Research activity
Phosphatases in cellular and molecular signalling in Plasmodium
It has become clear that the coordinated and reciprocal actions of kinases and phosphatases are fundamental regulation mechanisms for the development and growth of the malaria parasite. In many organisms Protein Phosphatase type 1 (PP1) is one of the major phosphatases and the most functionally and biochemically well-characterized. The PP1 catalytic (PP1c) subunit has diversified its function through the presence of regulatory proteins that form with the catalytic subunit highly specific enzymes (substrate specificity, localization/trafficking, control of the phosphatase activity of PP1c). In P. falciparum (Pf), the deadliest form of malaria, although the PP1c (PfPP1c) has been identified and despite its biological importance, very little has been reported on the regulatory subunits and on the molecular basis of the control of PfPP1c functions.
To fulfill its appropriate and precise function required during P. falciparum lifecycle, PfPP1c should be tightly and spatiotemporally controlled. On the basis of the well conserved sequence/structure of PP1c (from fungi to humans) and the high specificity of P. falciparum genome (~50-60% unknown proteins) and its distinctive cell division mechanism, it is conceivable that both conserved and specific regulators of PfPP1c are present and developed by the parasite.
Hence the general objective of our project is to characterize the regulatome of PfPP1 by mining the P. falciparum genome and by an in-depth approach using high-throughput yeast two-hybrid system. By investigating the regulators of PP1, we aim to provide fundamental insights into the regulation of PfPP1 at the molecular level. This will, in a long-term goal, permit to selectively modulate the specific signaling pathways to inhibit parasite growth.
Toxoplasma gondii is responsible for toxoplasmosis, a disease which can be fatal for the fetus of infected mothers and for immunocompromised patients. This parasite belongs to the Apicomplexa family which contains other parasites of medical importance such as Plasmodium (responsible for malaria) or Cryptosporidium (cryptosporidiosis). The pathogenicity of these parasites is based on their ability to divide in order to proliferate in the organism. They have therefore invented simplified division patterns in order to multiply efficiently and quickly while producing a large number of parasites. The mechanisms allowing the control and coordination of these modes of division are therefore essential to their ability to proliferate and therefore to the survival of these parasites in humans. We aim to expand our understanding on how these parasites are able to evolve flexible modes of division allowing them to proliferate in a large number of different organisms. We study the proteins that control and coordinate the division of these parasites, using T. gondii as a model Apicomplexa. We are particularly interested in transcription factors of the ApiAP2 family which coordinate specific expression profiles during the cell cycle. In addition, we also study the centrosome, which serves as a platform coordinating the cell cycle. These studies could lead us to better understand the mechanisms of division in this family of parasites in order to develop new molecules aiming at controlling their proliferation.
Vesicular trafficking pathways involved in ROP and MIC formation and exocytosis of dense granule proteins
Rhoptry and microneme organelles are formed de novo at each replication cycle by budding and fusion of vesicles emerging from the Golgi apparatus, which subsequently follow distinct secretory pathways to eventually form mature organelles. Recent studies have revealed some of the trafficking routes taken by the MIC and ROP proteins, by examining the functions of the endo-exocytic compartments, and have also identified transport molecules, such as the dynamin-like DrpB, the HOPS/CORVET complexes and the receptor TgSORTLR, required for organelle biogenesis. In contrast, biogenesis and secretion of dense granules (GRA) remain mostly unexplored. In general, despite being crucial for the development of the disease, the mechanisms regulating exchanges between the parasite and its external environment are poorly elucidated.For example, GRA proteins, which are released into the vacuolar space, play an essential role for the establishment of chronic toxoplasmosis by ensuring cyst formation into neuronal tissus. GRA and ROP proteins also represent key factors that modulate the immune response of infected cells. Using reverse genetics, proteomics, live imaging and high resolution microscopy, we recently identified novel parasite-specific effectors of MIC and ROP protein trafficking as well as key regulators of vesicle exocytosis to the parasite plasma membrane and vacuolar space.
Our goal is to unravel novel vesicular secretory pathways regulating parasite invasion, survival and virulence.
Subversion of dendritic cells by T.gondii
Dendritic cells (DCs) are key immune cells that initiate the pro-inflammatory response against Toxoplasma gondii infection, thereby preventing parasite dissemination and acute disease. Infected DCs secrete the pro-inflammatory cytokine IL-12, which triggers NK cells and CD4+ / CD8+ lymphocytes to secrete IFNg, a major cytokine that stimulates the cytotoxic response, responsible for parasite clearance and establishment of latent toxoplasmosis. In addition, DCs are professional antigen presenting cells that are specialized in loading peptides derived from exogenous and endogenous sources onto MHC class I and II molecules for presentation to CD8+ and CD4+ T cells, respectively. T. gondii has developed strategies to evade the immune response by secreting key parasitic factors into the host cytosol that modulate DCs activities, such as cytokine secretion and antigen presentation. These parasite effectors contained in rhoptries and dense granules interfere with key signaling pathways involved in the regulation of the pro-inflammatory response and block cellular defense mechanisms, such as IRG-mediated lysis of the parasitophorous vacuole (PV).
Our goal is to characterize how specific ROP and GRA proteins present at the PV membrane manipulate the host cell machinery involved in antigen cross-presentation on MHC I molecules and modulate endoplasmic reticulum homeostasis in infected DCs.
People involved in the project: Sabrina Marion (PI), Anais Poncet (PhD student) and Ludovic Huot (engineer).
Discovery of new antimalarial molecules through molecular and functional characterization of phosphatases specific to Plasmodium falciparum
Plasmodium falciparum (Pf), the deadliest agent of malaria, has developed resistance to almost all chemotherapeutics. It is necessary to understand the biology of this parasite in order to develop new drugs. In Pf, extensive research has now been started to study the Pf kinome and to examine whether targeting kinases could represent an effective mean for the treatment of the infection. However, the study of Pf phosphatases is still under-investigated. Amino acid sequence comparative analyses of Pf and Plasmodium berghei (Pb), a rodent malaria species, revealed that several of them are Plasmodium specific. Among these phosphatases, three were also suggested to be essential for blood stage parasites development of Pf. The present project is focused on the molecular and functional characterization of one of them and on the validation of this specific phosphatase as a new potential target for malaria. The gene has been cloned, annotated and expressed as a recombinant protein and its phosphatase activity has been demonstrated in vitro. Functional characterization in vivo was explored by conditional gene knock-out studies as well as by generating knock-in parasite lines to follow their trafficking during the parasite lifecycle (in Pf and Pb). Finally, we solved in silico the 3D structure of catalytic site of this phosphatase by homology modelling and identified a new set of potential specific inhibitors. A first series of pharmacomodulations allowed us to discover 13 new hits with IC50 ≤ 100 nM, including 3 with IC50 ≤ 50 nM. Cytotoxicity studies showed low cell toxicity on human HFF (Human Foreskin Fibroblasts) and HepG2 (human hepatocytes) cells with a selectivity index >100. Currently we are pursuing the pharmacomodulation of these hits, the molecule-target interaction and the in vivo efficacy in an animal model of malaria. Our project is supported by Inserm Transfert and SATT Nord.
