Proteomic and structural characterization of self-assembled vesicles from excretion/secretion products of Toxoplasma gondii

Abstract After the cell invasion, the parasite Toxoplasma gondii locates within a parasitophorous vacuole to proliferate. It continuously modifies the composition of the parasitophorous vacuole by the secretion of GRA and ROP proteins, some of which become inserted into the vacuole membrane, remain as soluble proteins or involved in the intravacuolar network. In this report, we analyze the excretion/secretion products and the vesicles released by extracellular tachyzoites, this structures were morphologically analyzed by electron microscopy and characterized by mass spectrometry. The structural analysis showed parasites secreting in vitro individual vesicles with similarities to ectosomes and exosomes and which characterized to self-assembly in vitro forming vesicle-tubular structures morphologically similar to the intravacuolar network from infected cells. The vesicle-tubular structures were recognized with antibodies against ROP2 and GRA2. In addition, analysis by Western blot evidenced proteins from the secretory organelles. A detailed proteomic analysis of exosomes, ectosomes and soluble proteins released in vitro is here reported. Presence of GRA proteins in secretions from resting extracellular parasites indicates that these molecules are not exclusively secreted within the parasitophorous vacuole of the infected cell as reported but they are constitutively excreted/secreted even in an extracellular condition. Data are available via ProteomeXchange with identifier PXD013767 . Significance Extracellular tachyzoites constitutively secrete components that previously were considered be secreted only within the parasitophorous vacuole, suggesting that in the infected host these molecules are in direct interaction with cells and molecules of the host cell including those of the immune response.