Toxoplasma gondii possesses an armada of secreted virulent factors that enable parasite invasion and survival into host cells. These factors are contained in specific secretory organelles, the rhoptries, micronemes and dense granules that release their content upon host cell recognition. Dense granules are secreted in a constitutive manner during parasite replication and play a crucial role in modulating host metabolic and immune responses. While the molecular mechanisms triggering rhoptry and microneme release upon host cell adhesion have been well studied, constitutive secretion remains a poorly explored aspect of T. gondii vesicular trafficking. Here, we investigated the role of the small GTPase Rab11A, a known regulator of exocytosis in eukaryotic cells. Our data revealed an essential role of Rab11A in promoting the cytoskeleton driven transport of dense granules and the release of their content into the vacuolar space. Rab11A also regulates transmembrane protein trafficking and localization during parasite replication, indicating a broader role of Rab11A in cargo exocytosis at the plasma membrane. Moreover, we found that Rab11A also regulates extracellular parasite motility and adhesion to host cells. In line with these findings, MIC2 secretion was altered in Rab11A-defective parasites, which also exhibited severe morphological defects. Strikingly, by live imaging we observed a polarized accumulation of Rab11A-positive vesicles and dense granules at the apical pole of extracellular motile and invading parasites suggesting that apically polarized Rab11A-dependent delivery of cargo regulates early secretory events during parasite entry into host cells. Toxoplasma gondii (T. gondii) is a highly prevalent parasite infecting a wide range of animals as well as humans. T. gondii secretes numerous virulent factors contained in specific organelles, termed the rhoptries, micronemes and dense granules. These factors are released upon host cell recognition and enable parasite invasion and subsequent development into an intracellular vacuole. In particular, dense granules contain critical effectors that modulate intrinsic defenses of infected host cells ensuring parasite survival and dissemination. The mechanisms regulating dense granule secretion have not been elucidated. In this study, we unraveled a novel role for the T. gondii GTPase Rab11A in promoting dense granule transport along the parasite cytoskeleton and their content release into the vacuolar space during parasite replication. We also found that T. gondii Rab11A regulates extracellular parasite motility and adhesion to host cells suggesting a broader role in distinct secretory pathways essential for parasite virulence.

Summary: Toxoplasma gondii possesses an armada of secreted virulent factors that enable parasite invasion and survival into host cells. These factors are contained in specific secretory organelles, the rhoptries, micronemes and dense granules that release their content upon host cell recognition. Dense granules are secreted in a constitutive manner during parasite replication and play a crucial role in modulating host metabolic and immune responses. While the molecular mechanisms triggering rhoptry and microneme release upon host cell adhesion have been well studied, constitutive secretion remains a poorly explored aspect of T. gondii vesicular trafficking. Here, we investigated the role of the small GTPase Rab11A, a known regulator of exocytosis in eukaryotic cells. Our data revealed an essential role of Rab11A in promoting the cytoskeleton driven transport of DG and the release of their content into the vacuolar space. Rab11A also regulates transmembrane protein trafficking and localization during parasite replication, indicating a broader role of Rab11A in cargo exocytosis at the plasma membrane. Moreover, we found that Rab11A also regulates extracellular parasite motility and adhesion to host cells. In line with these findings, MIC2 secretion was altered in Rab11A-defective parasites, which also exhibited severe morphological defects. Strikingly, by live imaging we observed a polarized accumulation of Rab11A-positive vesicles and dense granules at the apical pole of extracellular motile parasites suggesting that a Rab11A-dependent apically polarized transport of cargo regulates parasite motility.

