A Long-Chain Flavodoxin Protects from Oxidative Stress and Host Bacterial Clearance
Long-chain flavodoxins, ubiquitous electron shuttles containing flavin mononucleotide (FMN) as prosthetic group, play an important protective role against reactive oxygen species (ROS) in various microorganisms. Pseudomonas aeruginosa is an opportunistic pathogen which frequently has to face ROS toxicity in the environment as well as within the host. We identified a single ORF, hereafter referred to as fldP (for flavodoxin from P. aeruginosa), displaying the highest similarity in length, sequence identity and predicted secondary structure with typical long-chain flavodoxins. The gene was cloned and expressed in Escherichia coli. The recombinant product (FldP) could bind FMN and exhibited flavodoxin activity in vitro. Expression of fldP in P. aeruginosa was induced by oxidative stress conditions through an OxyR-independent mechanism, and an fldP-null mutant accumulated higher intracellular ROS levels and exhibited decreased tolerance to H2O2 toxicity compared to wild-type siblings. The mutant phenotype could be complemented by expression of a cyanobacterial flavodoxin. Overexpression of FldP in a mutT-deficient P. aeruginosa strain decreased H2O2-induced cell death and the hypermutability caused by DNA oxidative damage. FldP contributed to the survival of P. aeruginosa within cultured mammalian macrophages and in infected Drosophila melanogaster, which led in turn to accelerated death of the flies. Interestingly, the fldP gene is present in some but not all P. aeruginosa strains, constituting a component of the P. aeruginosa accessory genome. It is located in a genomic island as part of a self-regulated polycistronic operon containing a suite of stress-associated genes. The collected results indicate that the fldP gene encodes a long-chain flavodoxin, which protects the cell from oxidative stress, thereby expanding the capabilities of P. aeruginosa to thrive in hostile environments.
Vyšlo v časopise:
A Long-Chain Flavodoxin Protects from Oxidative Stress and Host Bacterial Clearance. PLoS Genet 10(2): e32767. doi:10.1371/journal.pgen.1004163
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.pgen.1004163
Souhrn
Long-chain flavodoxins, ubiquitous electron shuttles containing flavin mononucleotide (FMN) as prosthetic group, play an important protective role against reactive oxygen species (ROS) in various microorganisms. Pseudomonas aeruginosa is an opportunistic pathogen which frequently has to face ROS toxicity in the environment as well as within the host. We identified a single ORF, hereafter referred to as fldP (for flavodoxin from P. aeruginosa), displaying the highest similarity in length, sequence identity and predicted secondary structure with typical long-chain flavodoxins. The gene was cloned and expressed in Escherichia coli. The recombinant product (FldP) could bind FMN and exhibited flavodoxin activity in vitro. Expression of fldP in P. aeruginosa was induced by oxidative stress conditions through an OxyR-independent mechanism, and an fldP-null mutant accumulated higher intracellular ROS levels and exhibited decreased tolerance to H2O2 toxicity compared to wild-type siblings. The mutant phenotype could be complemented by expression of a cyanobacterial flavodoxin. Overexpression of FldP in a mutT-deficient P. aeruginosa strain decreased H2O2-induced cell death and the hypermutability caused by DNA oxidative damage. FldP contributed to the survival of P. aeruginosa within cultured mammalian macrophages and in infected Drosophila melanogaster, which led in turn to accelerated death of the flies. Interestingly, the fldP gene is present in some but not all P. aeruginosa strains, constituting a component of the P. aeruginosa accessory genome. It is located in a genomic island as part of a self-regulated polycistronic operon containing a suite of stress-associated genes. The collected results indicate that the fldP gene encodes a long-chain flavodoxin, which protects the cell from oxidative stress, thereby expanding the capabilities of P. aeruginosa to thrive in hostile environments.
Zdroje
1. ImlayJA (2008) Cellular defenses against superoxide and hydrogen peroxide. Annu Rev Biochem 77: 755–776.
2. MishraS, ImlayJ (2012) Why do bacteria use so many enzymes to scavenge hydrogen peroxide? Arch Biochem Biophys 525: 145–160.
3. ZhengM, DoanB, SchneiderTD, StorzG (1999) OxyR and SoxRS regulation of fur. J Bacteriol 181: 4639–4643.
4. FuldaS, HagemannM (1995) Salt treatment induces accumulation of flavodoxin in the cyanobacterium Synechocystis sp. PCC6803. J Plant Physiol 146: 520–526.
