Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA, 30602, USA.
Department of Cellular Biology, University of Georgia, Athens, GA, 30602, USA.
Mol Microbiol. 2018 Dec;110(6):973-994. doi: 10.1111/mmi.14131. Epub 2018 Oct 18.
Inorganic polyphosphate (polyP) is a polymer of three to hundreds of phosphate units bound by high-energy phosphoanhydride bonds and present from bacteria to humans. Most polyP in trypanosomatids is concentrated in acidocalcisomes, acidic calcium stores that possess a number of pumps, exchangers, and channels, and are important for their survival. In this work, using polyP as bait we identified > 25 putative protein targets in cell lysates of both Trypanosoma cruzi and Trypanosoma brucei. Gene ontology analysis of the binding partners found a significant over-representation of nucleolar and glycosomal proteins. Using the polyphosphate-binding domain (PPBD) of Escherichia coli exopolyphosphatase (PPX), we localized long-chain polyP to the nucleoli and glycosomes of trypanosomes. A competitive assay based on the pre-incubation of PPBD with exogenous polyP and subsequent immunofluorescence assay of procyclic forms (PCF) of T. brucei showed polyP concentration-dependent and chain length-dependent decrease in the fluorescence signal. Subcellular fractionation experiments confirmed the presence of polyP in glycosomes of T. brucei PCF. Targeting of yeast PPX to the glycosomes of PCF resulted in polyP hydrolysis, alteration in their glycolytic flux and increase in their susceptibility to oxidative stress.
无机多聚磷酸盐（polyP）是一种由高能磷酸酐键连接的三到数百个磷酸单元组成的聚合物，存在于从细菌到人类的各种生物中。大多数锥虫中的多聚磷酸盐都集中在液泡酸钙中，这是一种酸性钙储存库，拥有许多泵、交换器和通道，对其生存至关重要。在这项工作中，我们使用 polyP 作为诱饵，在 Trypanosoma cruzi 和 Trypanosoma brucei 的细胞裂解物中鉴定了超过 25 种假定的蛋白质靶标。对结合伴侣的基因本体分析发现，核仁蛋白和糖体蛋白的显著过表达。利用大肠杆菌外切多聚磷酸酶（PPX）的 polyP 结合结构域（PPBD），我们将长链 polyP 定位到了锥虫的核仁区和糖体区。基于 PPBD 与外源性 polyP 的预孵育以及随后对 T. brucei 的前鞭毛体（PCF）进行免疫荧光检测的竞争实验表明，荧光信号的强度随 polyP 浓度和链长的变化而降低。亚细胞分级实验证实了 T. brucei PCF 中的 polyP 存在于糖体中。将酵母 PPX 靶向到 PCF 的糖体中会导致 polyP 水解、糖酵解通量改变以及对氧化应激的敏感性增加。
