The Effect And Mechanism Of Mycobacterium Tuberculosis Transmembrane Protein Rv3737 On Macrophage Apoptosis

Objective:To observe the effect of Mycobacterium tuberculosis Rv3737 on the apoptosis of mycobacterium infected macrophages,and to explore the molecular mechanism of Rv3737 on the apoptosis of macrophages.Methods: Taking Mycobacterium smegmatis and Mycobacterium tuberculosis as experimental strains,Mycobacterium smegmatis overexpressing Rv3737(MS::p MV261＿Rv3737)and Mycobacterium tuberculosis knockout Rv3737(H37Rv △ Rv3737)and their empty strains MS::pMV261 and H37Rv-WT were constructed respectively.The overexpression strains and knockout strains were verified by Western Blot and Real-time PCR(q PCR).The knockout and overexpression strains were cultured in vitro to observe the effect of Rv3737 gene on the growth of Mycobacterium;Add MS:: p MV261＿Rv3737,MS:: p MV261 and H37 Rv △ Rv3737 and H37Rv-WT infected macrophages.The survival of mycobacteria in macrophages was calculated by colony forming unit(CFU);Macrophages were infected with MOI = 10.The expression of Annexin-V / PI was detected by flow cytometry to evaluate the effect of Rv3737 on the apoptosis of infected macrophages.The expression of apoptosis marker protein caspase 3,apoptosis representative protein caspase 8 in death receptor pathway,apoptosis representative protein caspase 9 in mitochondrial pathway and apoptosis representative protein caspase 12 in endoplasmic reticulum stress were detected by Western blot,To analyze the specific mechanism of Rv3737 regulating macrophage apoptosisResults: The in vitro growth curve showed that overexpression and knockout of Rv3737 did not affect the in vitro growth of Mycobacterium.The intracellular survival rate of MS::p MV261＿Rv3737 infected macrophages was significantly higher than that of MS::pMV261 in the control group;The intracellular survival rate of H37 Rv △ Rv3737 infected macrophages was significantly lower than that of H37Rv-WT,indicating that Rv3737 gene is conducive to the survival of mycobacteria in macrophages.Flow cytometry showed that Annexin-V(+)/ PI(+)decreased significantly after overexpression of Rv3737,and Western Blot showed that the shear of caspase 3 decreased significantly,indicating that Rv3737 significantly inhibited the apoptosis of Mycobacterium infected macrophages.The results of Western Blot showed that the shear of caspase 8,the key target molecule in death receptor mediated apoptosis,was significantly inhibited,but the expression of caspase 9and caspase 12 in endogenous and mitochondrial apoptosis pathway was not significantly abnormal.At the same time,further detection found that FADD and Fas,the upstream target molecules mediating death receptor apoptosis pathway,were significantly lower in the overexpression Rv3737 group than in the control group,on the contrary,the expression of FADD and Fas increased significantly after knockout of Mycobacterium tuberculosis Rv3737,indicating that transmembrane protein Rv3737 inhibits exogenous apoptosis mediated by death receptor pathway.Conclusion: Rv3737 inhibits the apoptosis of Mycobacterium infected macrophages through the exogenous apoptosis pathway mediated by death receptor,which helps to avoid the inherent immune attack of host cells and promote the survival and further infection of Mycobacterium in host cells.