Background Much remains to be known about the mechanisms by which O2-dependent host defenses mediate broad antimicrobial activity. Methodology/Principal Findings We show herein that reactive nitrogen species (RNS) generated by inducible nitric oxide (NO) synthase (iNOS) account for the anti-Burkholderia mallei activity of IFNγ-primed macrophages. Inducible NOS-mediated intracellular killing may represent direct bactericidal activity, because B. mallei showed an exquisite sensitivity to NO generated chemically. Exposure of B. mallei to sublethal concentrations of NO upregulated transcription of [Fe-S] cluster repair genes, while damaging the enzymatic activity of the [Fe-S] protein aconitase. To test whether [Fe-S] clusters are critical targets for RNS-dependent killing of B. mallei, a mutation was constructed in the NO-induced, [Fe-S] cluster repair regulator iscR. Not only was the iscR mutant hypersusceptible to iNOS-mediated killing, but its aconitase pool was readily oxidized by NO donors as compared to wild-type controls. Although killed by authentic H2O2, which also oxidizes [Fe-S] clusters, B. mallei appear to be resilient to NADPH oxidase-mediated cytotoxicity. The poor respiratory burst elicited by this bacterium likely explains why the NADPH oxidase is nonessential to the killing of B. mallei while it is still confined within phagosomes. Conclusions/Significance Collectively, these findings have revealed a disparate role for NADPH oxidase and iNOS in the innate macrophage response against the strict aerobe B. mallei. To the best of our knowledge, this is the first instance in which disruption of [Fe-S] clusters is demonstrated as cause of the bactericidal activity of NO congeners.
References
[1]
Lopez J, Copps J, Wilhelmsen C, Moore R, Kubay J, et al. (2003) Characterization of experimental equine glanders. Microbes Infect 5: 1125–1131.
[2]
Lehavi O, Aizenstien O, Katz LH, Hourvitz A (2002) Glanders-a potential disease for biological warfare in humans and animals. Harefuah 141 Spec No: 88–91, pp 119.
Reeves EP, Lu H, Jacobs HL, Messina CG, Bolsover S, et al. (2002) Killing activity of neutrophils is mediated through activation of proteases by K+ flux. Nature 416: 291–297.
[5]
Stuehr DJ, Marletta MA (1985) Mammalian nitrate biosynthesis: mouse macrophages produce nitrite and nitrate in response to Escherichia coli lipopolysaccharide. Proc Natl Acad Sci U S A 82: 7738–7742.
[6]
Fang FC (2004) Antimicrobial reactive oxygen and nitrogen species: concepts and controversies. Nat Rev Microbiol 2: 820–832.
[7]
Jones AL, Beveridge TJ, Woods DE (1996) Intracellular survival of Burkholderia pseudomallei. Infect Immun 64: 782–790.
[8]
Miyagi K, Kawakami K, Saito A (1997) Role of reactive nitrogen and oxygen intermediates in gamma interferon-stimulated murine macrophage bactericidal activity against Burkholderia pseudomallei. Infect Immun 65: 4108–4113.
[9]
Utaisincharoen P, Tangthawornchaikul N, Kespichayawattana W, Anuntagool N, Chaisuriya P, et al. (2000) Kinetic studies of the production of nitric oxide (NO) and tumour necrosis factor-α (TNF-α) in macrophages stimulated with Burkholderia pseudomallei endotoxin. Clin Exp Immunol 122: 324–329.
[10]
Utaisincharoen P, Tangthawornchaikul N, Kespichayawattana W, Chaisuriya P, Sirisinha S (2001) Burkholderia pseudomallei interferes with inducible nitric oxide synthase (iNOS) production: a possible mechanism of evading macrophage killing. Microbiol Immunol 45: 307–313.
[11]
Utaisincharoen P, Anuntagool N, Arjcharoen S, Limposuwan K, Chaisuriya P, et al. (2004) Induction of iNOS expression and antimicrobial activity by interferon (IFN)-β is distinct from IFN-γ in Burkholderia pseudomallei-infected mouse macrophages. Clin Exp Immunol 136: 277–283.
[12]
Breitbach K, Klocke S, Tschernig T, van Rooijen N, Baumann U, et al. (2006) Role of inducible nitric oxide synthase and NADPH oxidase in early control of Burkholderia pseudomallei infection in mice. Infect Immun 74: 6300–6309.
