He Xiaoxiao, Li Shengnan, Kaminskyj Susan G W
Department of Biology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
Eukaryot Cell. 2014 Feb;13(2):288-94. doi: 10.1128/EC.00334-13. Epub 2013 Dec 20.
Systemic fungal infections contribute to at least 10% of deaths in hospital settings. Most antifungal drugs target ergosterol (polyenes) or its biosynthetic pathway (azoles and allylamines), or beta-glucan synthesis (echinocandins). Antifungal drugs that target proteins are prone to the emergence of resistant strains. Identification of genes whose mutations lead to targeted resistance can provide new information on those pathways. We used Aspergillus nidulans as a model system to exploit its tractable sexual cycle and calcofluor white as a model antifungal agent to cross-reference our results with other studies. Within 2 weeks from inoculation on sublethal doses of calcofluor white, we isolated 24 A. nidulans adaptive strains from sectoring colonies. Meiotic analysis showed that these strains had single-gene mutations. In each case, the resistance was specific to calcofluor white, since there was no cross-resistance to caspofungin (echinocandin). Mutation sites were identified in two mutants by next-generation sequencing. These were confirmed by reengineering the mutation in a wild-type strain using a gene replacement strategy. One of these mutated genes was related to cell wall synthesis, and the other one was related to drug metabolism. Our strategy has wide application for many fungal species, for antifungal compounds used in agriculture as well as health care, and potentially during protracted drug therapy once drug resistance arises. We suggest that our strategy will be useful for keeping ahead in the drug resistance arms race.
在医院环境中，系统性真菌感染导致的死亡人数至少占10%。大多数抗真菌药物靶向麦角固醇（多烯类）或其生物合成途径（唑类和烯丙胺类），或β-葡聚糖合成（棘白菌素类）。靶向蛋白质的抗真菌药物容易出现耐药菌株。鉴定其突变导致靶向耐药的基因可为这些途径提供新信息。我们以构巢曲霉为模型系统，利用其易于处理的有性生殖周期，并以荧光增白剂作为模型抗真菌剂，将我们的结果与其他研究进行交叉参考。在接种亚致死剂量荧光增白剂后的2周内，我们从扇形菌落中分离出24株构巢曲霉适应性菌株。减数分裂分析表明，这些菌株具有单基因突变。在每种情况下，耐药性都对荧光增白剂具有特异性，因为对卡泊芬净（棘白菌素类）没有交叉耐药性。通过下一代测序在两个突变体中鉴定出突变位点。使用基因替换策略在野生型菌株中重新构建突变，证实了这些位点。其中一个突变基因与细胞壁合成有关，另一个与药物代谢有关。我们的策略广泛适用于许多真菌物种、用于农业以及医疗保健的抗真菌化合物，并且在耐药性出现后的长期药物治疗中可能也适用。我们认为我们的策略将有助于在耐药性军备竞赛中保持领先。
