The Toxoplasma gondii rhoptry protein ROP 2 is inserted into theparasitophorous vacuole membrane surrounding the intracellular parasiteand is exposed to the host cell cytoplasm

摘要

The origin of the vacuole membrane surrounding the intracellular protozoan parasite Toxoplasma gondii is not known. Although unique secretory organelles, the rhoptries, discharge during invasion of the host cell and may contribute to the formation of this parasitophorous vacuole membrane (PVM), no direct evidence for this hypothesis exists. Using a novel approach we have determined that parasite-encoded proteins are present in the PVM, exposed to the host cell cytoplasm. In infected cells incubated with streptolysin-O or low concentrations of digitonin, the host cell plasma membrane was selectively permeabilized without significantly affecting the integrity of the PVM. Antisera prepared against whole parasites or a parasite fraction enriched in rhoptries and dense granules reacted with the PVM in these permeabilized cells, indicating that parasite-encoded antigens were exposed on the cytoplasmic side of the PVM. Parasite antigens responsible for this staining of the PVM were identified by fractionating total parasite proteins by SDS-PAGE and velocity sedimentation, and then affinity purifying "fraction-specific" antibodies from the crude antisera. Proteins responsible for the PVM- staining,identified with fraction-specific antibodies, cofractionated with knownrhoptry proteins. The gene encoding one of the rhoptry proteins, ROP 2, wascloned and sequenced, predicting and integral membrane protein. Antibodiesspecific for ROP 2 reacted with the intact PVM. These results provide thefirst direct evidence that rhoptry contents participate in the formation ofthe PVM of T. gondii and suggest a possible role of ROP 2 in parasite-hostcell interactions.