Zinc Finger Protein ZNFX1 Promotes AMPK-mediated Autophagy Against Mycobacterium Tuberculosis By Stabilizing Prkaa2 MRNA

Tuberculosis,a chronic infectious disease caused by a single pathogen,holds the highest mortality rate worldwide.RNA-binding proteins（RBPs）are involved in autophagy—a key defense mechanism against Mycobacterium tuberculosis（Mtb）infection—by modulating RNA stability and forming intricate regulatory networks.However,the functions of host RBPs during Mtb infection remain relatively unexplored.ZNFX1,a conserved RBP critically involved in immune deficiency diseases and mycobacterial infections,is significantly upregulated in Mtb-infected macrophages.Here,we aimed to explore the immune regulatory functions of ZNFX1 during Mtb infection.We observed that Znfx1 knockout markedly compromised the multifaceted immune responses mediated by macrophages.This compromise resulted in reduced phagocytosis,suppressed macrophage activation,increased Mtb burden,progressive lung tissue injury,and chronic inflammation in Mtb-infected mice.Mechanistic investigations revealed that the absence of ZNFX1 inhibited autophagy,consequently mediating immune suppression.ZNFX1 critically maintained AMPK-regulated autophagic flux by stabilizing Prkaa2 mRNA,which encodes a key catalytic a subunit of AMPK,through its zinc finger region.This process contributed to Mtb growth suppression.These findings reveal a function of ZNFX1 in establishing anti-Mtb immune responses,enhancing our understanding of the roles of RBPs in tuberculosis immunity and providing a promising approach to bolster anti-tuberculosis immunotherapy.Methods1.Expression of ZNFX1 following Mycobacterium tuberculosis（Mtb）infection.Heatmap showing qPCR-identified expression of several zinc finger proteins in the study by Jonathan M.Budzik.ZNFX1 mRNA expression in various Mφ following H37Rv infection（MOI=2）was also identified using qPCR.ZNFX1 expression was detected through Western blot analysis in H37Rv-infected BMDM.ZNFX1 expression in lung tissues and lymph nodes（LN）of patients with chronic inflammation（CI）or TB was analysed using immuno-histochemistry and statistics.2.Mycobacterium tuberculosis（Mtb）load and tissue inflammation in H37Rv infected Znfx1^-/-miceIn vivo,Mtb load was assessed in the lungs and spleens of Znfx1^-/-mice at 1 and 4 weeks after H37Rv infection.Moreover,the lungs and spleens of Znfx1^-/-mice were assessed using HE staining,and MGCs in the spleen were subjected to statistical analysis.Assays were conducted to measure NO production in the lung and spleen 1 week after infection.Luminex multiplex assays were performed to measure cytokine expression in the peripheral blood of mice 1 week post H37Rv infection.ELISA assays were also conducted to measure cytokine expression in the lung and spleen of mice 4 weeks post H37Rv infection.3.Bone marrow derived macrophage（BMDM）immune function and macrophage numbers were assessed in Znfx1^-/-mice.Flow cytometry assays were conducted to measure the amounts of Ly6C+ CD11b+F4/80+ macrophages present in the lung,spleen,and lymph nodes of WT and Znfx1^-/-mice.Additionally,flow cytometry was used to assess red fluorescence positive WT and Znfx1^-/-BMDMs infected with H37Rv carrying the red fluorescence protein（RFP）gene at MOI=10 after 2 hours post-infection.CFU assays were performed to measure the amount of intracellular Mtb in H37Rvinfected wild type and Znfxl^-/-BMDM at MOI=5.qPCR was used to detect cytokine expression in Znfxl^-/-macrophages infected with H37Rv.Flow cytometry was utilized to evaluate the expression of CD80,CD86,MHC-II,and CD206 on the surface of H37Rv-infected wild type and Znfx1^-/-BMDM at MOI=2 after 24 hours post infection.4.ZNFX 1 modulation of Mφ immune function qPCR detection of ISG expression in Znfx1^-/-Mφ following stimulation with H37Rv,LPS and VSV,respectively;Western blot assays of STAT1 activation in H37Rv-infected Znfx1^-/-Mφ CFU assays of Mtb load in Znfx1^-/-Mφ treated with or without IFN-y;Western blot assay of the regulatory effects of ZNFX1 on p65、AKT、ERK、p38 signalling pathways activated after Mtb infection at MOI=5;Western blot assay of the regulatory effects of ZNFX1 on the activation of autophagy-associated signalling pathways including the mTOR pathway,AMPK and ULK1;Western blot assay of the regulatory effects of ZNFX1 on the levels of p62 and LC-3 transition;Double staining immunofluorescence assays of LC3 puncta in F4/80+Mφ in lung and spleen of WT and Znfx1^-/-mice following H37Rv infection;CFU assays of Mtb load in rapamycin treated WT and Znfx1^-/-BMDM.5.ZNFX1 affects Mφ immune function by modulating Prkaa2 expression and pAMPK activationHigh-throughput RNA-seq analysis of H37Rv-infected（MOI=2）WT and Znfx1-/BMDM at 6 h and 24 h post infection suggested Prkaa2 as a downstream target of ZNFX1;Identification