FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1966773944> ?p ?o ?g. }

• W1966773944 endingPage "7689" @default.
• W1966773944 startingPage "7682" @default.
• W1966773944 abstract "Aerobic metabolism generates biologically challenging reactive oxygen species (ROS) by the endogenous autooxidation of components of the electron transport chain (ETC). Basal levels of oxidative stress can dramatically rise upon activation of the NADPH oxidase-dependent respiratory burst. To minimize ROS toxicity, prokaryotic and eukaryotic organisms express a battery of low-molecular-weight thiol scavengers, a legion of detoxifying catalases, peroxidases, and superoxide dismutases, as well as a variety of repair systems. We present herein blockage of bacterial respiration as a novel strategy that helps the intracellular pathogen Salmonella survive extreme oxidative stress conditions. A Salmonella strain bearing mutations in complex I NADH dehydrogenases is refractory to the early NADPH oxidase-dependent antimicrobial activity of IFNγ-activated macrophages. The ability of NADH-rich, complex I-deficient Salmonella to survive oxidative stress is associated with resistance to peroxynitrite (ONOO-) and hydrogen peroxide (H2O2). Inhibition of respiration with nitric oxide (NO) also triggered a protective adaptive response against oxidative stress. Expression of the NDH-II dehydrogenase decreases NADH levels, thereby abrogating resistance of NO-adapted Salmonella to H2O2. NADH antagonizes the hydroxyl radical (OH·) generated in classical Fenton chemistry or spontaneous decomposition of peroxynitrous acid (ONOOH), while fueling AhpCF alkylhydroperoxidase. Together, these findings identify the accumulation of NADH following the NO-mediated inhibition of Salmonella's ETC as a novel antioxidant strategy. NO-dependent respiratory arrest may help mitochondria and a plethora of organisms cope with oxidative stress engendered in situations as diverse as aerobic respiration, ischemia reperfusion, and inflammation. Aerobic metabolism generates biologically challenging reactive oxygen species (ROS) by the endogenous autooxidation of components of the electron transport chain (ETC). Basal levels of oxidative stress can dramatically rise upon activation of the NADPH oxidase-dependent respiratory burst. To minimize ROS toxicity, prokaryotic and eukaryotic organisms express a battery of low-molecular-weight thiol scavengers, a legion of detoxifying catalases, peroxidases, and superoxide dismutases, as well as a variety of repair systems. We present herein blockage of bacterial respiration as a novel strategy that helps the intracellular pathogen Salmonella survive extreme oxidative stress conditions. A Salmonella strain bearing mutations in complex I NADH dehydrogenases is refractory to the early NADPH oxidase-dependent antimicrobial activity of IFNγ-activated macrophages. The ability of NADH-rich, complex I-deficient Salmonella to survive oxidative stress is associated with resistance to peroxynitrite (ONOO-) and hydrogen peroxide (H2O2). Inhibition of respiration with nitric oxide (NO) also triggered a protective adaptive response against oxidative stress. Expression of the NDH-II dehydrogenase decreases NADH levels, thereby abrogating resistance of NO-adapted Salmonella to H2O2. NADH antagonizes the hydroxyl radical (OH·) generated in classical Fenton chemistry or spontaneous decomposition of peroxynitrous acid (ONOOH), while fueling AhpCF alkylhydroperoxidase. Together, these findings identify the accumulation of NADH following the NO-mediated inhibition of Salmonella's ETC as a novel antioxidant strategy. NO-dependent respiratory arrest may help mitochondria and a plethora of organisms cope with oxidative stress engendered in situations as diverse as aerobic respiration, ischemia reperfusion, and inflammation. Oxidative stress engendered by the sustained synthesis of NO mediates cytotoxicity against a variety of eukaryotic and prokaryotic cells (1Hibbs Jr., J.B. Taintor R.R. Vavrin Z. Rachlin E.M. Biochem. Biophys. Res. Commun. 1988; 157: 87-94Crossref PubMed Scopus (1804) Google Scholar, 2De Groote M.A. Granger D. Xu Y. Campbell G. Prince R. Fang F.