Constitutive Calcium-independent Release of Toxoplasma gondii Dense Granules Occurs through the NSF/SNAP/SNARE/Rab Machinery

Abstract

The signals and the molecular machinery mediating release of dense matrix granules from pathogenic protozoan parasites are unknown. We compared the secretion of the endogenous dense granule marker GRA3 in Toxoplasma gondii with the release of a stably transfected foreign reporter, ␤-lactamase, that localizes to parasite dense granules. Both proteins were released constitutively in a calcium-independent fashion, as shown using both intact and streptolysin O-permeabilized parasites. N-Ethylmaleimide and recombinant bovine Rab-guanine dissociation inhibitor inhibited ␤-lactamase secretion in permeabilized parasites, whereas recombinant hamster N-ethylmaleimide-sensitive fusion protein and bovine ␣-SNAP augmented release. Guanosine 5-3-O-(thio)triphosphate, but not cAMP, augmented secretion in the presence but not in the absence of ATP. The T. gondii NSF/SNAP/SNARE/Rab machinery participates in dense granule release using parasite protein components that can interact functionally with their mammalian homologues.

The Toxoplasma gondii Dense Granule Protein GRA7 Is Phosphorylated upon Invasion and Forms an Unexpected Association with the Rhoptry Proteins ROP2 and ROP4

“…The ROP-containing strands have been proposed to function in delivery of ROPs to the PV membrane (21). GRA7 in these strands might also traffic to the PV membrane; however, given the constitutive release of GRAs into the PV (9,27), multiple routes to the PV membrane seem likely. Moreover, the continued presence of the GRA7 strands at 24 h into infection suggests functions other than delivery to the vacuole (Fig.…”

Toxoplasma gondii Rab6 Mediates a Retrograde Pathway for Sorting of Constitutively Secreted Proteins to the Golgi Complex

“…For pulse-chase analysis of GRA4, parasites were labeled for 10 min and chased after three washes for 0, 15, and 30 min. Parasites were freed from host cells by syringe passage in 1 ml of phosphate-buffered saline containing proteinase inhibitors (2) and centrifuged at 1400 ϫ g for 10 min at 4°C. Cell pellets were lysed in radioimmune precipitation assay buffer (24) containing Complete protease inhibitor mixture (Roche Molecular Biochemicals).…”

“…Micronemes and rhoptries are apically tethered and secreted during attachment and penetration of a host cell. Dense granules in Toxoplasma secrete aggregates of soluble and transmembrane proteins constitutively at a basal level and are stimulated for enhanced release following parasite invasion, suggesting a regulated, but Ca 2ϩ -independent, component (2). Immunohistochemical analysis indicates that the sorting of dense granule proteins from those targeted to micronemes and rhoptries occurs at the late Golgi cisternae or trans-Golgi network (TGN).…”

GRA Proteins of Toxoplasma gondii: Maintenance of Host-Parasite Interactions across the Parasitophorous Vacuolar Membrane

“…The cDNA of GRA3 encodes 2 methionines at the N-terminus followed by an open reading frame with a hydrophobic region of 22 amino acids flanked by charged residues consistent with a signal sequence [32]. The endogenous dense granule marker GRA3 is secreted constitutively in a calcium-independent fashion using T. gondii NSF/SNAP/SNARE/Rab machinery that can interact functionally with their mammalian homologues [33]. Recently, previously published sequence for GRA3 is actually an artificial chimera of 2 proteins of molecular weight 65 kDa, shares the C-terminus with published GRA3 and possesses no significant sequence similarity with any protein thus far deposited in Genbank [34].…”