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.

中文翻译：

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棘阿米巴Castellanii磷酸盐转运蛋白（AcPHS）对于维持无机磷酸盐的流入很重要，并且与滋养体的代谢过程有关。

棘阿米巴棘阿米巴是一种自由生活的阿米巴，是肉芽肿性阿米巴性脑炎和阿米巴性角膜炎的病原体。卡斯特氏菌可以作为滋养体或囊肿存在。滋养体是细胞的营养形式，与囊肿相比具有很大的感染能力，囊肿是休眠形式，可保护细胞免受环境变化的影响。磷酸盐转运蛋白是一组能够将无机磷酸盐从细胞外介质内化到内部的蛋白质。质膜磷酸盐转运蛋白负责维持磷酸盐的稳态，并在某些生物体中调节细胞的生长。这项工作的目的是对卡氏曲霉的质膜磷酸盐转运蛋白及其在细胞生长和代谢中的作用进行生物化学表征。为了测量无机磷酸盐（Pi）的吸收，滋养体在28°C的液体PYG培养基中生长2天。通过将完整细胞与0.5μCi的32Pi孵育1小时进行快速过滤来测量磷酸盐的吸收。Pi的运输是时间的线性函数，并表现出Michaelis-Menten动力学，Km = 88.78±6.86μMPi，Vmax = 547.5±16.9 Pi×h-1×10-6细胞。卡斯特氏菌呈现线性磷酸盐吸收长达1小时，细胞密度为1×105至2×106变形虫×ml-1。在酸性pH范围内，Pi吸收量高于在碱性范围内。活着的滋养体的耗氧量根据Pi在细胞外培养基中的添加而增加。当用FCCP处理细胞时，未观察到Pi对氧气流量的影响。增加的Pi浓度不仅增加了氧气消耗，还增加了细胞内ATP池。当这些细胞用FCCP处理或暴露于缺氧状态时，这些现象将被消除。总之，这些结果加强了以下假设：Pi是棘形棘阿米巴新陈代谢的关键营养素。