Global public health is currently confronted with a critical challenge in the form of antimicrobial resistance (Binder et al., 1999). Numerous studies suggest that bacterial cells may first develop increased tolerance before acquiring resistance, highlighting the importance of understanding the diverse tolerance mechanisms bacteria employ to survive antibiotic treatments (Levin-Reisman et al., 2017; Santi et al., 2021). Elucidating the role of metabolic pathways in conferring antibiotic resistance and tolerance is clinically relevant and therefore highly critical (Lopatkin et al., 2021). Aminoglycoside antibiotics display broad-spectrum activity against Gram-negative and Gram-positive bacteria by targeting their ribosomes. In this study, we have demonstrated that energy metabolism plays a crucial role in aminoglycoside tolerance, as knockout strains associated with the tricarboxylic acid cycle (TCA) and the electron transport chain (ETC) exhibited increased tolerance to aminoglycosides in the mid-exponential growth phase of
Escherichia coli cells. Given that aminoglycoside uptake relies on the ETC-driven electrochemical potential across the cytoplasmic membrane (Taber et al., 1987), our initial assumption was that these genetic perturbations would decrease the proton motive force and impair the aminoglycoside uptake. However, our results did not validate this assumption. We found no consistent metabolic changes, ATP levels, cytoplasmic pH variations, or membrane potential differences in the mutant strains compared to the wild type. Additionally, intracellular concentrations of fluorophore-labeled aminoglycoside 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 proteomics analyses highlighted a noteworthy upregulation of proteins linked to the TCA cycle in the mutant strains (
Figure 1A, 1B, 1C), 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 (
Figure 1D, 1E, 1F), 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.
Note: This study has been published in eLife (https://doi.org/10.7554/eLife.94903.1)
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Figure 1. The STRING visual network (Szklarczyk et al., 2023) depicts upregulated protein interactions of ΔsucA (A), ΔgltA (B) and ΔnuoI (C) mutants, and downregulated protein interactions for ΔsucA (D), ΔgltA (E), and ΔnuoI (F) mutants, compared to those of the wild-type strain.
