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Analysis of novel adherence factors for initial infection in Enterohemorrhagic Escherichia coli O157Sakai

肠出血性大肠杆菌O157Sakai初次感染的新依从因素分析

基本信息

批准号：
13670263
负责人：
TATSUNO Ichiro
金额：
$ 2.75万
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：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670263/
关键词：
O157 Adherence LEE transcriptional regulator Adherence O157:H7

项目摘要

Adherence of enterohemorrhagic Escherichia coli (EHEC) to intestinal epithelium is essential for initiation of the infection including diarrhea, and for the adherence, expression of the genes of the locus for enterocyte effacement (LEE) is thought to be crucial. To identify genes involved in modulating the adherent capacity, a collection of an EHEC O157 : H7 strain (O157Sakai) mutagenized by mini-Tn5Km2 were screened for their ability to increase the number of microcolonies (MC) on Caco-2 cells, and eight hyper adherent mutants were isolated. Analysis of the mini-Tn5Km2-flanked DNA sequences indicated that one possessed the insertion within an O157 antigen gene cluster, the other within the yhiF gene, and the remaining 6 mutants had their insertions in the yhiE gene. yhiE and yhiF products share amino acid homology (23% identity) to each other and with the LuxR family known as transcriptional regulatory proteins. The mutant having the insertion within the O157 antigen gene cluster, but not the other seven mutants, did not express the O157 side chain as determined by agglutination test and immunoblotting with polyclonal O157-specific antiserum. Importantly, the other mutants showed enhanced type III secretion. Their related mRNAs of LEE, but not ler mRNA, were also increased as compared with those in the wild-type. Indeed, when we introduced an inframe deletion into the yhiE or yhiF gene in O157Sakai, the capacity of the resultant mutants to adhere to Caco-2 cells was greatly increased. When one of the yhiE insertion mutants was orally inoculated into ICR mice, the number of bacteria shed into feces by day 14 was greater than that for wild-type. These results suggest that yhiE and yhiF are involved in the adherence of O157Sakai to epithelial cells as negative regulators for the expression of the genes required for the type III secretion system.

肠出血性大肠杆菌（EHEC）对肠上皮的粘附对于包括腹泻在内的感染的起始是必不可少的，并且对于粘附，肠上皮细胞消失（LEE）位点的基因的表达被认为是至关重要的。为了鉴定参与调节粘附能力的基因，收集经mini-Tn 5 Km 2诱变的EHEC O 157：H7菌株（O 157 Sakai），筛选其增加Caco-2细胞上的小菌落（MC）数目的能力，并分离出8个超粘附突变体。对突变体的DNA序列分析表明，其中一个突变体插入O 157抗原基因簇，另一个突变体插入yhiF基因，其余6个突变体插入yhiE基因。yhiE和yhiF产物彼此共享氨基酸同源性（23%同一性），并且与称为转录调节蛋白的LuxR家族共享氨基酸同源性（23%同一性）。通过凝集试验和用多克隆O 157特异性抗血清进行的免疫印迹测定，在O 157抗原基因簇内具有插入的突变体不表达O 157侧链，而其它7个突变体不表达。重要的是，其他突变体显示出增强的III型分泌。与野生型相比，LEE的相关mRNA也有所增加，但Ler mRNA没有增加。事实上，当我们在O 157 Sakai中的yhiE或yhiF基因中引入框内缺失时，所得突变体粘附Caco-2细胞的能力大大增加。当yhiE插入突变体之一经口接种到ICR小鼠中时，到第14天脱落到粪便中的细菌数量大于野生型。这些结果表明，yhiE和yhiF参与了O 157 Sakai粘附上皮细胞作为III型分泌系统所需基因表达的负调节剂。