Analysis of transcription factors determining azole resistance of Aspergillus fumigatus

由抗唑烟曲霉引起的侵袭性曲霉病的死亡率接近惊人的90%，使其成为一个具有急性意义的临床问题。早期对烟曲霉的研究表明，对唑的抗性是罕见的，抗性的遗传基础通常是由于编码唑靶蛋白羊毛甾醇α-14去甲基化酶的基因cyp51A的改变。我们小组最近的工作提供了证据，证明在cyp51A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abcG1（又名cdr1B）的表达与唑抗性有关。在这里，我们建议研究一种称为AtrR的关键转录调节因子，它协调调节cyp51A和abcG1的表达。AtrR是一种含有Zn2Cys6锌簇的因子，类似于其他真菌耐药转录调节因子。我们发现atrR基因的缺失消除了临床分离株的高水平唑耐药性。此外，利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点。我们已经产生了atrR的两个不同的高活性等位基因，它们驱动abcG1的表达升高并增强了对唑的抗性。这些数据表明，AtrR通常受到负面调节，可以通过不同的方式加以克服。我们认为，由于atrr依赖性靶基因（如abcG1和cyp51A）的表达增加，这种负调控系统的缺陷可能影响临床显著的唑耐药性。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方法对AtrR的调控进行解剖，以了解该因子如何诱导唑抗性。我们的最初目标是进行AtrR的结构/功能分析，并确定对该因子调控重要的蛋白质结构域。我们还将使用直接生化纯化来确定与AtrR相关并影响其功能的因素。其次，我们将使用包括黑斑羚转座子突变在内的正向遗传筛查，以一种公正的、功能性的方式识别AtrR调节因子。最后，我们将研究AtrR靶基因的作用，该基因编码一种称为RfeC的转录因子。初步数据表明，RfeC在AbcG1表达和唑耐药性中起重要作用。我们将研究atrR等位基因和rfeC（以及其他影响唑抗性的转录因子）之间的上位性关系，以建立控制唑抗性的调控体系。这些实验将阐明控制AtrR的生理网络，将麦角甾醇的生物合成与ABC转运体基因表达直接联系起来，并为该真菌病原体的抗唑性提供重要的新信息。

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.