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6399-6403Crossref PubMed Scopus (219) Google Scholar, 3MacMicking J.D. Nathan C. Hom G. Chartrain N. Fletcher D.S. Trumbauer M. Stevens K. Xie Q.W. Sokol K. Hutchinson N. Chen H. Mudgett J.S. Cell. 1995; 81: 641-650Abstract Full Text PDF PubMed Scopus (1280) Google Scholar). Because of its unpaired electron, NO directly reacts with metal prosthetic groups of cytochromes in the electron transport chain (ETC) 2The abbreviations used are: ETCelectron transport chainiNOSinducible nitric-oxide synthaseNDHNADH dehydrogenaseO2·¯superoxideOH·hydroxyl radicalONOO-peroxynitriteONOOHperoxynitrous acidROSreactive oxygen speciesRNSreactive nitrogen speciesSPI2Salmonella pathogenicity island 2WTwild typeIFNinterferonPBSphosphate-buffered salineXOxanthine oxidaseHXhypoxanthine. 2The abbreviations used are: ETCelectron transport chainiNOSinducible nitric-oxide synthaseNDHNADH dehydrogenaseO2·¯superoxideOH·hydroxyl radicalONOO-peroxynitriteONOOHperoxynitrous acidROSreactive oxygen speciesRNSreactive nitrogen speciesSPI2Salmonella pathogenicity island 2WTwild typeIFNinterferonPBSphosphate-buffered salineXOxanthine oxidaseHXhypoxanthine. and [Fe-S] clusters of dehydratases (4Cassina A. Radi R. Arch. Biochem. Biophys. 1996; 328: 309-316Crossref PubMed Scopus (607) Google Scholar, 5Beltran B. Mathur A. Duchen M.R. Erusalimsky J.D. Moncada S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14602-14607Crossref PubMed Scopus (337) Google Scholar). Alternatively, reactive nitrogen species (RNS) generated through the interaction of NO with O2 and superoxide (O2·¯) indirectly mediate cytotoxicity of this diatomic radical. The autooxidation of NO with O2 gives rise to RNS such as NO2· and N2O3 with potent oxidative and nitrosative activity. Independently, NO reacts with O2·¯ to generate ONOO-, a species capable of oxidizing amino acids, [Fe-S] clusters, and DNA (6Pryor W.A. Squadrito G.L. Am. J. Physiol. 1995; 268: L699-L722PubMed Google Scholar, 7Keyer K. Imlay J.A. J. Biol. Chem. 1997; 272: 27652-27659Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Despite its well-documented pro-oxidant functions, low concentrations of NO can paradoxically be cytoprotective. NO has been shown to both prevent oxidative damage of cardiocytes undergoing ischemia reperfusion and lessen the oxidative stress endured by mammalian host cells exposed to a variety of inorganic or organic peroxides (8Johnson 3rd, G. Tsao P.S. Lefer A.M. Crit. Care Med. 1991; 19: 244-252Crossref PubMed Scopus (210) Google Scholar, 9Wink D.A. Hanbauer I. Krishna M.C. DeGraff W. Gamson J. Mitchell J.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9813-9817Crossref PubMed Scopus (733) Google Scholar, 10Gorbunov N.V. Yalowich J.C. Gaddam A. Thampatty P. Ritov V.B. Kisin E.R. Elsayed N.M. Kagan V.E. J. Biol. Chem. 1997; 272: 12328-12341Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 11Joshi M.S. Ponthier J.L. Lancaster Jr., J.R. Free Radic. Biol. Med. 1999; 27: 1357-1366Crossref PubMed Scopus (137) Google Scholar). The mechanisms by which NO serves antioxidant roles remain, however, poorly understood. electron transport chain inducible nitric-oxide synthase NADH dehydrogenase superoxide hydroxyl radical peroxynitrite peroxynitrous acid reactive oxygen species reactive nitrogen species Salmonella pathogenicity island 2 wild type interferon phosphate-buffered saline xanthine oxidase hypoxanthine. electron transport chain inducible nitric-oxide synthase NADH dehydrogenase superoxide hydroxyl radical peroxynitrite peroxynitrous acid reactive oxygen species reactive nitrogen species Salmonella pathogenicity island 2 wild type interferon phosphate-buffered saline xanthine oxidase hypoxanthine. Investigations using prokaryotic microorganisms have elucidated several mechanisms by which NO protects against oxidative stress. In enteric bacteria, the OxyRS and SoxRS regulatory systems coordinate the expression of antioxidant defenses against H2O2 and O2·¯ (12Hidalgo E. Demple B. EMBO J. 1994; 13: 138-146Crossref PubMed Scopus (246) Google Scholar, 13Aslund F. Zheng M. Beckwith J. Storz G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6161-6165Crossref PubMed Scopus (434) Google Scholar). In addition to responding to oxyradicals, the [Fe-S] prosthetic group of SoxR and redox active cysteines of OxyR can be modified by NO, thereby activating a signal transduction cascade that stimulates transcription