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Isolation and characterization of novel virulence factors of enteropathogenic Escherichia coli

肠道病原性大肠杆菌新型毒力因子的分离和表征

基本信息

批准号：
10670250
负责人：
TOBE Toru
金额：
$ 2.05万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

The knew findings obtained from this project are follows ;1. The bfpTVW operon of enteropathogenic Escherichia coli (EPEC) is required for the transcriptional activation of the bfp operon, which encodes the major subunit and assembly machinery of bundle-forming pili (BFP). An immobilized T7-tagged BfpT fusion protein that binds specifically to upstream promoter sequences of bfpA and eae was used to 'fish out' from a promoter library other EPEC chromosomal fragments that are bound by the BfpT protein. One of promoters, thus selected, was positively regulated by bfpTVW and is not present in non-virulent Ecoli strains. This promoter was found to be located in a 4.9 kb EPEC specific sequence and upstream of orfl, which was revealed to encode a chaperone-like protein involving expression of BFP. Hence, orfl was designated trcA (bfpT-regulated chaperone-like protein gene) and the EPEC-specific region was LIM (Locus for improving microcolony formation).2. The complete nucleotide sequence and organization of the EAF plasmid of EPEC, strain B171 (Olll:NM), were determined. Besides the bfp and bfpTVW operons, the 69 kb EAF plasmid, henceforth designated pB171, contains potential virulence-associated genes. Of the newly identified ORFs, two ORFs which comprise a single operon were found that would encode proteins with high similarity to a C-terminal region of ToxB whose coding sequence is located on pO157, a large plasmid harbored by enterohemorrhagic E coli. Another ORF showed high similarity with trcA of EPEC. Two sites were found to be putative replication regions, one of that is similar to RepFIIA of p307 or F and the other to RepFIB of NR1. In addition to these, a third region was identified that contains plasmid maintenance genes. Insertion elements were scattered throughout the plasmid indicating the mosaic nature of the EAF plasmid and suggesting evolutionary events by which virulence genes may have been obtained.

从本项目中获得的已知发现如下：1。肠致病性大肠杆菌（EPEC）的bfpTVW操纵子是bfp操纵子转录激活所必需的，bfp操纵子编码纤毛形成皮利（BFP）的主要亚基和组装机制。使用特异性结合bfpA和eae的上游启动子序列的固定化T7标记的BfpT融合蛋白从启动子文库中“钓出”由BfpT蛋白结合的其它EPEC染色体片段。由此选择的启动子之一由bfpTVW正调控，并且不存在于非毒性大肠杆菌菌株中。发现该启动子位于4.9kb EPEC特异性序列和orfl的上游，其被揭示编码涉及BFP表达的伴侣样蛋白。因此，orf 1被命名为trcA（bfpT-regulated chaperone-like protein gene），EPEC特异性区域被命名为LIM（Locus for improving microcolony formation）.测定了EPEC菌株B171（Olll：NM）的EAF质粒的完整核苷酸序列和结构。除了bfp和bfpTVW操纵子之外，69 kb EAF质粒（此后命名为pB 171）含有潜在的毒力相关基因。在新鉴定的ORF中，发现了两个包含单个操纵子的ORF，其编码的蛋白质与ToxB的C-末端区域具有高度相似性，ToxB的编码序列位于肠出血性大肠杆菌所携带的大质粒pO 157上。另一个ORF与EPEC的trcA高度相似。发现两个位点是推定的复制区，其中一个类似于p307或F的RepFIIA，另一个类似于NR 1的RepFIB。除此之外，还鉴定了含有质粒维持基因的第三区域。插入元件分散在整个质粒中，表明EAF质粒的镶嵌性质，并表明可能已获得毒力基因的进化事件。