ABSTRACT Tuberculosis, caused by Mycobacterium tuberculosis, remains a major global health threat largely due to the pathogen’s remarkable ability to subvert host immune defenses. A key mechanism of this subversion is the type VII secretion systems, particularly the Esx-5 subsystem. The Esx-5 secretion system secretes substrates that are hypothesized to function as effector proteins, orchestrating host immune modulation and influencing the outcome of M. tuberculosis infections. While EsxN is a confirmed secreted effector of ESX-5, its precise biological function remains unknown. Our research aims to fill this gap by investigating how EsxN manipulates host innate immunity, particularly through the cGAS-STING axis. EsxN was overexpressed in Mycobacterium smegmatis (MsEsxN) using an acetamide-inducible vector. Wild-type and ISG15-deficient THP-1 macrophages were infected with MsEsxN or control strains (MOI = 10). Bacterial intracellular survival was quantified by CFU assay. Host cell viability was assessed via the CCK-8 kit. Cytosolic double-stranded DNA (dsDNA) was visualized using PicoGreen staining and confocal microscopy. Transcriptomes of infected cells were analyzed by RNA-seq. Type I interferon (IFN-I) /cytokine responses and pathway activation were validated by RT-qPCR and immunoblotting (TBK1/phospho-TBK1 p-TBK1, IRF3, ISG15, IL-1β). ISGylation was detected by ubiquitin-like modifier blotting. Statistical significance was determined by Student’s t -test (P < 0. 05). We demonstrate that the Esx-5 substrate EsxN (Rv1793) is a key M. tuberculosis virulence effector that drives immune evasion by hijacking the cGAS-STING-ISG15 axis. EsxN overexpression exhibited enhanced intracellular survival in human macrophages without affecting host viability. Crucially, EsxN triggers increased dsDNA leakage into the cytoplasm, amplifies IFN-I responses, and suppresses pro-inflammatory cytokines, enhancing bacterial intracellular survival. ISG15 deficiency abolished EsxN-driven effects, revealing that ISG15-dependent ISGylation is essential for this immune evasion. Our study uncovers a novel immune evasion strategy wherein M. tuberculosis exploits host DNA-sensing machinery to amplify immunosuppressive IFN-I responses via EsxN-ISG15 crosstalk, which suggests targeting ISG15 as a potential host-directed therapy against tuberculosis. IMPORTANCE Tuberculosis (TB) remains one of the world’s deadliest infectious diseases, killing over a million people annually. A major challenge in treating TB is understanding how the Mycobacterium tuberculosis evades our immune defenses to survive inside human cells. This study reveals that an M. tuberculosis protein, EsxN, acts as a “molecular trickster” by hijacking a key immune pathway-cGAS-STING and weakens the body’s ability to control TB infection, allowing the bacteria to survive better. Crucially, we identified ISG15 as an essential partner in this evasion strategy. These findings expose a hidden weapon used by M. tuberculosis and highlight ISG15 or DNA leakage as promising targets for new host-directed therapies.
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