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 (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-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 effect