INDUCED ACTIVATION OF THE TOXOPLASMA-GONDII NUCLEOSIDE TRIPHOSPHATE HYDROLASE LEADS TO DEPLETION OF HOST-CELL ATP LEVELS AND RAPID EXIT OF INTRACELLULAR PARASITES FROM INFECTED-CELLS

The nucleoside triphosphate hydrolase of Toxoplasma gondii is a potent apyrase. The protein is synthesized in large amounts and transported through the secretory pathway of the parasite and into the vacuolar sp ace in an oxidized and thereby enzymatically inactive form. Complete a ctivation of the purified enzyme is known to require dithiols (e.g. DT T); subcellular fractionation demonstrates that little if any (<5%) of the enzyme in the vacuolar space is active in the absence of DTT. Bot h native and epitope-tagged nucleoside triphosphate hydrolase (NTPase) were partially activated during immunoprecipitation, precluding preci se assessment of enzyme activity in the vacuolar space but suggesting that protein-protein interactions may trigger activation. When infecte d cells were treated with DTT, the NTPase was activated in a dose-resp onse fashion, as assessed by migration on SDS-polyacrylamide gel elect rophoresis and by an increase in enzymatic activity. After activation, enzyme activity decreased with time in the presence of DTT; this inac tivation was slowed by the presence of excess ATP. A rapid fall in hos t cell ATP was accompanied by an abrupt exit of parasites from cells. These results demonstrate that the oxidation/reduction status of the N TPase, the only parasite dense granule protein that contains disulfide bonds, is tightly controlled within the vacuolar space and may influe nce parasite exit from cells.