EspF acts as a molecular scaffold to facilitate TRIM25-mediated NLRP3 inflammasome activation during mycobacterial infection

Abstract

ABSTRACT The mycobacterial ESX-1 (Type VII) secretion system is essential for virulence and induces the NLRP3 inflammasome activation. However, the specific bacterial effectors involved in this process, beyond the well-characterized EsxA (ESAT-6), remain largely unidentified. Through systematic screening of ESX-1 effectors, we identified EspF as a potent and evolutionarily conserved activator of the NLRP3 inflammasome across pathogenic mycobacterial species, including Mycobacterium tuberculosis, M. bovis, and M. marinum. Our results demonstrate that EspF significantly augments the mature IL-1β and IL-18 release, ASC speck formation, caspase-1 activation, and gasdermin D (GSDMD)-mediated pyroptosis in THP-1 cells. Notably, infection with M. smegmatis or M. bovis BCG strain overexpressing EspF significantly enhanced NLRP3 inflammasome activation and pyroptosis, which were completely abolished in NLRP3-deficient cells. Mechanistically, EspF directly interacts with the NACHT and LRR domains of NLRP3. Furthermore, unbiased proteomic screening identified the host E3 ubiquitin ligase TRIM25 as an indispensable binding partner. In THP-1 cells, we confirmed the endogenous interaction and colocalization of the EspF–TRIM25–NLRP3 complex, demonstrating that EspF functions as a molecular scaffold that bridges TRIM25 to NLRP3. This proximity interaction, further validated in situ during mycobacterial infection, facilitates TRIM25-mediated K63-linked ubiquitination of NLRP3. In vivo, mice infected with M. bovis BCG strains overexpressing EspF exhibited exacerbated lung lesions, increased inflammatory cell infiltration, and higher bacterial burdens. Collectively, these findings reveal EspF-TRIM25-NLRP3 axis is a novel mechanism of mycobacterial pathogenesis that drives hyperinflammation to facilitate bacterial survival and dissemination.