Fighting the oxidative assault: The Trypanosoma cruzi journey to infection

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. Triparedoxina peroxidasa Ascorbato peroxidasa Triparedoxina Tripanotiona sintetasa Fe-Superóxido Dismutasa Life cycle of Trypanosoma cruzi 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. 8 min NBT2+ + O2•─ NBT+ + O2 2NBT+F+ + NBT 2+ Macrophage-T cruzi interactions 2 O2 + NADPH  2 O2• –+ NADP+ + H+ Rapid activation of NADP(H) oxidase during phagocytosis NBT DAPI Villalta et al, 1984

5. Inducible Nitric Oxide Synthase 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

6. Reactive Nitrogen Species Reactive Oxygen Species RNH2 O2 L-Arginine NADPH oxidase i NOS + 1e- - 5e- Nitric Oxide O2● _ ● NO - 1e- + 1e- SOD - ONOO peroxinitrite Hydrogen Peroxide H2O2 CO2 NO2¯ H+ Nitrite Peroxidase/H2O2 (MPO) - 1e- + 1e- ONOOH ONOOCO2¯ Hidroxil Radical Nitrogen dioxide ● NO2 OH¯ OH● + + 1e- - 1e- ●NO2 + CO3●¯ ●NO2+ OH● NO3¯ 2 H2O Nitrate Superoxide (radical anion) ●NO2 + CO3●¯

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

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

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. TR red In vitro peroxynitrite-oxidereductase activity of T. cruzi peroxiredoxins (10 3 M-1s-1) (10 3 M-1s-1) TcCPXRed NADPH T(SH)2 TXNox TR ONOOH ox 10 6 M-1s-1 + TcCPXOx TXNred NO2- TS2 NADP TR = trypanothione reductase T(SH)2= trypanothione TXN = tryparedoxin TcCPX= citosolic tryparedoxin peroxidase Trujillo, M et al., J. Biol. Chem. 279, 34175 (2004)

12. In vivo peroxynitrite-oxidereductase activity of T. cruzi TcMpx and tccpx peroxiredoxins 100 TcCPX IC50 480 M Control 80 TcMPX IC50 400 M TcAPX TcMPX 60 TcCPX [3H]-Thymidine Incorporation (%) 40 IC50 250 M 20 0 0 100 200 300 400 500 600 700 ONOO- (µM) Piacenza et al., Biochem J 2008, 410: 359-368.

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

14. Peroxynitrite-mediated protein oxidation Protection by peroxiredoxins Immuno-spin trapping Mason et al; 2002 - 2004 t1/2= months-years

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

16. OH R O• R Intraphagosomal peroxynitrite Peroxynitrite-derived intraphagosomal-T. cruzi protein radical detection ONOO- H+ OH •NO2 •OH + •NO2 NO 2 R 3-NO2-tyrosine Tyrosyl radical Tyrosine Alvarez et al., J. Biol. Chem. 286, 6627 (2011)

17. O NH H N 2 2 H C OCH 3 O T. cruzi CPX overexpressers •OH/•NO2 CO3●- O NH H N 2 Rhodamine Intracellular amastigote oxidant detection Dihydrorhodamine C OCH 3 O Alvarez et al., J. Biol. Chem. 286, 6627 (2011)

18. T. cruzi CPX overexpressers Tc CPX Wild type 120 100 80 Infección (%) * 60 40 20 Macrophage infection 0 ONOO- O2●- O2●- ONOO- Alvarez et al., J. Biol. Chem. 286, 6627 (2011)

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

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

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

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

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

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

26. First line of defenses: Superoxide Dismutases 2 O2• – + 2H+ O2 + H2O2 Prokaryotes Cu/Zn Mn Fe T. cruzi contains 4 isoforms : SOD-A/C mitochondrial SODB-1 Cytosol SODB-2 Glycosomes Most Eukaryotes Trypanosomatids

27. Studies with recombinant enzymes TbFeSODA TcFeSODB Cytosolic NO2-Tyr detection Inactivation due to Tyrosine nitration specific residues Mitochondrial Different enzyme sensitivities against peroxynitrite-dependent inactivation Fe-Superoxide Dismutases Mitochondrial Cytosolic Different cellular functions?

28. Intracellular probe oxidation Mitochondrial FeSOD-A and cell signaling SIN-1: gives intracellular and equimolar fluxes of NO and O2- Parasite viability • SOD-A inhibits peroxynitrite formation efficiently eliminating O2•- radicals

29. Role of FeSODs in cellular signaling and survival

30. Complement activation Ca2+ influx and mitochondrial dysfunction Programmed cell death in T. cruzi Death stimuli: Fresh human serum (FHS): FHS Control CTL HIS FHS Ctl TUNEL staining FHS DNA fragmentation Phosphatidyl serine exposure Piacenza et al., Proc. Natl. Acad. Sci. U.S.A. 98, 7301 (2001) Irigoín, F et al., Biochem. J (418)-595, 2009

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

32. HE Mito-Hidroetidio (Mitosox) Smith and Murphy, 2003 Mitochondrial FeSOD-A and cell signaling Intramitochondrial O2- detection: MitoSOX oxidation CL-Brener + DMNQ (superoxide generator)

33. Mitochondrial FeSOD-A and cell signaling • Parasite mitochondrial-O2- production triggers programmed cell death Piacenza et al., Biochem. J. 403, 323 (2007)

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

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

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

37. Cardiomyocyte-induced T. cruzi oxidative stress T. cruzi cardiomyocyte infection and detection of mitochondrial O2•- production by MitoSOX oxidation in intracellular amastigotes

38. Cardiomyocyte-induced T. cruzi oxidative stress Control Activated 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. International Colaboration Shane Wilkinson, UK Martin Taylor, UK John Kelly, UK Paola Zago, Argentina Miguel Basombrio, Argentina • Biochemistry Department • Gonzalo Peluffo • María Noel Alvarez • Alejandra Martínez • Madia Trujilo • Martín Hugo • Dolores Piñeyro • Carlos Robello • Rafael Radi Howard Hughes Medical Institute, USA National Institute of Health (NIH), USA PEDECIBA (Uruguay) CONICYT (Uruguay) PDT (Uruguay)

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

45. Activación Clásica

46. Activación Clásica Activación Alterna

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