Hodson Carla, Yang Ji, Hocking Dianna M, Azzopardi Kristy, Chen Qianyu, Holien Jessica K, Parker Michael W, Tauschek Marija, Robins-Browne Roy M
Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, The University of Melbourne, ParkvilleVIC, Australia.
Murdoch Childrens Research Institute, The Royal Children's Hospital, ParkvilleVIC, Australia.
Front Microbiol. 2017 Aug 11;8:1525. doi: 10.3389/fmicb.2017.01525. eCollection 2017.
Enterotoxigenic (ETEC) is the most common bacterial cause of diarrhea in children in developing countries, as well as in travelers to these countries. To cause disease, ETEC needs to produce a series of virulence proteins including enterotoxins, colonization factors and secretion pathways, which enable this pathogen to colonize the human small intestine and deliver enterotoxins to epithelial cells. Previously, a number of studies have demonstrated that CfaD, an AraC-like transcriptional regulator, plays a key role in virulence gene expression by ETEC. In this study, we carried out a transcriptomic analysis of ETEC strain, H10407, grown under different conditions, and determined the complete set of genes that are regulated by CfaD. In this way, we identified a number of new target genes, including , , , , , and ETEC_3214, whose expression is strongly activated by CfaD. Using promoter- reporters, primer extension and electrophoretic mobility shift assays, we characterized the CfaD-mediated activation of several selected target promoters. We also showed that the gut-associated environmental signal, sodium bicarbonate, stimulates CfaD-mediated upregulation of its virulence target operons. Finally, we screened a commercial small molecule library and identified a compound (CH-1) that specifically inhibited the regulatory function of CfaD, and by 2-D analoging, we identified a second inhibitor (CH-2) with greater potency.
产肠毒素大肠杆菌（ETEC）是发展中国家儿童腹泻以及前往这些国家的旅行者腹泻最常见的细菌病因。为了引发疾病，ETEC需要产生一系列毒力蛋白，包括肠毒素、定植因子和分泌途径，这些使得该病原体能够在人类小肠中定植并将肠毒素传递给上皮细胞。此前，多项研究表明，CfaD，一种类AraC转录调节因子，在ETEC毒力基因表达中起关键作用。在本研究中，我们对在不同条件下生长的ETEC菌株H10407进行了转录组分析，并确定了受CfaD调控的完整基因集。通过这种方式，我们鉴定了许多新的靶基因，包括、、、、、和ETEC_3214，其表达被CfaD强烈激活。使用启动子报告基因、引物延伸和电泳迁移率变动分析，我们对几个选定靶启动子的CfaD介导的激活进行了表征。我们还表明，肠道相关环境信号碳酸氢钠刺激CfaD介导的其毒力靶操纵子的上调。最后，我们筛选了一个商业小分子文库，鉴定了一种特异性抑制CfaD调节功能的化合物（CH-1），并且通过二维类似物分析，我们鉴定了一种效力更强的第二种抑制剂（CH-2）。