5. MazouniK, DomainF, ChauvatF, Cassier-ChauvatC (2003) Expression and regulation of the crucial plant-like ferredoxin of cyanobacteria. Mol Microbiol 49: 1019–1029.
6. SinghAK, LiH, ShermanLA (2004) Microarray analysis and redox control of gene expression in the cyanobacterium Synechocystis sp. PCC 6803. Physiol Plant 120: 27–35.
7. KrappAR, HumbertMV, CarrilloN (2011) The soxRS response of Escherichia coli can be induced in the absence of oxidative stress and oxygen by modulation of NADPH content. Microbiology 157: 957–965.
8. RedondoFJ, de la PenaTC, MorcilloCN, LucasMM, PueyoJJ (2009) Overexpression of flavodoxin in bacteroids induces changes in antioxidant metabolism leading to delayed senescence and starch accumulation in alfalfa root nodules. Plant Physiol 149: 1166–1178.
9. TognettiVB, PalatnikJF, FillatMF, MelzerM, HajirezaeiMR, et al. (2006) Functional replacement of ferredoxin by a cyanobacterial flavodoxin in tobacco confers broad-range stress tolerance. Plant Cell 18: 2035–2050.
10. LodeyroAF, CeccoliRD, Pierella KarlusichJJ, CarrilloN (2012) The importance of flavodoxin for environmental stress tolerance in photosynthetic microorganisms and transgenic plants. Mechanism, evolution and biotechnological potential. FEBS Lett 586: 2917–2924.
11. López-LlanoJ, MaldonadoS, JainS, LostaoA, Godoy-RuizR, et al. (2004) The long and short flavodoxins: II. The role of the differentiating loop in apoflavodoxin stability and folding mechanism. J Biol Chem 279: 47184–47191.
12. SanchoJ (2006) Flavodoxins: sequence, folding, binding, function and beyond. Cell Mol Life Sci 63: 855–864.
13. StoverCK, PhamXQ, ErwinAL, MizoguchiSD, WarrenerP, et al. (2000) Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406: 959–964.
14. BodeyGP, BolivarR, FainsteinV, JadejaL (1983) Infections caused by Pseudomonas aeruginosa. Rev Infect Dis 5: 279–313.
15. MorrisonAJJr, WenzelRP (1984) Epidemiology of infections due to Pseudomonas aeruginosa. Rev Infect Dis 6 Suppl 3: S627–642.
16. LyczakJB, CannonCL, PierGB (2002) Lung infections associated with cystic fibrosis. Clin Microbiol Rev 15: 194–222.
17. YeomJ, ParkW (2012) Pleiotropic effects of the mioC mutation on the physiology of Pseudomonas aeruginosa PAO1. FEMS Microbiol Lett 335: 47–57.
18. YeomJ, ParkW (2012) Biochemical characterization of ferredoxin-NADP(+) reductase interaction with flavodoxin in Pseudomonas putida. BMB Rep 45: 476–481.
19. BoratynGM, SchafferAA, AgarwalaR, AltschulSF, LipmanDJ, et al. (2012) Domain enhanced lookup time accelerated BLAST. Biol Direct 7: 12.
20. FillatMF, BorriasWE, WeisbeekPJ (1991) Isolation and overexpression in Escherichia coli of the flavodoxin gene from Anabaena PCC 7119. Biochem J 280 (Pt 1) 187–191.
21. PellettJD, BeckerDF, SaengerAK, FuchsJA, StankovichMT (2001) Role of aromatic stacking interactions in the modulation of the two-electron reduction potentials of flavin and substrate/product in Megasphaera elsdenii short-chain acyl-coenzyme A dehydrogenase. Biochemistry 40: 7720–7728.
22. MoreroNR, ArgarañaCE (2009) Pseudomonas aeruginosa deficient in 8-oxodeoxyguanine repair system shows a high frequency of resistance to ciprofloxacin. FEMS Microbiol Lett 290: 217–226.
23. SandersLH, SudhakaranJ, SuttonMD (2009) The GO system prevents ROS-induced mutagenesis and killing in Pseudomonas aeruginosa. FEMS Microbiol Lett 294: 89–96.
24. CookeMS, EvansMD, DizdarogluM, LunecJ (2003) Oxidative DNA damage: mechanisms, mutation, and disease. FASEB J 17: 1195–1214.
25. MoyanoAJ, LujánAM, ArgarañaCE, SmaniaAM (2007) MutS deficiency and activity of the error-prone DNA polymerase IV are crucial for determining mucA as the main target for mucoid conversion in Pseudomonas aeruginosa. Mol Microbiol 64: 547–559.
