Translation state array analysis in Aspergillus fumigatus

描述(由申请人提供)：摘要感染机会性真菌病原体烟曲霉仍然与不良结局有关。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和目前诊断方法识别感染的能力有限，导致治疗效果受损和高死亡率。在这笔赠款中，我们建议探索使用翻译状态阵列分析(TSAA)来增加我们对代谢重新编程的理解，代谢重新编程是该有机体适应37摄氏度生长的基础。我们的主要假设是，通过选择性地翻译特定的mRNAs，可以将37℃下的烟曲霉菌生长与25℃下的生长区分开来。这将使用TSAA进行测试，TSAA是一种基于微阵列的技术，它在基因组范围内评估mRNA与翻译机制的关联。目的我将使用TSAA来检验这一假设，即在37℃的生长诱导特定的mRNAs子集的翻译。TSAA将蔗糖梯度离心法分离核糖体与DNA阵列技术相结合，以测量单个mRNAs的翻译效率。烟曲霉菌将在25oC和37oC的温度下培养，菌丝提取物将在蔗糖梯度上分级。由于每个mRNA上的核糖体负载量与蛋白质产物的合成速度成正比，因此分级梯度将被分成两个池；一个池包含丰富的核糖体的mRNAs(代表翻译良好的mRNAs)，另一个池包含少量核糖体的mRNAs(代表翻译不足的mRNAs)。然后，来自这两个池的RNA将被用于询问烟曲霉微阵列，每个mRNA的翻译状态比率将被用作在每个温度下翻译mRNA的程度的指标。AIM II将验证候选基因，证明它们的mRNAs以一种温度依赖的方式经历核糖体负荷的变化，并且它们的编码产物受到温度的调节，并且可以在体内检测到。在37℃培养时上调的蛋白质有望有助于生物体在这种温度下的快速生长和整体适应，这有可能识别新的药物靶点或开发新的诊断方法。 项目简介：曲霉病有效治疗的主要障碍包括微生物在宿主体内的快速生长，以及目前诊断方法识别感染的能力有限，导致高水平的死亡率。这项研究的目标是使用一种最近发展的技术，翻译状态阵列分析，来识别在37℃下优先翻译成蛋白质的mRNAs，长期目标是识别可以作为新的治疗和/或诊断靶点的蛋白质。

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.