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Iron Homeostasis Affects Antibiotic-mediated Cell Death in Pseudomonas Species*

  • Jinki Yeom
    Affiliations
    From the Division of Environmental Science and Ecological Engineering, Korea University, Seoul, 136-713, South Korea and
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  • James A. Imlay
    Correspondence
    To whom correspondence may be addressed: Dept. of Microbiology, University of Illinois, Urbana, IL 61801. Tel.: 1-217-333-5812; Fax: 1-217-244-6697;
    Affiliations
    the Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801
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  • Woojun Park
    Correspondence
    To whom correspondence may be addressed: Division of Environmental Science and Ecological Engineering, Korea University, Seoul, 136-713, South Korea. Tel.: 82-2-3290-3067; Fax: 82-2-953-0737;
    Affiliations
    From the Division of Environmental Science and Ecological Engineering, Korea University, Seoul, 136-713, South Korea and
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  • Author Footnotes
    * This work was supported by Grant 20090091491 from the MEST/NRF to the Environment Biotechnology National Core Research Center and Grant 2009-0076488from the MEST/NRF program, South Korea.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1 and 2 and Figs. 1–9.
Open AccessPublished:May 17, 2010DOI:https://doi.org/10.1074/jbc.M110.127456
      Antibiotics can induce cell death via a variety of action modes, including the inhibition of transcription, ribosomal function, and cell wall biosynthesis. In this study, we demonstrated directly that iron availability is important to the action of antibiotics, and the ferric reductases of Pseudomonas putida and Pseudomonas aeruginosa could accelerate antibiotic-mediated cell death by promoting the Fenton reaction. The modulation of reduced nicotinamide-adenine dinucleotide (NADH) levels and iron chelation affected the actions of antibiotics. Interestingly, the deletion of the ferric reductase gene confers more antibiotic resistance upon cells, and its overexpression accelerates antibiotic-mediated cell death. The results of transcriptome analysis showed that both Pseudomonas species induce many oxidative stress genes under antibiotic conditions, which could not be observed in ferric reductase mutants. Our results indicate that iron homeostasis is crucial for bacterial cell survival under antibiotics and should constitute a significant target for boosting the action of antibiotics.

