Glutamate-mediated metabolic rewiring boosts CpxA/R-OmpF porin and proton motive force to resensitize antibiotic-resistant Escherichia coli to ceftazidime

Ceftazidime (CAZ) is a critically important broad-spectrum antibiotic widely used in clinical practice. However, the rapid emergence of bacterial resistance to CAZ poses a significant challenge in treating infections caused by multidrug-resistant pathogens. In this study, we employed a metabolism-reprogramming approach to characterize key features of laboratory-evolved CAZ-resistant Escherichia coli K12 and identified repressed glutamate metabolism as a reprogrammable target. Exogenous glutamate effectively resensitized both lab-evolved and clinically isolated multidrug-resistant E. coli strains to CAZ. The resensitization mechanism operates through two synergistic pathways driven by glutamate metabolic flux. First, glutamate conversion to inosine activates the inosine–CpxA–CpxR–OmpF regulatory axis, increasing outer membrane permeability. Second, glutamate entry into the pyruvate cycle restores the proton motive force (PMF), energizing the inner membrane. Together, increased outer membrane permeability and a restored PMF synergistically enhance intracellular accumulation of CAZ—by facilitating its entry through the widened OmpF porin and promoting its active uptake across the cytoplasmic membrane. This dual-mechanism strategy provides a novel two-pronged approach to overcoming CAZ resistance. Our findings underscore the potential of targeting bacterial metabolic pathways to restore susceptibility and extend the utility of existing antibiotics against resistant pathogens.
Keywords: Multidrug-resistant bacteria; E. coli; glutamate; CpxA/R-OmpF axis; proton motive force; metabolic state-reprogramming