Mitochondrial Peroxiredoxin Plays a Crucial Peroxidase-Unrelated Role during Infection: Insight into Its Novel Chaperone Activity
Two-cysteine peroxiredoxins are ubiquitous peroxidases that play various functions in cells. In Leishmania and related trypanosomatids, which lack catalase and selenium-glutathione peroxidases, the discovery of this family of enzymes provided the molecular basis for peroxide removal in these organisms. In this report the functional relevance of one of such enzymes, the mitochondrial 2-Cys peroxiredoxin (mTXNPx), was investigated along the Leishmania infantum life cycle. mTXNPx null mutants (mtxnpx−) produced by a gene replacement strategy, while indistinguishable from wild type promastigotes, were found unable to thrive in a murine model of infection. Unexpectedly, however, the avirulent phenotype of mtxnpx− was not due to lack of the peroxidase activity of mTXNPx as these behaved like controls when exposed to oxidants added exogenously or generated by macrophages during phagocytosis ex vivo. In line with this, mtxnpx− were also avirulent when inoculated into murine hosts unable to mount an effective oxidative phagocyte response (B6.p47phox−/− and B6.RAG2−/− IFN-γ−/− mice). Definitive conclusion that the peroxidase activity of mTXNPx is not required for parasite survival in mice was obtained by showing that a peroxidase-inactive version of this protein was competent in rescuing the non-infective phenotype of mtxnpx−. A novel function is thus proposed for mTXNPx, that of a molecular chaperone, which may explain the impaired infectivity of the null mutants. This premise is based on the observation that the enzyme is able to suppress the thermal aggregation of citrate synthase in vitro. Also, mtxnpx− were more sensitive than controls to a temperature shift from 25°C to 37°C, a phenotype reminiscent of organisms lacking specific chaperone genes. Collectively, the findings reported here change the paradigm which regards all trypanosomatid 2-Cys peroxiredoxins as peroxide-eliminating devices. Moreover, they demonstrate, for the first time, that these 2-Cys peroxiredoxins can be determinant for pathogenicity independently of their peroxidase activity.
Vyšlo v časopise:
Mitochondrial Peroxiredoxin Plays a Crucial Peroxidase-Unrelated Role during Infection: Insight into Its Novel Chaperone Activity. PLoS Pathog 7(10): e32767. doi:10.1371/journal.ppat.1002325
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.ppat.1002325
Souhrn
Two-cysteine peroxiredoxins are ubiquitous peroxidases that play various functions in cells. In Leishmania and related trypanosomatids, which lack catalase and selenium-glutathione peroxidases, the discovery of this family of enzymes provided the molecular basis for peroxide removal in these organisms. In this report the functional relevance of one of such enzymes, the mitochondrial 2-Cys peroxiredoxin (mTXNPx), was investigated along the Leishmania infantum life cycle. mTXNPx null mutants (mtxnpx−) produced by a gene replacement strategy, while indistinguishable from wild type promastigotes, were found unable to thrive in a murine model of infection. Unexpectedly, however, the avirulent phenotype of mtxnpx− was not due to lack of the peroxidase activity of mTXNPx as these behaved like controls when exposed to oxidants added exogenously or generated by macrophages during phagocytosis ex vivo. In line with this, mtxnpx− were also avirulent when inoculated into murine hosts unable to mount an effective oxidative phagocyte response (B6.p47phox−/− and B6.RAG2−/− IFN-γ−/− mice). Definitive conclusion that the peroxidase activity of mTXNPx is not required for parasite survival in mice was obtained by showing that a peroxidase-inactive version of this protein was competent in rescuing the non-infective phenotype of mtxnpx−. A novel function is thus proposed for mTXNPx, that of a molecular chaperone, which may explain the impaired infectivity of the null mutants. This premise is based on the observation that the enzyme is able to suppress the thermal aggregation of citrate synthase in vitro. Also, mtxnpx− were more sensitive than controls to a temperature shift from 25°C to 37°C, a phenotype reminiscent of organisms lacking specific chaperone genes. Collectively, the findings reported here change the paradigm which regards all trypanosomatid 2-Cys peroxiredoxins as peroxide-eliminating devices. Moreover, they demonstrate, for the first time, that these 2-Cys peroxiredoxins can be determinant for pathogenicity independently of their peroxidase activity.
