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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\n                      <jats:italic toggle=\"yes\">M. tuberculosis<\/jats:italic>\n                      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\n                      <jats:italic toggle=\"yes\">Mycobacterium smegmatis<\/jats:italic>\n                      (Ms_EsxN) using an acetamide-inducible vector. Wild-type and ISG15-deficient THP-1 macrophages were infected with Ms_EsxN 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-1\u03b2). ISGylation was detected by ubiquitin-like modifier blotting. Statistical significance was determined by Student\u2019s\n                      <jats:italic toggle=\"yes\">t<\/jats:italic>\n                      -test (\n                      <jats:italic toggle=\"yes\">P<\/jats:italic>\n                      &lt; 0.05). We demonstrate that the Esx-5 substrate EsxN (Rv1793) is a key\n                      <jats:italic toggle=\"yes\">M. tuberculosis<\/jats:italic>\n                      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\n                      <jats:italic toggle=\"yes\">M. tuberculosis<\/jats:italic>\n                      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.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Tuberculosis (TB) remains one of the world\u2019s deadliest infectious diseases, killing over a million people annually. A major challenge in treating TB is understanding how the\n                        <jats:italic toggle=\"yes\">Mycobacterium tuberculosis<\/jats:italic>\n                        evades our immune defenses to survive inside human cells. This study reveals that an\n                        <jats:italic toggle=\"yes\">M. tuberculosis<\/jats:italic>\n                        protein, EsxN, acts as a \u201cmolecular trickster\u201d by hijacking a key immune pathway-cGAS-STING and weakens the body\u2019s 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\n                        <jats:italic toggle=\"yes\">M. tuberculosis<\/jats:italic>\n                        and highlight ISG15 or DNA leakage as promising targets for new host-directed therapies.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.02488-25","type":"journal-article","created":{"date-parts":[[2026,2,25]],"date-time":"2026-02-25T14:01:37Z","timestamp":1772028097000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":1,"title":["EsxN drives ISG15-mediated dsDNA release to activate cGAS-STING signaling and promote mycobacterial survival"],"prefix":"10.1128","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1199-4912","authenticated-orcid":true,"given":"Qiao","family":"Zhang","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01kj4z117","id-type":"ROR","asserted-by":"publisher"}],"name":"State Key Laboratory Breeding Base of Eco-Environment and Bio-Resource of the Three Gorges Area, Key Laboratory of Eco-environments in Three Gorges Reservoir Region, Ministry of Education, School of Life Sciences, Institute of Modern Biopharmaceuticals, Southwest University","place":["Chongqing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-3763-1716","authenticated-orcid":true,"given":"Abulimiti","family":"Abudukadier","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01kj4z117","id-type":"ROR","asserted-by":"publisher"}],"name":"State Key Laboratory Breeding Base of Eco-Environment and Bio-Resource of the Three 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