Acanthamoeba castellanii is a free-living amoeba and the etiological agent of granulomatous amoebic
encephalitis and amoebic
keratitis. A. castellanii can be present as trophozoites or
cysts. The trophozoite is the vegetative form of the cell and has great infective capacity compared to the
cysts, which are the dormant form that protect the cell from environmental changes.
Phosphate transporters are a group of
proteins that are able to internalize
inorganic phosphate from the extracellular to intracellular medium. Plasma membrane
phosphate transporters are responsible for maintaining
phosphate homeostasis, and in some organisms, regulating cellular growth. The aim of this work was to biochemically characterize the plasma membrane
phosphate transporter in A. castellanii and its role in cellular growth and metabolism. To measure
inorganic phosphate (Pi) uptake, trophozoites were grown in liquid PYG medium at 28 °C for 2 days. The
phosphate uptake was measured by the rapid filtration of intact cells incubated with 0.5 μCi of 32Pi for 1 h. The Pi transport was linear as a function of time and exhibited Michaelis-Menten kinetics with a Km = 88.78 ± 6.86 μM Pi and Vmax = 547.5 ± 16.9 Pi × h-1 × 10-6 cells. A. castellanii presented linear
phosphate uptake up to 1 h with a cell density ranging from 1 × 105 to 2 × 106 amoeba × ml-1. The Pi uptake was higher in the acidic pH range than in the alkaline range. The oxygen consumption of living trophozoites increased according to Pi addition to the extracellular medium. When the cells were treated with
FCCP, no effect from Pi on the
oxygen flow was observed. The addition of increasing Pi concentrations not only increased oxygen consumption but also increased the intracellular
ATP pool. These phenomena were abolished when the cells were treated with
FCCP or exposed to
hypoxia. Together, these results reinforce the hypothesis that Pi is a key nutrient for Acanthamoeba castellanii metabolism.