Proton motive force as a driver of persister sensitization by adenosine.

Antibiotic tolerance and persistence contribute to the emergence of antimicrobial resistance, yet strategies to reverse these phenotypes remain limited. Our previous work revealed that the naturally occurring nucleoside adenosine can reverse antibiotic tolerance in diverse bacterial strains by modulating cellular energetics. Here, we define the mechanism underlying this potentiation, identifying adenosine metabolism as a driver of cytoplasmic alkalinization and proton motive force (PMF) generation. Using RNA sequencing, metabolite assays, and pH-sensitive fluorescent reporters, we show that adenosine is catabolized by purine nucleoside phosphorylase (deoD) to yield ribose-1-phosphate, which enters the pentose phosphate pathway. This metabolic flux stimulates the electron transport chain, leading to proton translocation, increased cytoplasmic pH, and enhanced PMF. Disruption of key metabolic enzymes (deoD, deoB, tktAB) or the inhibition of enolase abolishes both alkalinization and antibiotic sensitization. Using a respiratory-deficient mutant, we demonstrate that aerobic respiration is the primary driver of alkalinization and gentamicin potentiation, although adenosine can partially increase membrane potential independently of oxidative phosphorylation. These findings support a model in which adenosine metabolism promotes aminoglycoside uptake via PMF-driven transport, sensitizing tolerant bacteria to killing. Our work implicates the ribose moiety of adenosine as a key metabolic lever for reversing tolerance. Broader exploration of nucleoside-based adjuvants across bacterial species and antibiotic classes may reveal generalizable strategies to enhance antibiotic efficacy against recalcitrant infections.