Data-Independent Acquisition-Based Quantitative Proteomics, Nontargeted Metabolomics, and Protein–Ligand Interaction Reveal the Mechanism of Baicalin Inhibiting β - hemolytic/cytolytic Activities of Streptococcus agalactiae

Baicalin, a natural compound, inhibits Streptococcus agalactiae β-hemolytic/cytolytic (β-h/c) activity, but its molecular mechanism remains unclear. We integrated DIA quantitative proteomics, untargeted metabolomics, and protein–ligand interaction analysis to elucidate this mechanism. Ninety differentially expressed proteins and 21 differential metabolites were identified. KEGG enrichment showed 19 overlapping pathways, 8 involved in β-h/c biosynthesis. RT-qPCR confirmed significant downregulation of β-h/c biosynthetic genes. Molecular docking revealed the strongest binding of baicalin to serine/threonine kinase Stk1 (Vina score of −10.0), as confirmed by molecular dynamics simulation. The interaction was further validated by DARTS, DSF, and ITC. Site-directed mutagenesis identified Val93, Thr153, and Asp154 in Stk1’s kinase domain as key binding residues. In vitro phosphorylation assays showed that baicalin inhibited Stk1-mediated CovR phosphorylation at T65, Y70, and T85, which underlies its inhibition of S. agalactiae β-h/c activity. This study deepens understanding of baicalin’s mechanism and provides a basis for its development as a bacterial virulence inhibitor.