      Introduction

      Pseudomonas species perform key roles in the environment, including the degradation of natural and man-made chemicals and the establishment of important interactions with plants and animals (
      • Gómez M.I.
      • Prince A.
      ,
      • Gooderham W.J.
      • Hancock R.E.
      ,
      ). One pseudomonad, Pseudomonas aeruginosa, is a focus of particular study because it is the predominant opportunistic pathogen of cystic fibrosis patients (
      • Gómez M.I.
      • Prince A.
      ,
      • Thomas C.M.
      ). Clearance is difficult because of its unusually low susceptibility to antibiotic treatment (
      • Gooderham W.J.
      • Hancock R.E.
      ,
      • Page M.G.
      • Heim J.
      ). Several general factors contribute to this resistance, including the low permeability of the cellular envelope and the actions of multidrug efflux pumps (
      • Jeannot K.
      • Elsen S.
      • Köhler T.
      • Attree I.
      • van Delden C.
      • Plésiat P.
      ,
      • Lister P.D.
      • Wolter D.J.
      • Hanson N.D.
      ). In addition, quinolone treatment can be confounded by the outgrowth of subpopulations that have an altered lipid composition that is poorly permeable to hydrophilic quinolones (
      • Michéa-Hamzehpour M.
      • Furet Y.X.
      • Pechère J.C.
      ,
      • Norris M.J.
      • Rogers D.T.
      • Russell A.D.
      ,
      • Perz J.F.
      • Craig A.S.
      • Stratton C.W.
      • Bodner S.J.
      • Phillips Jr., W.E.
      • Schaffner W.
      ,
      • Rolston K.V.
      • Kontoyiannis D.P.
      • Yadegarynia D.
      • Raad I.I.
      ). Ominously, integrons that harbor genes encoding metallo-β-lactamase have also been found in the transposons and plasmids of Pseudomonas putida isolates (
      • Docquier J.D.
      • Riccio M.L.
      • Mugnaioli C.
      • Luzzaro F.
      • Endimiani A.
      • Toniolo A.
      • Amicosante G.
      • Rossolini G.M.
      ,
      • Poirel L.
      • Cabanne L.
      • Collet L.
      • Nordmann P.
      ), suggesting that these genetic elements might be transferred to P. aeruginosa and the Enterobacteriaceae (
      ). As many pseudomonads inhabit natural environments in which antibiotic exposure in possible, and because P. aeruginosa in particular comprises a public health hazard, Pseudomonas species are model strains for studying the development of antibiotic resistance.
      Until recently, bactericidal antibiotics were believed to kill cells by several well established mechanisms, typically involving the disruption of cell wall biosynthesis (ampicillin), the interruption of DNA replication (norfloxacin), or the overwhelming inhibition of protein synthesis (gentamicin and kanamycin) (
      • Dwyer D.J.
      • Kohanski M.A.
      • Collins J.J.
      ,
      • Kohanski M.A.
      • Dwyer D.J.
      • Hayete B.
      • Lawrence C.A.
      • Collins J.J.
      ). However, a system-analysis investigation conducted with Escherichia coli by Kohanski, Collins, and co-workers (
      • Dwyer D.J.
      • Kohanski M.A.
      • Collins J.J.
      ,
      • Kohanski M.A.
      • Dwyer D.J.
      • Hayete B.
      • Lawrence C.A.
      • Collins J.J.
      ,
      • Kohanski M.A.
      • Dwyer D.J.
      • Wierzbowski J.
      • Cottarel G.
      • Collins J.J.
      ) raised the possibility that in these organisms, bacteriocidal antimicrobials might additionally create oxidative stress, and that a part of their toxicity in aerobic habitats might be due to the accumulation of reactive oxygen species. Reactive oxygen species such as superoxide and hydrogen peroxide can block growth by inactivating key enzymes; additionally, they also are precursors of the hydroxyl radical (
      • Imlay J.A.
      • Linn S.
      ). The latter species is formed through the Fenton reaction (
      • Imlay J.A.
      • Linn S.
      ,
      • Touati D.
      ), in which unincorporated intracellular iron transfers an electron to hydrogen peroxide (Reaction 1).
      Fe2++H2O2Fe3++OH+HOFe3++ reductant Fe2+
      REACTIONS 1 AND 2


      The process is cyclical in vivo because intracellular reductants, including cysteine and reduced flavins, can reduce the oxidized iron back to its ferrous form (Reaction 2). The hydroxyl radical is powerful oxidant enough to react with either the base or sugar residues of DNA, leading to base modification and strand breakage. Because iron associates easily with nucleic acids, DNA is a common target, and, in fact, DNA damage is the cause of cell death when cells are stressed with either exogenous or endogenous H2O2 (
      • Touati D.
      ,
      • Woodmansee A.N.
      • Imlay J.A.
      ). Strikingly, E. coli was substantially protected against the lethal effects of antibiotics by cell-permeable iron chelators that inhibit the Fenton reaction (
      • Woodmansee A.N.
      • Imlay J.A.
      ).
      In this study, we tested whether oxidative stress is a significant component of antibiotic action against two Pseudomonas species, P. putida KT2440 and P. aeruginosa PAO1. Transcriptional profiling data confirmed that the expression of antioxidant enzymes was induced during exposure to a variety of antibiotics, and fluorescent probes indicated an increase in intracellular oxidants. DNA damage was detected, and cell death depended upon unincorporated iron and was facilitated by ferredoxin reductase, an iron-reducing enzyme that catalyzes reaction 2 above. Collectively, these data provide further support to the notion that antibiotics create oxidative stress, and they demonstrate that this phenomenon is not limited to enteric bacteria.

