The role of Aspergillus fumigatus SmiA transcription factor in the miltefosine resistance. The role of Aspergillus fumigatus SmiA transcription factor in the miltefosine resistance

By searching for new drugs against fungal pathogens, we found that miltefosine is active against Aspergillus fumigatus clinical isolates. A library of transcription factors (TF) null mutants was then challenged with this drug and we discovered a novel TF that confers resistance to miltefonise, named here SmiA. By using ChIP-seq, we searched for SmiA targets upon miltefosine treatment. Overall design: The SmiA-tagged strain was grown for 16 hours at 37oC in minimum liquid media at 220 rpm and then transferred to RPMI added with 12.5 ug/ml of miltefosine for 30 minutes (37oC 220 rpm). After crosslinking, the cells were washed and frozen for chromatin extraction and immunoprecipitation.

为筛选抗真菌病原体的新型药物，我们发现米替福新（Miltefosine）对烟曲霉（Aspergillus fumigatus）临床分离株具有抗菌活性。我们随后用该药物处理转录因子（Transcription Factor, TF）敲除突变体库，发现了一个全新的、可赋予菌株对米替福新抗性的转录因子，本文将其命名为SmiA。我们采用染色质免疫共沉淀测序（Chromatin Immunoprecipitation sequencing, ChIP-seq）技术，探究了米替福新处理条件下SmiA的靶标基因。
实验整体设计：将带有SmiA标签的菌株在37℃、220 rpm转速的基础液体培养基中培养16小时，随后转移至添加了12.5 μg/ml米替福新的RPMI培养基中，于37℃、220 rpm条件下处理30分钟。完成交联后，收集细胞并洗涤，冻存以备后续染色质提取与免疫共沉淀实验使用。