pH and glucose sensing in Trypanosoma brucei glycosomes

项目总结/摘要 非洲锥虫病，也称为非洲昏睡病，每年感染成千上万的人， 这是一个严重的疾病流行区，并伴随着持续的高社会和经济成本。现有治疗方法 锥虫病是可悲的不足，由于耐药性的出现，导致高 治疗失败率（在某些地区为30%），高毒性导致严重的发病率（10%）， 药物相关死亡率（5%）。因此，仍然迫切需要合理设计新的杀锥虫剂。 治疗，特别是那些通过靶向独特的锥虫生物学而使宿主毒性最小化的治疗。葡萄糖 代谢是非洲锥虫感染性生命周期阶段ATP的唯一来源， 与布氏锥虫一样，糖代谢的核心酶都位于糖体中，糖体是一种细胞器， 在寄生虫的哺乳动物宿主中发现。因此，糖体的功能和控制机制 控制糖体内的酶活性是药物设计的重要目标。我们已经证明 这种生物体中ATP的产生对糖体溶液中受环境影响的变化很敏感， 因此，表征糖体内环境是研究糖体内环境的必要步骤。 了解基本的糖酵解途径，并为开发抗- 靶向控制葡萄糖代谢的锥虫疗法。不过，目前缺乏这方面的信息 在最基本的层面上。在糖体内既没有pH值也没有葡萄糖， 受环境影响的动态范围是未知的，这反映了历史上缺乏 允许定量糖体内测量的方法。在这里，我们建议开发肽- 基于靶向小分子和重组蛋白的传感器以定量测定糖体内 pH值和葡萄糖水平。我们的初步数据表明，这种传感器可以被传递到糖体的 活的寄生虫开发的传感器可以随后修改用于测量其他糖体 溶质，包括ATP，并用于研究糖酵解的控制，作为对其他潜在的 重要的环境和发育条件，包括葡萄糖和二价盐浓度， 营养消耗和钙信号传导。由此产生的结果将阐明动态的机制， 糖体环境的调节，揭示了影响这种必需的活性的条件。 代谢途径，并介绍方法可能促进一系列新的途径来了解 测试寄生虫的新陈代谢值得注意的是，这项研究中开创的技术可以扩展到分析 其他致病性动质体寄生虫，如克氏锥虫和利什曼原虫属。这也是本地化 糖体中的ATP产生。此外，可以修改方法以评估细胞器内的 其他重要的亚细胞区室如线粒体，内质网， 和高尔基体。因此，这项工作的影响可能超出非洲锥虫的范围。

PROJECT SUMMARY/ABSTRACT 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.