转录因子Upc2调控ERG11基因转录介导热带假丝酵母菌对唑类药物耐药机制的研究

During the past two or three decades, the morbidity and mortality caused by Candida tropicalis are growing obviously, and what's more, its increasing serious resistance to azole antifungals also becomes a great challenge to clinical treatment. To date, the mechanisms of azole resistance in C. tropicalis have not been completely explained. Based on our previous experiments, a close relationship between the expression level of ERG11 gene and the susceptibility of azole antifungals was discovered. Therefore, we tend to further investigate the role of Upc2, a transcriptional factor of ERG11 gene, in mediating transcription of ERG11 and azole resistance of C. tropicalis. First of all, some transcription-relative structures,like the transcript start site and core transcription area of ERG11 promoter will be studied. Secondly, we tend to figure out the binding site that is combined to Upc2 by EMSA and ChIP experiments. Finally, the changes of Upc2 in azole-resitant C. tropicalis, including its expression level and mutations in coding area were investigated, to further elucidate its influence on transcriptional control of ERG11 gene, as well as the mechanisms of azole resistance in C.tropicalis. This study is not only useful for further understanding how the azole resistance happens and progresses in C. tropicalis, but also contributes to finding new therpeutic targets for azole-resistant C. tropicalis.

近二三十年来，热带假丝酵母菌的感染率和致病率逐年增加，对唑类抗真菌药物的耐药情况也日益严重，给临床治疗带来很大难度。目前，有关热带假丝酵母菌对唑类药物的耐药机制尚未研究透彻。本课题组在前期研究中发现，ERG11基因的过度表达与热带假丝酵母菌对唑类药物的敏感性之间存在密切关系。因此，本课题组拟通过对转录因子Upc2介导ERG11转录调控的研究，进一步阐明热带假丝酵母菌对唑类药物耐药的机制。首先明确ERG11启动子上转录起始位和核心启动区等与转录调控相关的结构，然后通过EMSA和ChIP实验探究Upc2与ERG11启动子的结合位点，最后分别从Upc2的RNA表达水平和编码区的突变两方面验证其调控ERG11的转录并介导热带假丝酵母菌对唑类药物耐药的机制。该课题的研究不仅有望从更深层的角度阐明热带假丝酵母菌唑类耐药株的发生发展过程，也有利于寻获治疗热带假丝酵母菌唑类耐药株的新药物靶点。

近年来，热带假丝酵母菌的临床感染率和致病率逐年增加，对唑类抗真菌药物的耐药情况也日益严重，给临床治疗带来很大难度。在本课题中我们发现，热带假丝酵母菌唑类耐药株过度表达ERG11基因和其转录因子Upc2基因。通过5’-Full RACE方法，我们明确了ERG11基因的转录起始位点，为ERG11转录调控的研究奠定了基础。通过对UPC2基因编码区测序，我们发现了一个新的突变位点G392E，并证实其可以引起ERG11基因的过度表达，以及对氟康唑和伊曲康唑耐药。该突变位点的发现可成为日后临床唑类耐药防治的靶点。