LentiniGaëlle, DuboisDavid, Maco Bohumil, Soldati-FavreDominique, Frenal Karine (2019), The roles of CEN2 and DLC8a in apical secretory organelles discharge of toxoplasma gondii, in Traffic
, 20(8), 583-600.
Dubois David, Soldati-favreDominique (2019), Biogenesis and Secretion of Micronemes in Toxoplasma gondii, in Cellular Microbiology
, 21( e13018), 1-14.
Darvill Nick, Dubois David J., Rouse Sarah L., Hammoudi Pierre-Mehdi, Blake Tom, Benjamin Stefi, Liu Bing, Soldati-Favre Dominique, Matthews Steve (2018), Structural Basis of Phosphatidic Acid Sensing by APH in Apicomplexan Parasites, in Structure
Hammoudi Pierre-Mehdi, Maco Bohumil, Dogga Sunil Kumar, Frénal Karine, Soldati-Favre Dominique (2018), Toxoplasma gondii TFP1 is an essential transporter family protein critical for microneme maturation and exocytosis A transporter essential for microneme secretion, in Molecular Microbiology
I. SUMMARY OF THE RESEARCH PLANBackgroundThe phylum Apicomplexa comprises numerous parasites of medical and veterinary importance. Among them, Toxoplasma gondii is an important pathogen infecting both humans and animals. It is responsible for latent toxoplasmosis infection in one third of the human population and also responsible for abortion in animals. In addition, this parasite is a model organism for the phylum being the most genetically and biochemically tractable and observable by different microscopy techniques. It is therefore the best-suited parasite to unravel the molecular details of the biogenesis of the unusual apicomplexan-specific secretory organelles called rhoptries.As an obligate intracellular parasite, invasion and subsequent establishment of a protected intracellular niche are critical events for the survival of T. gondii. The club-shaped rhoptry organelles, clustered together at the apical pole of the parasite are specifically involved in these two steps. The regulated secretion of the neck (RON proteins) and then bulb (ROP proteins) content during the invasion process not only assists in the penetration of the host cell and the formation of the parasitophorous vacuole but importantly also leads to the modulation of hot cellular functions and subversion of the host immune response [1, 2].The rhoptry organelles represent one hallmark of apicomplexan parasites. We, and others, have demonstrated that their apical positioning is absolutely essential for both invasion and establishment of an intracellular lifestyle and involves the acylated protein TgARO (Armadillo-Repeat Only) and the participation of the motor myosin F (MyoF) [3-5]. In parallel, the fine structure of the rhoptries revealed the presence of intramembranous particles (IMPs) periodically arranged in parallel rows mainly on the bulbous part of the rhoptries . They potentially represent cytoskeleton-interacting sites that could play a role in the maturation and/or in the apical docking of the rhoptries.Preliminary data indicates that some of the TgARO-interacting partners, the adenylate cyclase beta (TgACß) and the ARO interacting protein (TgAIP) , are the first known markers of an intermediate compartment of the rhoptries, located between the neck and the bulb, and appearing as a zone of intermediate density on ultrathin sections observed by transmission electron microscopy (TEM) . Functional results suggest that this zone could be involved in the clustering of the rhoptries.The apical docking of the rhoptries is actually crucial for the secretion of the rhoptry content at the very tip of the parasite. 3D reconstruction of the apical pole of invading T. gondii tachyzoites showed that only one rhoptry secretes its content at a time through the conoid and that only four rhoptry ducts can accommodate this apical space . In addition, vesicles aligned along the vertical microtubules have been visualized from the base of the conoid to the tip of the parasite where a circular structure similar to the porosomes described in mammalian cells can be observed .Working hypothesisThere is an abundant literature about the role of rhoptry proteins during invasion and the subsequent establishment of an intracellular lifestyle. However, very little is known about the organelle itself and more precisely about i) the biogenesis and possible recycling of the rhoptry organelles during cell division, ii) the building of the discrete subcompartments, ii) the organelle docking and bundling at the apical pole and iv) the triggers that lead to their secretion at the apical tip through a porosome-like structure.Recent studies and preliminary data from our two laboratories point out complementary competences that will be instrumental to address and unravel these questions.Specific aimsThis project aims at dissecting the biogenesis and recycling, positioning and clustering, as well as the mechanism of secretion of the apicomplexan-specific rhoptry organelles in Toxoplasma gondii.Our specific objectives are:Objective 1. Deciphering of the dynamics of rhoptry biogenesis and turnoverWe have identified 3 transmembrane proteins of the rhoptries that localized to different sub-domains (DHHC7 , TgTFP2 and TgTFP3). They will be crucial reagents for: i) getting a deeper insight into the ultrastructure and morphology of the rhoptries by electron microscopy (EM) techniques, ii) tracking the dynamics of the rhoptries biogenesis and their recycling by time-lapse microscopy and iii) determining their specific function during the maturation of the rhoptries by reverse genetic tools.Objective 2. Identification of the machineries that position and cluster the rhoptries at the apical poleWe have generated mutants of TgARO that are impaired in the apical docking of the rhoptries but are still clustered or impaired in both apical docking and clustering. We therefore plan to exploit these mutants to identify the machineries involved in these two functions by performing live proximal biotin labeling followed by mass spectrometry using the APEX2 technology . This reporter also enables the use of EM to resolve very precise localization of the proteins of interest and assess the morphology of the rhoptries in these mutants.Objective 3. Unravel the mechanism of rhoptry discharge at the apical tip during invasionWe have identified a protein at the extreme apical tip of the parasite (TgTAP1) that will be fused to the APEX2 reporter in the hope to i) identify the components of this secretion machinery by doing a proximity labeling followed by proteomic identification and then to ii) have a precise spatial and structural observation of the porosome-like structure.Expected valuesThis work will shed light on the process of rhoptry biogenesis leading to the formation of a unique secretory organelle, essential for the infectivity of the medically and veterinary relevant group of apicomplexan parasites. It will extend our knowledge on the molecular principles that orchestrate the building, maturation and localization of an organelle that ensure entry of the parasites into their target cell and their subsequent intracellular survival. In addition, this work aims at discovering new mechanisms involved in the process of rhoptry secretion for which we know almost nothing.