of antioxidant defenses such as Mn-superoxide dismutase, endonuclease IV, and hydroperoxidase I (14Nunoshiba T. DeRojas-Walker T. Wishnok J.S. Tannenbaum S.R. Demple B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9993-9997Crossref PubMed Scopus (278) Google Scholar, 15Hausladen A. Privalle C.T. Keng T. DeAngelo J. Stamler J.S. Cell. 1996; 86: 719-729Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 16Kim S.O. Merchant K. Nudelman R. Beyer Jr., W.F. Keng T. De-Angelo J. Hausladen A. Stamler J.S. Cell. 2002; 109: 383-396Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar). Alternatively, NO can trigger instant cytoprotection without evoking de novo protein synthesis. For instance, in the Gram-positive bacterium Bacillus subtilis, NO promotes antioxidant defenses by: 1) depleting free cysteine and thus limiting the availability of a Fenton fuel and 2) enhancing H2O2 consumption through the activation of KatA catalase (17Gusarov I. Nudler E. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13855-13860Crossref PubMed Scopus (223) Google Scholar). The ETC transfers reducing equivalents from a variety of substrates to respiratory cytochrome oxidases for the reduction of O2 to H2O. Discrete enzymatic components of the ETC such as NADH dehydrogenases and cytochromes couple oxidoreduction reactions with transport of H+ across the membrane, generating a proton gradient that drives ATP biosynthesis by F0/F1 ATPases and energizes transporters and organelles assembled in the membrane. The collapse of membrane potential and the subsequent fall in ATP synthesis appear to underlie the pathological effects associated with persistent inactivation of ETC redox centers (5Beltran B. Mathur A. Duchen M.R. Erusalimsky J.D. Moncada S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14602-14607Crossref PubMed Scopus (337) Google Scholar, 18Schweizer M. Richter C. Biochem. Biophys. Res. Commun. 1994; 204: 169-175Crossref PubMed Scopus (334) Google Scholar, 19Almeida A. Bolanos J.P. J. Neurochem. 2001; 77: 676-690Crossref PubMed Scopus (141) Google Scholar). Transient inhibition of respiration, on the other hand, can serve physiological roles by diverting O2 to alternative cellular and tissue usages (20Thomas D.D. Liu X. Kantrow S.P. Lancaster Jr., J.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 355-360Crossref PubMed Scopus (585) Google Scholar, 21Trimmer B.A. Aprille J.R. Dudzinski D.M. Lagace C.J. Lewis S.M. Michel T. Qazi S. Zayas R.M. Science. 2001; 292: 2486-2488Crossref PubMed Scopus (118) Google Scholar). In the process of reducing quinones and pumping H+ across the membrane, NADH dehydrogenases comprising the ETC complex I consume NADH. We hypothesize herein that inhibition of ETC can serve additional physiological roles by conveying NADH reducing equivalents to an adaptive response that protects against oxidative stress. To test this idea, the cytotoxicity of a variety of oxidative stress conditions was assessed in: 1) a Salmonella strain bearing mutations in NDH-I (Δnuo::km) and -II (Δndh::FRT) complex I NADH dehydrogenases and 2) wild-type Salmonella in which the ETC was inactivated following exposure to RNS. Bacterial Strains—Salmonella enterica serovar Typhimurium strain ATCC 14028s was used throughout this study as the wild type and as the background for the construction of mutant strains (Table 1 and supplemental Table S1). Mutations of the Salmonella chromosome were constructed following the one-step, λred-mediated gene replacement method described by Datsenko and Wanner (22Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11049) Google Scholar). To ensure that the phenotypes of the ETC complex I mutant were specific to the lack of NADH dehydrogenases, Δnuo::km and Δndh::FRT mutations were complemented with the low copy number pWSK29 vector expressing a wild-type ndh allele under the control of its native promoter. Wild-type Salmonella strain AV0554 expresses ndh under the pBAD18 promoter. Stationary phase wild-type Salmonella was tested after 16 h of culture in Luria-Bertani (LB) broth. Because of a delayed lag phase, complex I-deficient Salmonella strain AV0436 was grown for 20 h.TABLE 1Bacterial strains and plasmidsGenotypeSourceStrains Salmonella typhimurium strain 14028sWild-typeATCC AV0201ΔspiC::FRT(24McCollister B.D. Bourret