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Abstract：
Treatment of fungal infections associated with the filamentous fungus Aspergillus fumigatus is becoming more problematic as this organism is developing resistance to the main chemotherapeutic drug at an increasing rate. Azole drugs represent the current standard-of-care in the treatment of aspergillosis with this drug class acting by inhibiting a key step in the biosynthesis of the fungal sterol ergosterol. Azole compounds block the activity of the lanosterol α-14 demethylase, encoded by the cyp51A gene. A common route of azole resistance involves an increase in transcription of cyp51A. This transcriptional increase requires the function of a Zn2Cys6 DNA-binding domain-containing transcription activator protein called AtrR. AtrR was identified through its action as a positive regulator of expression of an ATP-binding cassette transporter (abcC/cdr1B here called abcG1). Using both deletion and alanine scanning mutagenesis, we demonstrate that a conserved C-terminal domain in A. fumigatus is required for the expression of abcG1 but dispensable for cyp51A transcription. This domain is also found in several other fungal pathogen AtrR homologs consistent with a conserved gene-selective function of this protein segment being conserved. Using RNA sequencing (RNA-seq), we find that this gene-specific transcriptional defect extends to several other membrane transporter-encoding genes including a second ABC transporter locus. Our data reveal that AtrR uses at least two distinct mechanisms to induce gene expression and that normal susceptibility to azole drugs cannot be provided by maintenance of wild-type expression of the ergosterol biosynthetic pathway when ABC transporter expression is reduced.
OBJECTIVE: Aspergillus fumigatus is the primary human filamentous fungal pathogen. The principal chemotherapeutic drug used to control infections associated with A. fumigatus is the azole compound. These drugs are well-tolerated and effective, but resistance is emerging at an alarming rate. Most resistance is associated with mutations that lead to overexpression of the azole target enzyme, lanosterol α-14 demethylase, encoded by the cyp51A gene. A key regulator of cyp51A gene expression is the transcription factor AtrR. Very little is known of the molecular mechanisms underlying the effect of AtrR on gene expression. Here, we use deletion and clustered amino acid substitution mutagenesis to map a region of AtrR that confers gene-specific activation on target genes of this transcription factor. This region is highly conserved across AtrR homologs from other pathogenic species arguing that its importance in transcriptional regulation is maintained across evolution.
OBJECTIVE: Aspergillus fumigatus is the primary human filamentous fungal pathogen. The principal chemotherapeutic drug used to control infections associated with A. fumigatus is the azole compound. These drugs are well-tolerated and effective, but resistance is emerging at an alarming rate. Most resistance is associated with mutations that lead to overexpression of the azole target enzyme, lanosterol α-14 demethylase, encoded by the cyp51A gene. A key regulator of cyp51A gene expression is the transcription factor AtrR. Very little is known of the molecular mechanisms underlying the effect of AtrR on gene expression. Here, we use deletion and clustered amino acid substitution mutagenesis to map a region of AtrR that confers gene-specific activation on target genes of this transcription factor. This region is highly conserved across AtrR homologs from other pathogenic species arguing that its importance in transcriptional regulation is maintained across evolution.
摘要：
与丝状真菌烟曲霉相关的真菌感染的治疗变得越来越成问题，因为这种生物正在以增加的速度发展对主要化疗药物的抗性。唑类药物代表了目前治疗曲霉病的标准药物，该类药物通过抑制真菌甾醇麦角甾醇生物合成的关键步骤而起作用。唑类化合物阻断羊毛甾醇α-14脱甲基酶的活性，由cyp51A基因编码。唑类抗性的常见途径涉及cyp51A转录的增加。这种转录增加需要含有Zn2Cys6DNA结合域的转录激活蛋白AtrR的功能。AtrR通过其作为ATP结合盒转运蛋白（abcC/cdr1B在此称为abcG1）表达的正调节剂的作用而被鉴定。使用缺失和丙氨酸扫描诱变，我们证明了烟曲霉中保守的C末端结构域对于abcG1的表达是必需的，但对于cyp51A转录是不必要的。该结构域也在其他几种真菌病原体AtrR同源物中发现，这与该蛋白质片段的保守基因选择功能一致。使用RNA测序（RNA-seq），我们发现这种基因特异性转录缺陷延伸到其他几个膜转运蛋白编码基因，包括第二个ABC转运蛋白基因座。我们的数据表明，AtrR使用至少两种不同的机制来诱导基因表达，并且当ABC转运蛋白表达降低时，维持麦角甾醇生物合成途径的野生型表达不能提供对唑类药物的正常敏感性。
目的：烟曲霉是主要的人类丝状真菌病原体。用于控制与烟曲霉有关的感染的主要化疗药物是唑类化合物。这些药物耐受性良好且有效,但是阻力正以惊人的速度出现。大多数抗性与导致唑类靶酶过表达的突变有关，羊毛甾醇α-14脱甲基酶，由cyp51A基因编码。cyp51A基因表达的关键调节因子是转录因子AtrR。对AtrR对基因表达的影响的分子机制知之甚少。这里,我们使用缺失和成簇氨基酸置换诱变来定位AtrR的一个区域,该区域赋予该转录因子靶基因的基因特异性激活.该区域在来自其他致病物种的AtrR同源物中高度保守，认为其在转录调控中的重要性在进化中得以维持。
目的：烟曲霉是主要的人类丝状真菌病原体。用于控制与烟曲霉有关的感染的主要化疗药物是唑类化合物。这些药物耐受性良好且有效,但是阻力正以惊人的速度出现。大多数抗性与导致唑类靶酶过表达的突变有关，羊毛甾醇α-14脱甲基酶，由cyp51A基因编码。cyp51A基因表达的关键调节因子是转录因子AtrR。对AtrR对基因表达的影响的分子机制知之甚少。这里,我们使用缺失和成簇氨基酸置换诱变来定位AtrR的一个区域,该区域赋予该转录因子靶基因的基因特异性激活.该区域在来自其他致病物种的AtrR同源物中高度保守，认为其在转录调控中的重要性在进化中得以维持。