Background: Butyrate is a microbiota-derived short-chain fatty acid with established roles in maintaining intestinal barrier integrity and regulating immune responses. Although its effects have been extensively investigated in immunocompetent models, systemic immune homeostasis in severely immunodeficient hosts remains incompletely understood. This exploratory study evaluated the impact of sodium butyrate (NaB) supplementation on tissue-associated microbial antigen detection and immune marker distribution along different organs in athymic nude mice. Methods: Female NU/J athymic mice were administered either regular drinking water (H₂O, n = 10) or NaB-supplemented water (150 mM, n = 5) for six weeks. Intestinal, liver, lung, and kidney tissues were collected for histopathological and immunohistochemical analyses. Expression of microbial antigens, including lipopolysaccharide (LPS) and lipoteichoic acid (LTA), as well as immune-associated markers (CD3, CD8a, CD68, CD278, and transglutaminase-2 [TGM2]), was assessed using a semiquantitative scoring system. Results: NaB supplementation did not significantly affect organ morphology but was associated with better weight gain. Histological examination revealed preserved tissue architecture in all organs, with only mild condensation of small intestinal crypts in NaB-treated mice. Immunohistochemical analysis showed consistently lower LPS expression in NaB-treated animals compared with controls in the small intestine, liver, and kidney, with a borderline reduction in the lungs. Reduced LPS detection was accompanied by significantly lower expression of the macrophage marker CD68 in the kidney. Conclusions: Oral NaB supplementation was associated with lower detection of bacterial antigens in tissues and reduced innate immune expression across multiple organs. These findings support a potential role for butyrate in limiting microbial expression and systemic inflammatory signaling in athymic mice.

Caractérisation fonctionnelle de la voie de sécrétion dépendante de Rab11A chez Toxoplasma gondii: Toxoplasma gondii possède une armada d’effecteurs parasitaires qui permettent l’invasion et la survie des parasites dans la cellule hôte. Ces facteurs sont contenus dans des organites sécrétoires spécifiques, les rhoptries (ROP), les micronèmes (MIC) et les granules denses (DG) qui libèrent leur contenu lors de l'adhésion et l'invasion active de l’hôte. Les protéines des DG (GRA) sont également sécrétées de manière dite « constitutive » lors de la réplication du parasite et jouent un rôle crucial dans la modulation de la réponse de l'hôte, assurant la survie et la dissémination du parasite. Alors que les mécanismes moléculaires régulant la libération des protéines ROP et MIC lors de l'invasion parasitaire ont été bien étudiés, la sécrétion constitutive des DG reste un aspect totalement inexploré du trafic vésiculaire de T. gondii. Au cours de cette thèse, nous avons d’abord étudié le rôle de la petite GTPase Rab11A, un régulateur de l’exocytose connu dans les cellules eucaryotes. Nous avons démontré que lors de la réplication parasitaire, TgRab11A régule le mouvement des DG dépendent de l’actine et stimule l’étape finale de leur l’exocytose au niveau de la membrane plasmique du parasite et donc la libération des protéines GRA dans l’espace vacuolaire et dans le cytosol de la cellule hôte. En outre, nous avons démontré une nouvelle fonction pour TgRab11A dans les premières étapes d’adhésion du parasite aux cellules hôtes et dans la motilité du parasite, et donc dans l’invasion des cellules hôtes. En accord avec ces résultats, la sécrétion de l'adhésine MIC2 est altérée chez les parasites extracellulaires inhibés dans l’activité de TgRab11A. De manière surprenante, les parasites extracellulaires mobiles envahissants la cellule hôte présentaient une accumulation apicale polarisée et focalisée des vésicules Rab11A-positives, suggérant un rôle pour TgRab11A dans les premiers évènements sécrétoires précoces déclenchés lors de l’invasion parasitaire. Collectivement, nos données ont révélé TgRab11A comme un régulateur crucial de la voie de sécrétion constitutive chez T. gondii. Dans une deuxième partie de cette thèse, nous avons caractérisé un nouveau partenaire de TgRab11A, contenant un domaine HOOK unique, que nous avons appelé TgHOOK. Nous avons constaté que cette protéine forme un complexe stable avec un homologue de la protéine Fused Toes (FTS) et une protéine interagissant avec HOOK (appelée HIP) nouvellement identifiée spécifique aux parasites coccidiens. HOOK et FTS sont deux régulateurs conservés du trafic endosomal connus pour favoriser le transport et/ou la fusion des vésicules chez d’autres eucaryotes. In T. gondii, nous avons constaté que le complexe TgHOOK-TgFTS-HIP s’accumule à l’extrémité apicale du parasite et favorise la sécrétion des protéines MIC, contribuant ainsi à l’invasion du parasite.