T.J. Gill R. Jones-Carson J. Vazquez-Torres A. J. Exp. Med. 2005; 202: 625-635Crossref PubMed Scopus (57) Google Scholar) AV0429Δcyo::FRTThis study AV0436Δnuo::km Δndh::FRTThis study AV0441Δnuo::km Δndh::FRT ΔspiC::FRTThis study AV0497ΔahpCF::FRTThis study AV0498Δnuo::km Δndh::FRT ΔahpCF::FRTThis study AV0552Δnuo::km Δndh::FRT pNDH (pWSK29::ndh)This study AV0553Δnuo::km Δndh::FRT pBNDH (pBAD18::ndh)This study AV0554pBNDH (pBAD18::ndh)This study AV0556pBAD (pBAD18)This studyPlasmids pKD13bla FRT ahp FRT oriR6K(22Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11049) Google Scholar) pCP20bla cat cI857 λPRflp pSC101 oriTS(52Cherepanov P.P. Wackernagel W. Gene. 1995; 158: 9-14Crossref PubMed Scopus (1384) Google Scholar) pWSK29bla lacZα oripSC101(53Wang R.F. Kushner S.R. Gene. 1991; 100: 195-199Crossref PubMed Scopus (1005) Google Scholar) pBAD18araC bla rrnB oriM13 oripBR322(54Guzman L.M. Belin D. Carson M.J. Beckwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (3923) Google Scholar) Open table in a new tab Macrophage Assays—C57BL/6 and congenic iNOS-/- (3MacMicking J.D. Nathan C. Hom G. Chartrain N. Fletcher D.S. Trumbauer M. Stevens K. Xie Q.W. Sokol K. Hutchinson N. Chen H. Mudgett J.S. Cell. 1995; 81: 641-650Abstract Full Text PDF PubMed Scopus (1280) Google Scholar) or gp91phox-/- (23Pollock J.D. Williams D.A. Gifford M.A. Li L.L. Du X. Fisherman J. Orkin S.H. Doerschuk C.M. Dinauer M.C. Nat. Genet. 1995; 9: 202-209Crossref PubMed Scopus (751) Google Scholar) mice were bred in our animal facility according to Institutional Animal Care and Use Committee guidelines. The anti-Salmonella activity of macrophages was assessed as previously described (24McCollister B.D. Bourret T.J. Gill R. Jones-Carson J. Vazquez-Torres A. J. Exp. Med. 2005; 202: 625-635Crossref PubMed Scopus (57) Google Scholar). Briefly, periodate-elicited peritoneal macrophages were cultured in RPMI 1640 medium (Cellgro, Herndon, VA) supplemented with 10% heat-inactivated fetal bovine serum (BioWhittaker, Walkersville, MD), 15 mm Hepes, 2 mm l-glutamine, 1 mm sodium pyruvate (Sigma-Aldrich), and 100 units· ml-1/100 mg·ml-1 of penicillin/streptomycin (Cellgro) as described (24McCollister B.D. Bourret T.J. Gill R. Jones-Carson J. Vazquez-Torres A. J. Exp. Med. 2005; 202: 625-635Crossref PubMed Scopus (57) Google Scholar). Selected groups of macrophages were treated with 200 units/ml of IFNγ (Invitrogen, St. Paul, MN) for 20 h before Salmonella infection. Macrophages were challenged with Salmonella at a multiplicity of infection (MOI) of 2 and after 25 min of infection the medium replaced with RPMI+ medium containing 18 μg/ml gentamicin. The cells were lysed at the indicated time points after infection and percent surviving bacteria recorded on LB agar plates. Synthesis of ROS and RNS by Macrophages—Macrophages isolated as above were infected with Salmonella at an MOI of 10. The monolayers were washed after 15 min of infection and the medium replaced with RPMI+ medium containing 6 μg/ml gentamicin and 100 μm luminol. Luminol chemiluminescence was used as an indicator of ONOO- production (25Radi R. Cosgrove T.P. Beckman J.S. Freeman B.A. Biochem. J. 1993; 290: 51-57Crossref PubMed Scopus (346) Google Scholar) by Salmonella-infected, IFNγ-primed macrophages. Synthesis of H2O2 was assessed by the horseradish peroxidase-mediated, luminol-dependent chemiluminescence in a reaction that contained 20 units/ml horseradish peroxidase and 100 μm luminol. Chemiluminescence was recorded at 37 °C on an Lmax® luminometer (Molecular Devices) at 5-min intervals for 1 h with an integration time of 4 s. Susceptibility to RNS and ROS in Vitro—Stationary phase wild-type and ETC complex I mutant Salmonella grown overnight in LB broth as above were diluted in PBS at a concentration of 5 × 105 cells/ml. The bacteria were incubated with 500 μm hypoxanthine (HX) (Sigma-Aldrich) and 0.1 units/ml xanthine oxidase (XO) (Roche Applied Science, Indianapolis, IN), 400 μm or 2.5 mm H2O2, or 750 μm ONOO- generator SIN1 (Molecular Probes, Eugene, OR). The HX/XO system generated 0.4 and 0.5 μm/min of O2·¯ and H2O2 as estimated by superoxide dismutase-inhibitable reduction of cytochrome c and horseradish peroxidase-catalyzed oxidation of phenol red, respectively (26Vazquez-Torres A. Jones-Carson J. Mastroeni P. Ischiropoulos