Translation state array analysis in Aspergillus fumigatus

烟曲霉翻译态阵列分析

• 批准号: 7470253
• 负责人: DAVID S ASKEW
• 金额: $ 22万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7470253
• 关键词: Affect; Aspergillosis; Aspergillus; Aspergillus fumigatus; Candidate Disease Gene; Centrifugation; Clinical; Condition; DNA; Development; Diagnostic; Diagnostic Procedure; Disease; Drug Delivery Systems; Environment; Epitopes; Equipment and supply inventories; Eukaryota; Eukaryotic Cell; Fractionation; Future; Gene Expression; Genes; Genome; Goals; Grant; Growth; Immunocompromised Host; In Situ; Individual; Infection; Invasive; Investigation; Life; Measures; Messenger RNA; Metabolic; Microarray Analysis; Northern Blotting; Numbers; Organism; Outcome; Pathogenesis; Polyribosomes; Population; Procedures; Protein Biosynthesis; Proteins; RNA; Rate; Ribosomes; Sensitivity and Specificity; Shock; Specificity of Diagnostic Test; Sucrose; Techniques; Technology; Temperature; Testing; Therapeutic Intervention; Translating; Translational Regulation; Translations; Treatment Efficacy; Western Blotting; abstracting; base; density; expectation; fitness; fungus; improved; in vivo; insight; mortality; mouse model; novel; novel diagnostics; novel therapeutics; pathogen; protein expression; rapid growth

DESCRIPTION (provided by applicant): Abstract Infections with the opportunistic fungal pathogen Aspergillus fumigatus continue to be associated with a poor outcome. Major obstacles to effective treatment include the rapid growth of the organism in the host environment and the limited ability of current diagnostic methods to identify the infection, resulting in impaired therapeutic efficacy and a high level of mortality. In this grant we propose to explore the use of translation state array analysis (TSAA) to increase our understanding of the metabolic reprogramming that is fundamental to the adaptation of this organism to growth at 37oC. Our overarching hypothesis is that the growth of A. fumigatus at 37oC can be distinguished from growth at 25oC by the selective translation of specific mRNAs. This will be tested using TSAA, a microarray based technology that evaluates mRNA association with the translational machinery on a genome wide scale. Aim I will use TSAA to test the hypothesis that growth at 37oC induces the translation of a specific subset of mRNAs. TSAA combines ribosome fractionation by sucrose gradient centrifugation with DNA array technology to measure the translational efficiency of individual mRNAs. A. fumigatus will be cultured at 25oC and 37oC, and hyphal extracts will be fractionated on a sucrose gradient. Since ribosome loading onto each mRNA is proportional to the rate of synthesis of the protein product, the fractionated gradient will be separated into two pools; one containing mRNAs with abundant ribosomes (representing well translated mRNA) and one containing mRNAs with few ribosomes (representing under translated mRNAs). RNA from the two pools will then be used to interrogate A. fumigatus micro-arrays, and the translation state ratio of each mRNA will be used as an indicator of how well an mRNA is translated at each temperature. Aim II will validate candidate genes by demonstrating that their mRNAs undergo changes in ribosome loading in a temperature dependent manner, and that their encoded products are modulated by temperature and can be detected in vivo. Proteins that are up-regulated at 37oC culture are expected to contribute to the rapid growth and overall fitness of the organism at this temperature, which has the potential to identify novel drug targets or the development of new diagnostics. PROJECT NARRATIVE: Major obstacles to the effective treatment of aspergillosis include the rapid growth of the organism in the host and the limited ability of current diagnostic methods to identify the infection, resulting in a high level of mortality. The goal of this study is to use a recently developed technique, translation state array analysis, to identify mRNAs that are preferentially translated into protein at 37oC, with the long term goal of identifying proteins that could serve as novel therapeutic and/or diagnostic targets.

描述（由申请人提供）：具有机会性真菌病原曲霉的抽象感染烟素继续与不良结果有关。有效治疗的主要障碍包括生物体在宿主环境中的快速生长以及当前诊断方法鉴定感染的能力有限，从而导致治疗功效受损和高水平的死亡率。在这笔赠款中，我们建议探索翻译状态阵列分析（TSAA）的使用，以增加我们对这种生物体对37oC生长至关重要的代谢重编程的理解。我们的总体假设是，通过特定mRNA的选择性翻译，可以将37oC处的烟曲霉的生长与25oC的生长区分开。这将使用TSAA（一种基于微阵列的技术）进行测试，该技术在基因组大规模上评估了与翻译机械的关联。目的，我将使用TSAA检验以下假设：37oC时生长诱导特定子集的MRNA的翻译。 TSAA通过蔗糖梯度离心与DNA阵列技术结合了核糖体分馏，以衡量单个mRNA的翻译效率。 A. fumigatus将在25oC和37oC上进行培养，菌丝提取物将在蔗糖梯度上分离。由于在每个mRNA上的核糖体负荷与蛋白质产物的合成速率成正比，因此分级梯度将分为两个池。一种含有大量核糖体（代表良好翻译的mRNA）和一个含有核糖体的mRNA（表示mRNA下的mRNA）的mRNA。然后，来自两个池的RNA将用于询问烟曲霉微阵列，每个mRNA的翻译状态比率将用作在每个温度下在每个温度下翻译mRNA的表现。 AIM II将通过证明其mRNA以温度依赖性方式进行核糖体负荷发生变化来验证候选基因，并通过温度调节其编码的产物，并且可以在体内检测到。在37oC培养物上上调的蛋白质预计有助于在该温度下的生物体快速生长和整体适应性，这有可能鉴定新的药物靶标或新诊断的发展。 项目叙述：有效治疗曲霉病的主要障碍包括宿主中生物体的快速生长以及当前诊断方法识别感染的能力有限，从而导致高死亡率。这项研究的目的是使用最近开发的技术，即翻译状态阵列分析，以识别优先转化为37oC蛋白质的mRNA，其长期目标是鉴定可以用作新的治疗和/或诊断靶标的蛋白质。