Vacuolar-type H+-ATPase regulates cytoplasmic pH in Toxoplasma gondii tachyzoites

Abstract

Cytoplasmic pH (pHi) regulation was studied in Toxoplasma gondii tachyzoites by using the fluorescent dye 2ʹ,7ʹ-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. Their mean baseline pHi (7.07±0.06; n = 5) was not significantly affected in the absence of extracellular Na+, K+ or HCO3- but was significantly decreased in a dose-dependent manner by low concentrations of N,Nʹ-dicyclohexylcarbodi-imide (DCCD), N-ethylmaleimide (NEM) or bafilomycin A1. Bafilomycin A1 also inhibited the recovery of tachyzoite pHi after an acid load with sodium propionate. Similar concentrations of DCCD, NEM and bafilomycin A1 produced depolarization of the plasma membrane potential as measured with bis-(1,3-diethylthiobarbituric)trimethineoxonol (bisoxonol), and DCCD prevented the hyperpolarization that accompanies acid extrusion after the addition of propionate, in agreement with the electrogenic nature of this pump. Confocal laser scanning microscopy indicated that, in addition to being located in cytoplasmic vacuoles, the vacuolar (V)-H+-ATPase of T. gondii tachyzoites is also located in the plasma membrane. Surface localization of the V-H+-ATPase was confirmed by experiments using biotinylation of cell surface proteins and immunoprecipitation with antibodies against V-H+-ATPases. Taken together, the results are consistent with the presence of a functional V-H+-ATPase in the plasma membrane of these intracellular parasites and with an important role of this enzyme in the regulation of pHi homoeostasis in these cells.

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

“…could result in a dramatic change in membrane potential across the parasite plasma membrane. In the case of Toxoplasma, however, the involvement of a [K ϩ ] gradient in establishing a membrane potential has been called into question (19) and we have, in fact, not detected any major difference in the parasites' membrane potential in extracellular or intracellular buffers (data not shown). To determine directly whether a [K ϩ ] gradient or a membrane potential across the parasite plasma membrane is involved in the parasites' ability to detect a change in the extracellular [K ϩ ], we performed egress experiments in the presence or absence of gramicidin, a H ϩ , Na ϩ , K ϩ ionophore, and valinomycin, a K ϩ ionophore.…”

Na+-dependent pH Regulation by the Amitochondriate Protozoan Parasite Giardia intestinalis

“…Determination of pH i from [2][3][4][5][6][7][8][9][10][11][12][13][14] C]DMO Distribution-G. intestinalis trophozoites were harvested by centrifugation and resuspended in MES-and HEPES-buffered saline of the required pH (pH 6.0 -7.5) at a density of ϳ6 ϫ 10 6 cells ml…”

“…Aliquots (100 l) of the equilibrated cell suspension were transferred, in triplicate, to microcentrifuge tubes containing 100 l of MES-and HEPES-buffered saline at the same pH containing [2][3][4][5][6][7][8][9][10][11][12][13][14] C] DMO (0.05 Ci) together with unlabeled DMO (to give a final DMO concentration of 1-100 M) and layered over 100 l of oil (a mixture of dibutyl phthalate and di-iso-octyl phthalate, 4:1, 1.03 g ml…”

A proton pumping pyrophosphatase in acidocalcisomes of Herpetomonas sp.

“…Kinetic studies have shown that acidocalcisomes maintain a low internal pH due to the presence of a V-H + -ATPase [22][23][24] and a V-H + -PPase [8,11,13,15,[25][26][27] which pump H + into the lumen of the organelle. They also posses a Ca 2+ -H + translocating ATPase, a Ca 2+ -H + exchanger and a Na + -H + exchanger which permit a complex regulation of these ions by the cell It was proposed that acidocalsisomes are involved in mechanisms of Ca 2+ signaling, osmoregulation, pH homeostasis and energy storage [16,28].…”