of Prkaa2 and its encoded protein AMPKa2 expression following H37Rv（MOI=2）in WT and Znfx1^-/-BMDM by qPCR and Western blot,respectively;Double staining immunofluorescence assays of p-AMPK in F4/80+ Mφin lung and spleen of WT and Znfxl^-/-mice following H37Rv infection;Flow cytometry assay of red fluorescence positive WT and Znfxl^-/-BMDM treated with EX229 followed by infection with H37Rv-RFP（MOI=10）;CFU assays of intracellular Mtb levels in H37Rv infected（MOI=5）WT and Znfx1^-/-BMDM and ZNFX1 silencing hMDM following EX229 treatment;Flow cytometry analysis of CD80,CD86 and MHC-II expressed on the surface of EX229 treated WT and Znfxl-/BMDM infected with H37Rv（MOI=2）.6.Effect of EX229 treatment on the antibacterial activity of Znfxl^-/-miceMtb load in lung and spleen was determined by CFU assays;assays of NO production in lung and spleen 1 week post infection;ELISA assays of cytokine expression in lung and spleen of mice 1 week post H37Rv infection;HE staining of lung and spleen and statistical analysis of MGCs in spleen.7.ZNFX1 regulates intracellular antibacterial activity by regulating Prkaa2 mRNA stabilityWestern blot assays of ZNFX1 in the cytosol,membrane and nuclear fractions of BMDM;Western blot assays of AMPKa and AMPKa2 levels in Znfxl^-/-BMDM treated with CHX and the degradation of treated with CHX,and the degradation rate of both proteins was analysed;qPCR detection of Prkaal and Prkaa2 in Znfxl-/BMDM treated with DRB;qPCR detection of Prkaa2 in ZNFX1-silencing hMDM treated with DRB;RIP assay using anti-ZNFX1 antibody and qPCR detection of the combination of ZNFX1 protein and Prkaal and Prkaa2 mRNA in BMDM,qPCR detection of Prkaa2 in WT and Znfx1^-/-BMDM infected with LV-Prkaa2;RNA pulldown assay to detect the combination of Prkaa2 mRNA and ZNFX1 protein in BMDM;RIP assay using anti-Flag antibody and qPCR to detect the combination of full-length or truncated ZNFX1 protein and Prkaa2 mRNA in 293T cells transfected with the different Znfxl plasmids;qPCR detection of Prkaa2 in 293T cells transfected with the different Znfx1 plasmids;CFU assay of Mtb load in WT and Znfx1^-/-BMDM transfected with the F2 variant of Znfx1.Results1.Demonstration of ZNFX1 expression in THP1,hMDM,BMDM cells and in lung and lymph node tissues of tuberculosis patients,H37Rv infection upregulates ZNFX1 expression;H37Rv infection of Znfxl^-/-mice indicated upregulation of Mtb load in lung and spleen tissues,lung inflammatory injury was significantly enhanced,an increasing number of multinucleated giant cells in the spleen.2.The secretion of NO in lung and spleen tissues was decreased;the expression of IFN-y,IL-12p70 and TNF-α in peripheral blood was suppressed;the expression of IFN-y and IL-12p70 in lung and spleen tissues was reduced.3.There was no significant difference in macrophage cells in lung,spleen and lymph node tissues of Znfx1^-/-mice,but the proliferation of intracellular Mtb and the expression of CD80,CD86 and MHC-II were attenuated;IL-12p35 mRNA was downregulated in Znfx1^-/-BMDM after H37Rv infection.4.ZNFX1 exerts its antibacterial activity not via manipulation the ISG pathway or responsiveness to IFN-y,but by modulation of the autophagic pathway;Znfx1-/BMDM is normally responsive to IFN-γ;Znfx1^-/-BMDM autophagic flux levels decreased;mTOR inhibitor can restore Znfx1^-/-BMDM intracellular Mtb load.5.Transcriptomic analysis demonstrated that ZNFX1 mainly affects the expression of the gene Prkaa2 encoding the autophagy-related protein AMPKα2;Reduced levels of autophagic flux in tissue macrophages of Znfx1^-/-mice.6.EX229 administration can rescue inflammatory cytokine secretion and tissue inflammation in lung and spleen of Znfnl^-/-mice;Western blotting of nuclear,membrane and cytosolic protein extracts showed that ZNFX1 localised to the cytoplasm and membrane;ZNFX1 does not interact with AMPKa2 and it’s upstream kinase LKB1;RIP and RNA pulldown showed that ZNFX1 zinc finger region can interact with Prkaa2 mRNA to regulate its mRNA stability;Over-expression of ZNFX1 zinc finger region can restore antibacterial activity in Znfx1^-/-BMDM.ConclusionOur study investigated the role of ZNFX1 in regulating immunity against Mtb infection,enabling Mtb to upregulate Znfnx1 expression regulating Mφ activity to suppress intracellular survival of Mtb independent of previously reported ISG signaling.ZNFX1 protein binding to Prkaa2 mRNA and maintains its stability modulates autophagic flux to affect the clearance of Mtb.Truncated ZNFX1 transfection in 293T proved that ZNFX1 functions through its zinc finger domain rather than SF1 helicase domain,and overexpressing ZNFX1 zinc finger domain in Znfx1^-/-Mφ restore its antibacterial activity.Our findings deepen the understanding of the immune mechanisms of TB and provide a potential novel approach to anti-TB immunotherapy.