蛋白质水平的烟曲霉cyp51A变异与唑类耐药分子机制研究

Aspergillus fumigatus is the most dominant pathogen of aspergillosis. Azoles are currently the most widely used antifungal agents. However, azole-resistant strains of A. fumigatus have increased during past years. The emergency of azole-resistant clinical strains of A. fumigatus is mainly associated with mutations in cyp51A gene. Previous studies indicated that the mechanism of azole resistance of Aspergillus is target-dependent. Therefore, the impact of the mutations on the structure and function of CYP51A protein is a key to clarify the mechanism of drug resistance. In our previous study, we obtained 4 A. fumigatus azole -resistant isolates by screening our clinical A. fumigatus collections. cyp51A genes of the azole -resistant isolates were sequenced and some mutations were identified. In addition, we established a new model of A. fumigatus CYP51A protein structure by homology modeling. We intend to analyze the structural characters of the cyp51A mutations based on this structure model. Meanwhile, we intend to predict the impact of those mutations on the binding of azoles to CYP51A protein. Furthermore, we obtained the in vitro expressed CYP51A proteins. Binding affinity of the wild-type and the mutant CYP51A proteins with azoles or with the substrate of CYP51A will be tested by Biacore technology, respectively. Taken together, we are trying to analyze the mechanism of azole resistance of A. fumigatus due to cyp51A mutations, on a level of protein structural and functional basis.

烟曲霉是最常见的致病曲霉，唑类是目前使用最广泛的抗真菌药物，但烟曲霉的唑类耐药株却逐年增加。临床烟曲霉唑类耐药株主要由cyp51A基因突变引起，研究提示耐药机制为靶位依赖性，但目前的研究多限于基因水平。因此，深入研究突变对CYP51A蛋白结构和功能的影响可能是阐明烟曲霉抗唑类药物的耐药机制的关键。在前期研究中，我们筛选得到了4株烟曲霉唑类临床耐药株，对cyp51A测序并分析了突变位点，并用同源建模方法建立了新的烟曲霉CYP51A蛋白结构模型。拟通过该模型分析各个突变在CYP51A蛋白结构上的意义，并预测这些突变对CYP51A蛋白与唑类结合的影响。在成功表达了烟曲霉CYP51A蛋白基础上，拟用Biacore技术检测野生型和突变型CYP51A蛋白与唑类结合的差异，以及突变对CYP51A蛋白结合底物功能的影响。目的是在蛋白质结构和功能层面，解析烟曲霉cyp51A突变导致唑类耐药的分子机制。

烟曲霉是最常见的致病曲霉，唑类是目前使用最广泛的抗真菌药物，但烟曲霉的唑类耐药株却逐年增加。临床烟曲霉唑类耐药株主要由cyp51A基因突变引起，研究提示耐药机制为靶位依赖性，但目前的研究多限于基因水平。因此，深入研究突变对CYP51A蛋白结构和功能的影响可能是阐明烟曲霉抗唑类药物的耐药机制的关键。在前期研究中，用同源建模方法建立了新的烟曲霉CYP51A蛋白结构模型。拟通过该模型分析各个突变在CYP51A蛋白结构上的意义，并预测这些突变对CYP51A蛋白与唑类结合的影响。在成功表达了烟曲霉CYP51A蛋白基础上，拟用Biacore技术检测野生型和突变型CYP51A蛋白与唑类结合的差异，以及突变对CYP51A蛋白结合底物功能的影响。通过临床和实验室研究，我们获得以下成果：①收集侵袭性曲霉病菌株120余株，筛选出5株多重唑类耐药株，国内外首次发现了G432A突变，发表SCI论著1篇报道；②根据酿酒酵母等CYP51蛋白晶体使用MOE构建烟曲霉CYP51A结构模型，模拟烟曲霉CYP51A蛋白与唑类药物结合，总结不同位点与唑类药物耐药的关系，分析临床突变株唑类耐药谱的内在机理，发表SCI论著1篇；③ 烟曲霉CYP51A蛋白体外表达，采用Biacore SPR技术，检测野生型CYP51A重组蛋白与与伊曲康唑的结合，但结果无差异；④ 烟曲霉耐药快速检测专利申请1项，就TR34/L98H/S297T/F495I进行快速的分子生物学检测；⑤ 动态模拟hgdeghog 途径中SMO与唑类药物的结合发表SCI论著1篇；⑥ 2015年国际真菌学会（ISHAM）做相关poster报告。