Intracellular trafficking of dense granule proteins in Toxoplasma gondii and experimental evidences for a regulated exocytosis

The dense granules of the intracellular protozoan Toxoplasma gondii are sec retory vesicles that play a major role in the structural modifications of t he parasitophorous vacuole (PV) in which the parasite develops, The biogene sis of dense granules as well as the regulatory mechanisms controlling thei r specific exocytosis are still poorly understood. In this paper, we analyz ed the secretory pathway of dense granule proteins (GRA proteins) in extrac ellular T. gondii through the effects of brefeldin A (BFA), Ultrastructural studies of BFA-treated parasites showed disassembly of the Golgi apparatus and accumulation of GRA proteins in a dilated vacuolar system connected to the nuclear envelope, BFA reversibly blocked the intracellular transport o f the newly synthesized GRA proteins in a dose-dependent manner (blockade o f 95 % at 1 mu g/ml of BFA). By contrast, discharge of GRA proteins from pr eformed dense granules was unaffected by BFA over a course of 60 min incuba tion. GRA protein secretion was dependent on incubation temperature as it o nly occurred above 26 degrees C and it could be stimulated by external fact ors. This stimulus might be provided by factor(s) present in the serum of t he extracellular medium, as incubation of parasites in serum-free medium re sulted in a dramatic decrease in protein secretion. Exocytosis can be resto red in a dose-dependent fashion by serum addition (maximal stimulatory acti vity in the 30-200 kDa range) and was optimal at an extracellular pH of 6.5 . Altogether, these results demonstrate that GRA proteins are exported thro ugh the Golgi apparatus via the classical secretory pathway and can be expe rimentally discharged from storage dense granules as regulated secretory pr oteins in response to specific stimulation, arguing in favor of a regulated component for dense granule exocytosis in I:gondii.