Analysis of transcription factors determining azole resistance of Aspergillus fumigatus

烟曲霉唑类抗性转录因子分析

• 批准号: 10451817
• 负责人: W Scott Moye-Rowley
• 金额: $ 50.82万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-06 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10451817
• 关键词: ATP-Binding Cassette Transporters; Acute; Affect; Agriculture; Alleles; Anabolism; Antibiotics; Antibodies; Antifungal Agents; Antimicrobial Resistance; Appearance; Aspergillosis; Aspergillus fumigatus; Azole resistance; Azoles; Binding Sites; Biochemical; Biological; Bypass; Candidate Disease Gene; Carrier Proteins; ChIP-seq; Clinic; Clinical; Coupled; Data; Defect; Development; Dissection; Drug resistance; Enzymes; Ergosterol; Gene Expression; Genes; Genetic; Genetic Epistasis; Genetic Screening; Genetic Transcription; Goals; Gold; High-Throughput DNA Sequencing; Histidine; Human; Hyperactivity; Infection; Lanosterol; Leucine; Light; Link; Mammalian Cell; Medicine; Molds; Molecular; Mutagenesis; Mutation; Organism; Participant; Patients; Pharmaceutical Preparations; Phenotype; Physiological; Physiology; Predisposition; Proteins; Regulation; Resistance; Resistance development; Role; Sterol Biosynthesis Pathway; Sterols; Structure; System; Tertiary Protein Structure; Testing; Transcriptional Activation; Virulence; Work; Zinc Cluster; antimicrobial drug; chemotherapy; chromatin immunoprecipitation; clinically significant; experimental study; fungus; gene network; insight; mortality; mutant; pathogenic fungus; pathogenic microbe; promoter; resistance allele; resistant Aspergillus; resistant strain; spelling; transcription factor; transcriptome sequencing

Invasive Aspergillosis caused by azole resistant A. fumigatus has a mortality rate nearing an alarming 90%, making this a clinical problem of acute significance. Early work on A. fumigatus suggested that azole resistance was rare and that the genetic basis of resistance was most often due to changes in a gene (cyp51A) encoding the azole target protein, lanosterol α-14 demethylase. Recent work from our groups has provided evidence that expression of an ATP-binding cassette (ABC) transporter-encoding gene called abcG1 (aka cdr1B) is linked to azole resistance in the absence of any changes at the cyp51A locus. Here we propose to investigate a key transcriptional regulator called AtrR that coordinately regulates expression of both cyp51A and abcG1. AtrR is a Zn2Cys6 zinc cluster-containing factor that resembles other fungal transcriptional regulators of drug resistance. We have found that loss of the atrR gene eliminated the high-level azole resistance seen in clinical isolates. Additional, preliminary data generated by use of chromatin immunoprecipitation coupled with high throughput DNA sequencing (ChIP-seq) and RNA-seq have shed light on direct and indirect targets of AtrR regulation. We have generated two different hyperactive alleles of atrR that drive elevated expression of abcG1 and enhanced azole resistance. These data suggest that AtrR is normally subject to negative regulation that can be overcome in different manners. We suggest that defects in this negative regulatory system may influence clinically significant azole resistance owing to increased expression of AtrR-dependent target genes (like abcG1 and cyp51A). The goal of this proposal is to employ a combined biochemical, molecular biological and genetic dissection of the regulation of AtrR in order to understand how this factor acts to induce azole resistance. Our initial goal is to carry out a structure/function analysis of AtrR and identify protein domains that are important for regulation of this factor. We will also use direct biochemical purification to identify factors that associate with AtrR and influence its function. Second, we will use forward genetic screening involving impala transposon mutagenesis to identify AtrR regulatory factors in an unbiased, functional manner. Finally, we will examine the role of an AtrR target gene that encodes a transcription factor called RfeC. Preliminary data indicate that RfeC is important in AbcG1 expression and azole resistance. We will examine the epistatic relationship between our atrR alleles and rfeC (as well as other azole resistance-affecting transcription factors) to establish the regulatory hierarchy controlling azole resistance. These experiments will illuminate the physiological network controlling AtrR that directly links ergosterol biosynthesis to ABC transporter gene expression and provide important new information about azole resistance in this fungal pathogen.

抗硫杆菌曲霉引起的侵袭性曲霉病的死亡率接近90％，这使得这是急性意义的临床问题。早期对烟曲霉的工作表明，硫醇耐药性很少见，耐药性的遗传基础通常是由于编码硫烷靶蛋白Lainosterolα-14脱甲基酶的基因（CYP51A）的变化所致。我们小组的最新工作提供了证据，表明ATP结合盒（ABC）转运蛋白编码基因称为ABCG1（aka CDR1B）在CYP51A基因座的任何变化的情况下与硫代抗性有关。在这里，我们建议研究一个称为ATRR的关键转录调节剂，该调节器协调调节CYP51A和ABCG1的表达。 ATRR是含有其他真菌转录耐药性调节剂的Zn2Cys6锌簇因子。我们发现，ATRR基因的丧失消除了在临床分离株中看到的高水平偏唑抗性。通过使用染色质免疫沉淀与高吞吐量DNA测序（CHIP-SEQ）和RNA-SEQ产生的其他初步数据，对ATRR调节的直接和间接靶标有启示。我们已经产生了ATRR的两个不同的多活跃等位基因，它们驱动ABCG1的表达升高并增强了唑的耐药性。这些数据表明，ATRR通常受到负面法规的约束，可以以不同的方式克服。我们建议，由于ATRR依赖性靶基因的表达增加（例如ABCG1和CYP51A），这种负调节系统中的缺陷可能会影响临床上显着的悬唑抗性。该提案的目的是采用ATRR调节的生化，分子生物学和遗传解剖的组合，以了解该因子如何作用于诱导硫唑抗性。我们的最初目标是对ATRR进行结构/功能分析，并确定对调节该因素很重要的蛋白质域。我们还将使用直接的生化纯化来确定与ATRR相关的因素并影响其功能。其次，我们将使用涉及Impala转座子诱变的正向遗传筛选以公正的功能方式鉴定ATRR调节因子。最后，我们将检查编码称为RFEC的转录因子的ATRR靶基因的作用。初步数据，表明RFEC在ABCG1表达和偶氮抗性中很重要。我们将研究我们的ATRR等位基因与RFEC（以及其他抗唑耐药的转录因子）之间的认识关系，以建立控制硫唑抗性的调节层次结构。这些实验将阐明控制ARTRR的物理网络，该物理网络将麦角固醇生物合成与ABC转运蛋白基因的表达联系起来，并提供有关这种真菌病原体中硫唑抗性的重要新信息。