Translation state array analysis in Aspergillus fumigatus

烟曲霉翻译态阵列分析

基本信息

批准号：
7561654
负责人：
DAVID S ASKEW
金额：
$ 19.5万
依托单位：
UNIVERSITY OF CINCINNATI
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-02-01 至 2011-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7561654
关键词：
Affect Aspergillosis Aspergillus Aspergillus fumigatus Candidate Disease Gene Centrifugation Clinical DNA Development Diagnostic Diagnostic Procedure Diagnostic tests Disease Drug Delivery Systems Environment Epitopes Equipment and supply inventories Eukaryota Fractionation Future Gene Expression Genes Goals Grant Growth Immunocompromised Host In Situ Individual Infection Investigation Life Measures Messenger RNA Metabolic Microarray Analysis Northern Blotting Organism Outcome Pathogenesis Polyribosomes Population Procedures Protein Biosynthesis Proteins RNA Ribosomes Sensitivity and Specificity Shock Sucrose Techniques Technology Temperature Testing Therapeutic Intervention Translating Translational Regulation Translations Treatment Efficacy Western Blotting abstracting base density effective therapy expectation fitness fungus genome-wide improved in vivo insight mortality mouse model novel novel diagnostics novel therapeutics pathogen protein expression rapid growth

项目摘要

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）：摘要机会性真菌病原体烟曲霉的感染仍然与不良结果相关。有效治疗的主要障碍包括微生物在宿主环境中的快速生长以及当前诊断方法识别感染的能力有限，从而导致治疗效果受损和死亡率较高。在这笔资助中，我们建议探索使用平移态阵列分析 (TSAA) 来增加我们对代谢重编程的理解，这是该生物体适应 37oC 生长的基础。我们的总体假设是，可以通过特定 mRNA 的选择性翻译来区分烟曲霉在 37°C 下的生长与在 25°C 下的生长。这将使用 TSAA 进行测试，TSAA 是一种基于微阵列的技术，可在基因组范围内评估 mRNA 与翻译机制的关联。目标我将使用 TSAA 来检验以下假设：37oC 下的生长会诱导特定 mRNA 子集的翻译。 TSAA 将蔗糖梯度离心的核糖体分级分离与 DNA 阵列技术相结合，以测量单个 mRNA 的翻译效率。烟曲霉将在 25oC 和 37oC 下培养，菌丝提取物将在蔗糖梯度上分级分离。由于装载到每个 mRNA 上的核糖体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；一种包含具有丰富核糖体的 mRNA（代表翻译良好的 mRNA），另一种包含具有少量核糖体的 mRNA（代表翻译不足的 mRNA）。然后，来自两个池的 RNA 将用于询问烟曲霉微阵列，并且每个 mRNA 的翻译状态比率将用作 mRNA 在每个温度下翻译情况的指标。 Aim II 将通过证明候选基因的 mRNA 以温度依赖的方式经历核糖体负载的变化以及它们的编码产物受温度调节并可以在体内检测来验证候选基因。在 37oC 培养物中上调的蛋白质预计将有助于生物体在此温度下的快速生长和整体适应度，这有可能识别新的药物靶点或开发新的诊断方法。项目叙述：有效治疗曲霉病的主要障碍包括宿主体内微生物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。本研究的目标是使用最近开发的技术（翻译状态阵列分析）来识别在 37oC 下优先翻译为蛋白质的 mRNA，长期目标是识别可作为新型治疗和/或诊断靶点的蛋白质。