Flo8调控 CDR1/CDR2基因表达在白念珠菌唑类药物耐药中作用机制研究

Candida albicans, the most common pathogen of causing invasive fungal infections, is characterized by high morbidity and mortality. What’s more, it is prone to developing resistance to azole antifungal drugs. In our previous studies, the high expression of efflux pump encoding genes CDR1 and CDR2 were detected in azole-resistant C.albicans isolates. However, the mechanism was not completely revealed. The detection of SILAC technology in proteomics indicated that the Flo8 expression in the resistant strain of azole was down-regulated than that of susceptible ones. On this basis, we constructed the flo8/flo8 knockout strain and found that it exhibited high resistance to azole drugs and high level expression of CDR1 and CDR2 gene. The interaction between Flo8 and genes CDR1/CDR2 was verified by ChIP experiment. Based on aforementioned results, we speculate that Flo8, a transcription factor, may regulate the expression of the CDR1 and CDR2 conferring to its azole resistance. This study aims to clarify the regulatory role of Flo8 on the activity of CDR1 and CDR2 promoter by validating the correlation between Flo8 expression and the expression of CDR1/CDR2 relevant to azole resistance in C.albicans strains, and finally we hope the mechanism of resistance to azole drugs in C. albicans would be revealed.

白念珠菌为侵袭性真菌感染最常见病原体，具有高感染率和高致死率特点，同时对唑类抗真菌药物易产生耐药。本课题组前期发现白念珠菌唑类耐药株中外排泵编码基因CDR1和CDR2高表达，但其机制未完全明确。蛋白质组学SILAC技术的检测提示唑类耐药株中Flo8表达较敏感株下调；在此基础上，我们又制备了flo8/flo8敲除株，发现其对唑类药物高度耐药并伴有CDR1和CDR2的高表达；进一步ChIP实验初步证实Flo8和CDR1/CDR2启动子区间有相互作用。基于已有的Flo8作为转录因子的报道和前期实验结果，我们推测Flo8可能作为转录因子调控CDR1和CDR2的表达，其改变与唑类耐药有关。本研究将明确Flo8对CDR1/CDR2启动子活性的调节作用，观察Flo8表达水平改变与CDR1/CDR2表达量及白念珠菌唑类药物耐药程度间的关联，从而帮助阐明白念珠菌唑类药物耐药的可能机制。

近年来，侵袭性真菌的感染率不断上升。白念珠菌是引起深部真菌感染最常见的病原体，具有高感染率和高致死率的特点。同时其对唑类抗真菌药物易产生耐药，增加了临床治疗的难度。. 本课题组以工程菌SN152为野生株，采用融合PCR结合同源重组技术构建了FLO8双拷贝基因敲除株、回复株和过表达株，并分析了其对三唑类药物的敏感性。发现FLO8基因和白念珠菌对唑类药物敏感性有关系：FLO8基因缺失会导致白念珠菌对氟康唑等唑类药物耐药；重新导入高表达FLO8基因，菌株重新变得对唑类药物敏感。进一步通过实时逆转录PCR和罗丹明6G外排实验，我们发现FLO8基因缺失会使白念珠菌ABC转运蛋白基因CDR1、CDR2表达升高，继而使菌株对氟康唑、伊曲康唑和伏立康唑等唑类药物的外排增加，最终导致菌株对唑类药物耐药；导入高表达FLO8基因后，菌株恢复对唑类药物的敏感性。通过Western-blot实验进一步验证了Flo8 低表达是通过上调 CDR1、CDR2表达而导致白念珠菌唑类耐药的。同时，本课题组通过对临床分离的白念珠菌FLO8基因突变位点的筛查，构建白念珠菌转录因子Flo8 G723R、T751D突变株，我们发现白念珠菌转录因子Flo8 G723R、T751D突变会增强毒力因子表达。此外，课题组进一步深入探究，得出转录因子Flo8与光滑念珠菌毒力之间存在关系。. 本研究从调控作用角度来验证Flo8与白念株菌CDR1、CDR2的转录调控以及唑类耐药之间的关系。同时也探究到转录因子Flo8与光滑念珠菌毒力之间存在关系，为临床治疗侵袭性真菌病感染寻找新的药物作用靶点提供有力的实验基础和理论依据。