title: 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.
abstract: 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 space in an oxidized and thereby enzymatically inactive form. Complete activation of the purified enzyme is known to require dithiols (e.g. DTT); subcellular fractionation demonstrates that little if any (<5%) of the enzyme in the vacuolar space is active in the absence of DTT. Both native and epitope-tagged nucleoside triphosphate hydrolase (NTPase) were partially activated during immunoprecipitation, precluding precise assessment of enzyme activity in the vacuolar space but suggesting that protein-protein interactions may trigger activation. When infected cells were treated with DTT, the NTPase was activated in a dose-response fashion, as assessed by migration on SDS-polyacrylamide gel electrophoresis and by an increase in enzymatic activity. After activation, enzyme activity decreased with time in the presence of DTT; this inactivation was slowed by the presence of excess ATP. A rapid fall in host cell ATP was accompanied by an abrupt exit of parasites from cells. These results demonstrate that the oxidation/reduction status of the NTPase, the only parasite dense granule protein that contains disulfide bonds, is tightly controlled within the vacuolar space and may influence parasite exit from cells.
authors.affiliation: Section of Infectious Diseases, Yale University School of Medicine, New Haven, Connecticut 06520-8022, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Silverman
authors.forename: H
authors.initials: H
authors.lastname: Qi
authors.forename: A
authors.initials: A
authors.lastname: Riehl
authors.forename: C
authors.initials: C
authors.lastname: Beckers
authors.forename: V
authors.initials: V
authors.lastname: Nakaar
authors.forename: K A
authors.initials: KA
authors.lastname: Joiner
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002522:Chlorocebus aethiops; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation; D043583:Nucleoside-Triphosphatase; D010084:Oxidation-Reduction; D011233:Precipitin Tests; D014122:Toxoplasma / Q000201:enzymology* / Q000502:physiology; D014709:Vero Cells
chemical_list: D000255:Adenosine Triphosphate; D017766:Acid Anhydride Hydrolases; D043583:Nucleoside-Triphosphatase; D004229:Dithiothreitol