Role of transcriptional regulation in Aspergillus fumigatus drug resistance

描述（由申请方提供）：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人群中的死亡率高达90%，主要是免疫功能降低的患者群体。A.烟曲霉菌对目前可用的抗真菌剂的耐药性，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滑念珠菌中的工作已经提供了基因转录作为耐药性的关键决定因素的明确暗示。本研究的目的是分析A.烟熏。虽然许多唑类耐药突变体是由编码唑类药物靶标的cyp51A基因内的变化引起的，但最近的分析表明，越来越多的唑类耐药突变体具有野生型cyp51A基因。这表明存在获得唑类药物耐药性的替代手段。我们已经建立了一个荧光素酶报告基因系统，允许识别A。烟曲霉启动子驱动唑诱导荧光素酶活性。该报告基因系统将用于检测一系列来自A. fumigatus的能力驱动唑诱导荧光素酶活性。将通过RNA测量和转录图谱验证唑诱导型表达。根据该信息，将进行缺失诱变以鉴定对于唑类药物攻击应答重要的启动子区域。这些DNA元件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增加表达的反式作用因子。这项工作的成功完成将为A.烟曲霉和重要的信息控制基因，响应药物的挑战。干扰耐药基因座的基因表达控制将阻止正常水平的药物耐受性的发展。A的增敏作用。烟曲霉对抗真菌药物的敏感性是这项工作的一个重要的长期目标。 公共卫生相关性：烟曲霉是人类主要的丝状真菌病原体，这种生物体对常见抗真菌药物的相对耐药性使情况复杂化。这项工作将启动分析基因转录的贡献，抗真菌药物耐药性的A。烟熏。

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.