Parasite Virulence - INSERM U1010, CNRS URA2581  


  HEADDr. SPAETH, Gerald / gspaeth@pasteur.fr
  MEMBERSDr. FORESTIER, Claire / Dr. MORALES, Miguel / Dr. FOUCHER, Aude / Mme PESCHER, Pascale / M. LECLERCQ, Olivier / M. GUESNON, Mickael / M. YAU, Wai-Lok / Mlle DACHER, Mariko / M. CAYLA, Mathieu / Mme ROUSELLE, Brigitte/ Mlle VEILLAULT, Sophie


  Annual Report

Our laboratory uses 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. We use gene knock out and over-expression strategies to elucidate the role of the Leishmania MAP kinases LmaMPK4, 7, and 10 in parasite environmental sensing. Utilizing epitope-tagged recombinant kinases expressed in transgenic parasites we revealed the cytoplasmic localization of these proteins, their amastigote-specific activity and phosphorylation, and the interaction of LmaMPK7 with heat shock proteins and LmaMPK10 with a putative orthologue of the FACT complex implicated in chromatin reorganization. Mutagenesis analysis allowed us to reveal a role of LmaMPK7 in the parasite-stress response, and to identify the parasite-specific C-terminal domain of LmaMPK10 as a negative regulatory element that inhibits the activity of this kinase. None of the three LmaMPKs could be studied by classical null mutant analsysis, suggesting that these proteins are essential for parasite viability. We currently establish conditional LmaMPK knock out mutants to overcome this limitation and to study the role of LmaMPKs in Leishmania differentiation and virulence.
Axis 2: Phosphoproteomic analysis of stage-specific signalling in Leishmania donovani. Utilizing Differential 2D gel electrophoresis (2D-DIGE) and LC-MS/MS, we identified over 600 phosphoproteins, quantified their abundance across the Leishmania stages, and mapped 108 phosphorylation sites. Our data reveal amastigote-specific phosphorylation of Leishmania HSPs as being an important factor in post-translational control of the parasite heat shock response. We identified a unique phosphorylation site in highly conserved Leishmania HSP90 and revealed the presence of STI-1/HOP-containing chaperone complexes that interact with ribosomal client proteins in an amastigote-specific manner. Mutagenesis analysis in conditional STI-1 null mutants uncovered two STI-1 phosphorylation sites that are essential for Leishmania survival. Phosphorylation of Leishmania chaperones thus represents a promising drug target via inhibition of heat-induced protein kinases.

Axis 3: Proteomic discovery of novel Leishmania protein kinases. Combining FPLC and isoelectric focussing with SDS-PAGE and in-gel kinase activity assay (IGKA), we established a powerful 2D screening that allowed us to reveal amastigote-specific phospho-transferase activities and to detect protein kinases that are able to use the Leishmania chaperone cyclophilin 40 and LmaMPK10 as substrates. The 2D IGKA thus will allow us to identify novel parasite protein kinases implicated in the parasite heat shock response and the activation of LmaMPKs, which will be exploited through the FP7 LEISHDRUG consortium (www.leishdrug.org) for anti-parasitic drug development.

Amastigote total proteins 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).

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

spaeth.jpg

Amastigote total proteins 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

Morales MA, Pescher P, Späth GF, 2009, "Leishmania major LmaMPK7 protein kinase activity inhibits intracellular growth of the pathogenic amastigote stage", Eukaryot Cell, Oct 2, [Epub ahead of print], PMID: 19801421.

Filipe-Santos O, Pescher P, Breart B, Lippuner C, Aebischer T, Glaichenhaus N, Späth GF, Bousso P, 2009, "A dynamic map of antigen recognition by CD4 T cells at the site of Leishmania major infection", Cell Host Microbe, Jul 23;6(1):23-33, PMID: 19616763.

Rotureau B, Morales MA, Bastin P, Späth GF, 2009, "The flagellum-MAP kinase connection in Trypanosomatids: a key sensory role in parasite signaling and development?", Cell Microbiol, Feb 4, [Epub ahead of print], PMID: 19207727.

Morales MA, Watanabe R, Laurent C, Lenormand P, Rousselle JC, Namane A, Späth GF, 2008, "Phosphoproteomic analysis of Leishmania donovani pro- and amastigote stages", Proteomics, Jan;8(2):350-63, PMID: 18203260

Morales MA, Renaud O, Faigle W, Shorte SL, Späth GF,2007, "Over-expression of Leishmania major MAP kinases reveals stage-specific induction of phosphotransferase activity", Int J Parasitol, Sep;37(11):1187-99, Epub Mar 30, PMID: 17481635.



  Web Site

More informations on our web site




Activity Reports 2009 - Institut Pasteur
If you have problems with this Web page, please write to rescom@pasteur.fr