A new pathway for azole resistance in Aspergillus fumigatus

性状（由申请方提供）：由唑类耐药A.烟曲霉具有惊人的12%的存活率，使得这成为具有严重意义的临床问题。对唑类耐药菌株的早期研究。烟曲霉的研究表明，唑类耐药性是一种相对罕见的现象，耐药性的遗传基础最常见的是由于编码唑类靶蛋白--羊毛甾醇α-14去甲基酶的基因（cyp 51 A）的变化。荷兰一个小组的广泛研究提供了令人信服的证据，表明在该国，大多数唑类耐药A。烟曲霉分离物含有单一复合突变，其由cyp 51 A启动子区的重复区（TR 34）和改变蛋白质的氨基酸序列的编码序列的改变（L98 H）组成。然而，来自英国的一个小组的最近研究表明，cyp 51 A基因的改变不太可能解释A.烟熏。超过50%的唑类耐药菌株 来自英国的患者具有野生型cyp 51 A，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cdr 1B的ATP结合盒（ABC）转运蛋白编码基因，该基因与几种患者分离株的唑类耐药性有关。cdr 1B的表达在唑类药物高度耐受的临床分离株中升高， cyp 51 A在序列上保持野生型。ABC转运蛋白编码基因的过表达导致唑类耐药已成为念珠菌唑类耐药的主要途径，我们相信这将成为念珠菌耐药的一个关键特征。烟熏。本申请将集中于cdr 1B基因的贡献，因为该ABC转运蛋白的表达在唑类耐药分离株中升高，并且cdr 1B的缺失产生唑类敏感表型。我们将直接比较野生型和含有与临床相关耐药性相关的各种cyp 51 A突变的cdr 1B Δ细胞的唑类耐药表型。我们还将使用免疫学探针评估cyp 51 A的表达，以确定这些突变等位基因是否影响蛋白质水平; 在A中不可用烟熏。我们将使用黑斑羚转座子诱变进行正向遗传筛选，以鉴定调节cdr 1B表达的基因。总之，这些方法将为A.提供了ABC转运蛋白和cyp 51 A基因的耐药性贡献的直接评估。

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 α-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 cdr1BΔ 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.