H. Fang F.C. J. Exp. Med. 2000; 192: 227-236Crossref PubMed Scopus (444) Google Scholar). The contribution of O2·¯ and H2O2 to the antimicrobial activity of the XO/HX system was estimated by adding 300 units/ml Cu-Zn superoxide dismutase or 25 units/ml catalase (Sigma-Aldrich), respectively. Percent survival was calculated after various times of exposure by recording the number of bacteria able to form a colony on LB plates. Selected groups of wild-type bacteria diluted at a concentration of 107 cells/ml in EG medium (0.811 mm MgSO4, 9.52 mm citric acid, 57.41 K2HPO4, 16.74 mm Na(NH4)HPO4, and 0.4% glucose, pH 7.0) were pre-adapted for 1 h with 750 μm spermine NONOate or SIN1 in the presence of 15 μg/ml of the protein synthesis inhibitor chloramphenicol. Control and RNS-adapted cells were diluted in PBS and challenged for 2 h with 400 μm H2O2 in the presence of chloramphenicol. About 35 μm NO donor spermine NONOate and SIN1 remained during H2O2 challenge. NADH/NAD+ Quantification—NADH and NAD+ were extracted in 0.2 m NaOH and 0.2 m HCl, respectively, from overnight cultures of Salmonella grown in LB medium as described above. The concentration of NADH/NAD+ was measured by the thiazolyl tetrazolium blue cycling assay (27San K.Y. Bennett G.N. Berrios-Rivera S.J. Vadali R.V. Yang Y.T. Horton E. Rudolph F.B. Sariyar B. Blackwood K. Metab. Eng. 2002; 4: 182-192Crossref PubMed Scopus (223) Google Scholar), and the concentration of nicotinamide was calculated by regression analysis of known standards. Nucleotide concentrations were corrected for bacterial density as estimated spectrophotometrically at A600, and the intracellular NADH concentration calculated based on a bacterial cell volume of 10-15 liters. Hydroxyl Radical Formation—Terephthalic acid was used to measure OH· via the fluorescence 2-hydroxyterephthalate adduct (λex = 315 nm; λem = 425 nm). The effect of NADH or NAD+ on the formation of 2-hydroxyterephthalate was monitored in a spectrofluorometer after the addition of 4 mm ONOO- into a solution of 2.5 mm of terephthalate in PBS, pH 7.0. Autofluorescence of NADH and NAD+ were subtracted from the final readings. Oxygen and H2O2 Consumption—Salmonella grown overnight in LB broth were cultured to 1 OD/ml in aerated EG medium, pH 7.0 at 37 °C. Consumption of oxygen and H2O2 was recorded with specific probes using a free radical analyzer (WPI Inc., Sarasota, FL). Selected samples were treated with the indicated concentrations of authentic ONOO- or 750 μm spermine NONOate or SIN1 1 min before analysis. Spectroscopy—Salmonella strain AV0429 lacking the cyo ubiquinol oxidase was grown overnight to stationary phase in LB broth. Inner membranes were prepared as described by Miller and Gennis (28Miller M.J. Gennis R.B. Methods Enzymol. 1986; 126: 87-94Crossref PubMed Scopus (23) Google Scholar) with minor modifications. Briefly, bacterial pellets were resuspended in 10 mm EDTA, 100 mm Tris/HCl buffer, pH 8.5. The cells were lysed by passing the suspension through a French Press Cell Disruptor (Thermo Electron Corporation, Milford, MA) three times at 18,000 psi at a flow rate of 5 ml/min. Cell debris were removed by centrifuging for 20 min at 10,000 × g in a Sorvall SL-50T rotor. The supernatant was centrifuged at 200,000 × g in a Beckman-type 70 Ti rotor for 1 h and the pellet solubilized in 75 mm potassium phosphate, 150 mm KCl, 5 mm EDTA, and 60 mm N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate (Sigma) buffer, pH 6.4. The solution was centrifuged at 200,000 × g for 1 h. Supernatants containing inner membranes were collected, and the protein concentration assayed using the BCA Protein Assay kit (Pierce). The protein was adjusted to 1.5 mg/ml in 75 mm potassium phosphate, 150 mm KCl, 5 mm EDTA, 10 mm ascorbate, and 60 mm N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate buffer, pH 6.4. Absorbance spectroscopy was collected in a Cary 50 Bio UV-Visible spectrophotometer. Selected groups of proteins were treated with 100 μm spermine NONOate for 30 min before the absorption spectra were collected. The spermine base was used as a negative control. Complex I-deficient Salmonella Is Hyper-resistant to the Oxidative Stress Encountered within IFNγ-primed Macrophages—The complex I-deficient Salmonella strain AV0436 (Δnuo::km Δndh::FRT), but not