Expression variance, biochemical and immunological properties of Toxoplasma gondii dense granule protein GRA7

Abstract

During intracellular stay, Toxoplasma gondii secretes dense granule proteins (GRA) which remodel the parasitophorous vacuole and are considered functional in parasite–host interrelation. Comparative analysis of parasites from mouse-virulent strain BK and an in vitro attenuated variant revealed that the level of GRA7 expression correlates with T. gondii virulence: proteome analysis and quantitation by immunoblot demonstrated a massive decrease in GRA7 steady-state synthesis parallel to the loss of virulence. Properties of GRA7 that are pertinent to its membrane targeting and to GRA7-directed immune resistance were studied in detail. GRA7 is exclusively membrane-associated in both parasites and infected host cells as demonstrated by subcellular fractionations. Triton X-114 partitioning of isolated parasites substantiated that GRA7 is an integral membrane protein, the hydrophobic stretch from amino acid 181 to 202 providing a possible membrane anchor. A fraction enriched for membranous material from infected host cells contained additional forms of GRA7 with reduced mobility in gel electrophoresis, indicating that the protein is modified after exocytosis from the parasite. By flow cytometric analysis, GRA7 was detected on the surface of intact host cells. An intracellular origin of surface-associated GRA7 seems likely since GRA7 released from extracellular parasites failed to label the host cell surface. Consistent with a role at a parasite–host interface, GRA7 proved to be a target antigen of the intracerebral immune response as evidenced by the presence of GRA7-specific antibodies in mouse cerebrospinal fluid during chronic infection.