Molecular Mechanism And Signaling Pathway Of P-type Atpase CtpG Mediating Mycobacterium Bovis BCG Resistance To Zn²⁺ Toxicity

Zinc is an essential trace element for almost all pathogens,but excess zinc is toxic to bacteria and therefore represents as an antibacterial mechanism of macrophages for intracellular pathogen.Mycobacterium tuberculosis and Mycobacterium bovis are the most successful representatives of intracellular pathogens with strong resistance to adverse environment stresses（including zinc toxicity）.However,the mechanisms and signaling pathways of these mycobacteria in response to and resistance to zinc toxicity remain largely unclear.In view of the high similarity of genomes between M.bovis and M.tuberculosis as well as the ubiquity of zinc toxicity in mycobacteria,this study used M. bovis BCG as the research object,identified the role of the P_1B-type ATPase CtpG in the process of combatting zinc toxicity and host immunity.The main experimental results obtained are briefly described as follows:（1）CtpG significantly enhances mycobacterial resistance to Zn²⁺toxicity in M.bovis BCG.By examining the growth difference among recombinant M.bovis BCG strains with different expression levels of ctpG under zinc toxicity condition,we found that deletion of ctpG significantly weakened the ability of M.bovis BCG to resist zinc toxicity,while complementing the wild-type gene ctpG could restore the resistance of the ctpG-deleted strain to zinc toxicity.Further,the zinc tolerance difference of these recombinant strains was analyzed.And we found ctpG expression could significantly enhance the resistance of M.bovis BCG to zinc toxicity.These results suggest that the expression of ctpG contributes to mycobacterial resistance to zinc toxicity in M.bovis BCG.（2）CtpG binds to zinc through the APC sequence,hydrolyzes ATP and promotes zinc efflux,then leads to the elevated survival of M.bovis BCG under zinc toxicity.Phylogenetic tree and amino acid sequence alignment analysis showed that CtpG had high similarity with zinc transporter,with multiple conserved domains,but slightly different zinc-binding sites.Among them,CtpG contains a unique APC site that binds zinc.Amino acid site-directed mutagenesis combined with enzymatic kinetics experiments comfired that the ATPase activity of both the wild-type protein CtpG and its mutant proteins CtpG（Mut）,CtpG（ΔAPC）had no significant difference under normal conditions;in the presence of zinc,zinc promoted the ATP hydrolysis activity of CtpG,but not the two mutant proteins,and other metal ions did not affect their ATPase activity.Further,CtpG could promote zinc efflux of M.bovis BCG under zinc toxicity conditions,and its efflux effect was significantly better than that of zinc-transporter Ctp C;however,under the same conditions,the mutant proteins CtpG（Mut）and CtpG（?APC）lost the ability to expel zinc.Subsequently,by comparatively assaying the survival differences of these recombinant strains under zinc toxicity conditions,the survival of ctpG-deleted strain and the corresponding complementary strains of the two mutant genes was significantly reduced under zinc toxicity conditions.Based on these results,CtpG is a novel zinc efflux protein through its APC sequence binding zinc,and hydrolyzes ATP,expel zinc outside the bacterial cell,and enhances M.bovis BCG resistance to zinc toxicity.（3）ctpG expression is induced by zinc and this process is dependent on transcription factor Cmt R.By analyzing ctpG expression in M.bovis BCG strains under zinc toxicity conditions,the expression of ctpG increased gradually with the addition of zinc concentration.And the induction fold of ctpG expression（about 50 times）was significantly higher than that of ctp C（about 10 times）.However,under the same conditions,other metal ions（except cadmium）did not affect ctpG expression of M.bovis BCG.Subsequently,RT-q PCR results confirmed that zinc could de-repress the inhibitory effect of transcription factor Cmt R on ctpG.Further,by analyzing the dependence of zinc-induced ctpG expression on Cmt R,we found that ctpG expression in the wild-type strain was significantly induced by zinc,while the expression of ctpG in cmt R-deleted strain was not obviously changed under the same conditions.These results suggest that zinc induces the expression of ctpG in M.bovis BCG and the process depends on Cmt R.（4）CtpG contributes to M.bovis BCG to inhibit zinc toxicity mediated by macrophage THP-1,and enhances the bacterial intracellular survival.By comparatively analyzing the content of free zinc in THP-1 cells infected with wild-type and ctpG-deleted strains and the co-localization between bacteria and free zinc,we found that free zinc content in ctpG-deleted strain infected macrophages was significantly increased,and the co-localization between ctpG-deleted strain and free zinc was more obvious.Further,RT-q PCR results confirmed that the ctpG-deleted strain and the mutant genes,ctpG（Mut）or ctpG（?APC）complementation strain stimulated the expression of genes encoded some zinc toxicity-related proteins MTF1,MT1 and MT2 in macrophages during infection.Finally,by comparatively analyzed the survival difference among ctpG-related recombinant strains in THP-1,we found that the survival ability of ctpG-deleted strain and its mutant complementary strains was significantly lower than that of the wild-type strain,while the survival of ctpG-overexpression strain was significantly higher.Therefore,these results suggest that CtpG can inhibit macrophages-mediated zinc toxicity and enhance the intracellular survival of M.bovis BCG.（5）CtpG enhances M.bovis BCG virulence in the infected mice.Using mouse model to comparatively analyze the survival difference among ctpG-related recombinant strains,we found that deletion of ctpG significantly reduced the survival of M.bovis BCG in mice,while overexpression of ctpG significantly enhanced the bacterial survival.Further,the pathological sections and RT-q PCR results confirmed that the high survival of the ctpG-overexpressing strain led to more inflammatory cell infiltration in the lungs and more inflammatory cytokines production in the spleen of the infected mice,while the ctpG-deleted strain lost the ability.Taken together,CtpG contributes to the virulence of M.bovis BCG.In conclusion,our study firstly found that P_1B-type ATPase CtpG is a new zinc efflux protein in M.bovis,and significantly enhances the bacterial ability to resist zinc toxicity.And we systematically elucidate the molecular mechanism and signaling pathway (Zn²⁺-Cmt R-CtpG)of mycobacterial responsing and resistance to zinc toxicity.Further,we identified the potential mechanism of CtpG enhancing bacterial survival in macrophages and mice.These works extand our understanding of the mechanisms of mycobacteria adaptation to environmental stress and resistance to zinc toxicity,and are expected to provide potential drug targets for the prevention and treatment of tuberculosis.