Quantitative Proteomic Analysis Reveals That Central Metabolic Pathways Influence Norfloxacin Susceptibility in Vibrio alginolyticus

Abstract

Reduced antibiotic susceptibility often involves reversible metabolic reprogramming. Here, data-independent acquisition (DIA)-based quantitative proteomics was employed to profile the response of Vibrio alginolyticus (V. alginolyticus) to norfloxacin (NOR) exposure, revealing a global metabolic downshift marked by prominent reductions in central carbon, energy, and amino acid pathways. High-throughput phenotypic screening identified 11 exogenous metabolites, including glycine (Gly) and glutamine (Gln), that significantly potentiated NOR-mediated growth inhibition. Mechanistically, physiological assays demonstrated that glycine supplementation perturbed cellular redox balance, accompanied by elevated reactive oxygen species (ROS) levels and a reduction in the intracellular reduced glutathione (GSH) pool under NOR challenge. Furthermore, mRNA expression analysis (qPCR) revealed that while NOR restricted transcripts of the downstream anaplerotic Gln-to-2-oxoglutarate axis, including the 2-oxoglutarate dehydrogenase complex and NAD+-dependent glutamate dehydrogenase, exogenous Gln supplementation effectively alleviated this transcriptional suppression. Collectively, these findings demonstrate that targeted metabolite supplementation overrides defensive low-flux states, forcing metabolic and transcriptional reactivation that intensifies antibiotic efficacy via ROS-mediated oxidative stress and impairment of antioxidant defense.