ZNFX1 promotes AMPK-mediated autophagy against Mycobacterium tuberculosis by stabilizing Prkaa2 mRNA
Honglin Liu, Zhenyu Han, Liru Chen, Jing Zhang, Zhanqing Zhang, Yaoxin Chen, Feichang Liu, Ke Wang, Jieyu Liu, Na Sai, Xinying Zhou, Chaoying Zhou, Shengfeng Hu, Qian Wen, Li Ma
Honglin Liu, Zhenyu Han, Liru Chen, Jing Zhang, Zhanqing Zhang, Yaoxin Chen, Feichang Liu, Ke Wang, Jieyu Liu, Na Sai, Xinying Zhou, Chaoying Zhou, Shengfeng Hu, Qian Wen, Li Ma
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Research Article Immunology Infectious disease

ZNFX1 promotes AMPK-mediated autophagy against Mycobacterium tuberculosis by stabilizing Prkaa2 mRNA

Abstract

Tuberculosis has the highest mortality rate worldwide for a chronic infectious disease caused by a single pathogen. RNA-binding proteins (RBPs) are involved in autophagy — a key defense mechanism against Mycobacterium tuberculosis (M. tuberculosis) infection — by modulating RNA stability and forming intricate regulatory networks. However, the functions of host RBPs during M. tuberculosis infection remain relatively unexplored. Zinc finger NFX1-type containing 1 (ZNFX1), a conserved RBP critically involved in immune deficiency diseases and mycobacterial infections, is significantly upregulated in M. tuberculosis–infected macrophages. Here, we aimed to explore the immunoregulatory functions of ZNFX1 during M. tuberculosis 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 M. tuberculosis burden, progressive lung tissue injury, and chronic inflammation in M. tuberculosis–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 protein kinase AMP-activated catalytic subunit alpha 2 mRNA, which encodes a key catalytic α subunit of AMPK, through its zinc finger region. This process contributed to M. tuberculosis growth suppression. These findings reveal a function of ZNFX1 in establishing anti–M. tuberculosis immune responses, enhancing our understanding of the roles of RBPs in tuberculosis immunity and providing a promising approach to bolster antituberculosis immunotherapy.

Authors

Honglin Liu, Zhenyu Han, Liru Chen, Jing Zhang, Zhanqing Zhang, Yaoxin Chen, Feichang Liu, Ke Wang, Jieyu Liu, Na Sai, Xinying Zhou, Chaoying Zhou, Shengfeng Hu, Qian Wen, Li Ma

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Figure 3

Regulation of macrophage activity by ZNFX1 and exploration of possible signaling mechanisms.

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Regulation of macrophage activity by ZNFX1 and exploration of possible s...
(A) Flow cytometry analysis of red fluorescence–positive WT and Znfx1–/– BMDMs infected with H37Rv carrying the red fluorescence protein (RFP) gene (i.e., H37Rv-RFP) at MOI = 10 at 2 hpi (n = 3). (B) Flow cytometry analysis of green fluorescence–positive WT and Znfx1–/– BMDMs incubated with FITC-conjugated latex beads (n = 4). (C) CFU assays of intracellular M. tuberculosis levels in H37Rv-infected WT and Znfx1–/– BMDMs at MOI = 5 (n = 4). (D) Flow cytometry analysis of CD80, CD86, MHC-II, and CD206 expression on the surface of H37Rv-infected WT and Znfx1–/– BMDMs at MOI = 2 at 24 hpi (n = 3). (E) Western blot assay of the regulatory effects of ZNFX1 on activation of signaling pathways following M. tuberculosis infection at MOI = 5. p-, phosphorylated. (F) Western blot assay of the regulatory effects of ZNFX1 on activation of the autophagy-associated mTOR signaling pathways. An unpaired 2-tailed t test (A and B) or a 2-way ANOVA with Holm-Šídák post hoc test (C–F) was used for statistical analysis. Data are presented as mean ± SD and are representative of at least 3 experiments with similar observations. **P < 0.01; ***P < 0.001; ****P < 0.0001.