Sph-mediated mtDNA release and autophagy-dependent ferroptosis contribute to Staphylococcus aureus-induced mastitis

Staphylococcus aureus ( S. aureus ), a major pathogen of mastitis, employs a diverse arsenal of virulence factors to invade and disrupt mammary tissues, thereby eliciting a complex and dysregulated inflammatory response. Sphingomyelin phosphodiesterase (Sph) has emerged as a key virulence determinant of S. aureus ; however, its potential role and underlying mechanisms in mastitis remain elusive. In this study, we demonstrate that Sph deficiency significantly suppresses S. aureus -induced ferroptosis. Mechanistically, Sph disrupts mitochondrial cristae through interaction with Mic60, a core component of the mitochondrial contact site and cristae-organizing system (MICOS). Sph-mediated cytosolic mitochondrial DNA (mtDNA) released is sensed by cyclic GMP-AMP synthase (cGAS), leading to activation of the STING signaling pathway, which further triggers the recruitment of GPX4 into the autophagy-lysosome pathway for degradation. Collectively, our study unveils a novel pathogenic mechanism whereby the S. aureus virulence factor Sph perturbs mitochondrial homeostasis, and engages the cGAS-STING axis to promote GPX4 degradation and ferroptosis, highlighting Sph as a potential therapeutic target for S. aureus -associated mastitis. S. aureus is a major etiological agent of mastitis, a prevalent inflammatory disease of the mammary gland that adversely affects animal health and milk production. During infection, S. aureus deploys a diverse repertoire of virulence factors to damage host cells and promote disease progression. Among these factors, sphingomyelin phosphodiesterase (Sph) has been implicated in host cell injury; however, its contribution to mastitis pathogenesis remains poorly understood. Here, we show that Sph perturbs mitochondrial homeostasis by interacting with Mic60 and disrupting mitochondrial cristae architecture, thereby triggering activation of the cGAS-STING signaling pathway. Activated STING subsequently promotes the autophagic degradation of GPX4, a key antioxidant enzyme that protects cells against lipid peroxidation, ultimately driving ferroptotic cell death. Collectively, our findings uncover a previously unrecognized mechanism, and identify Sph as a potential therapeutic target for S. aureus -associated mastitis.