影响病原性曲霉对唑类药物敏感性的新基因编码蛋白及其在耐药性中的作用机制

Azole-resistance in pathogenic Aspergillus spp., causing the poor prognosis and high mortality in aspergillosis patient, has been increased in recent years. Comprehensive study on resistant-mechanism of azole-resistance is particularly significant. Based on our previous research results in azole-resistance of Aspergillus fumigatus and on expounding the mechanism of voriconazole-resistance in Aspergillus flavus, especially on finding out that disruption of AFLA-216 gene in A. flavus and its homologue AFUA in A. fumigatus do increase antifungal susceptibility to triazole drugs, we hypothesize that AFLA-216 and AFUA gene as well as their encoding protein play the significant role in both azole-susceptibility and azole-resistance of Aspergillus spp.. In this proposal, the following issues will be conducted out: First, by using gene disruption and over-expression approaches, the contribution of AFLA-216 and AFUA gene to azole-susceptibility in Aspergillus spp. will be investigated; Second, by disrupting the AFLA-216 and AFUA gene in azole-resistant strains of both A. flavus and A. fumigatus, the mechanism reversing azole-resistance to azole-sensitivity in Aspergillus spp. will be studied; Third, by using Aspergillus spp. protein expression system, the recombinant protein encoded by AFLA-216 and AFUA gene will be expressed and purified, and the proteic structure of these recombinant proteins will be analyzed; in addition, the mechanism of action of these proteins as well as the encoding genes in influencing azole-susceptibility and azole-resistance will be further clarified by using digital gene expression Tag profiling procedure and protein-protein interaction strategies.

近年来，病原性曲霉对唑类药物的耐药性逐年增多并常引起患者治疗失败，深入研究其机制，具有重要意义。基于在病原性烟曲霉对唑类药物耐药性研究的原创性结果以及首先在国际上阐明黄曲霉对伏立康唑的耐药机制，尤其是在新近发现敲除黄曲霉AFLA-216及其烟曲霉同源基因AFUA可导致对三唑类药物高度敏感的基础上，我们推断AFLA-216和AFUA基因及其编码蛋白可显著影响病原性曲霉对三唑类药物的敏感性和耐药性。我们拟经本申请：一、以曲霉基因敲除和过表达等，研究AFLA-216和AFUA基因及其编码蛋白影响曲霉对三唑类药物敏感性的作用；二、敲除耐唑类药物的病原性曲霉的该基因，阐明其在逆转曲霉对唑类药物耐药性为敏感的作用；三、利用曲霉外源蛋白表达系统获得AFLA-216和AFUA蛋白并解析其结构，经表达谱测序、蛋白质相互作用及其对药物的影响等手段来揭示其功能，最终阐明影响曲霉对三唑类药物敏感性和耐药性的机制。

近年来，病原性曲霉对抗真菌药物的耐药性逐年增多并常引起患者治疗失败，开展病原性曲霉菌株药物敏感性监测，揭示临床菌株对常见抗真菌药物的敏感性，深入研究耐药临床菌株的耐药机制，并进一步开发耐药菌株检测方法，具有重要意义。因此，本课题组开展了如下工作：1）对新基因AFLA-216和AFUA进行了研究，结果显示黄曲霉AFLA-216和烟曲霉AFUA基因敲除不影响对常见抗真菌药物的敏感性；2）对研究过程中意外获得的一株对伏立康唑敏感性增高的烟曲霉的环化酶相关蛋白（Cyclase associated protein，CAP）基因敲除，证明CAP基因敲除可导致烟曲霉对三唑类药物敏感性增加、生长减慢、形态异常、应激的反应性增强、FK506和CAS的协同效应增强及毒力减弱；3）对发现的cyp51A基因中I454M/I455M突变进行了研究，结果证明cyp51A基因中I454M/I455M突变可以导致黄/烟曲霉对伏立康唑和艾沙康唑敏感性下降，揭示了曲霉的唑类药物新耐药机制；4）基于病原性曲霉菌株药物敏感性监测，初步制定了我国烟曲霉和黄曲霉的抗真菌药物流行病学临界值（Epidemiologic cutoff values，ECVs），揭示了我国曲霉菌株药物敏感性的特点，对于评价新型药物、指导临床用药具有良好的应用前景；5）基于唑类药物耐药机制研究，建立了检测唑类耐药烟曲霉菌株的Real-time PCR方法，可从敏感菌株与极低比例（1%）耐药菌株的混合感染中检测出耐药菌株的存在，该方法在临床唑类耐药菌株的检测上具有良好的应用前景；6）鉴定出响应唑类药物应激的转录因子ADS-1，证明了ADS-1通过调节cdr4和erg11基因表达水平来响应酮康唑应激，并进一步调节对三唑类药物耐药性的产生；7）对临床分离得到的耐两性霉素B（Amphotericin B，AMB）的黄曲霉菌株进行了研究，发现了耐AMB的黄曲霉菌株生长减慢、对氧化应激更敏感、毒力更弱，同时证明了内源性ROS水平增高是其对三唑类和棘白菌素类药物过度敏感的原因，结果对于理解包括病原性黄曲霉在内的病原真菌中，AMB的作用机制和耐药机制具有重要意义。