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

基本信息

批准号：
10088398
负责人：
W Scott Moye-Rowley
金额：
$ 19.75万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
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/10088398
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 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

项目摘要

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.

曲霉菌是人类主要的人类丝状真菌病原体。硫代药物代表曲霉病治疗的金标准，是唯一可以使用的药物住院。西欧有问题的发现表明抗唑的形式可以出现并经常传播烟曲霉。这些研究表明硫唑抗性的主要原因是编码靶标的基因中的化合物突变硫唑药物的酶，CYP51A。这些连接的突变包括在启动子序列（34个碱基对的串联重复：TR34）与氨基酸结合在CYP51A中的编码序列（L98H）中替换。包含此TR34的突变菌株 L98H CYP51A等位基因具有高度抗抗药物的耐药性，似乎没有适应性缺陷，领先抗性感染的高频。我们发现了一个新的转录因子称为与该TR34元素结合的ATRR，是正常CYP51A表达所必需的。重要的是，我们和其他人都发现hatrr无效突变体在小鼠中是无毒的感染的吸入模型。在这里，我们建议使用染色质免疫沉淀耦合使用高通量测序（CHIP-SEQ）数据集，我们已经生成了识别基因在影响病毒的ATRR的控制下。我们将使用鼠标感染模型来确定在感染小鼠肺中暴露了转录反应的ATRR调节基因。将进行纳米串技术，以允许测量真菌基因表达哺乳动物RNA的高背景。 ATRR目标基因将由其体内排序排序表达概况。我们将使用CRISPR技术和现有的破坏收集评估多达40个ATRR靶基因对体外上皮细胞损伤的作用测定。我们的目标是根据这些基因的体内表达谱以及对上皮细胞损伤的影响。从这些分析中，我们将选择多达8个目标基因使用小鼠吸入模型对病毒产生影响的破坏突变体。这工作将对ATRR介导的病毒因子的分子基础进行首次检查在这种感染肺部动物模型中，这对于发病机理至关重要。