Chemical proteomics reveals mechanisms of bacterial response to ROS mediated by antibiotics

The role of reactive oxygen species (ROS) in antibiotic-mediated bacterial killing and the emergence of resistance remains incompletely understood, particularly the identities and functional impacts of specific protein targets. To address this, we employed a site-centric chemoproteomics approach to map dynamic changes in the Escherichia coli cysteinome upon treatment with eight diverse antibiotics. Functional annotation revealed that these redox-sensitive proteins are enriched in crucial cellular processes such as amino acid biosynthesis and redox homeostasis. We further demonstrated that antibiotic-induced ROS directly regulate specific cysteine residues, C102 in MnmA, C116 in NemR, and C337 in TyrR, altering their functions through mechanisms including impaired enzymatic activity, induced dimerization, and modulated gene expression. These redox-mediated functional changes ultimately dictate E. coli's susceptibility to antibiotics. Our findings establish a direct molecular link between antibiotic-triggered ROS, specific cysteine modifications in key proteins, and bacterial survival, offering a new framework for strategies aimed at enhancing antibiotic efficacy.