Translation state array analysis in Aspergillus fumigatus

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局相关。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识别感染的能力有限，导致治疗效果受损和高死亡率。在这项资助中，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编程的理解，这是这种生物体适应37 ℃生长的基础。我们的总体假设是A.通过特异性mRNA的选择性翻译，可以将37 ℃下的烟曲霉生长与25 ℃下的烟曲霉生长区分开来。这将使用TSAA进行测试，TSAA是一种基于微阵列的技术，可在全基因组范围内评估mRNA与翻译机制的相关性。目的我将使用TSAA来检验在37 ℃下生长诱导特定mRNA子集翻译的假设。TSAA结合了蔗糖梯度离心的核糖体分离和DNA阵列技术来测量单个mRNA的翻译效率。A.烟曲霉将在25 ℃和37 ℃下培养，菌丝提取物将在蔗糖梯度上分级。由于核糖体加载到每个mRNA上与蛋白质产物的合成速率成比例，因此将分级梯度分成两个池;一个含有具有丰富核糖体的mRNA（代表翻译良好的mRNA），另一个含有具有很少核糖体的mRNA（代表翻译不足的mRNA）。然后将来自两个池的RNA用于询问A。在烟曲霉微阵列中，每个mRNA的翻译状态比率将用作mRNA在每个温度下翻译得有多好的指标。目的II将验证候选基因，证明他们的mRNA经历的变化，在核糖体负载的温度依赖性的方式，和他们的编码产物的温度调节，可以在体内检测。在37 ℃培养时上调的蛋白质预计有助于生物体在该温度下的快速生长和整体适应性，这有可能确定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微生物在宿主中的快速生长和当前诊断方法识别感染的有限能力，导致高死亡率。本研究的目的是使用最近开发的技术，翻译状态阵列分析，以识别在37 ℃优先翻译成蛋白质的mRNA，长期目标是识别可作为新的治疗和/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.