Martin Kirstee L, Smith Terry K
Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.
Biochem J. 2006 Jun 1;396(2):287-95. doi: 10.1042/BJ20051825.
PI (phosphatidylinositol) is a ubiquitous eukaryotic phospholipid which serves as a precursor for messenger molecules and GPI (glycosylphosphatidylinositol) anchors. PI is synthesized either de novo or by head group exchange by a PIS (PI synthase). The synthesis of GPI anchors has previously been validated both genetically and chemically as a drug target in Trypanosoma brucei, the causative parasite of African sleeping sickness. However, nothing is known about the synthesis of PI in this organism. Database mining revealed a putative TbPIS gene in the T. brucei genome and by recombinant expression and characterization it was shown to encode a catalytically active PIS, with a high specificity for myo-inositol. Immunofluorescence revealed that in T. brucei, PIS is found in both the endoplasmic reticulum and Golgi. We created a conditional double knockout of TbPIS in the bloodstream form of T. brucei, which when grown under non-permissive conditions, clearly showed that TbPIS is an essential gene. In vivo labelling of these conditional double knockout cells confirmed this result, showing a decrease in the amount of PI formed by the cells when grown under non-permissive conditions. Furthermore, quantitative and qualitative analysis by GLC-MS and ESI-MS/MS (electrospray ionization MS/MS) respectively showed a significant decrease (70%) in cellular PI, which appears to affect all major PI species equally. A consequence of this fall in PI level is a knock-on reduction in GPI biosynthesis which is essential for the parasite's survival. The results presented here show that PI synthesis is essential for bloodstream form T. brucei, and to our knowledge this is the first report of the dependence on PI synthesis of a protozoan parasite by genetic validation.
磷脂酰肌醇（PI）是一种普遍存在的真核生物磷脂，它作为信使分子和糖基磷脂酰肌醇（GPI）锚定物的前体。PI可以从头合成，也可以通过PI合酶（PIS）进行头部基团交换来合成。先前已经通过遗传学和化学方法验证了GPI锚定物的合成是非洲昏睡病致病寄生虫布氏锥虫的一个药物靶点。然而，对于这种生物体中PI的合成却一无所知。数据库挖掘在布氏锥虫基因组中发现了一个假定的TbPIS基因，通过重组表达和特性分析表明它编码一种具有催化活性的PIS，对肌醇具有高度特异性。免疫荧光显示，在布氏锥虫中，PIS存在于内质网和高尔基体中。我们在布氏锥虫的血流形式中创建了一个TbPIS的条件性双敲除株，当在非允许条件下生长时，清楚地表明TbPIS是一个必需基因。对这些条件性双敲除细胞进行体内标记证实了这一结果，表明在非允许条件下生长时，细胞形成的PI量减少。此外，分别通过气相色谱 - 质谱联用（GLC - MS）和电喷雾电离串联质谱（ESI - MS/MS）进行的定量和定性分析显示，细胞PI显著减少（70%），这似乎对所有主要的PI种类产生同等影响。PI水平下降的一个后果是GPI生物合成的连锁减少，而GPI生物合成对寄生虫的生存至关重要。这里呈现的结果表明，PI合成对于布氏锥虫的血流形式至关重要，据我们所知，这是通过基因验证首次报道原生动物寄生虫对PI合成的依赖性。
