No abstract

The biosynthesis and fate of 4 different dense granule proteins of Toxoplasma gondii were studied with 3 monoclonal antibodies raised against tachyzoites and 1 polyclonal antibody raised against a recombinant protein. These proteins have the following molecular weights: 27 kDa (GRA 1), 28 kDa (GRA 2), 30 kDa (GRA 3) and 40 kDa (GRA 4). All four proteins were found in dense granules by immunoelectron microscopy; in T. gondii-infected cells, they were found in the vacuolar network but, in addition, GRA 3 was also detected on the parasitophorous vacuole membrane. Therefore, dense granule contents undergo differential targeting when exocytosed in the parasitophorous vacuole. Metabolic labelling and immunoprecipitation showed that GRA 2 and GRA 3 were processed from lower molecular weight precursors, and that GRA 2 and GRA 4 incorporated [3H] glucosamine and are thus likely to be glycosylated.

Quantitative and qualitative analysis of lipids has been performed on a rhoptry fraction purified from Toxoplasma gondii tachyzoites. The lipid to protein ratio was estimated to be 0.26. The cholesterol to phospholipid ratio was unusually high at 1.48. Phosphatidylcholine was the major phospholipid; phosphatidylserine, phosphatidylinositol and sphingomyelin were absent whereas significant amounts of phosphatidic acid and lysophospholipids were found. This pelicular composition could be related to functional involvement of the organelle in host-cell invasion.

A striking feature of toxoplasmic seroconversion is the prominent and early IgM response to a low molecular weight antigen of 4-5 kDa. Two different monoclonal antibodies directed against the 4-5 kDa antigen have been generated and used to characterize this molecule. Using these monoclonal antibodies, we could demonstrate the surface localization of the low M(r) antigen by immunofluorescence and immuno-electron microscopy assays. By immunoblotting, we observed that one of the monoclonal antibodies was unable to recognize the 4-5 kDa antigen in tachyzoites propagated in cell culture, indicating an epitope variability between Toxoplasma gondii tachyzoites grown in vivo and in vitro. We discuss the implications of this latter finding in the design of diagnostic reagents.

Tachyzoites of Toxoplasma gondii have been shown to exocytose the contents of dense granules into the parasitophorous vacuole after host cell invasion. A monoclonal antibody specific for a 27-kDa protein was used to locate the dense granules by immunoelectron microscopy. The same antibody also reacted with the tubular network found in the parasitophorous vacuole, which confirmed that the dense granules were exocytosed by tachyzoites.