Identification of virulence determinants under the transcriptional control of AtrR in Aspergillus fumigatus

烟曲霉是人类主要的丝状真菌病原体。唑类药物代表 是治疗曲霉病的金标准，是唯一一种可以在不使用抗生素的情况下使用的药物。 住院在西欧的问题研究结果表明，唑类耐药形式 曲霉烟曲霉病会频繁出现和传播。这些研究表明， 唑类耐药的主要原因是靶基因编码的复合突变 唑类药物酶，cyp51A。这些连锁突变包括基因突变， 启动子序列（34个碱基对的串联重复：TR34）与氨基酸偶联， cyp51A中编码序列（L98H）的替换。含有该TR34的突变株 L98H cyp51A等位基因是高度唑类药物耐药的，并且似乎没有适合性缺陷，导致 耐药感染的高频率。我们发现了一种新的转录因子 称为AtrR，其与TR34元件结合，并且是正常cyp51A表达所必需的。 重要的是，我们和其他人都发现atrR缺失突变体在小鼠中是无毒的， 吸入感染模型。在这里，我们建议使用染色质免疫沉淀耦合 使用我们生成的用于识别基因的高通量测序（ChIP-seq）数据集 在影响毒力的AtrR的控制下。我们将使用小鼠感染模型来确定 在感染的小鼠肺中表现出转录反应的AtrR调节基因。 将采用Nanostring技术来允许测量真菌基因表达。 哺乳动物RNA的高背景。AtrR靶基因将按照其体内表达水平进行排序。 表达谱我们将使用CRISPR技术和现有的中断收集， 评估多达40种AtrR靶基因对体外上皮细胞损伤的作用 比色法我们的目标将是根据这些基因的体内表达谱优先考虑这些基因， 影响上皮细胞损伤。从这些分析中，我们将选择多达8个靶基因， 使用我们的小鼠吸入模型筛选破坏突变体对毒力的影响。这 这项工作将首次对AtrR介导的毒力因子的分子基础进行研究 在这个肺部感染的动物模型中，这些蛋白质是致病的关键。

Aspergillus fumigatus is the major human filamentous fungal pathogen. Azole drugs represent the gold standard in treatment of aspergillosis and are the only agent that can be used without hospitalization. Problematic findings in Western Europe have shown that azole resistant forms of A. fumigatus can arise and spread frequently. These studies have demonstrated that the primary cause of azole resistance is a compound mutation in the gene encoding the target enzyme for azole drugs, cyp51A. These linked mutations consist of an alteration in the promoter sequence (tandem duplication of 34 base pairs: TR34) coupled with an amino acid replacement in the coding sequence (L98H) in cyp51A. Mutant strains containing this TR34 L98H cyp51A allele are highly azole drug resistant and appear to have no fitness defect, leading to the high frequency of resistant infections. We have discovered a new transcription factor called AtrR that binds to this TR34 element and is required for normal cyp51A expression. Importantly, both we and others have found that atrR null mutants are avirulent in a mouse inhalation model of infection. Here we propose to use a chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) dataset that we have generated to identify genes under control of AtrR that impact virulence. We will use a mouse infection model to determine AtrR-regulated genes that exhibit transcriptional responses in the infected mouse lung. Nanostring technology will be employed to allow measurement of fungal gene expression in the high background of mammalian RNA. AtrR target genes will be rank ordered by their in vivo expression profile. We will use CRISPR technology and existing disruption collections to assess the role of up to 40 AtrR target genes for their effect on an in vitro epithelial cell damage assay. Our goal will be to prioritize these genes based on their in vivo expression profile and impact on epithelial cell damage. From these analyses, we will select up to 8 target gene disruption mutants to screen for an effect on virulence using our mouse inhalation model. This work will provide the first examination of the molecular basis of AtrR-mediated virulence factors that are critical for pathogenesis in this animal model of infected lungs.