Deciphering the core complex and regulatory elements of the rhoptry secretory machinery

Rhoptries are specialized secretory organelles at the heart of Apicomplexa pathogenesis. They inject parasite proteins directly into the cytoplasm of host cells not only for invasion but also for hijacking host functions crucial to establishing and sustaining infection. Rhoptries are connected to an apical vesicle (AV), which is docked to the parasite plasma membrane (PPM) by a complex machinery called the rhoptry secretory apparatus (RSA). This arrangement suggests the requirement for two membrane-fusion events during rhoptry secretion, yet little is known about the proteins that regulate them. Nd complex members (Nd6, Nd9, NdP1, NdP2, and Fer2) are essential for rhoptry exocytosis. Nd9, NdP1, and NdP2 are required to build the RSA, and Nd6 is linked to the exocytic site at the PPM/AV in Toxoplasma. These observations suggest a role for the Nd complex in the fusion event between the AV and the PPM. Here, we performed immunoprecipitation coupled with mass spectrometry to obtain a more comprehensive inventory of the rhoptry secretion machinery. We identified 9 additional NdPs, of which almost all are required for invasion of T. gondii in vitro, yet they are not essential. When tested for virulence in mice, the mutants appear highly attenuated, even the ones with no or mild in vitro defect. Like Nd6, most of these proteins are present at the exocytic site. A better visualization of their spatial organization is being achieved by iterative U-ExM. Two of the NdPs are predicted to be GTPases. One is an Arf-like GTPase and directly interacts with the predicted GAP-domain of Nd6. The other one, named NdP-GTPase, is a direct partner of Nd9. Phylogenetic analyses revealed that NdP-GTPase is part of an Alveolata-specific distinct clade that does not fall into any of the classical small GTPase sub-families. Further characterization of both Toxoplasma and Tetrahymena NdP-GTPases showed that they are present at the exocytic site, and their mutants are defective in rhoptry and mucocysts exocytosis, respectively. A GDP-locked mutant in Tetrahymena revealed the essentiality of GDP to GTP switch for mucocysts release upon stimulation. Similar experiments in Toxoplasma are currently ongoing. Collectively, these findings support the notion that rhoptry secretion is governed by GTPase activities, and certain Nd/NdP proteins may act as regulators or effectors of GTPase activity.