A broad distribution of the alternative oxidase in microsporidian parasites.
A broad distribution of the alternative oxidase in microsporidian parasites.
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DOI：
10.1371/journal.ppat.1000761
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发表时间：
2010-02-12
期刊：
影响因子：
6.7
通讯作者：
Keeling PJ
中科院分区：
文献类型：
--
作者：
Williams BA;Elliot C;Burri L;Kido Y;Kita K;Moore AL;Keeling PJ
微孢子虫是一类专性细胞内寄生真核生物，在最近发现高度还原的线粒体细胞器（称为有丝体）之前，它们被认为是线粒体的。对cucuuli脑囊虫全基因组的分析显示，细胞器中的一组蛋白质高度减少，主要与铁硫簇的组装有关。氧化磷酸化和克雷布斯循环蛋白不存在，这与小孢子虫和它们的核分裂体是厌氧的概念相一致，就像其他带有核分裂体的真核生物一样，比如贾第鞭毛虫。在这里，我们提供的证据开启了许多微孢子虫谱系中的有丝体不是完全厌氧的可能性。具体来说，我们已经在几个远亲微孢子虫物种的基因组中鉴定并表征了一个编码替代氧化酶（AOX）的基因，这是真核生物中典型的线粒体末端氧化酶，尽管这个基因在线虫的完整基因组中是缺失的。为了证实这些基因编码功能蛋白，我们在大肠杆菌中过量表达了地芽孢杆菌和人原锥虫的AOX基因，并以泛醇-1 （UQ-1）为底物分光光度法测定了AOX的活性。地棘球绦虫和人锥虫的AOX蛋白都以抗氰化物和抗霉素的方式减少了UQ-1，这种方式对ascofuranone（一种有效的锥虫体AOX抑制剂）敏感。AOX微孢子虫的生理作用可能是对糖酵解产生的还原性当量进行再氧化，其方式与在锥虫中观察到的类似。微孢子虫是专性细胞内寄生虫，在商业上具有重要意义的动物（如蜜蜂）中引起许多疾病，并引起重大的医学关注，特别是在免疫功能低下的人类中。虽然与真菌有关，但微孢子虫经历了一个快速适应细胞内环境的阶段，并在此过程中降低了其生物学的许多方面。值得注意的是，微孢子虫具有高度减少的线粒体（细胞的动力），反映在减少的能量代谢途径上。因此，它们可能只通过糖酵解过程产生ATP。在一些寄生虫中，这种糖酵解途径依赖于一个额外的步骤，涉及一种称为“替代氧化酶”的蛋白质。我们已经证明，这种蛋白质也存在于几种微孢子虫中。至关重要的是，这种蛋白质在人体中不存在，因此可以作为药物靶点加以利用。我们的实验表明，这种蛋白质可能在微孢子虫中广泛存在，并且对抗生素ascofuranone敏感，目前正在测试其作为引起昏睡病的病原体的潜在治疗方法。我们的研究结果表明，从昏睡病药物试验中收集到的知识有可能转移到一些微孢子虫病的治疗中。
Microsporidia are a group of obligate intracellular parasitic eukaryotes that were considered to be amitochondriate until the recent discovery of highly reduced mitochondrial organelles called mitosomes. Analysis of the complete genome of Encephalitozoon cuniculi revealed a highly reduced set of proteins in the organelle, mostly related to the assembly of iron-sulphur clusters. Oxidative phosphorylation and the Krebs cycle proteins were absent, in keeping with the notion that the microsporidia and their mitosomes are anaerobic, as is the case for other mitosome bearing eukaryotes, such as Giardia. Here we provide evidence opening the possibility that mitosomes in a number of microsporidian lineages are not completely anaerobic. Specifically, we have identified and characterized a gene encoding the alternative oxidase (AOX), a typically mitochondrial terminal oxidase in eukaryotes, in the genomes of several distantly related microsporidian species, even though this gene is absent from the complete genome of E. cuniculi. In order to confirm that these genes encode functional proteins, AOX genes from both A. locustae and T. hominis were over-expressed in E. coli and AOX activity measured spectrophotometrically using ubiquinol-1 (UQ-1) as substrate. Both A. locustae and T. hominis AOX proteins reduced UQ-1 in a cyanide and antimycin-resistant manner that was sensitive to ascofuranone, a potent inhibitor of the trypanosomal AOX. The physiological role of AOX microsporidia may be to reoxidise reducing equivalents produced by glycolysis, in a manner comparable to that observed in trypanosomes. Microsporidia are obligate intracellular parasites responsible for a number of diseases in commercially important animals (e.g. bees) and of significant medical concern, in particular in immunocompromised humans. Though related to fungi, microsporidia have undergone a rapid phase of adaption to the intracellular environment and have in the process reduced many aspects of their biology. Notably, microsporidia have highly reduced mitochondria (powerhouses of the cell) reflected in reduced energy metabolic pathways. Thus they likely produce ATP only through the process of glycolysis. In some parasites, this glycolytic pathway is dependent on an additional step involving a protein called the “alternative oxidase”. We have shown that this protein is also present in several species of microsporidia. Crucially, this protein is absent from humans and so can potentially be exploited as a drug target. Our experiments show that this protein is likely widespread in microsporidia, and is sensitive to the antibiotic ascofuranone, which is currently being tested as a potential treatment for the agent causing sleeping sickness. Our results suggest that knowledge gleaned from drug trials on sleeping sickness is potentially transferrable to the treatment of some cases of microsporidiosis.
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