      DISCUSSION

      Although antibiotics remain potent in anaerobic habitats, our data extend the observation that in aerobic habitats, they may impose an additional, oxidative stress upon target bacteria. Generally, bacteria require iron to survive as well as pseudomonads (
      • Woodmansee A.N.
      • Imlay J.A.
      ). To acquire iron from the soil environment, bacteria make biosynthetic iron chelators, which are known as siderophores. However, under conditions of excessive intracellular iron concentration, iron can cause bacterial cell death via the Fenton reaction (
      • Woodmansee A.N.
      • Imlay J.A.
      ). In such cases, the intracellular iron concentration must be adjusted clearly. Our study provides evidence to suggest that antibiotics can boost oxidative stress in the presence of specific iron concentrations. Our data directly showed that i) antibiotics generate oxidative stress, ii) iron concentration is an important factor in the mode of antibiotic action, iii) ferric reductase might accelerate bacterial cell death under antibiotic conditions, and iv) many oxidative stress defense and DNA repair genes are highly up-regulated in microarray analysis. These conclusions pertained both to the soil environment model strain, P. putida, and to the human pathogen, P. aeruginosa. Thus, oxidative stress appears to be a common mechanism of antibiotic-induced cellular death (
      • Kohanski M.A.
      • Dwyer D.J.
      • Hayete B.
      • Lawrence C.A.
      • Collins J.J.
      ,
      • Albesa I.
      • Becerra M.C.
      • Battán P.C.
      • Páez P.L.
      ,
      • Wang X.
      • Zhao X.
      ). It is far from clear why this is the case. One notion is that antibiotics somehow stimulate the intracellular formation of superoxide and/or hydrogen peroxide (
      • Kohanski M.A.
      • Dwyer D.J.
      • Hayete B.
      • Lawrence C.A.
      • Collins J.J.
      ,
      • Albesa I.
      • Becerra M.C.
      • Battán P.C.
      • Páez P.L.
      ). Because Pseudomonas strains synthesize pyocyanin (
      • Hernandez M.E.
      • Newman D.K.
      ,
      • Mavrodi D.V.
      • Bonsall R.F.
      • Delaney S.M.
      • Soule M.J.
      • Phillips G.
      • Thomashow L.S.
      ), a redox-cycling antibiotic that can generate reactive oxygen species, we tested whether it might be involved in the oxidative stress phenomena that are triggered by antibiotics. However, antibiotics continued to stimulate dihydrorhodamine oxidation in phzM and phzS mutants that cannot synthesize pyocyanin (supplemental Fig. 9) and antibiotics did not induce synthesis of pyocyanin (data not shown).
      So how might antibiotics stimulate intracellular oxidant formation? Oxidative stress must be secondary to the primary modes of action of antibiotics because mutations that circumvent the action of aminoglycosides upon ribosomes or of ampicillin upon penicillin-binding proteins also block cell death (
      • Eckert B.
      • Beck C.F.
      ). One clue might be the observation that oxidative injuries appear to be created by treatments that inhibit DNA replication or that activate addiction proteins, which causes programmed cell death in prokaryote cells (
      • Kohanski M.A.
      • Dwyer D.J.
      • Wierzbowski J.
      • Cottarel G.
      • Collins J.J.
      ,
      • Eckert B.
      • Beck C.F.
      ,
      • Davies B.W.
      • Kohanski M.A.
      • Simmons L.A.
      • Winkler J.A.
      • Collins J.J.
      • Walker G.C.
      ). A common element to these stresses may be that they, like ribosomal inhibitors, cause incomplete protein peptides to be formed. It seems possible that these peptides might associate with metabolic redox enzymes in a way that disrupts their function, perhaps causing them to transfer electrons to oxygen rather than to their physiological acceptor (
      • Davies B.W.
      • Kohanski M.A.
      • Simmons L.A.
      • Winkler J.A.
      • Collins J.J.
      • Walker G.C.
      ). However, it is important to note that the methods that have been used to document oxidative stress under these conditions have thus far been indirect, so the idea that H2O2 production is accelerated should not be regarded as proven (
      • Hassett D.J.
      • Imlay J.A.
      ). Further, given that ampicillin is known only to act directly as an inhibitor of cell wall biosynthesis, its effects do not easily fit the small peptide model. Thus, the immediate challenge in the field remains the discovery of a molecular connection between antibiotics and oxidative stress. Strikingly, our data indicate that antibiotic efficiency can be modulated by altering the iron concentration. The ferric iron reductase, FprB, was important for this effect. In principle, this opens the door to combination therapies that maximize antibiotic efficacy by elevating the levels of intracellular ferrous iron; conversely, mutants that are deficient in iron import and/or reduction might be expected to be outgrowers when populations are exposed to antibiotics that owe some of their potency to oxidative toxicity.

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