Despite the broad-spectrum antimicrobial activities of silver, its internal usage is restricted, owing to the toxicity. Strategies to enhance its efficacy are highly desirable but rely heavily on the understanding of its molecular mechanism of action. However, up to now, no direct silver-targeting proteins have been mined at a proteome-wide scale, which hinders systemic studies on the biological pathways interrupted by silver. Herein, we build up a unique system, namely liquid chromatography gel electrophoresis inductively coupled plasma mass spectrometry (LC-GE-ICP-MS), allowing 34 proteins directly bound by silver ions to be identified in Escherichia coli . By using integrated omic approaches, including metalloproteomics, metabolomics, bioinformatics, and systemic biology, we delineated the first dynamic antimicrobial actions of silver (Ag ~(+)) in E. coli , i.e., it primarily damages multiple enzymes in glycolysis and tricarboxylic acid (TCA) cycle, leading to the stalling of the oxidative branch of the TCA cycle and an adaptive metabolic divergence to the reductive glyoxylate pathway. It then further damages the adaptive glyoxylate pathway and suppresses the cellular oxidative stress responses, causing systemic damages and death of the bacterium. To harness these novel findings, we coadministrated metabolites involved in the Krebs cycles with Ag ~(+) and found that they can significantly potentiate the efficacy of silver both in vitro and in an animal model. Our study reveals the comprehensive and dynamic mechanisms of Ag ~(+) toxicity in E . coli cells and offers a novel and general approach for deciphering molecular mechanisms of metallodrugs in various pathogens and cells to facilitate the development of new therapeutics. The antimicrobial properties of silver are poorly understood. Tracking of the silver(I) proteome in Escherichia coli, in combination with metabolomics, bioinformatics and bioassays, delineates the dynamic antimicrobial actions of silver at a molecular level.
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