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

烟曲霉菌是人类丝状真菌的主要病原菌。唑类药物代表 治疗曲霉病的黄金标准，是唯一可以在没有 住院治疗。西欧的有问题的发现表明，唑类耐药形式 烟曲霉病可能会频繁发生和传播。这些研究表明， 产生唑类耐药性的主要原因是靶标编码基因的复合突变 用于唑类药物的酶，cyp51a。这些连锁突变包括 启动子序列(34个碱基对的串联重复：TR34)与一个氨基酸相连 替换cyp51a中的编码序列(L98H)。含有该TR34的突变株 L98H cyp51a等位基因对唑类药物高度耐药，似乎没有适应缺陷，领先 耐药感染的高频率。我们发现了一种新的转录因子 称为AtrR，它与这个TR34元件结合，是正常cyp51a表达所必需的。 重要的是，我们和其他人都发现atrr缺失突变体对小鼠是无毒的。 吸入性感染模型。在这里，我们建议使用染色质免疫沉淀耦合 使用我们为识别基因而生成的高通量测序(芯片序列)数据集 在AtrR的控制下，这会影响毒力。我们将使用小鼠感染模型来确定 受AtrR调节的基因，在感染的小鼠肺中表现出转录反应。 将采用纳米串技术来测量真菌基因在 哺乳动物RNA的高背景。AtrR靶基因将按其在体内的顺序进行排序 表达配置文件。我们将使用CRISPR技术和现有的中断收集来 评估多达40个AtrR靶基因在体外培养上皮细胞损伤中的作用 化验。我们的目标将是根据这些基因在体内的表达谱和 对上皮细胞损伤的影响。从这些分析中，我们将选择多达8个目标基因 利用我们的小鼠吸入模型，中断突变以筛选对毒力的影响。这 这项工作将首次对AtrR介导的毒力因子的分子基础进行检查 在这种肺部感染的动物模型中，它们对发病机制至关重要。

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.