Ferroptosis-Related Genes as Molecular Markers in Bovine Mammary Epithelial Cells Challenged with Staphylococcus aureus

<i>Staphylococcus aureus</i>-induced mastitis is a significant cause of economic losses in the dairy industry, yet its molecular mechanisms remain poorly defined. Although ferroptosis, a regulated cell death process, is associated with inflammatory diseases, its role in bovine mastitis is unknown. In this study, 11 <i>S. aureus</i> strains were isolated from milk samples obtained from cows with clinical or subclinical mastitis. Transcriptome analysis of Mac-T cells challenged with isolated <i>S. aureus</i> identified differentially expressed genes (DEGs). Enrichment analysis revealed significant associations between DEG clusters and traits related to bovine mastitis. KEGG pathway enrichment revealed ferroptosis, Toll-like receptor, and TNF signaling as significantly enriched pathways. Weighted gene co-expression network analysis (WGCNA) further prioritized ferroptosis-related genes (<i>HMOX1</i>, <i>SLC11A2</i>, <i>STEAP3</i>, <i>SAT1</i>, and <i>VDAC2</i>) involved in iron metabolism. Notably, the expression levels of <i>HMOX1</i> and <i>SAT1</i> were significantly increased in <i>S. aureus</i>-challenged Mac-T cells, and this upregulation was consistent with trends observed in transcriptome data from mother-daughter pairs of cows with subclinical mastitis caused by <i>S. aureus</i> infection. Furthermore, Ferrostatin-1 treatment significantly reduced the expression of <i>HMOX1</i> and <i>SAT1</i> in <i>S. aureus</i>-challenged cells, confirming the involvement of ferroptosis in this process. This study reveals that ferroptosis plays a key role in <i>S. aureus</i>-induced mastitis and highlights its potential as a target for molecular breeding strategies aimed at improving bovine mastitis resistance.