TRIM32 regulates Mycobacterium tuberculosis-induced pyroptosis and mitochondrial dysfunction by regulating TLR4 ubiquitylation.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), remains a major global health threat. The tripartite motif (TRIM) family of E3 ligases plays crucial roles in various cellular processes, yet the involvement of TRIM32 in TB remains poorly understood. This study explores the function of TRIM32 in M.tb infection and its underlying mechanisms. We found that M.tb infection significantly reduced TRIM32 expression in mouse macrophages, suggesting a regulatory role in TB progression. TRIM32 inhibited NLRP3 inflammasome activation and pyroptosis of M.tb-infected macrophages. Furthermore, TRIM32 overexpression mitigated mitochondrial dysfunction, evidenced by decreased reactive oxygen species (ROS) generation, inhibited mitochondrial ROS (mtROS), and restored mitochondrial membrane potential. Importantly, TRIM32 directly interacted with TLR4 and promoted its degradation via ubiquitination at the K48 site, a critical process for proteasomal degradation. The inhibitory effects of TRIM32 on pyroptosis and mitochondrial dysfunction were partially reversed by TLR4 overexpression, confirming TLR4 as a key mediator of TRIM32's actions. Our findings uncover TRIM32 as a critical regulator of mitochondrial health and pyroptosis in M.tb-infected macrophages through TLR4-mediated ubiquitylation, Moreover, silencing of endogenous TRIM32 aggravated NLRP3 inflammasome activation, pyroptosis and mitochondrial injury, and AAV-mediated TRIM32 overexpression alleviated lung pathology, lowered bacterial burden and improved survival in M.tb-infected mice, with these benefits being partially abrogated by TLR4 co-overexpression. Our findings identify TRIM32 as a candidate regulatory node of macrophage pyroptosis in TB that merits further preclinical evaluation.