烟曲霉唑类药物耐药突变体的筛选及其发生耐药的分子机制研究

Drug resistance of Aspergillus is a common problem in the clinical fungal infectious diseases. However, our knowledge of molecular mechanism of emergence of azole drug resistance in Aspergillus is rather limited. In vitro experiment, We observed small amounts of adaptive strains to ITZ after the clinical ITZ-sensitive Aspergillus fumigatus isolates inoculated on the medium with the high concentrations of ITZ, suggesting that drug resistance mutations exist in the nature. Following this idea, we performed the inducing experiment with the AF1160 strain spores. Eventually, a total of 92 adaptive strains were generated in our experiment and all of them were stably resistant to 16 μg/ml ITZ after re-streaking. After azole drug target cyp51A gene sequence analysis, we found only one adaptive strains was due to the cyp51A mutation which lead to targeted resistance; a G728A mutation as detected in AFUB_065450 gene in another adaptive strains based on single nucleotide polymorphism (SNP). Further, the goal of this study was to confirm the relation of AFUB_065450 gene and azole resistance by re-engineering the mutation in a wild type train using a gene replacement strategy, and verify the its function in the azole resistance in the animal model. Meanwhile, using SNP based on next generation sequencing to fast identify the mutation sites among 5-10 strains from the other adaptive strains and new genes associated with azole resistance. We will clarify the molecular mechanism of emergence of azoles resistance in Aspergillus fumigatus, providing great help in finding new targets of antifungal agents and novel drug therapies for fungal infetions.

病原菌曲霉耐药是临床治疗真菌感染面临的常见问题，但对曲霉耐药发生的分子机理知之甚少。前期体外实验发现伊曲康唑（ITZ）敏感的临床烟曲霉菌株在高浓度ITZ下有少量存活菌株，提示自然界中存在少量耐药个体。鉴于此，本项目通过高浓度伊曲康唑多次诱导，从临床烟曲霉菌株AF1160中分离到了92株对16 μg/ml ITZ稳定耐受的耐药菌株。药靶基因cyp51A测序结果显示仅一株菌是由已证实的cyp51A点突变导致耐药；另外一株菌经单核苷酸多态性（SNP）分析显示AFUB_065450基因发生点突变。因此，本课题将进一步用基因替换等分子手段鉴定AFUB_065450基因和耐药发生的相关性，并结合动物模型验证其在体内参与耐药的功能。同时选择5-10个其他耐药菌株检测SNP位点，并体外鉴定导致耐药发生的新基因。以期阐明曲霉唑类药物耐药发生分子机制，为开发新药物靶点和防治曲霉感染提供新思路。

病原菌曲霉耐药是临床治疗真菌感染面临的常见问题，但对曲霉耐药发生的分子机理知之甚少。前期体外实验发现伊曲康唑（ITZ）敏感的临床烟曲霉菌株在高浓度ITZ下有少量存活菌株，提示自然界中存在少量耐药个体。鉴于此，本项目通过高浓度伊曲康唑多次诱导，从临床烟曲霉菌株AF1160中分离到了92株对16 μg/ml ITZ稳定耐受的耐药菌株。药靶基因cyp51A测序结果显示仅一株菌是由已证实的cyp51A点突变导致耐药；另外一株菌经单核苷酸多态性（SNP）分析显示AFUB_065450基因发生点突变。因此，本课题将进一步鉴定AFUB_065450（Afcox10）基因和耐药发生的相关性，以及体外鉴定导致耐药发生的其它基因。经过本项目三年科学研究工作的开展，已经圆满完成了项目原计划的主要研究内容，特别是在Afcox10和耐药发生相关性以及它的耐药机制方面取得了重要的进展。同时意外发现在曲霉Afcyp51A基因中存在未证实和耐药发生相关的突变位点。通过本项目的开展，我们证实了未报道的基因Afcox10和曲霉耐药发生密切相关，其R243Q突变导致烟曲霉对伊曲康唑，伏立康唑抗真菌药的耐受性增加。主要是通过降低曲霉菌株胞内药物积累量这一途径使曲霉发生耐药。另外，我们通过体内体外实验也证实了曲霉Afcyp51A基因的新突变类型V436A, Y433N和曲霉唑类药物耐药发生也密切相关。这些研究成果揭示了和曲霉唑类耐药发生密切相关的新基因和新突变，为开发新药物靶点和防治曲霉感染提供新思路。