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

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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.