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      Pyruvate cycle increases aminoglycoside efficacy and provides respiratory energy in bacteria

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          Abstract

          <p id="d7424902e366">Exogenous metabolites have been documented to potentiate antibiotics to kill multidrug-resistant pathogens, but the mechanisms are largely unknown. The work presented here shows that intermetabolites from the TCA cycle and the phosphoenolpyruvate (PEP)-pyruvate-AcCoA pathway have the potential to improve targeting of these resistant microorganisms. The enzymes that connect the PEP-pyruvate-AcCoA pathway to the TCA cycle are essential for this potentiation, indicating both pathway and cycle can be merged to be considered a pyruvate cycle (P cycle). The P cycle operates routinely as a general mechanism for energy production and regulating the TCA cycle in <i>Escherichia coli</i> and <i>Edwardsiella tarda</i>. The results reported here provide insights into metabolite-facilitated targeting by antibiotics as well as bacterial energy metabolism and homeostasis. </p><p class="first" id="d7424902e375">The emergence and ongoing spread of multidrug-resistant bacteria puts humans and other species at risk for potentially lethal infections. Thus, novel antibiotics or alternative approaches are needed to target drug-resistant bacteria, and metabolic modulation has been documented to improve antibiotic efficacy, but the relevant metabolic mechanisms require more studies. Here, we show that glutamate potentiates aminoglycoside antibiotics, resulting in improved elimination of antibiotic-resistant pathogens. When exploring the metabolic flux of glutamate, it was found that the enzymes that link the phosphoenolpyruvate (PEP)-pyruvate-AcCoA pathway to the TCA cycle were key players in this increased efficacy. Together, the PEP-pyruvate-AcCoA pathway and TCA cycle can be considered the pyruvate cycle (P cycle). Our results show that inhibition or gene depletion of the enzymes in the P cycle shut down the TCA cycle even in the presence of excess carbon sources, and that the P cycle operates routinely as a general mechanism for energy production and regulation in <i>Escherichia coli</i> and <i>Edwardsiella tarda</i>. These findings address metabolic mechanisms of metabolite-induced potentiation and fundamental questions about bacterial biochemistry and energy metabolism. </p>

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          Most cited references46

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          A common mechanism of cellular death induced by bactericidal antibiotics.

          Antibiotic mode-of-action classification is based upon drug-target interaction and whether the resultant inhibition of cellular function is lethal to bacteria. Here we show that the three major classes of bactericidal antibiotics, regardless of drug-target interaction, stimulate the production of highly deleterious hydroxyl radicals in Gram-negative and Gram-positive bacteria, which ultimately contribute to cell death. We also show, in contrast, that bacteriostatic drugs do not produce hydroxyl radicals. We demonstrate that the mechanism of hydroxyl radical formation induced by bactericidal antibiotics is the end product of an oxidative damage cellular death pathway involving the tricarboxylic acid cycle, a transient depletion of NADH, destabilization of iron-sulfur clusters, and stimulation of the Fenton reaction. Our results suggest that all three major classes of bactericidal drugs can be potentiated by targeting bacterial systems that remediate hydroxyl radical damage, including proteins involved in triggering the DNA damage response, e.g., RecA.
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            Antibiotic efficacy is linked to bacterial cellular respiration.

            Bacteriostatic and bactericidal antibiotic treatments result in two fundamentally different phenotypic outcomes--the inhibition of bacterial growth or, alternatively, cell death. Most antibiotics inhibit processes that are major consumers of cellular energy output, suggesting that antibiotic treatment may have important downstream consequences on bacterial metabolism. We hypothesized that the specific metabolic effects of bacteriostatic and bactericidal antibiotics contribute to their overall efficacy. We leveraged the opposing phenotypes of bacteriostatic and bactericidal drugs in combination to investigate their activity. Growth inhibition from bacteriostatic antibiotics was associated with suppressed cellular respiration whereas cell death from most bactericidal antibiotics was associated with accelerated respiration. In combination, suppression of cellular respiration by the bacteriostatic antibiotic was the dominant effect, blocking bactericidal killing. Global metabolic profiling of bacteriostatic antibiotic treatment revealed that accumulation of metabolites involved in specific drug target activity was linked to the buildup of energy metabolites that feed the electron transport chain. Inhibition of cellular respiration by knockout of the cytochrome oxidases was sufficient to attenuate bactericidal lethality whereas acceleration of basal respiration by genetically uncoupling ATP synthesis from electron transport resulted in potentiation of the killing effect of bactericidal antibiotics. This work identifies a link between antibiotic-induced cellular respiration and bactericidal lethality and demonstrates that bactericidal activity can be arrested by attenuated respiration and potentiated by accelerated respiration. Our data collectively show that antibiotics perturb the metabolic state of bacteria and that the metabolic state of bacteria impacts antibiotic efficacy.
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              H2S: a universal defense against antibiotics in bacteria.

              Many prokaryotic species generate hydrogen sulfide (H(2)S) in their natural environments. However, the biochemistry and physiological role of this gas in nonsulfur bacteria remain largely unknown. Here we demonstrate that inactivation of putative cystathionine β-synthase, cystathionine γ-lyase, or 3-mercaptopyruvate sulfurtransferase in Bacillus anthracis, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli suppresses H(2)S production, rendering these pathogens highly sensitive to a multitude of antibiotics. Exogenous H(2)S suppresses this effect. Moreover, in bacteria that normally produce H(2)S and nitric oxide, these two gases act synergistically to sustain growth. The mechanism of gas-mediated antibiotic resistance relies on mitigation of oxidative stress imposed by antibiotics.
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                Author and article information

                Journal
                Proceedings of the National Academy of Sciences
                Proc Natl Acad Sci USA
                Proceedings of the National Academy of Sciences
                0027-8424
                1091-6490
                February 13 2018
                February 13 2018
                February 13 2018
                January 30 2018
                : 115
                : 7
                : E1578-E1587
                Article
                10.1073/pnas.1714645115
                5816162
                29382755
                4b5cc5e0-4e4a-4f17-983e-6ed3449587c6
                © 2018

                Free to read

                http://www.pnas.org/site/misc/userlicense.xhtml

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