烟曲霉FADB基因参与菌株耐药的潜在机理

FAD结合的氧化还原酶编码基因FADB (GenBank ID:Afu4g14630)的编码产物在烟曲霉中为一种与FAD结合的氧化还原酶，可能参与真菌的呼吸。为了探究其具体功能，本研究通过克隆烟曲霉FADB基因，构建烟曲霉FADB基因的敲除株，了解该基因对烟曲霉药物敏感性、渗透压、氧化压力物质敏感性的作用机理。采用高通量基因替换方法构建出FADB基因的敲除盒，筛选出符合的烟曲霉FADB敲除株ΔFADB。观察该突变株与野生株AF293对药物敏感性、氧化压力、渗透压物质敏感性的变化。利用E-test法检测ΔFADB对3种唑类药物(泊沙康唑、伊曲康唑、伏立康唑)的敏感性，结果证实ΔFADB对泊沙康唑的敏感性明显提高。在渗透压和氧化压力的培养试验中，与野生株AF293相比较，ΔFADB对氧化剂D-山梨醇和甲萘醌更敏感。加入泊沙康唑后，ΔFADB比野生株AF293产生的活性氧更多。说明烟曲霉FADB基因参与泊沙康唑抗烟曲霉机制并起到一定作用，该基因诱导的氧化应激反应可能是抗真菌药物杀伤真菌的作用机制之一，本研究为烟曲霉唑类耐药机制的研究提供了新的理论基础。

Potential contribution of FADB to drug resistance in Aspergillus fumigatus

Coding product of FADB gene in Aspergillus fumigatus is FAD binding oxidoreductase, which may be involved in the respiration of fungi. In this study, FADB gene knockout strain of A. fumigatus is constructed by cloning FADB gene, and mechanism of FADB gene responsible for the changes of drug sensitivity, osmotic pressure and oxidative stress sensitivity of A. fumigatus is investigated. The knockout box of FADB gene is constructed by high throughput gene replacement method, and the corresponding FADB knockout strain ΔFADB of A. fumigatus is selected. The changes of drug sensitivity, oxidative pressure and osmotic substance sensitivity of the mutant and wild type AF293 are observed. The results of detecting the sensitivity of ΔFADB to three kinds of azole by E-test prove that the sensitivity of ΔFADB to posaconazole is increased. In the cultivating tests under osmotic and oxidative pressure, ΔFADB was more sensitive to the oxidants D-sorbitol and menadione than the wild type AF293. ΔFADB produced more reactive oxygen species than wild AF293 when posaconazole was added. The FADB gene of A. fumigatus might play a role in the antifungal mechanism of posaconazole, and the induction of oxidative stress may be one of the mechanisms of antifungal drugs against fungi. The results of this study suggest that FADB might involve in azole resistance in A. fumigatus, and further mechanistic investigations is required in the future.