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The Programmed Cell Death Pathway of Trypanosomatid Parasites

The Programmed Cell Death Pathway of Trypanosomatid Parasites . Alain Debrabant, Ph.D. Laboratory of Bacterial Parasitic and Unconventional Agents Division of Emerging and Transfusion Transmitted Diseases OBRR/CBER / FDA. Life cycle of Leishmania. Differentiation. Dwyer, D.M. Growth.

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The Programmed Cell Death Pathway of Trypanosomatid Parasites

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  1. The Programmed Cell Death Pathway of Trypanosomatid Parasites Alain Debrabant, Ph.D. Laboratory of Bacterial Parasitic and Unconventional Agents Division of Emerging and Transfusion Transmitted Diseases OBRR/CBER / FDA

  2. Life cycle of Leishmania Differentiation Dwyer, D.M. Growth Promastigote Differentiation Sandfly vector Amastigote Human host Differentiation Macrophage Debrabant, A. Adapted from Paulo Pimenta Dwyer, D.M.

  3. N ? Promastigote Caspases N CAD (PCD) EndoG Amastigote Programmed Cell Death (PCD) Death signals Cell death Caspases N (apoptosis) Nucleases (CAD, EndoG)

  4. Objectives Identify the effector molecules of the Leishmania programmed cell death pathway Outcomes Better understanding of parasite growth and pathogenesis Effector molecules of trypanosomatid PCD pathways are potential new targets for drugs that would selectively trigger parasite death

  5. Programmed Cell Death Pathway in Leishmania 2 Mitochondria (kinetoplast) Death Signals . Change in mitochondrial membrane potential . Cytochrome C release . Growth arrest . Anti-leishmanial drugs . Oxidative stress (H2O2) ? ? 1 K 3 N Plasma membrane Cell death Nucleus . Anexin V positive . PI positive 5 . TUNEL positive . DNA ladder 4 Activation of protease(s) “Caspase-like” activities 1-5: possible sequence of events N: nucleus; K: kinetoplast

  6. Similarity: 95% Localization of LdMCs LdMC1 LdMC2 ~52 kDa proteins LdMC enzymatic activity LdMC Acidocalcisome Merge Phase Metacaspases are stored in acidocalcisomes + H2O2 - H2O2 Metacaspases involved in Leishmania PCD ? Anti-LdMC Anti-TcPPase Activity TUNEL assay Caspase substrates Trypsin substrates • Insensitive to caspase inhibitors (e.g. Z-VAD-fmk) • Inhibited by serine/cysteine protease inhibitors • (e.g. leupeptin, antipain, TLCK) - H2O2 + H2O2 LdMC are probably involved in Leishmania PCD Metacaspase have “trypsin-like” activity Leishmania donovani metacaspases

  7. Programmed Cell Death Pathway in Leishmania 2 Mitochondria (kinetoplast) Death Signals . Change in mitochondrial membrane potential . Cytochrome C release . Growth arrest . Anti-leishmanial drugs . Camptothecin . H2O2 ? ? 1 K 3 N Plasma membrane Cell death Nucleus . Anexin V positive . PI positive 5 . TUNEL positive . DNA ladder 4 Activation of protease(s) “Caspase-like” activity Metacaspase activity 1-5: possible sequence of events N: nucleus; K: kinetoplast

  8. Overexpression of EndoG in Leishmania Leishmania EndoG + H2O2 Untreated Phase TUNEL Single copy gene 54 kDa protein Control 29% similarity with human EndoG Constitutively transcribed in both parasite stages (RT-PCR) Counts 45 7.6 6.4 43 6 76 13 36 Ld EndoG+ Fluorescence intensity Parasites overexpressing EndoG spontaneously undergo PCD Localization of Leishmania endoG EndoG RNAi in Trypanosoma brucei + H2O2 Untreated Mitotracker Red HA Merge Phase Control Control HA CtrlInd EndoG-HA Northern Ld EndoG+ r RNA EndoG RNAi -tubulin EndoG-HA is associated with Leishmania mitochondria Parasites with reduced expression of EndoG are more resistant to H2O2 induced PCD Leishmania EndonucleaseG

  9. Summary Goal: Identify effectors molecules of Leishmania PCD pathway Results: We identified and characterized two effector molecules of the Leishmania PCD pathway (metacaspases and endonuclease-G) Outcomes: 1. Better understanding of Leishmania growth and pathogenesis 2. Effector molecules of PCD represent new drug targets

  10. Acknowledgments DETTD / CBER Nancy Lee (Metacaspase) S. Gannavaram (EndoG) A. Selvapandiyan Robert Duncan Hira Nakhasi University of Illinois Roberto Docampo Summer Students Chetan Vedvyas Vaibhav Bist NIH / Laboratory of Parasitic Diseases Sylvie Bertholet Shaden Kamhawi

  11. Thank You

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