26. MatheeK, CiofuO, SternbergC, LindumPW, CampbellJI, et al. (1999) Mucoid conversion of Pseudomonas aeruginosa by hydrogen peroxide: a mechanism for virulence activation in the cystic fibrosis lung. Microbiology 145 (Pt 6) 1349–1357.
27. OchsnerUA, VasilML, AlsabbaghE, ParvatiyarK, HassettDJ (2000) Role of the Pseudomonas aeruginosa oxyR-recG operon in oxidative stress defense and DNA repair: OxyR-dependent regulation of katB-ankB, ahpB, and ahpC-ahpF. J Bacteriol 182: 4533–4544.
28. LiberatiNT, UrbachJM, MiyataS, LeeDG, DrenkardE, et al. (2006) An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants. Proc Natl Acad Sci U S A 103: 2833–2838.
29. LutterEI, FariaMM, RabinHR, StoreyDG (2008) Pseudomonas aeruginosa cystic fibrosis isolates from individual patients demonstrate a range of levels of lethality in two Drosophila melanogaster infection models. Infect Immun 76: 1877–1888.
30. Limmer S, Haller S, Drenkard E, Lee J, Yu S, et al. Pseudomonas aeruginosa RhlR is required to neutralize the cellular immune response in a Drosophila melanogaster oral infection model. Proc Natl Acad Sci U S A 108: 17378–17383.
31. KiewitzC, TümmlerB (2000) Sequence diversity of Pseudomonas aeruginosa: impact on population structure and genome evolution. J Bacteriol 182: 3125–3135.
32. SpencerDH, KasA, SmithEE, RaymondCK, SimsEH, et al. (2003) Whole-genome sequence variation among multiple isolates of Pseudomonas aeruginosa. J Bacteriol 185: 1316–1325.
33. MatheeK, NarasimhanG, ValdesC, QiuX, MatewishJM, et al. (2008) Dynamics of Pseudomonas aeruginosa genome evolution. Proc Natl Acad Sci U S A 105: 3100–3105.
34. WinsorGL, LamDK, FlemingL, LoR, WhitesideMD, et al. (2011) Pseudomonas Genome Database: improved comparative analysis and population genomics capability for Pseudomonas genomes. Nucleic Acids Res 39: D596–600.
35. SmaniaAM, SeguraI, PezzaRJ, BecerraC, AlbesaI, et al. (2004) Emergence of phenotypic variants upon mismatch repair disruption in Pseudomonas aeruginosa. Microbiology 150: 1327–1338.
36. FelizianiS, LujánAM, MoyanoAJ, SolaC, BoccoJL, et al. (2010) Mucoidy, quorum sensing, mismatch repair and antibiotic resistance in Pseudomonas aeruginosa from cystic fibrosis chronic airways infections. PLOS One 5: e12669.
37. Løbner-OlesenA, BoyeE (1992) Different effects of mioC transcription on initiation of chromosomal and minichromosomal replication in Escherichia coli. Nucleic Acids Research 20: 3029–3036.
38. BirchOM, HewitsonKS, FuhrmannM, BurgdorfK, BaldwinJE, et al. (2000) MioC is an FMN-binding protein that is essential for Escherichia coli biotin synthase activity in vitro. J Biol Chem 275: 32277–32280.
39. PuanKJ, WangH, DairiT, KuzuyamaT, MoritaCT (2005) fldA is an essential gene required in the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis. FEBS Lett 579: 3802–3806.
40. PomposielloPJ, BennikMH, DempleB (2001) Genome-wide transcriptional profiling of the Escherichia coli responses to superoxide stress and sodium salicylate. J Bacteriol 183: 3890–3902.
41. ZhengM, WangX, TempletonLJ, SmulskiDR, LaRossaRA, et al. (2001) DNA microarray-mediated transcriptional profiling of the Escherichia coli response to hydrogen peroxide. J Bacteriol 183: 4562–4570.
42. SchmidtKD, TümmlerB, RömlingU (1996) Comparative genome mapping of Pseudomonas aeruginosa PAO with P. aeruginosa C, which belongs to a major clone in cystic fibrosis patients and aquatic habitats. J Bacteriol 178: 85–93.
43. SilbyMW, WinstanleyC, GodfreySA, LevySB, JacksonRW (2011) Pseudomonas genomes: diverse and adaptable. FEMS Microbiol Rev 35: 652–680.
44. HassettDJ, CohenMS (1989) Bacterial adaptation to oxidative stress: implications for pathogenesis and interaction with phagocytic cells. FASEB J 3: 2574–2582.
