Leibniz Institute for Natural Product Research and Infection Biology, Hans-Knöll-Institute, Jena, Germany.
BMC Genomics. 2010 Jan 15;11:32. doi: 10.1186/1471-2164-11-32.
Aspergillus fumigatus is a thermotolerant human-pathogenic mold and the most common cause of invasive aspergillosis (IA) in immunocompromised patients. Its predominance is based on several factors most of which are still unknown. The thermotolerance of A. fumigatus is one of the traits which have been assigned to pathogenicity. It allows the fungus to grow at temperatures up to and above that of a fevered human host. To elucidate the mechanisms of heat resistance, we analyzed the change of the A. fumigatus proteome during a temperature shift from 30 degrees C to 48 degrees C by 2D-fluorescence difference gel electrophoresis (DIGE). To improve 2D gel image analysis results, protein spot quantitation was optimized by missing value imputation and normalization. Differentially regulated proteins were compared to previously published transcriptome data of A. fumigatus. The study was augmented by bioinformatical analysis of transcription factor binding sites (TFBSs) in the promoter region of genes whose corresponding proteins were differentially regulated upon heat shock.
91 differentially regulated protein spots, representing 64 different proteins, were identified by mass spectrometry (MS). They showed a continuous up-, down- or an oscillating regulation. Many of the identified proteins were involved in protein folding (chaperones), oxidative stress response, signal transduction, transcription, translation, carbohydrate and nitrogen metabolism. A correlation between alteration of transcript levels and corresponding proteins was detected for half of the differentially regulated proteins. Interestingly, some previously undescribed putative targets for the heat shock regulator Hsf1 were identified. This provides evidence for Hsf1-dependent regulation of mannitol biosynthesis, translation, cytoskeletal dynamics and cell division in A. fumigatus. Furthermore, computational analysis of promoters revealed putative binding sites for an AP-2alpha-like transcription factor upstream of some heat shock induced genes. Until now, this factor has only been found in vertebrates.
Our newly established DIGE data analysis workflow yields improved data quality and is widely applicable for other DIGE datasets. Our findings suggest that the heat shock response in A. fumigatus differs from already well-studied yeasts and other filamentous fungi.
烟曲霉是一种耐热的人类致病性霉菌，也是免疫功能低下患者侵袭性曲霉病（IA）最常见的病因。它的优势基于许多未知的因素。烟曲霉的耐热性是被认为与致病性有关的特征之一。它使真菌能够在高达甚至高于发热宿主的温度下生长。为了阐明耐热机制，我们通过二维荧光差异凝胶电泳（DIGE）分析了烟曲霉从 30°C 温度转移到 48°C 时的蛋白质组变化。为了改善 2D 凝胶图像分析结果，通过缺失值插补和归一化优化了蛋白质斑点定量。将差异调节的蛋白质与烟曲霉先前发表的转录组数据进行比较。通过分析热休克时差异调节基因的启动子区域中转录因子结合位点（TFBS），对研究进行了补充。
通过质谱（MS）鉴定了 91 个差异调节的蛋白质斑点，代表 64 种不同的蛋白质，它们表现出连续的上调、下调或振荡调节。许多鉴定出的蛋白质参与蛋白质折叠（伴侣）、氧化应激反应、信号转导、转录、翻译、碳水化合物和氮代谢。在差异调节蛋白中，有一半检测到转录水平改变与相应蛋白之间存在相关性。有趣的是，鉴定出了一些以前未描述的热休克调节因子 Hsf1 的潜在靶标。这为 Hsf1 依赖的烟曲霉甘露醇生物合成、翻译、细胞骨架动力学和细胞分裂的调节提供了证据。此外，对启动子的计算分析显示，一些热激诱导基因的上游存在一个 AP-2alpha 样转录因子的潜在结合位点。到目前为止，这种因子只在脊椎动物中发现过。
我们新建立的 DIGE 数据分析工作流程提高了数据质量，并且广泛适用于其他 DIGE 数据集。我们的研究结果表明，烟曲霉的热应激反应与已经研究得很好的酵母和其他丝状真菌不同。
