Carvalho-Kelly 2020 J Bioenerg Biomembr

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Carvalho-Kelly LF, Pralon CF, Rocco-Machado N, Nascimento MT, Carvalho-de-Araujo AD, Meyer-Fernandes JR (2020) Acanthamoeba castellanii phosphate transporter (AcPHS) is important to maintain inorganic phosphate influx and is related to trophozoite metabolic processes. J Bioenerg Biomembr 52:93-102.

» PMID: 31965457

Carvalho-Kelly LF, Pralon CF, Rocco-Machado N, Nascimento MT, Carvalho-de-Araujo AD, Meyer-Fernandes JR (2020) J Bioenerg Biomembr

Abstract: 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.

Keywords: Achantamoeba castellanii, Energetic metabolism, PHO84 inorganic phosphate transporter Bioblast editor: Plangger M

Labels: MiParea: Respiration 

Organism: Protists 

Preparation: Intact cells 

Regulation: Ion;substrate transport, Phosphate  Coupling state: ET 

HRR: Oxygraph-2k 

Labels, 2020-01