The transsulfuration pathway metabolite α-ketobutyrate drives RIPK1-lactate axis-dependent autophagy to alleviate Staphylococcus aureus infection.

Breastfeeding anchors infant immunity and long-term health, but its benefits are threatened by Staphylococcus aureus (S. aureus) mastitis, an increasingly prevalent condition driven by antimicrobial resistance and therapeutic limitations. Beyond compromising maternal wellness, mastitis threatens the safety and continuity of breast milk, highlighting a critical need for innovative intervention strategies. Herein, we demonstrated that α‑ketobutyrate (α-KB), a metabolite of the transsulfuration pathway, mitigated S. aureus‑induced inflammation, oxidative stress, and blood-milk barrier (BMB) disruption both in vivo and in vitro. α‑KB enhanced macroautophagic/autophagic responses, marked by increased ATG5, BECN1 (beclin 1), and LC3-II:LC3-I conversion and reduced SQSTM1/p62, through a RIPK1-lactate-TFEB axis. Specifically, it directly bound and stabilized RIPK1, elevated lactate production, and drove TFEB nuclear translocation to activate macroautophagy/autophagy and promote intracellular bacterial clearance. Molecular docking and molecular dynamics simulations suggested stable α‑KB and RIPK1 binding via hydrophobic and hydrogen bond interactions; RIPK1 knockout abolished α‑KB-induced autophagy and lactate generation, effects rescued by lactate supplementation. This study identifies a novel immunometabolic circuit linking a metabolite to RIPK1-lactate-TFEB-mediated autophagy, offering therapeutic potential against antibiotic‑resistant S. aureus mastitis and presenting a new paradigm for safeguarding breastfeeding quality and infant health.Abbreviations: 3-MA: 3-methyladenine; α-KB: α‑ketobutyrate; ATG5: autophagy related 5; BMB: blood-milk barrier; CETSA: cellular thermal shift assay; DARTS: drug affinity responsive target stability; IL1B: interleukin 1 beta; IL6: interleukin 6; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MST: microscale thermophoresis; NOS2: nitric oxide synthase 2; OCLN: occludin; PBS: phosphate-buffered saline; PTGS2: prostaglandin-endoperoxide synthase 2; Rapa: rapamycin; RIPK1: receptor interacting serine/threonine kinase 1; RT-PCR: real-time polymerasechain reaction;S. aureus:Staphylococcus aureus; SEM: standard error of the mean; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TJP1: tight junction protein 1; TNF: tumor necrosis factor; WT: wild-type.