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

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.