[13]
Johnston RB Jr (2001) Clinical aspects of chronic granulomatous disease. Curr Opin Hematol 8: 17–22.
[14]
Segal BH, Ding L, Holland SM (2003) Phagocyte NADPH oxidase, but not inducible nitric oxide synthase, is essential for early control of Burkholderia cepacia and Chromobacterium violaceum infection in mice. Infect Immun 71: 205–210.
[15]
Brett PJ, Burtnick MN, Su H, Nair V, Gherardini FC (2007) iNOS activity is critical for the clearance of Burkholderia mallei from infected RAW 264.7 murine macrophages. Cell Microbiol.
[16]
Figurski DH, Helinski DR (1979) Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A 76: 1648–1652.
[17]
Craig FF, Coote JG, Parton R, Freer JH, Gilmour NJ (1989) A plasmid which can be transferred between Escherichia coli and Pasteurella haemolytica by electroporation and conjugation. J Gen Microbiol 135: 2885–2890.
[18]
MacMicking JD, Nathan C, Hom G, Chartrain N, Fletcher DS, et al. (1995) Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell 81: 641–650.
[19]
Pollock JD, Williams DA, Gifford MA, Li LL, Du X, et al. (1995) Mouse model of X-linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production. Nat Genet 9: 202–209.
[20]
De Groote MA, Ochsner UA, Shiloh MU, Nathan C, McCord JM, et al. (1997) Periplasmic superoxide dismutase protects Salmonella from products of phagocyte NADPH-oxidase and nitric oxide synthase. Proc Natl Acad Sci U S A 94: 13997–14001.
[21]
McCollister BD, Bourret TJ, Gill R, Jones-Carson J, Vazquez-Torres A (2005) Repression of SPI2 transcription by nitric oxide-producing, IFNγ-activated macrophages promotes maturation of Salmonella phagosomes. J Exp Med 202: 625–635.
[22]
Pick E (1986) Microassays for superoxide and hydrogen peroxide production and nitroblue tetrazolium reduction using an enzyme immunoassay microplate reader. Methods Enzymol 132: 407–421.
[23]
Voskuil MI, Schnappinger D, Visconti KC, Harrell MI, Dolganov GM, et al. (2003) Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program. J Exp Med 198: 705–713.
[24]
Rowland CA, Lertmemongkolchai G, Bancroft A, Haque A, Lever MS, et al. (2006) Critical role of type 1 cytokines in controlling initial infection with Burkholderia mallei. Infect Immun 74: 5333–5340.
[25]
Nathan C, Shiloh MU (2000) Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens. Proc Natl Acad Sci U S A 97: 8841–8848.
[26]
Stevens JM, Ulrich RL, Taylor LA, Wood MW, Deshazer D, et al. (2005) Actin-binding proteins from Burkholderia mallei and Burkholderia thailandensis can functionally compensate for the actin-based motility defect of a Burkholderia pseudomallei bimA mutant. J Bacteriol 187: 7857–7862.
[27]
Ribot WJ, Ulrich RL (2006) The animal pathogen-like type III secretion system is required for the intracellular survival of Burkholderia mallei within J774.2 macrophages. Infect Immun 74: 4349–4353.
[28]
Denis M (1991) Interferon-gamma-treated murine macrophages inhibit growth of tubercle bacilli via the generation of reactive nitrogen intermediates. Cell Immunol 132: 150–157.
[29]
Vazquez-Torres A, Jones-Carson J, Balish E (1995) Nitric oxide production does not directly increase macrophage candidacidal activity. Infect Immun 63: 1142–1144.
[30]
Brunelli L, Crow JP, Beckman JS (1995) The comparative toxicity of nitric oxide and peroxynitrite to Escherichia coli. Arch Biochem Biophys 316: 327–334.
[31]
De Groote MA, Granger D, Xu Y, Campbell G, Prince R, et al. (1995) Genetic and redox determinants of nitric oxide cytotoxicity in a Salmonella typhimurium model. Proc Natl Acad Sci U S A 92: 6399–6403.
[32]
Drapier JC, Hibbs JB Jr (1986) Murine cytotoxic activated macrophages inhibit aconitase in tumor cells. Inhibition involves the iron-sulfur prosthetic group and is reversible. J Clin Invest 78: 790–797.
