The antibacterial efficacy of nitroxoline against multidrug resistant Escherichia coli associated with copper binding

Nitroxoline (NIT) has been approved for the treatment of uncomplicated urinary tract infections (UTIs) for more than half a century, yet its antimicrobial properties remain incompletely understood. Here, we determined the intricate connections between NIT's metal-chelating capabilities and its antibacterial activity. Metal ion binding characteristics were measured by Ultraviolet-visible (UV-vis) spectroscopy. Biochemical assays and molecular docking studies were performed to elucidate the underlying mechanism. We found that NIT could interact with a diverse array of metal ions, including Cu²⁺, Fe²⁺, Zn²⁺ and Mn²⁺. While, the addition of Cu²⁺ significantly decreased NIT's antibacterial effect against uropathogenic Escherichia coli (UPEC) strain J96 and multidrug resistant E coli B2, with the minimum inhibitory concentration (MIC) increased from 8 mg/L to 64 mg/L. Mechanically, NIT significantly decreased the intracellular copper ion levels and reduced bacterial transmembrane electrical potential. Furthermore, NIT promoted production of nitric oxide, peroxynitrite (ONOO-), and reactive oxygen species (ROS). However, the interaction of Cu²⁺ with NIT suppressed the induced generation of ROS but not the generation of ONOO- in E coli, suggesting that the antibacterial activity of NIT arose from multiple functional groups within its molecular structure. Moreover, NIT triggered intracellular acidification concomitant with enhanced glucose uptake, yet paradoxically suppressed ATP generation, suggesting a potential uncoupling between glycolytic flux and oxidative phosphorylation. Finally, the action of NIT was predicted to bind to the CuB-metal redox centers of cytochrome bo(3) ubiquinol oxidase through molecular docking analyses. Collectively, these data illuminate the antibacterial activity of NIT as a potent copper-related metalloantibiotic against UPEC.