A new pathway for azole resistance in Aspergillus fumigatus

烟曲霉唑类抗性的新途径

基本信息

批准号：
9089985
负责人：
W Scott Moye-Rowley
金额：
$ 17.89万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-06-15 至 2017-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9089985
关键词：
ATP-Binding Cassette Transporters Acute Alleles Allergic Amino Acid Sequence Antifungal Agents Appearance Aspergillosis Aspergillus fumigatus Azole resistance Azoles Candida Cell membrane Cells Chemistry Chronic Clinic Clinical Code Country Data Development Disease Drug Efflux Drug Tolerance Drug effect disorder Drug resistance Elements Europe Gene Duplication Genes Genetic Genetic Screening Genetic Transcription Goals Health Healthcare Systems Human Hypersensitivity In Vitro Incidence Individual Infection Laboratories Lanosterol Lesion Link Malaria Molecular Mutagenesis Mutation Mycoses Natural regeneration Netherlands Oral Organism Pathway interactions Patients Pharmaceutical Preparations Phenotype Play Population Populations at Risk Promoter Regions Proteins Reporting Research Resistance Role Route Site Survival Rate System Testing Time Trans-Activators Tuberculosis United Kingdom United States Work antimicrobial drug base clinically relevant cost effective therapy genetic analysis interest intravenous administration mortality mutant overexpression pathogen promoter research study resistance mechanism

项目摘要

DESCRIPTION (provided by applicant): Invasive Aspergillosis caused by azole resistant A. fumigatus has an alarming 12% survivability making this a clinical problem of acute significance. Early work on azole resistant isolates of A. fumigatus suggested that azole resistance was a relatively rare occurrence and that the genetic basis of resistance was most often due to changes in a gene (cyp51A) encoding the azole target protein, lanosterol ¿-14 demethylase. Extensive studies from a group in the Netherlands provided compelling evidence that in that country, most azole resistant A. fumigatus isolates contained a single compound mutation consisting of a duplicated region of the cyp51A promoter region (TR34) and a change in the coding sequence altering the amino acid sequence of the protein (L98H). However, more recent studies from a group in the United Kingdom indicated that alterations in the cyp51A gene were unlikely to explain all azole resistance in A. fumigatus. More than 50% of azole resistant isolates from UK patients have wild-type versions of cyp51A, inconsistent with changes at this gene explaining drug tolerance. In 2013, the UK group identified an ATP-binding cassette (ABC) transporter-encoding gene called cdr1B as being associated with azole resistance in several patient isolates. Expression of cdr1B was elevated in highly azole tolerant clinical isolates while the cyp51A remained wild-type in sequence. Overexpression of ABC transporter-encoding genes resulting in azole tolerance has been the predominant route of azole resistance in the Candida species and we believe this will emerge as a key feature of drug resistance in A. fumigatus. This application will focus on the contributions of the cdr1B gene as expression of this ABC transporter is elevated in azole resistance isolates and loss of cdr1B produces an azole sensitive phenotype. We will directly compare the azole resistance phenotypes of wild-type and cdr1B¿ cells containing various cyp51A mutations that are associated with clinically relevant drug resistance. We will also evaluate expression of cyp51A using immunological probes to determine if these mutant alleles influence protein levels; information that is currently unavailable in A. fumigatus. We will carry out a forward genetic screen using impala transposon mutagenesis to identify genes that modulate cdr1B expression. Together, these approaches will provide important new information into the molecular basis of azole resistance in A. fumigatus and provide the direct assessment of drug resistance contributions from ABC transporters and the cyp51A gene.

描述（由申请人提供）：由唑类抗性烟曲霉引起的侵袭性曲霉病具有令人震惊的 12% 存活率，这使其成为具有重要意义的临床问题。对唑类抗性烟曲霉分离株的早期研究表明，唑类抗性是相对罕见的情况。耐药性的遗传基础通常是由于编码唑类靶蛋白羊毛甾醇的基因 (cyp51A) 发生变化¿ -14 去甲基化酶。来自荷兰的一个小组的广泛研究提供了令人信服的证据，表明在该国，大多数唑类抗性烟曲霉分离株含有单一复合突变，该突变由 cyp51A 启动子区域 (TR34) 的重复区域和然而，英国一组最近的研究表明，cyp51A 基因的改变不太可能解释这一现象。烟曲霉中所有唑类耐药超过 50% 的唑类耐药菌株。来自英国患者的 cyp51A 具有野生型版本，这与解释药物耐受性的该基因的变化不一致。2013 年，英国研究小组发现了一种名为 cdr1B 的 ATP 结合盒 (ABC) 转运蛋白编码基因与一些患者的唑类耐药性相关。患者分离株中 cdr1B 的表达在高度唑类耐受的临床分离株中升高。 cyp51A 序列保持野生型，导致唑类耐受的 ABC 转运蛋白编码基因的过度表达是念珠菌属中唑类抗性的主要途径，我们相信这将成为烟曲霉耐药性的一个关键特征。本申请将重点关注 cdr1B 基因的贡献，因为该 ABC 转运蛋白的表达在唑类抗性分离株中升高，并且 cdr1B 的缺失会产生唑类敏感表型。将直接比较野生型和cdr1B的唑类抗性表型¿含有与临床相关耐药性相关的各种 cyp51A 突变的细胞我们还将使用免疫探针评估 cyp51A 的表达，以确定这些突变等位基因是否影响当前的蛋白质水平；我们将利用黑斑羚转座子诱变进行正向遗传筛选，以鉴定调节 cdr1B 表达的基因，这些方法将为烟曲霉中唑类抗性的分子基础提供重要的新信息，并提供直接的信息。评估 ABC 转运蛋白和 cyp51A 基因的耐药性贡献。