Induced Activation of the Toxoplasma gondiiNucleoside 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.

The Opportunistic Pathogen Toxoplasma gondii Deploys a Diverse Legion of Invasion and Survival Proteins

“…NTPase isoforms are apyrases, which were demonstrated to function in intracellular replication of the parasite based on antisense interruption of expression (73). Also, the full scale activation of NTPase with dithiols may contribute to parasite exit from the host cell (74). It is likely that several of the novel secretory products identified herein are also derived from the DGs.…”

Possible Effects of Microbial Ecto-Nucleoside Triphosphate Diphosphohydrolases on Host-Pathogen Interactions

“…Exit of parasites in response to thiol compounds is Ca 2ϩ dependent, suggesting that ATP depletion releases Ca 2ϩ stores that are controlled by ATP (141). These data all suggest that the activation of a secreted NTPDase must be tightly regulated by the parasite (131). Recently, it has been shown that the reducing agent glutaredoxin (GRX) is secreted by the parasite as replication increases.…”

“…However, since parasitic infections generate host nitric oxide and other free radicals, which results in oxidative and nitrative stress (165), it may be that T. gondii itself is responsible for the reduction and therefore activation of NTPDase 3. Upon activation of the enzyme by exogenous thiols, the level of ATP in the host cell is rapidly depleted, and the parasites exit the host cell within a minute of thiol treatment (131). Exit of parasites in response to thiol compounds is Ca 2ϩ dependent, suggesting that ATP depletion releases Ca 2ϩ stores that are controlled by ATP (141).…”

“…Apart from purine salvage, NTPDase activity may be directly related to virulence by influencing intracellular replication and exit from host cells (131). When NTPDase 3 is secreted by the parasite, it remains largely oxidized despite the reducing environment of the host cell.…”

The Loss of Cytoplasmic Potassium upon Host Cell Breakdown Triggers Egress of Toxoplasma gondii

“…Similarly, the treatment of Toxoplasma-infected cells with the strong reducing agent DTT 1 results in the rapid egress of the parasites (11). The mechanism of action of DTT is not clear but may involve the artificial activation of parasitic nucleoside triphosphate hydrolases (12).…”