Identification and role of thiols in Toxoplasma gondii egress.

The nucleoside triphosphate hydrolase of Toxoplasma gondii is a potent apyrase that is secreted into the parasitophorous vacuole where it appears to be essentially inactive in an oxidized form. Recent evidence shows that nucleoside triphosphate hydrolase can be activated by dithiothreitol in vivo. On reduction of the enzyme, there is a rapid depletion of host cell ATP. Previous results also demonstrate a dithiothreitol induced egress of parasites from the host cell with a concurrent Ca2+ flux, postulated to be a consequence of the release of ATP-dependent Ca2+ stores within the tubulovesicular network of the parasitophorous vacuole. Reduction of the nucleoside triphosphate hydrolase appears crucial for its activation; however, the exact mechanism of reduction/activation has not been determined. Using a variety of techniques, we show here that glutathione promoters activate a Ca2+ flux and decrease ATP levels in infected human fibroblasts. We further show the in vitro activation of nucleoside triphosphate hydrolase by endogenous reducing agents, one of which we postulate might be secreted into the PV by T. gondii. Our findings suggest that the reduction of the parasite nucleoside triphosphate hydrolase, and ultimately parasite egress, is under the control of the parasites themselves.

Bleomycin affects cell wall anchorage of mannoproteins in Saccharomyces cerevisiae.

Bleomycin induces strand breakage in DNA through disruption of glycosidic linkages. We investigated the ability of bleomycin to damage yeast cell walls, which are composed primarily of carbohydrate. Bleomycin treatment of intact yeast cells facilitated enzymatic conversion of yeasts to spheroplasts. Bleomycin treatment also altered anchorage of mannoproteins to the cell wall matrix in intact cells or isolated cell walls. Cell surface mannoproteins were labelled with 125I, and their solubilization was monitored. Seventeen hour treatments with bleomycin released some of the label directly into treatment supernatants and facilitated extraction of mannoproteins by dithiothreitol and lytic enzymes. Bleomycin treatments as short as 10 min caused changes in extraction of mannoproteins from intact cells. Specifically, cell wall anchorage of several mannoproteins was affected by the drug. There were drug-induced changes in extractability of mannoproteins with apparent molecular weights of 96,000, 80,000, 61,000, 41,000, 31,500, and 21,000 (determined after deglycosylation with endo-N-acetylglucosaminidase H). The similarity of results obtained in the presence and absence of cycloheximide, the appearance of cell wall effects after only 10 min of treatment, and the similarity of effects in intact cells and isolated cell walls are consistent with direct drug-induced damage and inconsistent with a mechanism dependent on expression of bleomycin-damaged genes or other intracellular mediators. The results are consistent with bleomycin-mediated increases in cell wall permeability through disruption of glycosidic cross-linking structures in the cell wall.