A novel lipid (phosphatidylthreonine)-regulated calcium homeostasis in Toxoplasma gondii

弓形虫中一种新型脂质（磷脂酰苏氨酸）调节的钙稳态

• 批准号: 245364597
• 负责人: Dr. Nishith Gupta
• 金额: --
• 依托单位: Arbeitsgruppe Organische und Bioorganische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/245364597?language=en
• 关键词: novel; lipid; phosphatidylthreonine; regulated; calcium

The major membrane glycerophospholipid classes, described thus far, include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine (PtdSer) and phosphatidylinositol. Our recent work has demonstrated the natural occurrence and genetic origin of an exclusive and rather abundant lipid phosphatidylthreonine (PtdThr) in a widespread and clinically relevant eukaryotic model parasite Toxoplasma gondii. Of note is the fact that PtdThr is a natural homolog of the otherwise-universal PtdSer, which is long known to control Ca2+ homeostasis in mammalian cells. Our earlier work showed that targeted genetic disruption of phosphatidylthreonine synthase (PTS) impairs the lytic cycle and virulence of the parasite due to unforeseen attenuation of the consecutive events of motility, egress and invasion. Using a calcium biosensor, we observed that loss of PtdThr causes a dysregulation of cytosolic calcium that in turn translates into a defective gliding motility. In this proposal, we aim to examine the mechanistic regulation of calcium homeostasis by PtdThr using an approach spanning across the disciplines of biochemistry, gene engineering, cell biology and synthetic chemistry. In brief, we will determine the levels of calcium in the cytosol and endoplasmic reticulum (i.e. site of PtdThr and PtdSer synthesis) of the PTS-mutant by expressing a gene-encoded sensor. Assays involving genetic or chemical modulation of calcium homeostasis, and genetic restoration of lipid perturbation in the PtdThr-mutant will be done to establish a role of lipids in calcium regulation. In parallel, we will identify PtdSer/PtdThr-binding proteins underlying Ca2+ regulation by click-chemistry and mass spectrometry, followed by making of selected parasite mutants and phenotyping. Equally, subcellular distribution of PtdThr and PtdSer will be detected using synthetic chemical probes and customized lipid biosensors via high-resolution imaging. Not least, physiological importance of the key PtdThr species in T. gondii will be investigated by chemical complementation of the parasite cultures. Upon completion, we shall understand the roles of PtdThr (and PtdSer) in calcium homeostasis during asexual reproduction of T. gondii, which can eventually be exploited to inhibit the parasite growth.

迄今为止描述的主要膜甘油磷脂类包括磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸（PtdSer）和磷脂酰肌醇。我们最近的工作已经证明了一个独特的和相当丰富的脂质磷脂酰苏氨酸（PtdThr）在一个广泛的和临床相关的真核模式寄生虫弓形虫的自然发生和遗传起源。值得注意的是，PtdThr是另一种通用PtdSer的天然同系物，长期以来已知PtdSer控制哺乳动物细胞中的Ca 2+稳态。我们早期的工作表明，有针对性的遗传破坏磷脂酰苏氨酸合酶（PTS）损害的溶解周期和寄生虫的毒力，由于不可预见的衰减的连续事件的运动，出口和入侵。使用钙生物传感器，我们观察到PtdThr的损失导致细胞溶质钙的失调，这反过来又转化为有缺陷的滑行运动。在这项提案中，我们的目标是研究的机制调节钙稳态PtdThr使用跨学科的生物化学，基因工程，细胞生物学和合成化学的方法。简而言之，我们将通过表达基因编码的传感器来确定PTS突变体的胞质溶胶和内质网（即PtdThr和PtdSer合成的位点）中的钙水平。将进行涉及钙稳态的遗传或化学调节以及PtdThr突变体中脂质扰动的遗传恢复的测定，以确定脂质在钙调节中的作用。与此同时，我们将通过点击化学和质谱法鉴定PtdSer/PtdThr结合蛋白的Ca 2+调节，然后进行选定的寄生虫突变体和表型分析。同样，PtdThr和PtdSer的亚细胞分布将使用合成化学探针和定制的脂质生物传感器通过高分辨率成像进行检测。尤其是，T.将通过寄生虫培养物的化学补充来研究弓形虫。完成后，我们将了解PtdThr（和PtdSer）在T.弓形虫，最终可以用来抑制寄生虫的生长。