Conducted jointly by CEA and CEA, this research spotlights the role of TgREMIND in Toxoplasma gondii infection.
The Essentials: Conducted jointly by CEA and CEA, this research spotlights the role of TgREMIND in Toxoplasma gondii infection.
Rodrigue Houngue, researcher at I2BC; I2BC; LBBM (Commissariat à l'énergie atomique et aux énergies alternatives).
Thesis defended in 2023 at the doctoral school Doctoral School of Structure and Dynamics of Living Systems (Gif-sur-Yvette, Essonne; 2015-....).
This research was carried out under a Franco-Beninese joint supervision (cotutelle), ensuring simultaneous grounding in local field realities and international academic standards.
Referenced in the ABES/STAR network, this thesis meets the rigor standards of French higher education.
Context and Research Question
Toxoplasma gondii, a parasite of the Apicomplexa phylum, is a pathogen responsible for toxoplasmosis, an infection that affects both animals and humans. The virulence and intracellular survival of T. gondii depend largely on specific secretory organelles, notably micronemes, rhoptries and dense granules. These organelles play a fundamental role in the invasion of host cells. Virulence factors, such as MIC, ROP/RON and GRA proteins, are contained within these structures and are crucial for infection. Understanding the intracellular trafficking and biogenesis of these organelles is therefore essential for developing therapeutic strategies against toxoplasmosis.
The protein TgREMIND (T. gondii REgulator of Membrane Interacting Domain) has been identified as a key regulator in the biogenesis of secretory organelles. This regulator interacts with other trafficking factors, such as TgVPS35 and TgSORT, and is involved in the secretion of virulence factors. Studying TgREMIND makes it possible to explore the molecular mechanisms underlying T. gondii infection, thereby opening perspectives for targeting these mechanisms in the development of treatments against toxoplasmosis.
Methodology
The research was structured around the identification and characterization of TgREMIND. A bioinformatics approach made it possible to determine that TgREMIND contains an F-BAR domain, known for its ability to induce vesicle formation by binding to membranes. A second domain, named REMIND, was identified, but its functions remain to be defined. The binding of TgREMIND to phosphoinositides was confirmed through Lipid Overlay assays, indicating a predicted interaction with cell membranes.
To explore TgREMIND's interaction partners, GST pull-down experiments were conducted. These tests revealed that TgREMIND interacts with various lipid factors and proteins associated with dense granules and rhoptries. Confocal microscopy was used to visualize the expression of TgREMIND in subcellular compartments, highlighting its cytoplasmic distribution and its presence in Golgi and post-Golgi compartments.
An inducible knock-out mutant of TgREMIND was created to study the consequences of the absence of this protein. Morphological analyses were carried out to observe changes in rhoptry structure and the presence of dense granules. Secretion tests made it possible to assess the impact on virulence and the parasites' capacity to invade cells.
Key Findings
Analysis of the results revealed that TgREMIND plays a crucial role in intracellular trafficking and the biogenesis of secretory organelles in T. gondii. The absence of TgREMIND resulted in notable morphological changes to the rhoptries, a disappearance of dense granules, and inhibition of the secretion of their contents. These alterations led to a significant inability of the parasites to invade host cells, thereby compromising their capacity to cause toxoplasmic infection.
The native size of TgREMIND was determined to be approximately 140 kDa using western blot techniques. Complementation experiments on the TgREMIND mutant showed that restoring TgREMIND corrected the observed defects, confirming its role in the biogenesis of secretory organelles.
Particular attention was also paid to TgSTART, a lipid partner of TgREMIND. The characterization of TgSTART was carried out using transgenic parasites and a TgSTART knockdown mutant, providing additional information on lipid interactions within the cell.
Discussion and Outlook
The results obtained in this research underscore the importance of TgREMIND in the functioning of secretory organelles in T. gondii, and consequently, in the parasite's virulence. Identifying TgREMIND's interaction partners and its role in organelle biogenesis provides a better understanding of the molecular mechanisms underlying T. gondii infection.
The implications of these discoveries are significant for the development of treatments against toxoplasmosis. By targeting the biogenesis and secretion pathways of secretory organelles, it becomes possible to design innovative therapies that could reduce the parasite's virulence. The collaboration between CEA and the University of Abomey-Calavi, along with validation by ABES/STAR, reinforces the credibility of the results presented.
Future studies could explore the mechanisms of action of TgREMIND in greater detail, as well as its interactions with other proteins involved in intracellular trafficking. Furthermore, research on TgSTART and other lipid partners could provide additional avenues for developing new therapeutic approaches against toxoplasmosis and other parasitic infections.
Accéder à l'étude complète
Sources and Access
Rodrigue Houngue. TgREMIND est essentiel pour la biogénèse des organites sécrétoires de Toxoplasma gondii et pour l'infection de l'hôte. Biologie cellulaire. Université Paris-Saclay; Université d'Abomey-Calavi UAC (Bénin), 2023. Français. ⟨NNT : 2023UPASL029⟩. ⟨tel-04072013⟩