Role of transcriptional regulation in Aspergillus fumigatus drug resistance

描述（由申请人提供）：烟曲霉是人类主要的丝状真菌病原体。侵袭性曲霉病在特定患者群体中死亡率高达90%，主要是在免疫功能下降的患者群体中。烟曲霉对目前可用的抗真菌药物具有很高的内在耐药性，再加上这些药物的数量有限，使得耐药性成为曲霉病临床治疗中特别严重的问题。先前在真菌病原体如白色念珠菌和光丝念珠菌的研究已经清楚地表明基因转录是耐药性的关键决定因素。本研究的目的是分析烟曲霉对唑的抗性的转录贡献。虽然许多抗唑突变是由编码唑类药物靶点的cyp51A基因的变化引起的，但最近的分析表明，越来越多的抗唑突变具有野生型cyp51A基因。这表明存在其他途径获得对唑类药物的耐药性。我们建立了一个荧光素酶报告基因系统，以鉴定烟曲霉启动子驱动唑诱导的荧光素酶活性。该报告基因系统将用于检测一系列烟曲霉启动子融合物驱动唑诱导荧光素酶活性的能力。唑诱导的表达将通过RNA测量和转录作图来验证。根据这些信息，将进行删除诱变以确定启动子中对唑类药物应答重要的区域。这些DNA元素的鉴定将作为未来工作的起点，旨在发现在这种重要的抗真菌药物存在下导致表达增加的反式作用因子。这项工作的成功完成将为烟曲霉的表达分析提供一系列有用的质粒，并为药物应答基因的控制提供重要信息。对耐药位点基因表达控制的干扰将阻止正常水平的耐药发展。增加烟曲霉对抗真菌药物的敏感性是这项工作的重要长期目标。

DESCRIPTION (provided by applicant): Aspergillus fumigatus is the major filamentous fungal pathogen in humans. Invasive aspergillosis has up to a 90% mortality rate in particular patient populations, primarily among patient groups with reduced immune function. The high intrinsic resistance of A. fumigatus to currently available antifungal agents, coupled with the limited number of these drugs, makes drug resistance an especially acute issue in clinical treatment of aspergillosis. Previous work in fungal pathogens such as Candida albicans and Candida glabrata has provided clear implication of gene transcription as a key determinant of drug resistance. The goal of this proposal is to analyze the transcriptional contribution to azole tolerance in A. fumigatus. While many azole resistant mutants are caused by changes within the cyp51A gene encoding the target of azole drugs, more recent analyses indicate that an increasing number of azole tolerant mutants have wild-type cyp51A genes. This suggests the presence of alternative means of acquiring resistance to azole drugs. We have generated a luciferase reporter gene system to allow the identification of A. fumigatus promoters that drive azole inducible luciferase enzyme activity. This reporter gene system will be used to test a series of promoter fusions from A. fumigatus for the ability to drive azole inducible luciferase activity. Azole inducible expression will be verified by RNA measurements and transcriptional mapping. From this information, deletion mutagenesis will be carried out to identify regions of the promoter(s) important for the response to azole drug challenge. Identification of these DNA elements will serve as the starting point for future work aimed at discovering the trans-acting factors responsible for increased expression in the presence of this important antifungal drug. Successful completion of this work will provide both a useful series of plasmids for expression analysis in A. fumigatus and important information about control of genes that respond to drug challenge. Interference with control of gene expression of drug resistance loci will prevent normal levels of drug tolerance from developing. Increased sensitization of A. fumigatus to antifungal drugs is an important long term goal of this work. PUBLIC HEALTH RELEVANCE: Aspergillus fumigatus is the major human filamentous fungal pathogen, a situation complicated by the relative resistance of this organism to common antifungal drugs. This work will initiate analysis of the contribution of gene transcription to antifungal drug resistance in A. fumigatus.