Vol. 13, Issue 7, 2397-2409, July 2002

Biogenesis of Nanotubular Network in Toxoplasma Parasitophorous Vacuole Induced by Parasite Proteins

Corinne Mercier,^* Jean-François Dubremetz, Béatrice Rauscher,^* Laurence Lecordier, L. David Sibley,^§ and Marie-France Cesbron-Delauw^*

 ^*Centre National de la Recherche Scientifique FRE 2383, Bâtiment CERMO, Université Joseph Fourier, Grenoble, France 38041;  Centre National de la Recherche Scientifique Unité Mixte Recherche 5539, Université Montpellier II, Montpellier, 34095 France;  Laboratory of Molecular Parasitology, Université Libre de Bruxelles, Gosselies, 6041 Belgium; and  ^§Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110

The intracellular parasite Toxoplasma gondii develops within a nonfusogenic vacuole containing a network of elongated nanotubules that form connections with the vacuolar membrane. Parasite secretory proteins discharged from dense granules (known as GRA proteins) decorate this intravacuolar network after invasion. Herein, we show using specific gene knockout mutants, that the unique nanotubule conformation of the network is induced by the parasite secretory protein GRA2 and further stabilized by GRA6. The vacuolar compartment generated by GRA2 knockout parasites was dramatically disorganized, and the normally tubular network was replaced by small aggregated material. The defect observed in gra2 parasites was evident from the initial stages of network formation when a prominent cluster of multilamellar vesicles forms at a posterior invagination of the parasite. The secretory protein GRA6 failed to localize properly to this posterior organizing center in gra2 cells, indicating that this early conformation is essential to proper assembly of the network. Construction of a gra6 mutant also led to an altered mature network characterized by small vesicles instead of elongated nanotubules; however, the initial formation of the posterior organizing center was normal. Complementation of the gra2 knockout with mutated forms of GRA2 showed that the integrity of both amphipathic alpha-helices of the protein is required for correct formation of the network. The induction of nanotubues by the parasite protein GRA2 may be a conserved feature of amphipathic alpha-helical regions, which have also been implicated in the organization of Golgi nanotubules and endocytic vesicles in mammalian cells.