CMPK2 Regulates Anti-tuberculosis Immunity By Promoting Phagosome-lysosome Fusion In Macrophages

Background:Tuberculosis(TB),caused by the bacterium Mycobacterium Tuberculosis(Mtb),is one of the world’s oldest infectious diseases.Host innate immunity and adaptive immunity are involved in the anti-tuberculosis infection process,and many immune cells play an important role.Among them,macrophages are an important part of innate immunity,and their role in Mtb infection can not be ignored.Cytidylate kinase 2(CMPK2)is involved in mitochondrial genome replication and regulates the activation of inflammatory bodies by influencing mitochondrial DNA replication.At the same time,CMPK2 is involved in the maintenance of mitochondrial homeostasis,and the imbalance of mitochondrial homeostasis will lead to neurodegenerative diseases.In addition,CMPK2,as an interferon stimulator,plays an important role in the immune process of human immunodeficiency virus and Dengue virus infection,but the effects and mechanisms of CMPK2 on Mtb infection are still lacking.Methods:1.The expression of CMPK2 in macrophages after Mtb infection was detected by Western blot and qRT-PCR;2.The effect of CMPK2 on the intracellular load of macrophages was detected by colony counting experiment;3.qRT-PCR was used to detect the replication and transcription of mitochondrial DNA,mRNA of pro-inflammatory cytokines,mRNA of key glycolysis enzymes,mRNA of IFN-β and ISG in macrophages infected with Mtb;4.The effect of CMPK2 on the secretion of pro-inflammatory cytokines in macrophages infected with Mtb was detected by ELISA;5.The effect of CMPK2 on mitochondrial reactive oxygen species in Mtb infected macrophages was detected by flow cytometry;6.The effects of CMPK2 on apoptosis and autophagy of macrophages infected with Mtb were detected by Western blot;7.The effects of CMPK2 on the amount of ATP produced and lactic acid secreted by macrophages infected with Mtb were detected by kit method;8.The localization of CMPK2 in macrophages was detected by nuclear plasma separation and immunofluorescence;9.Transcriptome sequencing was used to analyze the effect of silencing CMPK2 on gene transcription in Mtb infected macrophages;10.Western blot was used to detect the early endosomal marker protein VAMP3 expression in macrophages after silencing CMPK2,and immunofluorescence was used to detect the co-localization of VAMP3 and Mtb;11.Western blot was used to detect the expression of LAMP-1,a marker protein of lysosome,in macrophages after silencing CMPK2,and immunofluorescence was used to detect the co-localization of LAMP-1 and Mtb;12.qRT-PCR and Western blot were used to detect the expression of TFEB in macrophages after silencing CMPK2.Results:1.The expression of CMPK2 is increased in macrophages infected with Mtb;2.CMPK2 inhibits the survival of Mycobacterium tuberculosis in macrophages;3.CMPK2 is localized in cytoplasm and mitochondria of macrophages;4.The regulation of the antibacterial effect of CMPK2 on macrophages does not depend on its enzyme function;5.The regulation of the antibacterial effect of CMPK2 on macrophages does not depend on its ISG function;6.CMPK2 is not involved in regulating apoptosis and autophagy of macrophages infected with Mtb;7.CMPK2 promoted the expression of VAMP3 and increased the colocalization of endosomes and Mtb;8.CMPK2 promoted the expression of LAMP-1 and increased the colocalization of lysosome and Mtb.Conclusions:In this study,it was found that after Mtb infection of macrophages,CMPK2 promoted the fusion of bacteria-containing phagosomes and lysosomes by up-regulating the expression of VAMP3 and LAMP-1,the marker of lysosomes,so as to enhance the ability of lysosomes to kill Mtb.