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 established conditional LmaMPK knock out mutants to overcome this limitation and currently 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.

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

1. Hem S, Gherardini PF, Osorio y Fortéa J, Hourdel V, Morales MA, Watanabe R, Pescher P, Kuzyk MA, Smith D, Borchers CH, Zilberstein D, Helmer-Citterich M, Namane A, Späth GF, 2010, "Identification of Leishmania-specific protein phosphorylation sites by LC-ESI-MS/MS and comparative genomics analyses", Proteomics Nov;10(21):3868-83.

2. Yau WL, Blisnick T, Taly JF, Helmer-Citterich M, Schiene-Fischer C, Leclercq O, Li J, Schmidt-Arras D, Morales MA, Notredame C, Romo D, Bastin P, Späth GF, 2010,"Cyclosporin A treatment of Leishmania donovani reveals stage-specific functions of cyclophilins in parasite proliferation and viability", PLoS Negl Trop Dis. Jun 29;4(6):e729.

3. Morales MA, Watanabe R, Dacher M, Chafey P, Osorio y Fortéa J, Scott DA, Beverley SM, Ommen G, Clos J, Hem S, Lenormand P, Rousselle JC, Namane A, Späth GF, 2010, "Phosphoproteome dynamics reveal heat-shock protein complexes specific to the Leishmania donovani infectious stage", Proc Natl Acad Sci U S A. May 4;107(18):8381-6.

4. Morales MA, Pescher P, Späth GF, 2010, "Leishmania major MPK7 protein kinase activity inhibits intracellular growth of the pathogenic amastigote stage", Eukaryot Cell. 2010 Jan;9(1):22-30.

5. Späth GF, Schlesinger P, Schreiber R, Beverley SM, 2009, "A novel role for Stat1 in phagosome acidification and natural host resistance to intracellular infection by Leishmania major", PLoS Pathog. Apr;5(4):e1000381.