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

烟曲霉 AtrR 转录控制下毒力决定簇的鉴定

• 批准号: 9914775
• 负责人: W Scott Moye-Rowley
• 金额: $ 25.07万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-24 至 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9914775
• 关键词: ATP-Binding Cassette Transporters; Agriculture; Alleles; Amino Acids; Animal Model; Aspergillosis; Aspergillus fumigatus; Attenuated; Azole resistance; Azoles; Base Pairing; Binding; Biological Assay; CRISPR/Cas technology; Candida albicans; Code; Collection; Complex; Coupled; Data; Data Set; Defect; Disease; Drug resistance; Elements; Enzymes; Epithelial Cells; Exhibits; Expression Profiling; Frequencies; Fungal Genes; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Goals; Gold; High-Throughput Nucleotide Sequencing; Hospitalization; Human; In Vitro; Incidence; Infection; Inhalation; Link; Lung; Maps; Measurement; Mediating; Messenger RNA; Methods; Modeling; Molecular; Mus; Mutation; Organism; Pathogenesis; Pathogenicity; Pattern; Pharmaceutical Preparations; Play; Positioning Attribute; Predisposition; Promoter Regions; Publishing; RNA; Resistance; Role; Specific qualifier value; Technology; Testing; Tissues; Transcript; Transcriptional Regulation; Virulence; Virulence Factors; Western Europe; Work; base; cell injury; chromatin immunoprecipitation; dosage; experimental study; fitness; fungus; in vivo; insight; mortality; mouse model; mutant; nano-string; novel; novel strategies; overexpression; pathogen; pathogenic fungus; resistant strain; response; transcription factor; transcriptome sequencing

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.

烟曲霉是主要的人类丝状真菌病原体。唑类药物代表 治疗曲霉病的黄金标准，是唯一可以在不使用曲霉病的情况下使用的药物 住院治疗。西欧的问题发现表明，唑类耐药形式 烟曲霉可以频繁出现和传播。这些研究表明， 唑类耐药的主要原因是编码靶标基因的复合突变 唑类药物的酶，cyp51A。这些连锁突变包括 与氨基酸偶联的启动子序列（34 个碱基对的串联重复：TR34） cyp51A 中编码序列 (L98H) 的替换。含有该TR34的突变株 L98H cyp51A 等位基因对唑类药物具有高度耐药性，并且似乎没有健康缺陷，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，它与 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 无效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偶联 使用我们生成的用于识别基因的高通量测序 (ChIP-seq) 数据集 受 AtrR 控制，影响毒力。我们将使用小鼠感染模型来确定 AtrR 调节的基因在受感染的小鼠肺部表现出转录反应。 纳米线技术将用于测量真菌基因表达 哺乳动物 RNA 的高背景。 AtrR 靶基因将按照其体内情况进行排序 表达谱。我们将利用 CRISPR 技术和现有的破坏集合来 评估多达 40 个 AtrR 靶基因对体外上皮细胞损伤的影响 化验。我们的目标是根据这些基因的体内表达谱和 对上皮细胞损伤的影响。从这些分析中，我们将选择最多 8 个目标基因 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 这项工作将首次检查 AtrR 介导的毒力因子的分子基础 这对于这种肺部感染动物模型的发病机制至关重要。