ESX-5 component EccB₅ targets GSDMD to drive pyroptosis and promote Mtb dissemination

Mycobacterium tuberculosis (Mtb) promotes tuberculosis pathogenesis in part by exploiting host pyroptosis, yet bacterial factors that directly engage the pyroptotic executioner machinery remain poorly defined. Here we identify the ESX-5 component EccB₅ as a mycobacterial virulence factor that binds gasdermin D (GSDMD) and potentiates pathogenic pyroptosis. A proteome-wide screen using GSDMD as bait revealed EccB₅ as a specific interactor, mapped to the EccB₅ C terminus and residue R406. Purified protein assays demonstrated micromolar affinity direct binding between EccB₅ and GSDMD, with a dissociation constant of 5.22 μM by surface plasmon resonance. In vivo, conditional ATc-inducible knockdown of eccB₅ in the Mtb H37Rv strain markedly reduces lung pathology, bacterial burden, and host cell death in mice. Conversely, ectopic expression of EccB₅ in nonpathogenic Mycobacterium smegmatis is sufficient to trigger robust macrophage pyroptosis and amplify inflammatory responses in mice. Mechanistically, EccB₅ enhances canonical inflammasome-associated GSDMD processing and promotes NLRP3-dependent GSDMD-N membrane association and oligomerization, without evidence of caspase-3/GSDME activation or noncanonical inflammasome engagement. Genetic ablation of host GSDMD abolishes EccB₅-driven pyroptotic responses, establishing GSDMD as an essential host target. Together, these findings define an ESX5-linked virulence axis that directly couples mycobacterial infection to the execution phase of pyroptosis, thereby facilitating Mtb dissemination and disease progression. Targeting the EccB₅-GSDMD interface provides a mechanistically grounded strategy for host-directed therapy in tuberculosis.