Parasite Virulence - INSERM Avenir, CNRS URA2581  


  HEADDr. SPAETH, Gerald / gspaeth@pasteur.fr
  MEMBERSDr. FORESTIER, Claire / Dr. MORALES, Miguel / Dr. SCHMIDT-ARRAS, Dirk / Mme PESCHER, Pascale / Mme WATANABE, Reiko / M. LECLERCQ, Olivier/ M. GUESNON, Mickael / M. YAU, Wai-Lok / Mlle DACHER, Mariko/ Mme KACER, Martine/ Mlle VEILLAULT, Sophie


  Annual Report

Our laboratory use genetic and proteomic methods to study the molecular basis of virulence of the proto-zoan parasite Leishmania. Three axes are pursued:

Axis 1: The role of Leishmania MAP kinases in virulence and pathogenesis. Leishmania undergoes multiple differentiation steps in response to extracellular signals that adapt parasite virulence for survival in the insect vector and the human host. We use gene knock out and over-expression strategies to elucidate the role of the Leishmania extracellular-regulated/mitogen activated protein kinases LmaMPK4, 7, and 10 in parasite environmental sensing. Utilizing epitope-tagged recombinant kinases expressed in transgenic parasites we could reveal the cytoplasmic localization of these proteins, their amastigote-specific phosphorylation and activity, and their interaction with members of the heat shock protein family. We will elucidate the functions and interactions of these proteins during the infectious cycle with the major aim to gain insight into the mechanism of stage-specific gene regulation and signaling in Leishmania. Given the implication of LmaMPK signalling in the amastigote stage, these kinases and their substrates may contribute substantially to parasite virulence and therefore may represent new targets for therapeutic intervention.
Axis 2: Phosphoproteomic analysis of stage-specific signalling in Leishmania donovani. Despite the relevance of the Leishmania amastigote stage for pathogenesis, our understanding on the mechanisms underlying development of this stage and its intra-cellular survival remains poorly investigated. We elucidate the signalling networks associated with Leishmania differentiation analyzing purified phosphoproteins by Differential 2D gel electro-phoresis (DIGE) and LC-MS-MS/iTRAQ. To date, we have identified over 600 putative phosphoproteins and have quantified their abundance across the Leishmania life cycle. Current efforts are focused on the identification of the phosphorylation sites and the determination of the phosphorylation stoichiometry in pro- amd amastigote stages. The major objective is to reveal amastigote-specific phosphorylation events and use recombinant phosphoproteins for the identi-fication of novel amastigote-specific protein kinases. 
Axis 3: Exploiting the Leishmania kinome for drug development. Our previously published work on Leishmania MAP kinases and novel approaches recently developed in our laboratory, including phosphor-proteomic analysis and 2D in-gel kinase assay (see figure), are exploited through the FP7 LEISHDRUG consortium (www.leishdrug.org) for antiparasitic drug development. The LEISHDRUG consortium comprises 14 teams and is organized in three clusters with each two interactive scientific work packages that together follow the major stages of the drug development process, including (i) identification of hit compounds and target kinases, (ii) hit-to-lead validation, and (iii) lead characterization. 

Keywords: Leishmania, virulence, signaling, MAP kinases, phosphoproteomics

Virpar.jpg

Amastigote total protein resolved by two-dimensional gel electrophoresis and revealed by silver-staining (2DE, left). Kinase activities were identified by in-gel kinase assay and revealed by autoradiography (IGKA, right).



  Publications

  1. Morales M.A., Watanabe R., Laurent C., Lenormand P., Rousselle J.C., Namane A. and RG.F. Späth,, “Phosphoproteomic analysis of Leishmania donovani pro- and amastigote stages”, Proteomics, 2008 18:350-363.

  2. Späth G.F., McDowell M.A., and S.M. Beverley SM, “Leishmania major intracellular survival is not altered in SHP-1 deficient me(v) or CD45(-/-) mice”, Exp Parasitol. 2008 Jul 19[Epub ahead print].

  3. Frevert U., Späth G.F. and H. Yee, “Exoerythrocytic development of Plasmodium gallinaceum in the White Leghorn chicken”. Int J Parasitology, 2008, 38:655-72.

  4. Morales M.A., Renaud O., Faigle W., Shorte S.L. and G. F. Späth, 2007, “ Over-expression of Leishmania major MAP kinases reveals stage-specific induction of phosphotransferase activity”, Int. J. of Parasitology, , 11:1187-99.

  5. Arevalo I, Tulliano G, Quispe A, Spaeth G, Matlashewski G, Llanos-Cuentas A, Pollack H, 2007, “Role of imiquimod and parenteral meglumine antimoniate in the initial treatment of cutaneous leishmaniasis”, Clin. Infect. Dis., 44:1549-54.



  Web Site

More informations on our web site




Activity Reports 2009 - Institut Pasteur
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