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Fighting the oxidative assault: The Trypanosoma cruzi journey to infection Dra. Lucia Piacenza Dpto. De Bioquimica, Facultad de Medicina y Center for Free.

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Presentation on theme: "Fighting the oxidative assault: The Trypanosoma cruzi journey to infection Dra. Lucia Piacenza Dpto. De Bioquimica, Facultad de Medicina y Center for Free."— Presentation transcript:

1 Fighting the oxidative assault: The Trypanosoma cruzi journey to infection Dra. Lucia Piacenza Dpto. De Bioquimica, Facultad de Medicina y Center for Free Radical and Biomedical Research Montevideo, Uruguay

2 Life cycle of Trypanosoma cruzi Triparedoxina peroxidasa Ascorbato peroxidasa Triparedoxina Tripanotiona sintetasa Fe-Superóxido Dismutasa Adapted from The Trypanosoma cruzi Proteome Atwood III et al; Science Vol 309; 2005

3 Central rol of macrophages in the control of T. cruzi infections Phagocytes: First line of defense against invading T. cruzi

4 Macrophage-T cruzi interactions Villalta et al, min Rapid activation of NADP(H) oxidase during phagocytosis 2 O 2 + NADPH 2 O 2 – + NADP + + H + NBT DAPI NBT 2+ + O 2 NBT + + O 2 2NBT + F + + NBT 2+

5 Activators:IFN-γ, TNF-α, IL-1β, IL-6, pathogen-derived products (viral, bacterial and protozoa) Inhibitors:IL-4, IL-10, IL-13, TGF-β Apoptotic cell removal Inducible Nitric Oxide Synthase

6 NO Nitric Oxide NO 2 ¯ Nitrite Nitrogen dioxide NO 2 NO 3 ¯ Nitrate RNH 2 L-Arginine - 1e - i NOS Reactive Nitrogen Species Peroxidase/H 2 O 2 (MPO) - 5e - Superoxide (radical anion) + Hydrogen Peroxide OH Hidroxil Radical H2O2H2O2 2 H 2 O O2O2 Reactive Oxygen Species NADPH oxidase OH ¯ SOD + 1e - O2 _O2 _ peroxinitrite NO 2 + CO 3 ¯ ONOO ONOOH NO 2 + OH H+H+ CO 2 ONOOCO 2 ¯ - NO 2 + CO 3 ¯

7 Szabo, Ischiropoulos and Radi, Nature Reviews (2007) Peroxynitrite toxicity

8 Intraphagosomal Oxidants Resting Macrophage 2 hrs infection Piacenza et al., Curr. Opin. Microbiol. 12, 415 (2009)

9 Activated macrophage 2 hrs infection Piacenza et al., Curr. Opin. Microbiol. 12, 415 (2009) Intraphagosomal Oxidants- Our hypothesis

10 Antioxidant Defense in T. cruzi Piacenza et al., Curr. Opin. Microbiol. 12, 415 (2009)

11 NADPH NADP + TR ox TR red T(SH) 2 TS 2 TXN ox TXN red TcCPX Red TcCPX Ox ONOOH NO 2 - In vitro peroxynitrite-oxidereductase activity of T. cruzi peroxiredoxins TR = trypanothione reductase T(SH) 2 = trypanothione TXN = tryparedoxin TcCPX= citosolic tryparedoxin peroxidase 10 6 M -1 s -1 (10 3 M -1 s -1 ) Trujillo, M et al., J. Biol. Chem. 279, (2004)

12 ONOO - (µM) Control TcAPX TcCPX TcMPX [ 3 H]-Thymidine Incorporation (%) TcCPX IC M TcMPX IC M IC M In vivo peroxynitrite-oxidereductase activity of T. cruzi TcMpx and tccpx peroxiredoxins Piacenza et al., Biochem J 2008, 410:

13 ControlControl + peroxynitrite TcCPX + peroxynitrite TcCPX T. cruzi overexpressers are resistant to peroxynitrite cytotoxicity

14 Peroxynitrite-mediated protein oxidation Protection by peroxiredoxins Immuno-spin trapping Masonet al; t 1/2 = months-years

15 Intraphagosomal peroxynitrite Peroxynitrite-derived intraphagosomal-T. cruzi protein radical detection Alvarez et al., J. Biol. Chem. 286, 6627 (2011) Immune spin-trap of DMPO-protein nitrone adducts

16 Intraphagosomal peroxynitrite Alvarez et al., J. Biol. Chem. 286, 6627 (2011) Peroxynitrite-derived intraphagosomal-T. cruzi protein radical detection OH R Tyrosine R O Tyrosyl radical NO 2 R OH NO 2 3-NO 2 -tyrosine ONOO - OH + NO 2 H+H+

17 O NH 2 H 2 N C O OCH 3 H H 2 N C O 3 O NH OH/ NO 2 CO 3 - Dihydrorhodamine Rhodamine T. cruzi CPX overexpressers Alvarez et al., J. Biol. Chem. 286, 6627 (2011) Intracellular amastigote oxidant detection

18 T. cruzi CPX overexpressers Infección (%) Tc CPX Wild type O 2 - ONOO - O 2 - * ONOO - Macrophage infection Alvarez et al., J. Biol. Chem. 286, 6627 (2011)

19 T. cruzi CPX overexpressers Balb-c infections In vivo Days post-infection Trypomastigotes / 50 fields Alvarez et al., J. Biol. Chem. 286, 6627 (2011) Increase infectivity of TcCPX overexpressers

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21 Does enzymes of T. cruzi antioxidant network are involved in infectivity?

