|Parasite Virulence - INSERM U1010, CNRS URA2581|
|HEAD||Dr. SPAETH, Gerald / firstname.lastname@example.org|
|MEMBERS||Dr. 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
Our laboratory uses genetic and proteomic methods to study the molecular basis of virulence of the proto-zoan parasite Leishmania. Three axes are pursued:
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 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
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.
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Activity Reports 2009 - Institut Pasteur
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