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

肠出血性大肠杆菌（EHEC）粘附在肠上皮上对于包括腹泻在内的感染的开始是必不可少的，而对于粘附，肠细胞清除（LEE）位点的基因表达被认为是至关重要的。为了确定调节粘附能力的基因，我们筛选了一株经mini-Tn5Km2诱变的EHEC O157: H7菌株（O157Sakai），以检测其增加Caco-2细胞上微菌落（MC）数量的能力，并分离出8个高粘附突变体。对mini- tn5km2侧的DNA序列分析表明，1个突变体插入O157抗原基因簇，1个突变体插入yhiF基因簇，其余6个突变体插入yhiE基因簇。yhiE和yhiF产品彼此具有氨基酸同源性（23%同源性），并与转录调节蛋白LuxR家族具有同源性。经凝集试验和O157特异性抗血清免疫印迹检测，插入O157抗原基因簇的突变体不表达O157侧链，而其他7个突变体不表达O157侧链。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，它们的LEE相关mRNA增加，而ler mRNA未增加。事实上，当我们在O157Sakai的yhiE或yhiF基因中引入帧内缺失时，所产生的突变体粘附Caco-2细胞的能力大大增加。将其中一种yhiE插入突变体口服接种到ICR小鼠体内，第14天排入粪便中的细菌数量多于野生型。这些结果表明，yhiE和yhiF作为III型分泌系统所需基因表达的负调节因子参与O157Sakai粘附上皮细胞。

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.