EspF Acts as a Molecular Scaffold to Facilitate TRIM25-Mediated NLRP3 Inflammasome Activation During Mycobacterial Infection

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. In this study, we identified the ESX-1 effectors EspF as a critical and conserved regulator of the NLRP3 inflammasome across pathogenic mycobacterial species. Our results demonstrate that EspF significantly augments the mature IL-1β and IL-18 release, caspase-1 activation, and gasdermin D (GSDMD)-mediated pyroptosis. Notably, infection with M. smegmatis or M. bovis BCG strains 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 a critical partner recruited by EspF to NLRP3, which mediated K63-linked NLRP3 ubiquitination. 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.