isogenic strains bearing single mutations (not shown), was hyper-resistant to the antimicrobial activity of IFNγ-primed macrophages (Fig. 1a). A 100-fold survival advantage was manifested shortly after infection and lasted for at least 6 h, a period in which NADPH oxidase-dependent defenses prevail (26Vazquez-Torres A. Jones-Carson J. Mastroeni P. Ischiropoulos H. Fang F.C. J. Exp. Med. 2000; 192: 227-236Crossref PubMed Scopus (444) Google Scholar). The intracellular advantage of strain AV0436 is specific to the lack of ETC NADH dehydrogenases because complementation with the low copy plasmid pNDH expressing the wild-type ndh allele restored susceptibility to macrophage oxidative killing (Fig. 1a). Survival of wild-type and complex I-deficient Salmonella was similar in gp91phox-/- macrophages unable to sustain a respiratory burst (Fig. 1b). Although to a lesser degree than defects in the NADPH oxidase, a mutation in the inducible NO synthase also ameliorated the early differences in intracellular survival between wild-type and complex I-deficient Salmonella. Together, these findings indicate that the lack of ETC NADH dehydrogenases not only protects Salmonella against host-derived oxyradicals but also lessens synergistic toxicity of ROS and NO congeners. Aerobic organisms exploit an assortment of mechanisms for detoxification or avoidance of ROS (29Fang F.C. Nat. Rev. Microbiol. 2004; 2: 820-832Crossref PubMed Scopus (1214) Google Scholar). The antioxidant repertoire is especially varied in intracellular pathogens residing within professional phagocytes capable of generating massive amounts of ROS in the NADPH oxidase-dependent respiratory burst. To test whether the hyper-resistance of complex I-deficient Salmonella might be related to the Salmonella pathogenicity island 2 (SPI2) type III secretion system that ameliorates exposure of Salmonella to reactive species such as H2O2 and ONOO- (30Vazquez-Torres A. Xu Y. Jones-Carson J. Holden D.W. Lucia S.M. Dinauer M.C. Mastroeni P. Fang F.C. Science. 2000; 287: 1655-1658Crossref PubMed Scopus (451) Google Scholar, 31Chakravortty D. Hansen-Wester I. Hensel M. J. Exp. Med. 2002; 195: 1155-1166Crossref PubMed Scopus (267) Google Scholar), a ΔspiC::FRT allelle that inactivates SPI2 secretion and effector functions (32Uchiya K. Barbieri M.A. Funato K. Shah A.H. Stahl P.D. Groisman E.A. EMBO J. 1999; 18: 3924-3933Crossref PubMed Scopus (290) Google Scholar, 33Freeman J.A. Rappl C. Kuhle V. Hensel M. Miller S.I. J. Bacteriol. 2002; 184: 4971-4980Crossref PubMed Scopus (70) Google Scholar, 34Yu X.J. Ruiz-Albert J. Unsworth K.E. Garvis S. Liu M. Holden D.W. Cell Microbiol. 2002; 4: 531-540Crossref PubMed Scopus (61) Google Scholar) was combined with Δnuo::km Δndh::FRT alleles generating strain AV0441 (NDH-I- NDH-II- SPI2-). Strain AV0441 was as resistant to early macrophage oxidative killing as its complex I-deficient isogenic control (Fig. 1c). Poor NADPH oxidase-dependent killing of complex I-deficient Salmonella cannot be explained by reduced synthesis of ONOO- by IFNγ-primed macrophages (Fig. 1d). Moreover, unstimulated macrophages produced similar amounts of H2O2 in response to wild-type or complex I-deficient Salmonella (Fig. 1d). Salmonella to NADPH oxidase-mediated intracellular killing might be associated with resistance to ROS and RNS. Strain AV0436 was found to be refractory to the antimicrobial activity of the O2·¯/H2O2-generating HX/XO system (Fig. 2a). Catalase, but not Cu-Zn superoxide dismutase, abrogated the killing of wild-type Salmonella by HX/XO (Fig. 2b), suggesting that complex I-deficient Salmonella is resistant to H2O2. Supporting this notion, complex I-deficient bacteria survived exposure to authentic H2O2, becoming susceptible upon expression of the NDH-II-complementing pNDH plasmid (Fig. 2c). Analogous to the NADH-mediated potentiation of oxidative stress previously associated with respiratory arrest in rapidly growing Escherichia coli (35Pacelli R. Wink D.A. Cook J.A. Krishna M.C. DeGraff W. Friedman N. Tsokos M. Samuni A. Mitchell J.B. J. Exp. Med. 1995; 182: 1469-1479Crossref PubMed Scopus (222) Google Scholar, 36Woodmansee A.N. Imlay J.A. J. Biol. Chem. 2002; 277: 34055-34066Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), the log phase complex I-deficient Salmonella was susceptible