A new pathway for azole resistance in Aspergillus fumigatus

描述(由申请人提供)：由唑类抗药性烟曲霉菌引起的侵袭性曲霉病具有令人震惊的12%的存活率，这使这成为一个具有严重意义的临床问题。对唑类抗药性烟曲霉菌株的早期研究表明，对唑类抗药性是一种相对罕见的现象，耐药性的遗传基础通常是由于编码唑靶蛋白羊毛甾醇α-14去甲基酶的基因(Cyp51a)的改变。荷兰一个研究小组的广泛研究提供了令人信服的证据，表明在该国，大多数对唑耐药的烟曲霉菌分离株包含一个单一的复合突变，该突变由cyp51a启动子区域(TR34)的重复区域和编码序列的变化改变蛋白质的氨基酸序列(L98H)组成。然而，英国一个研究小组最近的研究表明，cyp51a基因的变化不太可能解释烟曲霉菌对唑类药物的所有耐药性。50%以上的唑类耐药菌株 来自英国的患者具有野生型cyp51a，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的三磷酸腺苷结合盒(ABC)转运子编码基因，该基因与几种患者分离株的唑类耐药性有关。高度耐唑临床分离株中cdr1B的表达升高 Cyp51a序列保持野生型。ABC转运蛋白编码基因的过度表达导致了对唑类的耐药，这是念珠菌耐药的主要途径，我们相信这将成为烟曲霉菌耐药的一个关键特征。这一应用将集中在cdr1B基因的贡献上，因为这种ABC转运蛋白在唑类耐药分离株中的表达增加，并且cdr1B的缺失产生了一种唑类敏感的表型。我们将直接比较野生型和cdr1BΔ细胞中含有与临床相关耐药相关的各种cyp51a突变的唑类耐药表型。我们还将使用免疫探针评估cyp51a的表达，以确定这些突变的等位基因是否影响蛋白质水平；目前的信息是 在烟曲霉菌中不可用。我们将使用黑斑羚转座子突变进行正向遗传筛选，以确定调节cdr1B表达的基因。综上所述，这些方法将为了解烟曲霉唑耐药性的分子基础提供重要的新信息，并为直接评估ABC转运蛋白和cyp51a基因的耐药性贡献提供依据。

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.