22 T. cruzi strains and virulence Piacenza et al., Int. J. Parasitol. 39, 1455 (2009) Biochemical and molecular diversity of T. cruzi populations

23 TCC CL-WT T. cruzi strain analysis Piacenza et al., Int. J. Parasitol. 39, 1455 (2009) Virulent vs attenuated

24 Antioxidant enzyme contents Specific antibodies used TcCPXTcMPXTcTS TcCPXTcMPXTcTS Contenido relativo de enzima 3 days epimastigotes Metacyclic trypomastigotes Piacenza et al., Int. J. Parasitol. 39, 1455 (2009) Antioxidant enzyme evaluated: TcCPX TcMPX TcAPX TcTS TcTR

25 Trypomastigotes/100 fields Relative enzyme content (metacyclic trypomastigote) Antioxidant enzymes: virulence factors Piacenza et al., Int. J. Parasitol. 39, 1455 (2009)

26 First line of defenses: Superoxide Dismutases 2 O 2 – + 2H + O 2 + H 2 O 2 Cu/ZnMn Fe Most Eukaryotes Prokaryotes Trypanosomatids T. cruzi contains 4 isoforms : SOD-A/C mitochondrial SODB-1 Cytosol SODB-2 Glycosomes

27 Fe-Superoxide Dismutases Different cellular functions? TcFeSODB TbFeSODA NO 2 -Tyr detection Inactivation due to Tyrosine nitration specific residues Different enzyme sensitivities against peroxynitrite-dependent inactivation Cytosolic Mitochondrial Studies with recombinant enzymes Mitochondrial Cytosolic

28 Mitochondrial FeSOD-A and cell signaling SIN-1: gives intracellular and equimolar fluxes of NO and O 2 - SOD-A inhibits peroxynitrite formation efficiently eliminating O 2 - radicals Intracellular probe oxidation Parasite viability

29 Role of FeSODs in cellular signaling and survival

30 Programmed cell death in T. cruzi TUNEL staining Death stimuli: Fresh human serum (FHS): Control FHS CTL HIS FHS DNA fragmentation Phosphatidyl serine exposure Ctl FHS Piacenza et al., Proc. Natl. Acad. Sci. U.S.A. 98, 7301 (2001) Irigoín, F et al., Biochem. J (418)-595, 2009 Complement activation Ca 2+ influx and mitochondrial dysfunction

31 Mitochondrial FeSOD-A and cell signaling Control TcSODA IHS FHS HIS FHS bp Mitochondrial Fe-SODA overexpression Piacenza et al., Biochem. J. 403, 323 (2007)

32 CL-Brener + DMNQ (superoxide generator) Intramitochondrial O 2 - detection: MitoSOX oxidation Mitochondrial FeSOD-A and cell signaling Smith and Murphy, 2003 HE Mito-Hidroetidio (Mitosox)

33 Mitochondrial FeSOD-A and cell signaling Parasite mitochondrial-O 2 - production triggers programmed cell death Piacenza et al., Biochem. J. 403, 323 (2007)

34 Peritoneal macrophages Souza EM et al., 2003 Cell and Tissue Research. In vivo hearth infection(14 dpi) In vitro cardiomiocytes infection Apoptotic parasites in the vertebrate host Chronic infection Death stimuli unknown

35 Mitochondrial respiration Piacenza et al., Curr. Opin. Microbiol. 12, 415 (2009)

36 Ctl DMNQ (1 mM) SPM-NO (3 mM) CL-Brener TcSOD-A Mitochondrial FeSOD-A and cell signaling Intramitochondrial NO-dependent MitoSOX oxidation: SOD-A protects NO-derived mitochondrial parasite dysfunction Control TcSODA

37 T. cruzi cardiomyocyte infection and detection of mitochondrial O 2 - production by MitoSOX oxidation in intracellular amastigotes Cardiomyocyte-induced T. cruzi oxidative stress

38 ControlActivated Cardiomyocyte-derived NO as a cellular mediator in the control of parasite proliferation

39 Conclusions, hypothesis and perspectives Intraphagosomal peroxynitrite is a potent cytotoxin against internalized T. cruzi Wild type strains of high virulence have increase expression of antioxidant enzymes Levels of mitochondrial FeSOD could modulate parasite fate during chronic infection

40 Conclusions, hypothesis and perspectives Evaluation of the behavior in chronic models of infection of stable overepressers of TcSODA, TcSODB, TcCPX and TcMPX In chronic infection we postulate NO as a key player In the control and progression of the disease Evaluate cardiomyocyte-derived NO as a cell mediator for the signaling of amastigote apoptosis in vivo

41 Howard Hughes Medical Institute, USA National Institute of Health (NIH), USA PEDECIBA (Uruguay) CONICYT (Uruguay) PDT (Uruguay) Biochemistry Department Gonzalo Peluffo María Noel Alvarez Alejandra Martínez Madia Trujilo Martín Hugo Dolores Piñeyro Carlos Robello Rafael Radi International Colaboration Shane Wilkinson, UK Martin Taylor, UK John Kelly, UK Paola Zago, Argentina Miguel Basombrio, Argentina

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43 Different mitochondrial membrane potential in infective T. cruzi stages obtained from infected cardiomiocytes

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45 Activación Clásica

46 Activación Alterna Activación Clásica

47 Eficiencia en el establecimineto de la infección 1- Capacidad de las células inmunes de establecer de forma temprana una respuesta proinflamatoria con inducción de la iNOS en macrófagos 2- Niveles de enzimas antioxidantes en los tripomastigotas metacíclicos 3- Presencia de parásitos apoptóticos en el inóculo infectivo


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