Isolation and characterization of novel virulence factors of enteropathogenic Escherichia coli

已知的研究结果如下：1.致病性大肠杆菌(EPEC)的bfpTVW操纵子是转录激活BFP操纵子所必需的，该操纵子编码形成菌毛(BFP)的主要亚基和组装机制。一种固定的T7标记的BfpT融合蛋白专门与bfpA和EAE的上游启动子序列结合，用于从启动子文库中筛选出与BfpT蛋白结合的其他EPEC染色体片段。这样选择的一个启动子受到bfpTVW的正调控，在无毒株中不存在。该启动子位于4.9kb的EPEC特异性序列中，位于orf1的上游，编码一个与BFP表达有关的伴侣样蛋白。因此，orf1被命名为trcA(bfpT调节的伴侣样蛋白基因)，EPEC特异性区域为LIM(促进微集落形成的基因)。测定了EPEC菌株B171(Ol11：NM)的EAF质粒的全序列和组织结构。除了bfp和bfpTVW操纵子外，69 kb的EAF质粒，此后命名为pB171，包含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，它们编码的蛋白质与ToxB的C末端区域高度相似，其编码序列位于肠出血性大肠杆菌携带的大质粒pO157上。另一个ORF与EPEC的trcA有很高的相似性。发现两个可能的复制区，其中一个与p307或F的RepFIIA相似，另一个与NR1的RepFIB相似。除此之外，还发现了第三个含有质粒维持基因的区域。插入元件散布在整个质粒中，表明EAF质粒的嵌合体性质，并表明可能已经获得了毒力基因的进化事件。

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.