The Kennedy phospholipid biosynthesis pathways are refractory to genetic disruption in Plasmodium berghei and therefore appear essential in blood stages
The Kennedy phospholipid biosynthesis pathways are refractory to genetic disruption in Plasmodium berghei and therefore appear essential in blood stages
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DOI：
10.1016/j.molbiopara.2010.05.006
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发表时间：
2010-10-01
影响因子：
1.5
通讯作者：
Gannoun-Zaki, Laila
中科院分区：
文献类型：
--
作者：
Dechamps, Sandrine;Wengelnik, Kai;Gannoun-Zaki, Laila
磷脂酰胆碱（PC）和磷脂酰乙醇胺（PE）是疟原虫的主要膜磷脂（PL），可通过从头（Kennedy）COP-胆碱和CDP-乙醇胺途径以及CDP-二酰甘油依赖性途径产生。Kennedy途径起始于外源性胆碱和乙醇胺，涉及胆碱激酶（CK）和乙醇胺激酶（EK），随后是胆碱-磷酸胞苷酰转移酶（CCT）和乙醇胺-磷酸胞苷酰转移酶（ECT），其催化COP-胆碱和CDP-乙醇胺的形成。最后，在疟原虫中，PC和PE显然是由共同的胆碱/乙醇胺磷酸转移酶（CEPT）合成的。在这里，我们已经研究了肯尼迪途径的基本性质，伯氏疟原虫，啮齿动物疟疾寄生虫。伯氏疟原虫CEPT、CCT、ECT和CK酶的序列分析揭示了酶活性所必需的所有催化结构域和必需残基和基序的存在。构建体被设计用于在伯氏疟原虫中产生cept、cct、ect和ck基因的基因敲除和GFP融合体。我们发现，所有四个基因是一贯难敲除尝试。同时，用GFP成功标记这些蛋白质证明了lad是可靶向的，并表明这些基因在伯氏疟原虫血液期寄生虫中是必需的。CCT、Ea和CK的GFP融合蛋白存在于细胞质中，而GFP-CEPT主要定位于内质网中。这些结果表明，COP-胆碱和CDP-乙醇胺从头途径是伯氏疟原虫无性发育所必需的，并且与PC和PE的其他可能来源无关。(C)2010 Elsevier BV保留所有权利。
Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the main membrane phospholipids (PLs) of Plasmodium parasites and can be generated by the de novo (Kennedy) COP-choline and CDP-ethanolamine pathways and by the CDP-diacylglycerol dependent pathway. The Kennedy pathways initiate from exogenous choline and ethanolamine involving choline kinase (CK) and ethanolamine kinase (EK), followed by the choline-phosphate cytidylyltransferase (CCT) and ethanolamine-phosphate cytidylyltransferase (ECT) that catalyse the formation of COP-choline and CDP-ethanolamine. Finally, in Plasmodium, PC and PE are apparently synthesized by a common choline/ethanolamine-phosphotransferase (CEPT). Here, we have studied the essential nature of the Kennedy pathways in Plasmodium berghei, a rodent malaria parasite. Sequence analysis of the P. berghei CEPT, CCT, ECT and CK enzymes revealed the presence of all catalytic domains and essential residues and motifs necessary for enzymatic activities. Constructs were designed for the generation of gene knockout and GFP-fusions of the cept, cct, ect and ck genes in P. berghei. We found that all four genes were consistently refractory to knockout attempts. At the same time, successful tagging of these proteins with GFP demonstrated that the lad were targetable and indicated that these genes are essential in P. berghei blood stage parasites. GFP-fusions of CCT, Ea and CK were found in the cytosol whereas the GFP-CEPT mainly localised in the endoplasmic reticulum. These results indicate that both COP-choline and CDP-ethanolamine de novo pathways are essential for asexual P. berghei development and are non-redundant with other possible sources of PC and PE. (C) 2010 Elsevier B.V. All rights reserved.