Carbon Sources Tune Antibiotic Susceptibility in Pseudomonas aeruginosa via Tricarboxylic Acid Cycle Control

Abstract

SUMMARY Metabolically dormant bacteria present a critical challenge to effective antimicrobial therapy because these bacteria are genetically susceptible to antibiotic treatment but phenotypically tolerant. Such tolerance has been attributed to impaired drug uptake, which can be reversed by metabolic stimulation. Here, we evaluate the effects of central carbon metabolite stimulations on aminoglycoside sensitivity in the pathogen Pseudomonas aeruginosa. We identify fumarate as a tobramycin potentiator that activates cellular respiration and generates a proton motive force by stimulating the tricarboxylic acid (TCA) cycle. In contrast, we find that glyoxylate induces phenotypic tolerance by inhibiting cellular respiration with acetyl-coenzyme A diversion through the glyoxylate shunt, despite drug import. Collectively, this work demonstrates that TCA cycle activity is important for both aminoglycoside uptake and downstream lethality and identifies a potential strategy for potentiating aminoglycoside treatment of P. aeruginosa infections.

Antibiotic efficacy — context matters

“…Experiments using the Seahorse XF Analyzer have captured real-time increases to cellular respiration by bactericidal antibiotics [33-35], complementing real-time measurements of overflow ROS production using electrochemical [36] or genetically encoded biosensors [33]. Interestingly, integrated analyses of transcriptomic and metabolomic data have revealed TCA cycle activity to be critical for antibiotic lethality, independent from drug uptake [37]. Collectively, these studies, and several others [8,38-45], demonstrate that changes to bacterial metabolism participate in lethality for many bactericidal antibiotics.…”

“…It is our view that much of the misunderstanding over antibiotic lethality is derived from the difficulty in experimentally separating the lethal contributions of essential gene product inhibition from those of downstream mechanisms. It may be possible to distinguish between these biochemically, as the lethality driven by secondary processes can be interrupted by inhibiting protein translation and cell respiration [34] or by metabolic shunting [37]. Cell death emerging from stress-induced death processes was recently studied in an antibiotic-free system using a historically significant fusion protein [64].…”

“…Under anoxia, nitrate frequently participates in anaerobic respiration as the terminal electron acceptor and can potentiate antibiotic killing [33]; it is not yet known if nitrate and other terminal electron acceptors also generate toxic metabolic byproducts that contribute to antibiotic death processes. Cellular respiration likely affects antibiotic efficacy due to its roles in metabolism and energy production, but may also contribute to lethality by facilitating drug import [37,71] or stimulating intracellular alkalization [72]. Collectively, these studies implicate respiratory activity as an important mediator of the lethal processes induced by antibiotics.…”

“…Recent studies using flow cytometry and high-throughput assays have revealed how specific carbon metabolites or amino acids may enhance antibiotic death processes by fueling TCA cycle activity, increasing cellular respiration and inducing a proton motive force, promoting both drug uptake and subsequent lethality [37,71,75,76]. Interestingly, flux through the glyoxylate shunt was shown to suppress aminoglycoside potentiation by metabolites such as fumarate despite measureable drug uptake, implicating an active metabolic component to antibiotic lethality [37]. This is supported by recent chemogenetic screens revealing collateral antibiotic sensitivity under nutrient limitation [77].…”

<b><i>Pseudomonas aeruginosa</i></b> and <b><i>Klebsiella</i></b> <b><i>pneumoniae</i></b> Adaptation to Innate Immune Clearance Mechanisms in the Lung

“…In addition to the oxidant stress generated by bacterial metabolism, which for P. aeruginosa is preferentially oxidative, there are numerous sources of oxidant stress in vivo. In a clinical setting such as CF, organisms are continually exposed to antimicrobial agents, which, despite their specific bacterial targets, wind up killing through the production of toxic oxidants [23]. Low-level exposure to aminoglycosides, as well as the fluoroquinolones that induce DNA damage, presents a major inducer of oxidant stress within the bacteria and inhibits bacterial growth.…”