Zdroje
1. CastroHTomásAM 2008 Peroxidases of Trypanosomatids. Antioxid Redox Signal 10 1593 1606
2. RheeSGWooHA 2011 Multiple Functions of Peroxiredoxins: Peroxidases, Sensors and Regulators of the Intracellular Messenger H2O2, and Protein Chaperones. Antioxid Redox Signal 15 781 794
3. Krauth-SiegelRLCominiMASchleckerT 2007 The trypanothione system. FlohéLHarrisJR Peroxiredoxin Systems New York Springer 231 251
4. NogocekeEGommelDUKiessMKaliszHMFlohéL 1997 A unique cascade of oxidoreductases catalyses trypanothione-mediated peroxide metabolism in Crithidia fasciculata. Biol Chem 378 827 836
5. JangHHLeeKOChiYHJungBGParkSK 2004 Two enzymes in one; two yeast peroxiredoxins display oxidative stress-dependent switching from a peroxidase to a molecular chaperone function. Cell 117 625 635
6. MoonJCHahYSKimWYJungBGJangHH 2005 Oxidative stress-dependent structural and functional switching of a human 2-Cys peroxiredoxin isotype II that enhances HeLa cell resistance to H2O2-induced cell death. J Biol Chem 280 28775 28784
7. WilkinsonSRTempertonNJMondragonAKellyJM 2000 Distinct mitochondrial and cytosolic enzymes mediate trypanothione-dependent peroxide metabolism in Trypanosoma cruzi. J Biol Chem 275 8220 8225
8. CastroHSousaCSantosMCordeiro-da-SilvaAFlohéL 2002 Complementary antioxidant defense by cytoplasmic and mitochondrial peroxiredoxins in Leishmania infantum. Free Radic Biol Med 33 1552 1562
9. BarrSDGedamuL 2003 Role of peroxidoxins in Leishmania chagasi survival. Evidence of an enzymatic defense against nitrosative stress. J Biol Chem 278 10816 10823
10. LinYCHsuJYChiangSCLeeST 2005 Distinct overexpression of cytosolic and mitochondrial tryparedoxin peroxidases results in preferential detoxification of different oxidants in arsenite-resistant Leishmania amazonensis with and without DNA amplification. Mol Biochem Parasitol 142 66 75
11. PiacenzaLPeluffoGAlvarezMNKellyJMWilkinsonSR 2008 Peroxiredoxins play a major role in protecting Trypanosoma cruzi against macrophage- and endogenously-derived peroxynitrite. Biochem J 410 359 368
12. IyerJPKaprakkadenAChoudharyMLShahaC 2008 Crucial role of cytosolic tryparedoxin peroxidase in Leishmania donovani survival, drug response and virulence. Mol Microbiol 68 372 391
13. WilkinsonSRHornDPrathalingamSRKellyJM 2003 RNA interference identifies two hydroperoxide metabolizing enzymes that are essential to the bloodstream form of the african trypanosome. J Biol Chem 278 31640 31646
14. AcestorNMasinaSIvesAWalkerJSaraviaNG 2006 Resistance to oxidative stress is associated with metastasis in mucocutaneous leishmaniasis. J Infect Dis 194 1160 1167
15. WalkerJAcestorNGongoraRQuadroniMSeguraI 2006 Comparative protein profiling identifies elongation factor-1beta and tryparedoxin peroxidase as factors associated with metastasis in Leishmania guyanensis. Mol Biochem Parasitol 145 254 264
16. PiacenzaLAlvarezMNPeluffoGRadiR 2009 Fighting the oxidative assault: the Trypanosoma cruzi journey to infection. Curr Opin Microbiol 12 415 421
17. PiacenzaLZagoMPPeluffoGAlvarezMNBasombrioM 2009 Enzymes of the antioxidant network as novel determiners of Trypanosoma cruzi virulence. Int J Parasitol 39 1455 1464
