Effects of Extracellular Potassium on Acid Release and Motility Initiation in Toxoplasma gondii

Abstract

The internal pH (pHi) of Toxoplasma gondii was estimated by measuring the accumulation of the weak base 9-aminoacridine in buffers with various ionic compositions. The pHi of the metabolizing parasite increased when the extracellular K+ was elevated in alkaline medium or when the external pH (pHe) was substantially increased in medium employing high external K+ (90 mM). The parasite in mouse peritoneal fluid, or in potassium sulfate buffer (pH 8.2), where the pHi was demonstrated to be increased to 7.9, became motile when acidic buffer was substituted for the original suspension medium. This acid-induced independent movement subsided within 5 min but was repeatedly induced if the pHe was serially lowered to 6.0. Basic buffers, on the other hand, abolished motility when applied to the moving parasites. Nigericin, which is known to collapse pH gradients across the membrane, also abolished motility.

Inhibition of cytoplasmic and organellar protein synthesis in Toxoplasma gondii. Implications for the target of macrolide antibiotics.

“…Inhibition of protein synthesis was measured in extracellular tachyzoites using ionic conditions optimized as described in the text. The effect of monovalent cations on tachyzoite motility (14), infectivity (15), and protein synthesis ( 16) has been previously noted. 107 extracellular parasites were added to l-ml aliquots of a series of modified pH 7.2 AISS buffers (containing 0.1% BSA), in which the KCl/NaCl ratio was varied, but the sum of the NaCl and KCl concentrations kept constant at 150 mM.…”

Functional Dissection of Toxoplasma gondii Perforin-like Protein 1 Reveals a Dual Domain Mode of Membrane Binding for Cytolysis and Parasite Egress

“…Following 2-day growth, parasites in high passage flasks had egressed, whereas parasites in low passage flasks remained intracellular. Egressed parasites were filter-purified in Endo buffer (44.7 mM K 2 SO 4 , 106 mM sucrose, 10 mM MgSO 4 , 20 mM Tris-H 2 SO 4 (pH 8.2), 5 mM glucose, 3.5 mg/ml bovine serum albumin) (44), and a red-green invasion assay was performed as previously described (45). Low passage flasks were washed twice with room temperature Endo buffer, scraped, and syringe-passed prior to filter purification in Endo buffer and subsequent invasion assay.…”

Targeted Deletion of MIC5 Enhances Trimming Proteolysis of Toxoplasma Invasion Proteins

“…Indirect immunofluorescence assays were performed on intracellular parasites as described previously (27). To examine protein localization during invasion, parasites were syringed, filtered, washed, and concentrated in room temperature "Endo" buffer (a potassium-rich solution that arrests parasite motility) (20) before being applied to HFF grown overnight on 8-well chamber slides for 20 min at 37°C. The Endo buffer was replaced with 37°C invasion medium (DMEM supplemented with 20 mM HEPES and 3% fetal bovine serum), and parasites were allowed to invade for 1 min at 37°C before fixation as described previously (27).…”