Chemoproteomics in Live Bacteria Identifies Covalent Interactome of Epigallocatechin-3-Gallate (EGCG) Underlying Its Multimodal Antibacterial Mechanism.

Abstract

Epigallocatechin-3-gallate (EGCG), the most bioactive catechin of green tea, exhibits remarkable antibacterial activity against a wide range of bacteria, including multidrug-resistant strains. However, its precise molecular targets and mechanisms of action remain poorly understood. Herein, we have designed YnEGCG, a clickable and cell-permeable activity-based probe of EGCG, to comprehensively investigate its molecular targets through in situ chemoproteomics. YnEGCG enabled the fluorescence visualization and mass-spectrometric identification of covalently interacting proteins from live E. coli. Quantitative proteomics identified over 600 proteins, with approximately 10% exhibiting remarkably high enrichment (H:L > 50). These included critical bacterial enzymes such as DNA gyrase, DNA polymerase, ATP synthase, ribosomal proteins, etc., and several previously unidentified targets. Further, we experimentally validated that EGCG inhibits DNA gyrase activity as well as de novo protein synthesis. Taken together, our in situ chemoproteomics studies revealed that EGCG binds to critical bacterial enzymes, uncovering previously unknown antibacterial targets and providing insights into its broad-spectrum action.