18. HarderSBenteMIsermannKBruchhausI 2006 Expression of a mitochondrial peroxiredoxin prevents programmed cell death in Leishmania donovani. Eukaryot Cell 5 861 870
19. LiuBLiuYMotykaSAAgboEEEnglundPT 2005 Fellowship of the rings: the replication of kinetoplast DNA. Trends Parasitol 21 363 369
20. SelaDYaffeNShlomaiJ 2008 Enzymatic mechanism controls redox-mediated protein-DNA interactions at the replication origin of kinetoplast DNA minicircles. J Biol Chem 283 32034 32044
21. BeattieLPeltanAMaroofAKirbyABrownN 2010 Dynamic imaging of experimental Leishmania donovani-induced hepatic granulomas detects Kupffer cell-restricted antigen presentation to antigen-specific CD8 T cells. PLoS Pathog 6 e1000805
22. MotykaSADrewMEYildirirGEnglundPT 2006 Overexpression of a cytochrome b5 reductase-like protein causes kinetoplast DNA loss in Trypanosoma brucei. J Biol Chem 281 18499 18506
23. WieseM 1998 A mitogen-activated protein (MAP) kinase homologue of Leishmania mexicana is essential for parasite survival in the infected host. Embo J 17 2619 2628
24. HuynhCSacksDLAndrewsNW 2006 A Leishmania amazonensis ZIP family iron transporter is essential for parasite replication within macrophage phagolysosomes. J Exp Med 203 2363 2375
25. SpäthGFEpsteinLLeaderBSingerSMAvilaHA 2000 Lipophosphoglycan is a virulence factor distinct from related glycoconjugates in the protozoan parasite Leishmania major. Proc Natl Acad Sci U S A 97 9258 9263
26. MisslitzAMottramJCOverathPAebischerT 2000 Targeted integration into a rRNA locus results in uniform and high level expression of transgenes in Leishmania amastigotes. Mol Biochem Parasitol 107 251 261
27. CastroHBuddeHFlohéLHofmannBLünsdorfH 2002 Specificity and kinetics of a mitochondrial peroxiredoxin of Leishmania infantum. Free Radic Biol Med 33 1563 1574
28. LoschenGFlohéLChanceB 1971 Respiratory chain linked H2O2 production in pigeon heart mitochondria. FEBS Lett 18 261 264
29. MontemartiniMKaliszHMHechtHJSteinertPFlohéL 1999 Activation of active-site cysteine residues in the peroxiredoxin-type tryparedoxin peroxidase of Crithidia fasciculata. Eur J Biochem 264 516 524
30. FlohéLBuddeHBrunsKCastroHClosJ 2002 Tryparedoxin peroxidase of Leishmania donovani: molecular cloning, heterologous expression, specificity, and catalytic mechanism. Arch Biochem Biophys 397 324 335
31. BuchnerJGrallertHJakobU 1998 Analysis of chaperone function using citrate synthase as nonnative substrate protein. Methods Enzymol 290 323 338
32. WilkinsonSRMeyerDJKellyJM 2000 Biochemical characterization of a trypanosome enzyme with glutathione-dependent peroxidase activity. Biochem J 352 Pt 3 755 761
33. SchleckerTSchmidtADirdjajaNVonckenFClaytonC 2005 Substrate specificity, localization, and essential role of the glutathione peroxidase-type tryparedoxin peroxidases in Trypanosoma brucei. J Biol Chem 280 14385 14394
34. KönigJFairlambAH 2007 A comparative study of type I and type II tryparedoxin peroxidases in Leishmania major. FEBS J 274 5643 5658
35. DiechtierowMKrauth-SiegelRL 2011 A tryparedoxin-dependent peroxidase protects African trypanosomes from membrane damage. Free Radic Biol Med 51 856 868
