pH and glucose sensing in Trypanosoma brucei glycosomes

布氏锥虫糖体中的 pH 和葡萄糖传感

• 批准号: 9077835
• 负责人: KENNETH A CHRISTENSEN
• 金额: $ 18.39万
• 依托单位: BRIGHAM YOUNG UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-12-12 至 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9077835
• 关键词: Affect; Africa South of the Sahara; African; African Trypanosomiasis; Area; Biology; Blood Circulation; Calcium Signaling; Carbon; Cells; Cessation of life; Chagas Disease; Consumption; Cues; Data; Development; Drug Design; Drug resistance; Endoplasmic Reticulum; Environment; Enzymes; Failure; Fluorescent Probes; Foundations; Glucose; Glycolysis; Glycosome; Goals; Golgi Apparatus; HIV; Health; Housing; Human; Individual; Leishmania; Leishmaniasis; Life; Measurement; Mediating; Metabolic; Metabolic Pathway; Metabolism; Methodology; Mission; Mitochondria; Monitor; Morbidity - disease rate; Nutrient; Organelles; Organism; Parasites; Parasitic Diseases; Pathway interactions; Peptides; Pharmaceutical Preparations; Pharmacotherapy; Production; Proteins; Public Health; Quantitative Evaluations; Recombinant Proteins; Recombinants; Regulation; Research; Sepsis; Series; Sodium Chloride; Solutions; Source; Staging; Techniques; Temperature; Testing; Toxic effect; Treatment Failure; Trypanosoma; Trypanosoma brucei brucei; Trypanosoma cruzi; Trypanosomiasis; Work; base; deprivation; design; economic cost; environmental change; enzyme activity; enzyme mechanism; extracellular; glucose metabolism; glucose sensor; hexokinase; in vivo; killings; mortality; novel strategies; peroxisome; ratiometric; response; sensor; small molecule; social; solute; sugar; targeted treatment; therapeutic target

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.

描述（由申请人提供）：非洲锥虫病，也称为非洲昏睡病，每年在流行地区感染数万人，并伴随着持续的高社会和经济成本。现有的锥虫病治疗方法严重不足，这是由于出现耐药性导致高治疗失败率（某些地区为30%），以及高毒性导致显著的发病率（10%）和药物相关死亡率（5%）。因此，仍然迫切需要合理设计新的杀锥虫疗法，特别是通过靶向独特的锥虫生物学来最小化宿主毒性的那些。葡萄糖代谢是非洲锥虫布氏锥虫感染性生命周期阶段的ATP的唯一来源，并且对糖代谢起中心作用的酶被容纳在糖体中，糖体是在寄生虫的哺乳动物宿主中未发现的细胞器。因此，糖体功能和控制糖体内酶活性的机制是药物设计的重要目标。我们已经证明，在这种生物体中的ATP生产是敏感的糖体溶液条件，包括pH值的环境影响的变化。因此，表征的intraglycosomal环境是一个必要的步骤，在了解基本的糖酵解途径，并奠定了基础，为发展的抗锥虫疗法，目标控制葡萄糖代谢。然而，目前缺乏最基本的信息。糖体内的pH值和葡萄糖均未定量，其受环境影响的动态范围未知，这反映了历史上缺乏允许定量糖体内测量的方法。在这里，我们建议开发肽靶向的小分子和重组蛋白为基础的传感器，以定量测定糖内pH值和葡萄糖水平。我们的初步数据表明，这样的传感器可以交付给活寄生虫的糖体。开发的传感器可以随后进行修改，用于测量其他糖体溶质，包括ATP，并用于研究控制糖酵解作为对其他潜在重要的环境和发育条件的响应，包括葡萄糖和二价盐浓度，营养消耗和钙信号。由此产生的研究结果将阐明糖体环境的动态调节机制，揭示影响这一重要代谢途径活性的条件，并引入可能促进一系列新方法来理解和测试寄生虫代谢的方法。值得注意的是，这项研究中开创的技术可以扩展到其他致病性动质体寄生虫，如克氏锥虫和利什曼原虫的分析。也定位于糖体中ATP的产生。此外，可以修改的方法，以评估在其他重要的亚细胞区室，如线粒体，内质网和高尔基体的细胞器内环境。因此，这项工作的影响可能超出非洲锥虫的范围。