45. WeiQ, MinhPN, DotschA, HildebrandF, PanmaneeW, et al. (2012) Global regulation of gene expression by OxyR in an important human opportunistic pathogen. Nucleic Acids Res 40: 4320–4333.
46. MaddocksSE, OystonPC (2008) Structure and function of the LysR-type transcriptional regulator (LTTR) family proteins. Microbiology 154: 3609–3623.
47. HayesJD, FlanaganJU, JowseyIR (2005) Glutathione transferases. Annu Rev Pharmacol Toxicol 45: 51–88.
48. RostiJ, BartonCJ, AlbrechtS, DupreeP, PaulyM, et al. (2007) UDP-glucose 4-epimerase isoforms UGE2 and UGE4 cooperate in providing UDP-galactose for cell wall biosynthesis and growth of Arabidopsis thaliana. Plant Cell 19: 1565–1579.
49. CresnarB, PlaperA, BreskvarK, Hudnik-PlevnikT (1998) cDNA sequence and deduced amino acid sequence of a fungal stress protein induced in Rhizopus nigricans by steroids. Biochem Biophys Res Commun 250: 664–667.
50. FanousA, HeckerM, GorgA, ParlarH, JacobF (2010) Corynebacterium glutamicum as an indicator for environmental cobalt and silver stress–a proteome analysis. J Environ Sci Health B 45: 666–675.
51. GrifantiniR, ToukokiC, ColapricoA, GryllosI (2011) Peroxide stimulon and role of PerR in group A Streptococcus. J Bacteriol 193: 6539–6551.
52. BrykR, LimaCD, Erdjument-BromageH, TempstP, NathanC (2002) Metabolic enzymes of mycobacteria linked to antioxidant defense by a thioredoxin-like protein. Science 295: 1073–1077.
53. CifaniN, PompiliB, AnileM, PatellaM, DisoD, et al. (2013) Reactive-oxygen-species-mediated P. aeruginosa killing is functional in human cystic fibrosis macrophages. PLOS One 8: e71717.
54. LauGW, BritiganBE, HassettDJ (2005) Pseudomonas aeruginosa OxyR is required for full virulence in rodent and insect models of infection and for resistance to human neutrophils. Infect Immun 73: 2550–2553.
55. JacobsMA, AlwoodA, ThaipisuttikulI, SpencerD, HaugenE, et al. (2003) Comprehensive transposon mutant library of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 100: 14339–14344.
56. KovachME, ElzerPH, HillDS, RobertsonGT, FarrisMA, et al. (1995) Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166: 175–176.
57. ChoiKH, SchweizerHP (2006) mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa. Nat Protoc 1: 153–161.
58. HoangTT, Karkhoff-SchweizerRR, KutchmaAJ, SchweizerHP (1998) A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212: 77–86.
59. ShinM (1971) Ferredoxin-NADP reductase from spinach. Methods Enzymol 23: 440–447.
60. DeLongJM, PrangeRK, HodgesDM, ForneyCF, BishopMC, et al. (2002) Using a modified ferrous oxidation-xylenol orange (FOX) assay for detection of lipid hydroperoxides in plant tissue. J Agric Food Chem 50: 248–254.
61. SedmakJJ, GrossbergSE (1977) A rapid, sensitive, and versatile assay for protein using Coomassie brilliant blue G250. Anal Biochem 79: 544–552.
62. Rodríguez-RojasA, BlázquezJ (2009) The Pseudomonas aeruginosa pfpI gene plays an antimutator role and provides general stress protection. J Bacteriol 191: 844–850.
63. FleiszigSM, Wiener-KronishJP, MiyazakiH, VallasV, MostovKE, et al. (1997) Pseudomonas aeruginosa-mediated cytotoxicity and invasion correlate with distinct genotypes at the loci encoding exoenzyme S. Infect Immun 65: 579–586.
Štítky
Genetika Reprodukčná medicínaČlánok vyšiel v časopise
PLOS Genetics
2014 Číslo 2
- Je „freeze-all“ pro všechny? Odborníci na fertilitu diskutovali na virtuálním summitu
- Gynekologové a odborníci na reprodukční medicínu se sejdou na prvním virtuálním summitu
Najčítanejšie v tomto čísle
- Genome-Wide Association Study of Metabolic Traits Reveals Novel Gene-Metabolite-Disease Links
- A Cohesin-Independent Role for NIPBL at Promoters Provides Insights in CdLS
- Classic Selective Sweeps Revealed by Massive Sequencing in Cattle
- Arf4 Is Required for Mammalian Development but Dispensable for Ciliary Assembly