Metabolic activation of pyruvate cycle by protocatechualdehyde triggers oxidative killing of ampicillin-resistant Escherichia coli

ABSTRACT Antimicrobial resistance has emerged as a major global public health threat, underscoring the urgent need for novel therapeutic strategies. In this study, we demonstrate that protocatechualdehyde (PA), a natural compound derived from Salvia miltiorrhiza , exhibits potent and time-dependent bactericidal activity against ampicillin-resistant Escherichia coli . PA was also effective against AmpC β-lactamase-expressing strains and clinically isolated multidrug-resistant strains of E. coli , Pseudomonas aeruginosa , and Klebsiella pneumoniae , notably by exerting a combined effect with aminoglycoside antibiotics to overcome resistance. Mechanistically, integrated metabolomic and functional analyses reveal that PA induces a profound metabolic reprogramming in ampicillin-resistant Escherichia. coli , characterized by the hyperactivation of central carbon metabolism, with pyruvate catabolism serving as a critical hub. This forced metabolic flux leads to a surge in intracellular ATP and NADH, ultimately driving an overload of the electron transport chain and a lethal burst of reactive oxygen species (ROS). Genetic and chemical inhibition of the pyruvate dehydrogenase complex attenuates both ROS production and the bactericidal effect, confirming the causal link between metabolic disruption and bacterial death. PA treatment markedly improved survival and reduced bacterial burden in a murine systemic infection model, suggesting its therapeutic potential for infections. These findings provide a foundational rationale for developing PA-based therapeutics or derivatives to combat multidrug-resistant Gram-negative infections, particularly in combination with aminoglycoside antibiotics. IMPORTANCE The rising prevalence of multidrug-resistant Gram-negative pathogens is limiting treatment options. This study identifies the natural compound PA as an effective bactericidal agent against ampicillin-resistant and clinically relevant multi-drug-resistant (MDR) Escherichia coli and other Gram-negative species. Importantly, we elucidate a previously unreported mechanism whereby PA hijacks bacterial central metabolism, specifically pyruvate metabolism, leading to metabolic overactivation, accumulation of NADH and ATP, and ultimately lethal reactive oxygen species (ROS) production. Furthermore, under the combined effect of PA and aminoglycoside antibiotics, their minimum inhibitory concentrations are reduced against resistant strains. These findings support the therapeutic potential of PA as either a standalone or adjunctive treatment for drug-resistant infections. This work emphasizes the value of targeting bacterial metabolism as a viable strategy to combat antimicrobial resistance.