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Infection and Immunity, May 2003, p. 2598-2606, Vol. 71, No. 5
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.5.2598-2606.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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

Ichiro Tatsuno,1 Keiji Nagano,2 Kazuki Taguchi,2 Li Rong,1 Hiroshi Mori,2 and Chihiro Sasakawa1*

Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639,1 Laboratory of Microbiology, Department of Public Health Pharmacy, Gifu Pharmaceutical University, Gifu 502-8585, Japan2

Received 21 October 2002/ Returned for modification 8 January 2003/ Accepted 11 February 2003


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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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INTRODUCTION
 
Enterohemorrhagic Escherichia coli (EHEC) are members of a class of pathogenic E. coli that cause a range of illnesses, including nonbloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. Although the mechanisms involved in infection of intestines in humans are still to be elucidated, the pathogenesis of EHEC (represented by strain O157:H7) has been shown to have several characteristics, including the production of Shiga toxins, the ability to produce an attaching and effacing (A/E) intestinal lesion, and the ability to induce enterohemolytic activity (6, 17, 32, 35).

Among these characteristics, the ability to induce A/E intestinal lesions is shared by enteropathogenic E. coli (EPEC) (32). The attachment of both pathogens to intestinal epithelial cells, leading to the formation of A/E lesions, depends on the presence of the locus of enterocyte effacement (LEE) region, which contains a subset of genes encoding intimin (eae), its receptor (tir/espE), a type III secretion machine, and the type III secreted proteins, such as EspA, EspB, and EspD (9, 15, 22, 25). In vitro assays have indicated that the bacterial colonization takes place through at least three steps: on the initial attachment to the host cells, EHEC and EPEC elongate filaments which consist of EspA proteins and make a bridge (known as the translocon and assembled by the filament and the membrane pore mediated by EspB and EspD) between the bacterial cell and host cell; in the second step, Tir and other effector proteins are injected into the host epithelial cells through the translocon; and in the last step, the bacteria intimately attach to and develop microcolonies (MC) on epithelial cells and induce condensation of F-actin underneath the MC, leading to A/E lesions (12, 13, 23, 24, 27, 28, 32, 41).

Nevertheless, the mode of initial attachment of EHEC appears to be partially distinct from that of EPEC. In the initial step, EPEC seems to initiate its adherence to the intestinal epithelial cells through the binding of bundle-forming pili (BFP) to the target intestinal epithelial cells. Thus, the presence of BFP in the case of EPEC is believed to contribute to the colonization on epithelial cells (12).

To understand the initial adherence of EHEC to epithelial cells, Tatsuno et al. recently performed random transposon (mini-Tn5Km2) mutagenesis using the O157Sakai strain, which was originally isolated from a large outbreak in 1996 in Sakai City, Japan, and discovered that the sites of insertion which caused no adherence phenotype were all allocated to the genes in the LEE (41). Further, when Tatsuno et al. mutated the toxB gene on pO157 of O157Sakai, the bacterial adherence capacity declined to 15 to 40% of the wild-type level, a result which was accompanied by a decrease in the levels of EspA, EspB, EspD, and Tir production (42). These studies, therefore, have suggested that the adherence factors encoded by the genes in the LEE of EHEC play major roles in bacterial adherence to the host cells, at least under conditions in vitro.

Evaluations of the genomic database of strain O157Sakai have led to predictions that the chromosome possesses at least 14 putative fimbrially associated loci in the form of various lengths of gene clusters, although most of them have not yet been reported to be involved in bacterial adherence (19). Torres et al. (43) were unable to identify the long polar fimbriae encoded in one of the 14 putative fimbrially associated loci on the wild-type O157:H7 strain and observed only a modest reduction in the adherence of the fimbrial mutant. The reports prompted us to test the possibility that O157Sakai utilizes some as-yet-undefined adherence factors in vivo, whereas such factors might be repressed in vitro. In these contexts, we decided to isolate increased-adherence mutants, such as those relieved from putative repression, by analyzing 2,000 mini-Tn5Km2 mutants which were previously collected (41).


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MATERIALS AND METHODS
 
Bacterial strains, media, and tissue culture. The O157:H7 strain RIMD 0509952 (referred to as O157Sakai) was originally isolated from a patient in the biggest outbreak to occur in 1996 in Sakai City, Japan (26, 45). EHEC was maintained as described previously (41). Caco-2 cells were maintained as reported previously (41).

Construction of mini-Tn5Km2 mutants. In this study, a collection of mini-Tn5Km2 mutants previously constructed was used. Briefly, to obtain a selectable marker for conjugation, plasmid pSC101 (39), encoding a tetracycline resistance gene, was introduced into the O157Sakai strain by electroporation with a Gene Pulser electroporator (Bio-Rad Laboratories, Hercules, Calif.) in a 10% glycerol solution at 2.5 kV and 25 µF. The resulting O157Sakai/pSC101 strain, which was named O157T, was verified to have retained the phenotype of the parent in terms of adherence properties, including the levels of intimin expression and secretion of EspA, EspB, and Tir into culture medium from immunoblottings with specific antibodies for intimin, EspA, EspB, and Tir proteins (data not shown). The mini-Tn5Km2-bearing plasmid pUT mini-Tn5Km2 (11) was introduced into O157T from an E. coli K-12 derivative, SM17 {lambda}pir (10), by conjugation, and the transposon was inserted randomly into the chromosome. The insertion mutants thus obtained were purified on agar plates, and the individual clones were kept in 50% glycerol in Luria broth (LB) at -80°C. In addition, strain O157T was used as a wild type throughout this study.

