Role of transcriptional regulation in Aspergillus fumigatus drug resistance

描述(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的死亡率高达90%，主要是在免疫功能降低的患者群体中。烟曲霉菌对现有抗真菌药物的高度耐受性，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的一个特别尖锐的问题。以前对白色念珠菌和光滑念珠菌等真菌病原体的研究表明，基因转录是耐药的关键决定因素。这项建议的目的是分析在烟曲霉菌中对唑类耐药性的转录贡献。虽然许多唑耐药突变株是由编码唑类药物靶标的cyp51a基因的变化引起的，但最近的分析表明，越来越多的唑耐药突变株具有野生型cyp51a基因。这表明存在获得对唑类药物抗药性的替代方法。我们已经建立了一个荧光素酶报告基因系统，可以用来鉴定驱动唑诱导的荧光素酶活性的烟曲霉启动子。该报告基因系统将被用来测试烟曲霉菌的一系列启动子融合驱动唑诱导的荧光素酶活性的能力。将通过RNA测量和转录作图来验证Azole的诱导表达。根据这些信息，将进行缺失突变，以确定对唑类药物挑战的反应重要的启动子(S)区域。对这些DNA元件的鉴定将作为未来工作的起点，旨在发现在存在这种重要的抗真菌药物时，导致表达增加的反式作用因素。这项工作的成功完成将为烟曲霉菌的表达分析提供一系列有用的质粒，并为控制响应药物挑战的基因提供重要信息。对耐药基因表达控制的干扰将阻止正常水平的药物耐受的发展。增加烟曲霉菌对抗真菌药物的敏感度是这项工作的一个重要的长期目标。 公共卫生相关性：烟曲霉是人类丝状真菌的主要病原体，这种细菌对常见抗真菌药物的相对耐药性使情况变得复杂。这项工作将开始分析基因转录在烟曲霉抗真菌耐药性中的作用。

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.