Metabolic disruption impairs ribosomal protein levels, resulting in enhanced aminoglycoside tolerance

Abstract

Aminoglycosides, a class of antibiotics, have been in use for decades, displaying broad-spectrum activity against Gram-negative and Gram-positive bacteria. They target ribosomes and disrupt protein synthesis. Although their use declined due to newer antibiotics with lower toxicity, increasing drug resistance has renewed interest in aminoglycosides. Herein, we have demonstrated that energy metabolism plays a crucial role in aminoglycoside tolerance, as knockout strains with deleted genes associated with the tricarboxylic acid cycle (TCA) and the electron transport chain (ETC) exhibited increased tolerance to aminoglycosides in the mid-exponential growth phase ofEscherichia colicells. Our initial hypothesis posited that genetic perturbations would lead to a reduction in the proton motive force, subsequently affecting the uptake of aminoglycosides. This hypothesis is based on the prevailing notion that aminoglycoside uptake is dependent on the distinctive and energy-driven electrochemical potential across the cytoplasmic membrane. However, our results did not support this hypothesis. Despite genetic perturbations in mutant strains, we found no consistent metabolic changes, ATP levels, cytoplasmic pH variations, or membrane potential differences compared to wild-type strains. Additionally, intracellular concentrations of fluorophore-labeled gentamicin remained similar across all strains. To uncover the mechanism responsible for the observed tolerance in mutant strains, we employed untargeted mass spectrometry to quantify the proteins within these mutants and subsequently compared them to their wild-type counterparts. Our comprehensive analysis, which encompassed protein-protein association networks and functional enrichment, unveiled a noteworthy upregulation of proteins linked to the TCA cycle in the mutant strains, suggesting that these strains compensate for the perturbation in their energy metabolism by increasing TCA cycle activity to maintain their membrane potential and ATP levels. Furthermore, our pathway enrichment analysis shed light on local network clusters displaying downregulation across all mutant strains, which were associated with both large and small ribosomal binding proteins, ribosome biogenesis, translation factor activity, and the biosynthesis of ribonucleoside monophosphates. These findings offer a plausible explanation for the observed tolerance of aminoglycosides in the mutant strains. Altogether, this research has the potential to uncover mechanisms behind aminoglycoside tolerance, paving the way for novel strategies to combat such cells.