Axis 1: The role of extracellular-regulated protein kinases (ERK/MAPK) in virulence and pathogenesis of the intracellular parasite Leishmania
Across all eukaryotic species, highly conserved extracellular-regulated protein kinases (MAPK/ERK) sense environmental conditions and modulate gene expression in response to nutritional and physiological changes. Conceivably, this signal transduction pathway may play a crucial role in virulence of microbial pathogens that are often adapted for survival in multiple host systems. We utilize the protozoan parasites Leishmania major and Leishmania donovani as a model system to elucidate the relevance of the parasite MAPK signaling pathway in intracellular infection. In recent work published by our lab we provided evidence for the stage-specific phosphorylation and activity of the parasite MAP kinases (LmaMPKs) LmaMPK4, 7, and 10 in the pathogenic amastigote stage. 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 potential implication of LmaMPKs in the development of the pathogenic amastigote stage, these kinases and their substrates may contribute substantially to parasite virulence and therefore may represent new targets for therapeutic intervention.
Axis 2: Quantitative assessment of amastigote-specific signaling in Leishmania donovani by phosphoproteomics
Despite the relevance of the Leishmania amastigote stage for pathogenesis, our understanding on the mechanisms underlying development of this stage and its intracellular survival remains only poorly investigated. The most significant insight into amastigote-specific biology and physiology results from proteomic studies, which revealed only few significant changes in protein abundance during the early stage differentiation, and little stage-specific proteins were detected (3 – 9%). These data raise the important question how Leishmania stably maintains the promastigote and amastigote phenotypes throughout the infectious cycle in the presence of a nearly constitutive proteome. Conceivably, post-translational modifications of these common proteins, such as protein phosphorylation, may play an important role in stage-specific modulation of protein activities and interactions, which may regulate the distinct physiological and morphological features of the parasite developmental stages. We investigate the signaling networks underlying Leishmania differentiation analyzing purified phosphoproteins by Differential 2D gel electrophoresis (DIGE) and LC-MS-MS/iTRAQ in collaboration with the proteomics platforms of the Insitut Pasteur and the University of Victoria by. To date, we have identified over 600 putative phospho-proteins 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 phospho-proteins for the identification of novel amastigote-specific protein kinases.
Axis 3: Exploiting the Leishmania kinome for drug development.
Through the FP7 LEISHDRUG consortium (www.leishdrug.org), we will uses a highly interdisciplinary approach to reveal Leishmania signaling molecules associated with amastigote virulence, with the major aim to exploit parasite-specific pathways for anti-leishmanial drug development. The LEISHDRUG consortium comprises 14 teams world-wide and is largely based on our previously published work on Leishmania MAP kinases and novel approaches recently developed in our laboratory, including phospho-proteomics analysis and 2D in-gel kinase assay. The consortium is based on 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.
Axis 4: Exploiting Leishmania glycolipid virulence determinants to stimulate early protective immunity through CD1d-mediated NK T cell activation.
We investigate how Leishmania virulence factors interact with the host immune system. This project is focused on the recognition of the Leishmania virulence factor lipophopshoglycan by CD1d-mediated antigen presentation. Antigen-presenting molecules of the CD1 family recognize the structural uniqueness of microbial lipoglycans and initiate a rapid, cytotoxic T-cell response. We previously reported that a subset of highly liver-enriched “innate-like” lymphocytes, Natural Killer T (NK T) cells, participate in the early inflammatory response to visceral L.donovani infection. We identified the major L.donovani surface glycoconjugate lipophosphoglycan (LPG) as a potential microbial glycolipid antigen, which binds with high affinity to the antigen-presenting molecule CD1d and stimulates a robust Th1-response in CD1d-restricted NK T cells. NK T cell deficient CD1d-/- mice showed a defect in granuloma formation and increased parasite burden. These data suggest an important role for CD1d-mediated presentation of microbial glycolipids in innate antimicrobial resistance and immunopathogenesis. Our current efforts are focused on (1) the isolation of the Leishmania-responsive NKT cell subset, (2) the analysis of LPG intracellular trafficking in infected host cells and its potential processing in the endo-lysosomal compartment, (3) the identification of the immunodominant LPG carbohydrates, and (4) the validation of LPG and synthetic glycolipids in immunoprotection.