of esx H and complementation of the mutant gene esx H(Mut3)(Mut3:the Zn²⁺binding site was mutant)significantly reduced theantioxidant survival ability as well as the intracellular Zn²⁺content of M.bovis BCG.By contrast,complementation of the wildtype gene esx H could restore the bacterialantioxidant survival ability and the intracellular Zn²⁺content.Exogenous Zn²⁺addition into the medium partially rescued the antioxidant ability of the mycobacteria.Andoverexpression of esx H could enhance the antioxidant survival ability of the cmt R-deleted strain in M.bovis BCG.Taken together,Esx H facilitates the Zn²⁺acquisition and enhances the antioxidant survival ability of M.bovis BCG,and theantioxidant survival ability regulated by Cmt R depends on activating the expression of the esx-3 operon gene esx H.Finally,EMSA and RT-q PCR assays revealed that Zn²⁺could inhibit the DNA-binding activity of Cmt R and significantly induce the expression of cmt R in M.bovis BCG.Therefore,these results suggest that Cmt R regulatesmycobacterial antioxidant survival ability via the Cmt R-Zur-Esx H-Zn²⁺signaling pathway.(3)Cmt R contributes to M.bovis BCG ability to inhibit ROS production and phagosome maturation in macrophages,and enhances the mycobacterial survival in the mouse.By analyzing the MFI(mean fluorescence intensity)of ROS probe inmacrophages infected with recombinant strains of M.bovis BCG with flow cytometry,we found the cmt R-deleted strain and the cmt R(C24S)complementation strain could significantly increase the MFI of ROS dye in macrophages.And then,the MFI of theearly and late marker of phagosome maturation in the infected macrophages was detectedby confocal assays,respectively.We observed the cmt R-overexpressing strain strikingly lowed the MFI of the phagosome maturation markers while cmt R-deleted strain increased the MFI of markers in macrophages.Taken together,these results suggest the expression of cmt R in M.bovis BCG can inhibit ROS production and phagosome maturation inmacrophages.Furthermore,Cmt R increased the mycobacterial survival in macrophages by using macrophages models.Finally,mouse experiments confirmed overexpression of cmt R significantly enhanced M.bovis BCG ability to survive in the lung of the infected mouse.By contrast,knockout of the gene obviously reduced the bacterial survival.Therefore,our study successfully characterized a novel redox sensor Cmt R and dissected its regulatory signaling pathway and molecular mechanism on themycobacterial antioxidant survival,and identified its role in combating the host's killing.These efforts have provided a new glance for mycobacterial antioxidant strategies and deepened our understanding of the pathogen-host interaction mechanism.