LAUSR

CFDC was the first FDA-approved siderophore cephalosporin antibiotic in 2019 and is known for its Trojan horse tactics and broad antimicrobial activity against Gram-negative bacteria. However, its antibacterial mechanism is not fully understood, and whether it has an impact on in vivo iron ion homeostasis remains unknown. ABSTRACT This study aimed to investigate the antibacterial mechanism of cefiderocol (CFDC) using data-independent acquisition quantitative proteomics combined with cellular and molecular biological assays. Numerous differentially expressed proteins related to the production of NADH, reduced cofactor flavin adenine dinucleotide (FADH2), NADPH and reactive oxygen species (ROS), iron-sulfur cluster binding, and iron ion homeostasis were found to be upregulated by CFDC. Furthermore, parallel reaction monitoring analysis validated these results. Meanwhile, we confirmed that the levels of NADH, ROS, H2O2, and iron ions were induced by CFDC, and the sensitivity of Escherichia coli to CFDC was inhibited by the antioxidant vitamin C, N-acetyl-l-cysteine, and deferoxamine. Moreover, deferoxamine also suppressed the H2O2 stress induced by CFDC. In addition, knockout of the NADH-quinone oxidoreductase genes (nuoA, nuoC, nuoE, nuoF, nuoG, nuoJ, nuoL, nuoM) in the respiratory chain attenuated the sensitivity of E. coli to CFDC far beyond the effects of cefepime and ceftazidime; in particular, the E. coli BW25113 ΔnuoJ strain produced 60-fold increases in MIC to CFDC compared to that of the wild-type E. coli BW25113 strain. The present study revealed that CFDC exerts its antibacterial effects by inducing ROS stress by elevating the levels of NADH and iron ions in E. coli. IMPORTANCE CFDC was the first FDA-approved siderophore cephalosporin antibiotic in 2019 and is known for its Trojan horse tactics and broad antimicrobial activity against Gram-negative bacteria. However, its antibacterial mechanism is not fully understood, and whether it has an impact on in vivo iron ion homeostasis remains unknown. To comprehensively reveal the antibacterial mechanisms of CFDC, data-independent acquisition quantitative proteomics combined with cellular and molecular biological assays were performed in this study. The findings will further facilitate our understanding of the antibacterial mechanism of CFDC and may provide a theoretical foundation for controlling CFDC resistance in the future.