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.