Analysis of transcription factors determining azole resistance of Aspergillus fumigatus

由氮唑耐药烟曲霉菌引起的侵袭性曲霉病的死亡率接近令人震惊的90%，这使这成为一个具有紧迫意义的临床问题。对烟曲霉的早期研究表明，对唑类的耐药性是罕见的，耐药性的遗传基础最常见的是编码唑靶蛋白羊毛甾醇α-14去甲基酶的基因(Cyp51a)的变化。我们团队最近的工作提供了证据，证明在cyp51a基因没有任何变化的情况下，名为Abcg1(又名cdr1B)的ATP结合盒(ABC)转运体编码基因的表达与唑类耐药性有关。在这里，我们建议研究一种名为AtrR的关键转录调控因子，它协调调节cyp51a和Abcg1的表达。AtrR是一种含锌锌簇的因子，与其他真菌耐药转录调控因子相似。我们发现，atrR基因的缺失消除了临床分离株中出现的高水平的唑类耐药性。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调节的直接和间接靶标。我们已经产生了两个不同的atrR高活性等位基因，它们驱动Abcg1的高表达和增强的唑类耐药性。这些数据表明，AtrR通常受到负面调控，可以通过不同的方式克服这些负面调控。我们认为，这种负性调控系统的缺陷可能通过增加AtrR依赖的靶基因(如Abcg1和cyp51a)的表达而影响临床上显著的唑类耐药性。这项建议的目的是采用生化、分子生物学和遗传学相结合的方法剖析AtrR的调节，以了解该因素如何作用于诱导唑类耐药。我们最初的目标是对AtrR的结构/功能进行分析，并确定对该因子的调节至关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相关的因素并影响其功能。其次，我们将使用包括黑斑羚转座子突变的正向遗传筛选，以无偏见、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的表达和唑类耐药中起重要作用。我们将检查我们的atrR等位基因与RFEC(以及其他影响唑类耐药性的转录因子)之间的上位关系，以建立控制唑类耐药性的调控体系。这些实验将阐明控制AtrR的生理网络，该网络直接将麦角甾醇的生物合成与ABC转运蛋白基因的表达联系起来，并为该真菌对唑类耐药性的研究提供重要的新信息。

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.