to H2O2 (Fig. 2d). Because ONOO-, which is similarly formed in response to wild-type and complex I-deficient Salmonella (Fig. 1d), constitutes an intrinsic component of the early anti-Salmonella arsenal of IFNγ-treated macrophages (26Vazquez-Torres A. Jones-Carson J. Mastroeni P. Ischiropoulos H. Fang F.C. J. Exp. Med. 2000; 192: 227-236Crossref PubMed Scopus (444) Google Scholar, 31Chakravortty D. Hansen-Wester I. Hensel M. J. Exp. Med. 2002; 195: 1155-1166Crossref PubMed Scopus (267) Google Scholar), the susceptibility of Salmonella strain AV0436 to the ONOO- generator SIN1 was also studied. As for H2O2, the viability of stationary phase, complex I-deficient Salmonella was unaffected after exposure to SIN1 (Fig. 2e). These data demonstrate that Salmonella lacking complex I of the ETC are resistant to a variety of biologically active peroxides. Antioxidant Activity of NADH—As anticipated, Salmonella strain AV0436 lacking NDH-I and NDH-II contained higher NADH/NAD+ ratios than wild-type controls (Fig. 3a). The intracellular concentration of NADH in wild-type and complex I-deficient bacteria were 167 and 484 μm, respectively, whereas their NAD+ levels were 885 and 887 μm. The complex I-deficient Salmonella complemented with the pNDH plasmid contained 206 μm NADH and 763 μm NAD+. NADH could serve antioxidant functions by enhancing peroxidatic activity of alkylhydroperoxidases (37Storz G. Jacobson F.S. Tartaglia L.A. Morgan R.W. Silveira L.A. Ames B.N. J. Bacteriol. 1989; 171: 2049-2055Crossref PubMed Scopus (206) Google Scholar, 38Bryk R. Griffin P. Nathan C. Nature. 2000; 407: 211-215Crossref PubMed Scopus (563) Google Scholar). To test this hypothesis, a ΔahpCF::FRT mutation deleting both subunits of the alkylhydroperoxidase reductase was combined with nuo:km and Δndh alleles inactivating NDH-I and NDH-II NADH dehydrogenases. The lack of ahpCF increased by 5- and 2-fold the killing of complex I-deficient Salmonella by 750 μm SIN1 or 400 μm H2O2, respectively (Fig. 3b). NADH may also provide direct antioxidant activity, because NADH was oxidized by ONOOH (Fig. 3c) and a combination of H2O2 and Fe2+ (Fig. 3d). In contrast, NADH was not consumed by ONOO- or H2O2 (Fig. 3c). Together, these data indicate that NADH can scavenge OH· derived from H2O2 and ONOOH. Accordingly, NADH, but not NAD+, prevented ONOOH-induced synthesis of hydroxyterephthalate (Fig. 3e), which is a specific signature of OH· (39Barreto J.C. Smith G.S. Strobel N.H. McQuillin P.A. Miller T.A. Life Sci. 1995; 56: PL89-PL96PubMed Google Scholar). Inhibition of the ETC by RNS Elicits an Adaptive Response against Oxidative Stress—Because defects in the ETC promote resistance of Salmonella to oxidative stress following exposure to a variety of ROS and RNS (Fig. 2), we tested whether the pharmacological blockage of respiration following NO treatment may also trigger an antioxidant adaptive response. As expected by its ability to inhibit metal centers of cytochromes of the ETC (35Pacelli R. Wink D.A. Cook J.A. Krishna M.C. DeGraff W. Friedman N. Tsokos M. Samuni A. Mitchell J.B. J. Exp. Med. 1995; 182: 1469-1479Crossref PubMed Scopus (222) Google Scholar, 36Woodmansee A.N. Imlay J.A. J. Biol. Chem. 2002; 277: 34055-34066Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), 750 μm NO donor spermine NONOate arrested Salmonella respiration (Fig. 4a). ONOO-, which has been shown to inhibit ETC complex I in mitochondria (4Cassina A. Radi R. Arch. Biochem. Biophys. 1996; 328: 309-316Crossref PubMed Scopus (607) Google Scholar), also inhibited the respiration of Salmonella (Fig. 4b). NO conferred instant cytoprotection against 2.5 mm (Fig. 4c) or 400 μm H2O2 (Fig. 4e). The protective effects were nonetheless transitory because the viability of NO-adapted Salmonella started to decline after 50 min of exposure to 2.5 H2O2 (Fig. 4c). The reversible nitrosylation of terminal cytochromes provides a mechanism for the transitory protective effects associated with NO. Because the bd-type ubiqu" @default.
• W1966773944 created "2016-06-24" @default.
• W1966773944 creator A5001158218 @default.
• W1966773944 creator A5004056687 @default.
• W1966773944 creator A5039362941 @default.
• W1966773944 creator A5041314705 @default.
• W1966773944 creator A5047797144 @default.
• W1966773944 creator A5067351258 @default.
