<i>Streptococcus agalactiae</i> suppresses milk protein synthesis in bovine mammary epithelial cells via <i>PGK1</i> ‐mediated inhibition of the <i>JAK2/STAT5</i> signalling pathway

Background Streptococcus agalactiae is a major pathogen of bovine mastitis. Phosphoglycerate kinase 1 ( PGK1 ) is considered a multifunctional virulence factor in bacterial pathogens and plays a novel role in host–pathogen interaction: PGK1 facilitates energy acquisition and immune evasion in intracellular group B streptococcus infection; PGK1 enhances systemic dissemination by facilitating evasion of host immunity in S. pyogenes infection; PGK1 ‐derived S. pneumoniae promotes epithelial adhesion and lung colonisation. Understanding the mechanisms underlying mammary epithelial cell damage and milk synthesis inhibition is critical for developing mastitis prevention strategies. Aim To establish an intracellular infection model of S. agalactiae in bovine mammary epithelial cells ( BMECs ) to investigate the effects on apoptosis and elucidate mechanisms disrupting milk synthesis with a focus on PGK1 and the JAK2 / STAT5 pathway. Methods BMECs were infected with S. agalactiae (multiplicity of infections: 20–100, 2–8 h). Cytotoxicity, apoptosis and cytoskeletal changes were assessed. PGK1 expression, apoptosis markers and milk synthesis mediators ( JAK2 / STAT5 / ELF5 / CSN2 ) were analysed via qRT ‐ PCR and western blot. PGK1 knockdown and JAK2 inhibitor (50 μM AG490 for 24 h [ DMSO vehicle control]) treatment validated the pathway involved. Major Findings S. agalactiae infection caused time‐ and dose‐dependent cytotoxicity (maximal LDH release: ~40% increase, P &lt; 0.05; cell viability reduction: ~35%, P &lt; 0.05 at an MOI = 100 for 8 h). Infection induced cytoskeleton rearrangement and apoptosis (apoptosis rate, 25.3% vs. control, 5.1%, P &lt; 0.05) and upregulated PGK1 expression. PGK1 knockdown reduced apoptosis by ~50% ( P &lt; 0.05). S. agalactiae downregulated JAK2 / STAT5 / ELF5 ( P &lt; 0.05) and impaired milk protein synthesis. CSN2 expression was decreased by ~60% ( P &lt; 0.05) and partially rescued by PGK1 knockdown but exacerbated by AG490 . Implications PGK1 ‐mediated apoptosis and JAK2 / STAT5 / ELF5 suppression are key mechanisms in S. agalactiae inhibiting milk synthesis. Molecular targets for mitigating mastitis‐induced milk loss were identified, supporting dairy safety and productivity strategies.