Meneau Isabelle, Coste Alix T, Sanglard Dominique
Institute of Microbiology, University of Lausanne and University Hospital Center, CH-1011 Lausanne.
Institute of Microbiology, University of Lausanne and University Hospital Center, CH-1011 Lausanne
Med Mycol. 2016 Aug 1;54(6):616-27. doi: 10.1093/mmy/myw005. Epub 2016 Mar 1.
Aspergillus fumigatus can cause severe fatal invasive aspergillosis in immunocompromised patients but is also found in the environment. A. fumigatus infections can be treated with antifungals agents among which azole and echinocandins. Resistance to the class of azoles has been reported not only from patient samples but also from environmental samples. Azole resistance mechanisms involve for most isolates alterations at the site of the azole target (cyp51A); however, a substantial number of isolates can also exhibit non-cyp51A-mediated mechanisms.We aimed here to identify novel A. fumigatus genes involved in azole resistance. For this purpose, we designed a functional complementation system of A. fumigatus cDNAs expressed in a Saccharomyces cerevisiae isolate lacking the ATP Binding Cassette (ABC) transporter PDR5 and that was therefore more azole-susceptible than the parent wild type. Several genes were recovered including two distinct ABC transporters (atrF, atrI) and a Major Facilitator transporter (mdrA), from which atrI (Afu3g07300) and mdrA (Afu1g13800) were not yet described. atrI mediated resistance to itraconazole and voriconazole, while atrF only to voriconazole in S. cerevisiae Gene inactivation of each transporter in A. fumigatus indicated that the transporters were involved in the basal level of azole susceptibility. The expression of the transporters was addressed in clinical and environmental isolates with several azole resistance profiles. Our results show that atrI and mdrA tended to be expressed at higher levels than atrF in normal growth conditions. atrF was upregulated in 2/4 of azole-resistant environmental isolates and was the only gene with a significant association between transporter expression and azole resistance. In conclusion, this work showed the potential of complementation to identify functional transporters. The identified transporters were suggested to participate in azole resistance of A. fumigatus; however, this hypothesis will need further approaches to be verified.
烟曲霉可在免疫功能低下的患者中引起严重的致命性侵袭性曲霉病，但在环境中也有发现。烟曲霉感染可用抗真菌药物治疗，其中包括唑类和棘白菌素类。不仅在患者样本中，而且在环境样本中都已报道了对唑类的耐药性。唑类耐药机制在大多数分离株中涉及唑类靶点（cyp51A）位点的改变；然而，相当数量的分离株也可表现出非cyp51A介导的机制。我们的目的是鉴定参与唑类耐药的新型烟曲霉基因。为此，我们设计了一个烟曲霉cDNA的功能互补系统，该系统在缺乏ATP结合盒（ABC）转运蛋白PDR5的酿酒酵母分离株中表达，因此比亲本野生型对唑类更敏感。回收了几个基因，包括两个不同的ABC转运蛋白（atrF、atrI）和一个主要易化子转运蛋白（mdrA），其中atrI（Afu3g07300）和mdrA（Afu1g13800）尚未见报道。atrI介导对伊曲康唑和伏立康唑的耐药，而atrF仅介导酿酒酵母中对伏立康唑的耐药。烟曲霉中每个转运蛋白的基因失活表明这些转运蛋白参与了唑类敏感性的基础水平。在具有几种唑类耐药谱的临床和环境分离株中研究了转运蛋白的表达。我们的结果表明，在正常生长条件下，atrI和mdrA的表达水平往往高于atrF。atrF在2/4的唑类耐药环境分离株中上调，并且是转运蛋白表达与唑类耐药之间具有显著关联的唯一基因。总之，这项工作显示了互补法在鉴定功能性转运蛋白方面的潜力。已鉴定的转运蛋白被认为参与了烟曲霉的唑类耐药；然而，这一假设需要进一步的研究方法来验证。
