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.
Discovering the composition and the roles of the T.gondii nuclear pore
The nuclear pore complex (NPC) is a key structure in eukaryotes regulating nuclear-cytoplasmic transport as well as a wide range of cellular processes. Each NPC is composed of multiple copies of approximately 30 different proteins known as nucleoporins (NUPs), for which the composition and structure have been largely characterized in yeast, mammals and Trypanosoma brucei. Strikingly, the description of the putative components of the NPC in T. brucei led to the discovery of a conserved protein arrangement that spans the eukaryotic kingdom. However, the composition of the NPC has not been examined in apicomplexan parasites. We discovered potential NUPs in T. gondii by the use of affinity purification. Most of these proteins did not share primary sequence conservation with known eukaryotic NUPs. We are now exploring the role of these proteins in multiple process such as gene regulation, cell cycle regulation and chromatin sequestration.
Personnel involved in the project: Mathieu Gissot (PI), Ludovic Huot and Thomas Mouveaux.
Factors controlling virulence genes expression
Tachyzoites are responsible of the acute phase of the disease and are able to invade and grow in any mammalian nucleated cell. Therefore, they are crucial to the T. gondii pathogenesis. Apicomplexan specific organelles such as the micronemes and rhoptries contain proteins enabling invasion and virulence. Specifically, micronemes proteins are known to mediate attachment, reorientation and ultimately invasion of the host cell. For example, micronemes proteins AMA1 and MIC2 are known to be important for invasion. Rhoptry proteins are involved in creation of the parasitophorous vacuole and virulence. Indeed, ROP16, ROP18 and ROP5 are exported in the host cell cytoplasm or nucleus and are shown to play critical roles in virulence. During the cell cycle, expression of micronemes and rhoptry proteins is tightly regulated so these proteins are produced « just in time » for their export and packaging into the newly formed micronemes and rhoptries in the daughter cell. However, the determinants controlling the expression of those proteins are unknown. These factors are governing the expression of proteins critical for the ability of the parasite to invade and control the host cell.
Our goal is to identify and characterize the crucial regulators of the tachyzoite virulence by discovering the factors that control the expression of the rhoptries and micronemes transcripts and proteins.
Personnel involved in the project: Mathieu Gissot (PI), Kevin Lesage, Ludovic Huot and Thomas Mouveaux.
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).