• W1966773944 date "2008-03-01" @default.
• W1966773944 modified "2023-09-30" @default.
• W1966773944 title "Nitric Oxide Evokes an Adaptive Response to Oxidative Stress by Arresting Respiration" @default.
• W1966773944 cites W1415018605 @default.
• W1966773944 cites W1505685265 @default.
• W1966773944 cites W1532066573 @default.
• W1966773944 cites W1534977663 @default.
• W1966773944 cites W1568326433 @default.
• W1966773944 cites W1588367297 @default.
• W1966773944 cites W1668019941 @default.
• W1966773944 cites W1886833444 @default.
• W1966773944 cites W1967187479 @default.
• W1966773944 cites W1970584869 @default.
• W1966773944 cites W1970727664 @default.
• W1966773944 cites W1970935798 @default.
• W1966773944 cites W1971245738 @default.
• W1966773944 cites W1994389040 @default.
• W1966773944 cites W1996048275 @default.
• W1966773944 cites W1997745291 @default.
• W1966773944 cites W2001190819 @default.
• W1966773944 cites W2001207916 @default.
• W1966773944 cites W2007013508 @default.
• W1966773944 cites W2011233979 @default.
• W1966773944 cites W2014361215 @default.
• W1966773944 cites W2022377100 @default.
• W1966773944 cites W2023822923 @default.
• W1966773944 cites W2028585334 @default.
• W1966773944 cites W2030566353 @default.
• W1966773944 cites W2032890845 @default.
• W1966773944 cites W2039045136 @default.
• W1966773944 cites W2047201070 @default.
• W1966773944 cites W2054941036 @default.
• W1966773944 cites W2058062846 @default.
• W1966773944 cites W2066515050 @default.
• W1966773944 cites W2066732573 @default.
• W1966773944 cites W2069292335 @default.
• W1966773944 cites W2073066847 @default.
• W1966773944 cites W2084808295 @default.
• W1966773944 cites W2085551318 @default.
• W1966773944 cites W2085773333 @default.
• W1966773944 cites W2095213403 @default.
• W1966773944 cites W2108012416 @default.
• W1966773944 cites W2111176922 @default.
• W1966773944 cites W2112896586 @default.
• W1966773944 cites W2116137883 @default.
• W1966773944 cites W2118341013 @default.
• W1966773944 cites W2122515902 @default.
• W1966773944 cites W2123507727 @default.
• W1966773944 cites W2135004245 @default.
• W1966773944 cites W2139045536 @default.
• W1966773944 cites W2143971149 @default.
• W1966773944 cites W2160084442 @default.
• W1966773944 cites W2162429416 @default.
• W1966773944 cites W2164004338 @default.
• W1966773944 cites W2317659478 @default.
• W1966773944 cites W98049954 @default.
• W1966773944 doi "https://doi.org/10.1074/jbc.m708845200" @default.
• W1966773944 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18198179" @default.
• W1966773944 hasPublicationYear "2008" @default.
• W1966773944 type Work @default.
• W1966773944 sameAs 1966773944 @default.
• W1966773944 citedByCount "81" @default.
• W1966773944 countsByYear W19667739442012 @default.
• W1966773944 countsByYear W19667739442013 @default.
• W1966773944 countsByYear W19667739442014 @default.
• W1966773944 countsByYear W19667739442015 @default.
• W1966773944 countsByYear W19667739442016 @default.
• W1966773944 countsByYear W19667739442017 @default.
• W1966773944 countsByYear W19667739442018 @default.
• W1966773944 countsByYear W19667739442019 @default.
• W1966773944 countsByYear W19667739442020 @default.
• W1966773944 countsByYear W19667739442021 @default.
• W1966773944 countsByYear W19667739442022 @default.
• W1966773944 countsByYear W19667739442023 @default.
• W1966773944 crossrefType "journal-article" @default.
• W1966773944 hasAuthorship W1966773944A5001158218 @default.
• W1966773944 hasAuthorship W1966773944A5004056687 @default.
• W1966773944 hasAuthorship W1966773944A5039362941 @default.
• W1966773944 hasAuthorship W1966773944A5041314705 @default.
• W1966773944 hasAuthorship W1966773944A5047797144 @default.
• W1966773944 hasAuthorship W1966773944A5067351258 @default.
• W1966773944 hasBestOaLocation W19667739441 @default.
• W1966773944 hasConcept C105702510 @default.
• W1966773944 hasConcept C12554922 @default.
• W1966773944 hasConcept C134018914 @default.
• W1966773944 hasConcept C182215343 @default.
• W1966773944 hasConcept C185592680 @default.
• W1966773944 hasConcept C2776151105 @default.
• W1966773944 hasConcept C33507282 @default.
• W1966773944 hasConcept C519581460 @default.

• next