Increased Adherence to Caco-2 Cells Caused by Disruption of the yhiE and yhiF Genes in Enterohemorrhagic Escherichia coli O157:H7

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Abstract
Adherence of enterohemorrhagic Escherichia coli (EHEC) to intestinal epithelium is essential for initiation of infections, including diarrhea, and expression of the genes of the locus of enterocyte effacement (LEE) is thought to be crucial for adherence. To identify genes involved in modulating the adherent capacity, bacteria collected from 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 mutants with increased adherence were isolated. Analysis of the mini-Tn5Km2-flanked DNA sequences indicated that one possessed the insertion within an O157 antigen gene cluster, another possessed the insertion within the yhiF gene, and the remaining six mutants had their insertions in the yhiE gene. yhiE and yhiF products share amino acid homology (23% identity) to each other and with members of the LuxR family, which are known as transcriptional regulatory proteins. The mutant having the insertion within the O157 antigen gene cluster did not express the O157 side chain (as determined by agglutination test and immunoblotting with polyclonal O157-specific antiserum), unlike the other seven mutants. Importantly, the other mutants showed enhanced type III secretion. Levels of the related mRNAs of genes of the LEE, but not that of ler mRNA, were also increased compared with those in the wild type. Indeed, when we introduced an in-frame 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 the 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.

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