The unicellular eukaryote Toxoplasma gondii commonly parasitizes humans and other vertebrates, and approximately one-third of the human population is chronically infected. This opportunistic pathogen is an obligate intracellular parasite that belongs to the phylum of Apicomplexa, which also includes Cryptosporidium and Plasmodium species and numerous other human and veterinary pathogens. For an obligate intracellular parasite, host cell invasion is a vital event to survive, replicate and spread. Rhoptries are specialized apical secretory organelles conserved in the Apicomplexa phylum that plays a critical role in establishing the intracellular niche. Rhoptry proteins fall into two categories based on their localization in the sub-compartments of the organelle: rhoptry neck proteins (RONs) and rhoptry bulb proteins (ROPs). Some of these proteins critically participate in invasion (RONs) and subversion of host cellular function (ROPs). In addition to proteins, rhoptries contain lipids and membranous materials that contribute to the formation of the parasitophorous vacuole membrane to sustain invasion and to hijack host functions crucial for maintaining the infection. During invasion, a set of rhoptry proteins form a RON complex that is indispensable for the formation of the moving junction (MJ), a structure at the interface between host and parasite plasma membrane which is critical for parasite invasion and serves as a support to propel it inside the host. The thesis project was aiming to identify novel components implicated in rhoptry discharge. The first part of the thesis focused on TGGT1_321650 which was previously identified as a substrate of ASP3, an aspartyl protease essential for rhoptry secretion. It encodes a rhoptry neck protein, RON13 predicted to be fitness-conferring. RON13 possesses a transmembrane spanning segment, a predicted S/T kinase domain, and a large C-terminal extension. Biochemical characterization of the enzyme and resolution of its 3D structure at atomic resolution by cryo-electron microscopy reveal that RON13 is an atypical S/T kinase. Phenotypical investigations showed that parasites lacking RON13 or expressing an inactive RON13 kinase can secrete their rhoptry content but cannot form the MJ, resulting in a severe defect in invasion. Comparative phosphoproteomics between WT and RON13-depleted parasites led to the identification of numerous RON13 substrates. Mainly ROPs and RONs are phosphorylated by RON13, including the RON complex. Collectively, RON13 activity is instrumental for RON complex assembly at the MJ, justifying its role in invasion.

Recent studies based notably on cryo-electron tomography, have shed light on the structural and molecular players of the rhoptry secretion apparatus, however, the molecular mechanisms that timely and spatially orchestrate organelle discharge are far from being fully understood. To fill up in part this gap of knowledge, we scrutinized 7 predicted fitness-conferring proteins found in the rhoptry fraction by localization of organelle proteins by isotope tagging (LOPIT). The second part of the thesis was dedicated to the characterization of TGGT1_242820, which codes for a 22 kDa Rhopty Associated Protein (RAP1). RAP1 is located at the rhoptry bulb and at the rhoptry tip and shows a dynamic localization following invasion. Conditional depletion of RAP1 results in a severe block in rhoptry discharge leading to an impairment in invasion.