Analysis of transcription factors determining azole resistance of Aspergillus fumigatus

耐唑A.烟曲霉的死亡率接近惊人的90%，使其成为具有严重意义的临床问题。早期的工作A。烟曲霉的研究表明，唑类耐药性是罕见的，抗性的遗传基础最常见的是由于编码唑类靶蛋白--羊毛甾醇α-14脱甲基酶的基因（cyp 51 A）的变化。我们小组最近的工作提供了证据表明，在cyp 51 A位点没有任何变化的情况下，ATP结合盒（ABC）转运蛋白编码基因abcG 1（又名cdr 1B）的表达与唑类耐药有关。在这里，我们建议调查一个关键的转录调节称为AtrR，协调调节cyp 51 A和abcG 1的表达。AtrR是一个含锌2Cys 6锌簇的因子，类似于其他真菌耐药性的转录调节因子。我们发现，atrR基因的缺失消除了临床分离株中观察到的高水平唑类耐药。此外，通过使用染色质免疫沉淀结合高通量DNA测序（ChIP-seq）和RNA-seq产生的初步数据揭示了AtrR调控的直接和间接靶标。我们已经产生了两种不同的atrR过度活跃的等位基因，驱动abcG 1的表达升高和唑类耐药性增强。这些数据表明，AtrR通常受到可以以不同方式克服的负调控。我们认为，在这个负调控系统的缺陷可能会影响临床显着的唑类药物耐药，由于AtrR依赖的靶基因（如abcG 1和cyp 51 A）的表达增加。本提案的目标是采用生物化学，分子生物学和遗传学相结合的解剖AtrR的调节，以了解该因子如何诱导唑类耐药。我们的初步目标是进行AtrR的结构/功能分析，并确定蛋白质结构域，这是重要的调节这个因素。我们还将使用直接生化纯化来鉴定与AtrR相关并影响其功能的因子。其次，我们将使用正向遗传筛选涉及黑斑羚转座子诱变，以确定AtrR的调节因子在一个公正的，功能性的方式。最后，我们将研究编码转录因子RfeC的AtrR靶基因的作用。初步数据表明RfeC在AbcG 1表达和唑类耐药性中是重要的。我们将研究我们的atrR等位基因和rfeC（以及其他唑类耐药影响转录因子）之间的上位性关系，以建立控制唑类耐药的调控层次。这些实验将阐明控制AtrR的生理网络，直接将麦角甾醇生物合成与ABC转运蛋白基因表达联系起来，并提供有关这种真菌病原体中唑类抗性的重要新信息。

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.