Effect of Extracellular Ions on Motility and Cell Entry in Toxoplasma gondii

Abstract

Toxoplasma gondii tachyzoites were quiescent in mouse peritoneal fluid or in K2SO4 buffer at pH 8.2. They became consistently motile when K+ was replaced by other monovalent or divalent cations at a constant pH (pH = 8.2). They also became motile when Cl- was substituted for SO4(2-). Nitrate or SCN-, can also be substituted for Cl- to a certain extent. Tachyzoites showed independent movement for more than 15 min in KCl, and for about 5 min in the other buffers at pH 8.2 after which they were exhausted and stopped. These tachyzoites could not then be further stimulated to motility by renewal of the suspension buffer. Infection of monolayer cells was demonstrated only with parasites which were motile during inoculation. The highest infectivity was thus obtained either with freshly collected tachyzoites or with those preincubated in K2SO4 buffer for 30 min at 37 degrees C at alkaline pH and thus not yet exhausted for motility. Approximately 34 to 38% of these latter organisms were seen to enter cells when they were inoculated into cultures immediately after being resuspended in MEM for 30 min at 37 degrees C. Conversely, those whose motility had been exhausted by the preincubation in buffers other than K2SO4, pH 8.2 could not enter monolayer cells. Additionally, parasites were unable to enter cells when inoculated into cultures in K2SO4 buffer at alkaline pH; instead they remained quiescent on the surface of the monolayer cells, suggesting that Toxoplasma enters the host cells by active invasion.

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.…”

Toxoplasma Rhoptry Protein 16 (ROP16) Subverts Host Function by Direct Tyrosine Phosphorylation of STAT6

“…To investigate the kinetics of STAT6 activation, a potassium buffer shift was used to synchronize invasion as described elsewhere (15,16). Briefly, tachyzoites were harvested, washed, and resuspended in Endo buffer (44.7 mM K 2 SO 4 , 106 mM sucrose, 10 mM Mg 2 SO 4 , 20 mM Tris (pH 8.2), 5 mM glucose, and 3.5 mg/ml BSA).…”

Ionophore-Resistant Mutants of Toxoplasma gondii Reveal Host Cell Permeabilization as an Early Event in Egress

“…Gliding motility was assessed indirectly by immunofluorescence analysis of the major Toxoplasma surface antigen SAG1 in trails (7). Parasites were immobilized in K 2 SO 4 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, and 3.5 mg of BSA/ml [10]) and were seeded onto a tissue culture-treated LabTek chamber slide. After 15 min at 37°C, the K 2 SO 4 buffer was removed and replaced with either the same medium or HBSS c and the parasites were incubated for 2 min more at 37°C.…”