36. CastroHRomaoSCarvalhoSTeixeiraFSousaC 2010 Mitochondrial redox metabolism in trypanosomatids is independent of tryparedoxin activity. PLoS One 5 e12607
37. HillebrandHSchmidtAKrauth-SiegelRL 2003 A second class of peroxidases linked to the trypanothione metabolism. J Biol Chem 278 6809 6815
38. TrujilloMBuddeHPiñeyroMDStehrMRobelloC 2004 Trypanosoma brucei and Trypanosoma cruzi tryparedoxin peroxidases catalytically detoxify peroxynitrite via oxidation of fast reacting thiols. J Biol Chem 279 34175 34182
39. RequejoRHurdTRCostaNJMurphyMP 2010 Cysteine residues exposed on protein surfaces are the dominant intramitochondrial thiol and may protect against oxidative damage. FEBS J 277 1465 1480
40. DolaiSYadavRKPalSAdakS 2008 Leishmania major ascorbate peroxidase overexpression protects cells against reactive oxygen species-mediated cardiolipin oxidation. Free Radic Biol Med 45 1520 1529
41. ParkJWPiszczekGRheeSGChockPB 2011 Glutathionylation of peroxiredoxin I induces decamer to dimers dissociation with concomitant loss of chaperone activity. Biochemistry 50 3204 3210
42. SanchezYLindquistSL 1990 HSP104 required for induced thermotolerance. Science 248 1112 1115
43. HübelAKrobitschSHöraufAClosJ 1997 Leishmania major Hsp100 is required chiefly in the mammalian stage of the parasite. Mol Cell Biol 17 5987 5995
44. QueitschCHongSWVierlingELindquistS 2000 Heat shock protein 101 plays a crucial role in thermotolerance in Arabidopsis. Plant Cell 12 479 492
45. VoosW 2009 Mitochondrial protein homeostasis: the cooperative roles of chaperones and proteases. Res Microbiol 160 718 725
46. KellyJMWardHMMilesMAKendallG 1992 A shuttle vector which facilitates the expression of transfected genes in Trypanosoma cruzi and Leishmania. Nucleic Acids Res 20 3963 3969
47. RomaoSCastroHSousaCCarvalhoSTomásAM 2009 The cytosolic tryparedoxin of Leishmania infantum is essential for parasite survival. Int J Parasitol 39 703 711
48. BuffetPASulahianAGarinYJNassarNDerouinF 1995 Culture microtitration: a sensitive method for quantifying Leishmania infantum in tissues of infected mice. Antimicrob Agents Chemother 39 2167 2168
49. CastroHSousaCNovaisMSantosMBuddeH 2004 Two linked genes of Leishmania infantum encode tryparedoxins localised to cytosol and mitochondrion. Mol Biochem Parasitol 136 137 147
Štítky
Hygiena a epidemiológia Infekčné lekárstvo LaboratóriumČlánok vyšiel v časopise
PLOS Pathogens
2011 Číslo 10
- Parazitičtí červi v terapii Crohnovy choroby a dalších zánětlivých autoimunitních onemocnění
- Očkování proti virové hemoragické horečce Ebola experimentální vakcínou rVSVDG-ZEBOV-GP
- Koronavirus hýbe světem: Víte jak se chránit a jak postupovat v případě podezření?
Najčítanejšie v tomto čísle
- Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Regulates Cell Stress Response and Apoptosis
- Biochemical and Structural Insights into the Mechanisms of SARS Coronavirus RNA Ribose 2′-O-Methylation by nsp16/nsp10 Protein Complex
- The SARS-Coronavirus-Host Interactome: Identification of Cyclophilins as Target for Pan-Coronavirus Inhibitors
- Herpesvirus Telomerase RNA (vTR) with a Mutated Template Sequence Abrogates Herpesvirus-Induced Lymphomagenesis