pH and glucose sensing in Trypanosoma brucei glycosomes

描述(申请人提供)：非洲锥虫病，也被称为非洲昏睡病，在流行地区每年感染数万人，并伴随着持续的高社会和经济损失。现有的锥虫病治疗方法严重不足，这既是因为出现耐药性，导致治疗失败率高(某些地区为30%)，也是因为高毒性导致显著发病率(10%)和与药物有关的死亡率(5%)。因此，仍然需要合理设计新的杀锥虫疗法，特别是那些通过靶向独特的锥虫生物学将宿主毒性降至最低的疗法。葡萄糖代谢是非洲布氏锥虫(Trypanosoma Brucei)感染生命周期阶段三磷酸腺苷的唯一来源，糖代谢中心的酶位于糖体中，糖体是这种寄生虫的哺乳动物宿主(S)中没有的细胞器。因此，糖体功能和控制糖体内酶活性的机制都是药物设计的重要目标。我们已经证明，这种生物体中的ATP生产对糖体溶液条件(包括pH)的环境影响变化很敏感。因此，确定糖体内环境的特征是了解基本的糖酵解途径的必要步骤，并将为开发以控制葡萄糖代谢为目标的抗锥虫体疗法奠定基础。然而，目前在最基本的层面上缺乏这方面的信息。糖体内的pH和葡萄糖都没有被量化，它们受环境影响的动态范围也是未知的(S)，这一缺乏反映了历史上缺乏能够对糖体内进行定量测量的方法。在这里，我们建议开发以多肽为靶点的小分子和重组蛋白传感器，以定量测定糖体内的pH和葡萄糖水平。我们的初步数据表明，这种传感器可以传递到活体寄生虫的糖体上。开发的传感器随后可以被改进用于测量包括ATP在内的其他糖体溶质，并用于研究糖酵解的控制作为对其他潜在的重要环境和发育条件的反应，包括葡萄糖和二价盐浓度、营养耗竭和钙信号。由此产生的发现将阐明糖体环境的动态调节机制，揭示影响这一基本代谢途径活动的条件，并引入可能有助于了解和测试寄生虫新陈代谢的一系列新方法。值得注意的是，这项研究中开创的技术可以扩展到对其他致病动体寄生虫的分析，如克氏锥虫和利什曼原虫。这也使糖体中的三磷酸腺苷的产生局部化。此外，还可以对方法进行改进，以评估其他重要亚细胞亚室的细胞器内环境，如线粒体、内质网和高尔基体。因此，这项工作可能会产生超越非洲锥虫病的影响(S)。

DESCRIPTION (provided by applicant): African trypanosomiasis, also called African sleeping sickness, infects tens of thousands of individuals yearly in endemic areas, and is accompanied by continuing high social and economic cost. Existing treatments for trypanosomiasis are woefully inadequate, due both to the emergence of drug resistance that results in high treatment failures rates (30% in some areas), and high toxicity resulting in significant morbidity (10%) and drug-related mortality (5%). There thus remains an outstanding need for rational design of new trypanocidal therapies, particularly those that minimize host toxicity by targeting unique trypanosome biology. Glucose metabolism is the sole source of ATP for the infectious lifecycle stage of the African trypanosome, Trypanosoma brucei, and enzymes central to sugar metabolism are housed in the glycosome, an organelle not found in the parasite's mammalian host(s). Hence, both glycosome function and the control mechanisms governing enzyme activity inside the glycosome are important targets for drug design. We have demonstrated that ATP production in this organism is sensitive to environmentally-influenced changes in glycosomal solution conditions, including pH. Characterizing the intraglycosomal environment is therefore a necessary step in understanding essential glycolytic pathways, and would lay the groundwork for development of anti-trypanosome therapies that target control of glucose metabolism. However, this information is currently lacking at the most basic level. Neither pH nor glucose has been quantified inside the glycosome and their environmentally influenced dynamic range(s) are unknown, a paucity that reflects the historical lack of methodologies to allow quantitative intraglycosomal measurement. Here we propose development of peptide-targeted small molecule and recombinant protein-based sensors to quantitatively determine intraglycosomal pH and glucose levels. Our preliminary data indicates that such sensors can be delivered to the glycosomes of live parasites. Developed sensors can be subsequently modified for measurement of other glycosomal solutes, including ATP, and used to investigate control of glycolysis as a response to other potentially important environmental and developmental conditions, including glucose and divalent salt concentrations, nutrient depletion, and calcium signaling. Resulting findings will illuminate the mechanisms of dynamic regulation of the glycosomal environment, reveal conditions that influence the activity of this essential metabolic pathway, and introduce methodologies likely facilitate a series of new approaches to understanding and testing parasite metabolism. Notably, techniques pioneered in this study can be extended to analysis of other pathogenic kinetoplastid parasites, such as Trypanasoma cruzi and Leishmania spp. that also localize ATP production in glycosomes. In addition, the methodologies can be modified to evaluate the intraorganellar environment in other important subcellular compartments such as the mitochondria, endoplasmic reticulum, and Golgi apparatus. The work is therefore likely to have impact(s) beyond African trypanosomiasis.