Unit: Parasite Virulence
Director: Gerald Spaeth
Our group "G5 Parasite Virulence" uses genetic, proteomic and immunological methods to study the molecular base of virulence and persistence of Leishmania, an important human pathogen which produces serious diseases in endemic areas around the world. The infectious cycle of Leishmania includes several extra- and intracellular stages that are adapted for survival in the insect vector and the vertebrate host by differential expression of virulence factors. Our group analyzes the molecular mechanism of the regulation of these factors and their interaction with the innate immune system. The projects are aimed to identify and validate target molecules for the development of novel anti-leishmanial strategies, and to establish genetically attenuated parasites for vaccination.
1) The role of Leishmania mitogen-activated protein kinases in parasite virulence
Miguel Morales, Reiko Watanabe, Gerald Spaeth
The main axis of our research is focused on the question how Leishmania adapts its virulence to the mammalian host by perceiving environmental signals during the infection. The final objective of this study is to target the corresponding signaling pathways for the development of alternative and more effective anti-leishmanial therapies. We utilize transgenic and gene knock out strategies to elucidate the role of Leishmania ERK/MAPK (extracellular-regulated/mitogen activated protein kinases) in parasite pathogenicity. We established three transgenic lines of L.major and L.donovani, over-expressing the kinases MPK4, 7, and 10, coupled to GFP. This cellular system allowed us to show an induction of the phosphotransferase activity of these kinases during differentiation of the pathogenic amastigote stage. Mouse and macrophage infection assays revealed a role of MPK4 in parasite virulence, and implicated MPK7 in the regulation of intracellular parasite proliferation. We initiated studies on the sub-cellular localization of the MPKs using confocal microscopy, and use co-immunoprecipitation assays to identify molecular interaction partners of these molecules. We plan to identify the substrates of MPKs by peptide array screening and analysis of the phospho-proteome. We currently establish knock out mutants of the three MPKs. The null mutant lines will allow us to further elucidate the role of the MPKs in Leishmania virulence and to identify target genes of these signaling pathways by comparative proteomics. This project will allow important insight into how the host environment regulates the phenotype of microbial pathogens, with broad applicability to other parasitic systems, including Plasmodium falciparum, Trypanosoma cruzi, and Trypanosoma brucei, all of which alternate between insect and vertebrate hosts during their infectious cycles.
Immune-recognition of Leishmania lipoglycans by the CD1d-NK T cell immune axis
Claire Forestier, Pascale Pescher, Gerald Spaeth
Visceral leishmaniasis (VL) caused by the protozoan parasite Leishmania donovani afflicts over 2 million people worldwide and leads to hepato-splenomegaly with ultimately fatal outcome if left untreated. Even though protozoan, bacterial, and viral liver pathogens account for millions of deaths each year, only little is known on liver-specific immunity and pathways responsible for tissue-specific pathogen recognition. We recently 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 the first bona fide 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.
Photo 1: Transgenic Leishmania major expressing a GFP-tagged MAP kinase MPK7. Cells were stained with anti- tubulin (red) and anti-GFP (green) antibodies.
Photo 2: Liver section of control and CD1d-deficient mice 4 weeks after Leishmania donoivani infection. CD1d mice show a defect in granuloma formation and increased parasite burden (parasites are marked by the arrow head).
Keywords: Leishmania, MAP kinase, virulence, glycolipids, innate immunity