Worledge, C. S. | bioRxiv Science

bioRxiv2025

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 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, though 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.

microbiology↗

bioRxiv2025

Regulation of Epithelial HIF by Probiotic Escherichia coli

The gastrointestinal tract is home to trillions of microorganisms that interact with their host in profound ways, including regulation of immune, endocrine, and neurological functions. One mechanism by which these microbes interact with their eukaryotic host is through the generation of short-chain fatty acids (SCFAs), which are metabolized by the intestinal epithelium creating a state of "physiologic hypoxia". This hypoxia, in turn, results in stabilization and activation of hypoxia-inducible factor (HIF), a transcription factor family shown to support gut barrier function and homeostasis, in the intestinal epithelium. The association between HIF and intestinal homeostasis has been long understood, as both genetic and pharmacologic potentiation of the HIF signaling pathway has been shown to promote barrier function both in vitro and in vivo. Although it has been previously established that pathogenic bacteria regulate HIF stabilization and activity in the intestinal epithelium independent of SCFA metabolism, it is not clear whether this property extends to noninfectious and/or commensal bacterial species. Here, we demonstrate that nonpathogenic, commensal strains of Escherichia coli stabilize HIF in intestinal epithelial cells in vitro. Further, we show that HIF is transcriptionally active in these cells and drives a "pro-barrier" transcriptional program. This property was found to be dependent on bacterial aerobic respiration, as genetic elimination of E. coli aerobic respiration abolished HIF stabilization and the subsequent transcriptional phenotype. Finally, we observed induction of tissue hypoxia in vivo using antibiotic-treated mice colonized with wild-type, but not respiration-deficient, E. coli. These findings demonstrate a novel ability for probiotic E. coli to regulate intestinal homeostasis through activation of HIF and suggest that this mechanism might be leveraged in as a novel therapeutic to combat intestinal inflammation, such as that observed during inflammatory bowel disease (IBD).

cell biology↗