[33]
Tortora V, Quijano C, Freeman B, Radi R, Castro L (2007) Mitochondrial aconitase reaction with nitric oxide, S-nitrosoglutathione, and peroxynitrite: mechanisms and relative contributions to aconitase inactivation. Free Radic Biol Med 42: 1075–1088.
[34]
Hausladen A, Fridovich I (1994) Superoxide and peroxynitrite inactivate aconitases, but nitric oxide does not. J Biol Chem 269: 29405–29408.
[35]
Castro L, Rodriguez M, Radi R (1994) Aconitase is readily inactivated by peroxynitrite, but not by its precursor, nitric oxide. J Biol Chem 269: 29409–29415.
[36]
Gardner PR, Costantino G, Szabo C, Salzman AL (1997) Nitric oxide sensitivity of the aconitases. J Biol Chem 272: 25071–25076.
[37]
Shiloh MU, MacMicking JD, Nicholson S, Brause JE, Potter S, et al. (1999) Phenotype of mice and macrophages deficient in both phagocyte oxidase and inducible nitric oxide synthase. Immunity 10: 29–38.
[38]
LaCourse R, Ryan L, North RJ (2002) Expression of NADPH oxidase-dependent resistance to listeriosis in mice occurs during the first 6 to 12 hours of liver infection. Infect Immun 70: 7179–7181.
[39]
Stevens MP, Wood MW, Taylor LA, Monaghan P, Hawes P, et al. (2002) An Inv/Mxi-Spa-like type III protein secretion system in Burkholderia pseudomallei modulates intracellular behaviour of the pathogen. Mol Microbiol 46: 649–659.
[40]
Vazquez-Torres A, Jones-Carson J, Balish E (1996) Peroxynitrite contributes to the candidacidal activity of nitric oxide-producing macrophages. Infect Immun 64: 3127–3133.
[41]
Schell MA, Ulrich RL, Ribot WJ, Brueggemann EE, Hines HB, et al. (2007) Type VI secretion is a major virulence determinant in Burkholderia mallei. Mol Microbiol 64: 1466–1485.
[42]
Swanson MS, Molofsky AB (2005) Autophagy and inflammatory cell death, partners of innate immunity. Autophagy 1: 174–176.
[43]
Molofsky AB, Byrne BG, Whitfield NN, Madigan CA, Fuse ET, et al. (2006) Cytosolic recognition of flagellin by mouse macrophages restricts Legionella pneumophila infection. J Exp Med 203: 1093–1104.
[44]
Zamboni DS, Kobayashi KS, Kohlsdorf T, Ogura Y, Long EM, et al. (2006) The Birc1e cytosolic pattern-recognition receptor contributes to the detection and control of Legionella pneumophila infection. Nat Immunol 7: 318–325.
[45]
MacMicking JD, Taylor GA, McKinney JD (2003) Immune control of tuberculosis by IFN-γ-inducible LRG-47. Science 302: 654–659.
[46]
Gutierrez MG, Master SS, Singh SB, Taylor GA, Colombo MI, et al. (2004) Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages. Cell 119: 753–766.
[47]
Brett PJ, Burtnick MN, Snyder DS, Shannon JG, Azadi P, et al. (2007) Burkholderia mallei expresses a unique lipopolysaccharide mixture that is a potent activator of human Toll-like receptor 4 complexes. Mol Microbiol 63: 379–390.
[48]
Lopez-Jaramillo P, Ruano C, Rivera J, Teran E, Salazar-Irigoyen R, et al. (1998) Treatment of cutaneous leishmaniasis with nitric-oxide donor. Lancet 351: 1176–1177.
[49]
Vazquez-Torres A, Fang FC (1999) Therapeutic applications of NO in infection. In: Fang FC, editor. Nitric oxide and infection. New York: Kluwer Acad/Plenum Publishers. pp. 475–488.
[50]
Yabuuchi E, Kosako Y, Oyaizu H, Yano I, Hotta H, et al. (1992) Proposal of Burkholderia gen. nov. and transfer of seven species of the genus Pseudomonas homology group II to the new genus, with the type species Burkholderia cepacia (Palleroni and Holmes 1981) comb. nov. Microbiol Immunol 36: 1251–1275.
[51]
Bergman MA, Cummings LA, Alaniz RC, Mayeda L, Fellnerova I, et al. (2005) CD4+-T-cell responses generated during murine Salmonella enterica serovar Typhimurium infection are directed towards multiple epitopes within the natural antigen FliC. Infect Immun 73: 7226–7235.
