Mycobacterial ESAT-6 triggers cGAS-STING pathway via the GSDMD-NT dependent mitochondria dysfunction

<i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) remains a global health threat due to its ability to subvert host immune responses. Emerging evidence suggests that <i>Mtb</i> infection induces the production of type I interferons (IFNs), during which cGAS directly binds to <i>Mtb</i> genomic DNA. Here, we demonstrate that ESAT-6, a secreted virulence factor of <i>Mtb</i>, potently induces type I interferon (IFN) responses through a cGAS/STING-dependent signaling axis. Genetic ablation and pharmacological inhibition of cGAS or STING abolished ESAT-6-induced IFN-β production. Mechanistically, ESAT-6 induces mitochondrial dysfunction, characterized by mitochondrial DNA (mtDNA) release and mitochondrial ROS (mtROS) production. Further investigation revealed that ESAT-6 triggers GSDMD cleavage, followed by the formation of GSDMD-NT. GSDMD-NT then translocates to mitochondria, disrupting their integrity and promoting mtDNA and mtROS release. GSDMD deficiency prevented ΔΨm loss, mtDNA/mtROS release, and IFN-β responses. Therefore, our findings establish a novel ESAT-6-GSDMD-mtDNA axis that drives type I IFN responses, providing critical insights into <i>Mtb</i> immune evasion strategies.