METABOLIC DISRUPTION IMPAIRS RIBOSOMAL PROTEIN LEVELS, RESULTING IN ENHANCED AMINOGLYCOSIDE TOLERANCE
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eLife assessment
The findings of this study are valuable as they challenge the dogma regarding the link between lowered bacterial metabolism and tolerance to aminoglycosides. The authors propose that the well-known tolerance to AG of mutants such as those of complexes I and II is not due to a decrease in the proton motive force and thus antibiotic uptake. The results presented here are solid but incomplete and the conclusions require additional experimental support.
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Abstract
Aminoglycoside antibiotics display broad-spectrum activity against Gram-negative and Gram-positive bacteria by targeting their ribosomes. Herein, 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 energy-driven electrochemical potential across the cytoplasmic membrane, our initial expectation was that these genetic perturbations would decrease the proton motive force (PMF), subsequently affecting the uptake of aminoglycosides. However, our results did not corroborate 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 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 during the mid-exponential growth phase, 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.
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eLife assessment
The findings of this study are valuable as they challenge the dogma regarding the link between lowered bacterial metabolism and tolerance to aminoglycosides. The authors propose that the well-known tolerance to AG of mutants such as those of complexes I and II is not due to a decrease in the proton motive force and thus antibiotic uptake. The results presented here are solid but incomplete and the conclusions require additional experimental support.
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Reviewer #1 (Public Review):
Summary:
In this study, the authors investigate the tolerance of aminoglycosides in E. coli mutants deleted in the Krebs cycle and respiratory chain enzymes. The motivation for this study is unclear. Transport of aminoglycosides is pmf-dependent, as the authors correctly note, and knocking out energy-producing components leads to tolerance of aminoglycosides, this has been well established. In S. aureus, clinically relevant "small colony" strains selected for in the course of therapy with aminoglycosides acquire null mutations in the biosynthesis of heme or ubiquinone, and have been studied in detail. In E. coli, such knockouts have not been reported in clinical isolates, probably due to severe fitness costs. At the same time, single-cell analysis has shown that individual cells with a decrease in the …Reviewer #1 (Public Review):
Summary:
In this study, the authors investigate the tolerance of aminoglycosides in E. coli mutants deleted in the Krebs cycle and respiratory chain enzymes. The motivation for this study is unclear. Transport of aminoglycosides is pmf-dependent, as the authors correctly note, and knocking out energy-producing components leads to tolerance of aminoglycosides, this has been well established. In S. aureus, clinically relevant "small colony" strains selected for in the course of therapy with aminoglycosides acquire null mutations in the biosynthesis of heme or ubiquinone, and have been studied in detail. In E. coli, such knockouts have not been reported in clinical isolates, probably due to severe fitness costs. At the same time, single-cell analysis has shown that individual cells with a decrease in the expression of Krebs cycle enzymes are tolerant of antibiotics and have lower ATP (Manuse et al., PLoS Biol 19: e3001194). The authors of the study under review report that knocking out ICD, isocitrate dehydrogenase that catalyzes the rate-limiting step in the Krebs cycle, has little effect on aminoglycoside tolerance and actually leads to an increase in the level of ATP over time. This observation does not seem to make much sense and contradicts previous reports, specifically that E. coli ICD is tolerant of antibiotics and, not surprisingly, produces Less ATP (Kabir and Shimizu, Appl Micro-biol Biotechnol. 2004; 65(1):84-96; Manuse et al., PLoS Biol 19: e3001194). Mutations in other Krebs cycle enzymes, unlike ICD, do lead to a dramatic increase in tolerance of aminoglycosides according to the paper under review. This is all very confusing.Apart from the confusing data, it is not clear what useful information may be obtained from the choice of the experimental system. The authors examine exponentially growing cells of E. coli for tolerance of aminoglycosides. The population at this stage of growth is highly susceptible to aminoglycosides, and only some rare persister cells can survive. However, the authors do not study persisters. A stationary population of E. coli is tolerant of aminoglycosides, and this is clinically relevant, but this is not the subject of the study.
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Reviewer #2 (Public Review):
Summary:
This interesting study challenges a dogma regarding the link between bacterial metabolism decrease and tolerance to aminoglycosides (AG). The authors demonstrate that mutants well-known for being tolerant to AG, such as those of complexes I and II, are not so due to a decrease in the proton motive force (PMF) and thus antibiotic uptake, as previously reported in the literature.Strengths:
This is a complete study. These results are surprising and are based on various read-outs, such as ATP levels, pH measurement, membrane potential, and the uptake of fluorophore-labeled gentamicin. Utilizing a proteomic approach, the authors show instead that in tolerant mutants, there is a decrease in the levels of proteins associated with ribosomes (targets of AG), causing tolerance.Weaknesses:
The use of a …Reviewer #2 (Public Review):
Summary:
This interesting study challenges a dogma regarding the link between bacterial metabolism decrease and tolerance to aminoglycosides (AG). The authors demonstrate that mutants well-known for being tolerant to AG, such as those of complexes I and II, are not so due to a decrease in the proton motive force (PMF) and thus antibiotic uptake, as previously reported in the literature.Strengths:
This is a complete study. These results are surprising and are based on various read-outs, such as ATP levels, pH measurement, membrane potential, and the uptake of fluorophore-labeled gentamicin. Utilizing a proteomic approach, the authors show instead that in tolerant mutants, there is a decrease in the levels of proteins associated with ribosomes (targets of AG), causing tolerance.Weaknesses:
The use of a single high concentration of aminoglycoside: my main comment on this study concerns the use of an AG concentration well above the MIC (50 µg/ml or 25 µg/ml for uptake experiments), which is 10 times higher than previously used concentrations (Kohanski, Taber) in study showing a link with PMF. This significant difference may explain the discrepancies in results. Indeed, a high concentration of AG can mask the effects of a metabolic disruption and lead to less specific uptake. However, this concentration highlights a second molecular level of tolerance. Adding experiments using lower concentrations (we propose 5 µg/ml to compare with the literature) would provide a more comprehensive understanding of AG tolerance mechanisms during a decrease in metabolism.Another suggestion would be to test iron limitation (using an iron chelator as DIP), which has been shown to induce AG tolerance. Can the authors demonstrate if this iron limitation leads to a decrease in ribosomal proteins? This experiment would validate their hypothesis in the case of a positive result. Otherwise, it would help distinguish two types of molecular mechanisms for AG tolerance during a metabolic disruption: (i) PMF and uptake at low concentrations, (ii) ribosomal proteins at high concentrations.
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