Screening the mini-Tn5Km2 mutant library for adherent ability. The adherence of strain O157 derivatives to Caco-2 cells was evaluated as previously described (41). Briefly, the insertion mutants were grown in Dulbecco's modified Eagle's medium (DMEM)-glycerol overnight at 37°C and inoculated at a multiplicity of infection of approximately 50:1 onto semiconfluent Caco-2 cell monolayers grown on 96-well microtiter plates filled with DMEM with 10% fetal calf serum, which was replaced with fresh DMEM-glycerol. Bacteria and cells were incubated for 2 h, washed five times with 0.1 ml of sterile phosphate-buffered saline (PBS), and incubated with fresh DMEM-glycerol for another 2.5 h. These incubation times were determined to be the minimum for visualization of MC formed by O157Sakai in the large screening. The monolayers were fixed and stained with Giemsa's solution for microscopic evaluation. Bacterial clusters on Caco-2 cells consisting of eight or more bacteria were considered MC. To quantitate the adherence capacity of the mutants, bacteria freshly grown in DMEM-glycerol at 37°C for 2 h were used to infect Caco-2 cells on coverslips in a 24-well plastic plate for 1.5 h. Then these infected monolayers were washed five times with sterile PBS (1 ml) and incubated with fresh DMEM-glycerol for another 2.5 h. The numbers of MC were scored as the sum of 20 microscopic fields. The 20 fields were chosen concentrically from the center of a cover glass. This protocol was used for all adherence assays unless otherwise described.

Cloning mini-Tn5Km2 fragments. The EcoRI, EcoRV, or PstI digest of total DNA from each strain was ligated to EcoRI-, EcoRV-, or PstI-digested pBluescript II KS(+) (37) and used to transform E. coli DH5{alpha} by selecting for kanamycin-resistant transformants. Clones of the EcoRI, EcoRV, or PstI fragment containing the mini-Tn5Km2 insert were obtained. The region flanking mini-Tn5Km2 was sequenced by primer to the T3 or T7 promoter on pBluescript II KS(+).

Length of LPS. Alterations in the length of lipopolysaccharides (LPS) of O157Sakai derivatives were investigated using an agglutination assay with antiserum specific for O157 (Denka Seiken Co., Ltd., Tokyo, Japan) and the electrophoretic profiles of LPS in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as previously described (21).

Protein secretion and expression. The secretion and expression of EspA, EspB, and Tir were analyzed as described previously (41).

Northern blotting. Bacterial cells were freshly grown at 37°C for 2 h in 10 ml of DMEM containing 0.45% glycerol. At an optical density at 600 nm of ~0.27, the cells were harvested and total cellular RNA was prepared. The total RNA (2, 0.5, 0.125, or 0.3125 µg) was blotted onto a Hybond-N+ membrane (Amersham) by spotting as described previously (36). The 563-bp EcoRV-SacII fragment containing the internal region of the espB structural gene and the 395-bp SspI fragment containing the entire ler structural gene were used as probes for espB and ler mRNA, respectively. Plasmid pIC28 was constructed by cloning the 8.3-kbp EcoRI fragment (which contains the region from the 3' terminal of escC to the escR gene and mini-Tn5Km2 and which was derived from the chromosome of strain B9-F9, having a mini-Tn5Km2 insertion in escC at the LEE) into pBluescript II KS(+). The original pIC28-derived 4.7-kbp PstI fragment (which contains the region from the 3' terminal of escC to the 3' terminal of escU and was derived from pIC28) was used as a probe for genes encoding the type III secretion system. Probes to detect mRNA of the other genes were amplified by PCR, and the primers used are listed in Table 1. The membrane was hybridized to the DNA probe labeled with a BrightStar Psorelen-Biotin nonisotopic labeling kit (Ambion) and washed, and the signals were visualized with a BrightStar BioDetect nonisotopic detection kit (Ambion).


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  		TABLE 1. The sequences of primers used as described in Fig. 6

Plasmid constructions. A set of oligonucleotides, primer G1-C5 forward (5'-AAACCTGAAGACATGAATGC-3')-primer G1-C5 reverse (5'-GCTACCTTCATCCACTAATG-3'), were used to amplify DNA fragments of 2,187 bp corresponding to the yhiE gene and additional flanking regions from chromosomal DNA of strain O157Sakai by PCR with Takara ExTaq (Takara Shuzo Co., Ltd., Otsu, Japan). The amplified product was cloned into the vector pGEM-T (Promega) to make the plasmid pIC132, in which the yhiE gene is oriented in the opposite direction to the lacUV5 promoter.

Plasmid pIC133 was constructed by cloning the DNA fragments of 1,000 bp (corresponding to the yhiF gene amplified from chromosomal DNA of strain O157Sakai) by PCR using a set of oligonucleotides, primer C8-C8-F(11K) (5'-CTATCAGGGCCGACTGCTCG-3')-primer C8-C8-R(12K) (5'-ATTGATCAATCGTTCACACC-3') into the pGEM-T vector (Promega). The yhiF gene is oriented in the same direction as the lacUV5 promoter. A plasmid having the yhiF gene oriented differently from the lacUV5 promoter was also constructed and was designated pIC134.

Construction of an in-frame yhiE yhiF espADB deletion mutant. Bacteria were cultured at 30°C for all constructions.

Two sets of oligonucleotides, primer 1 (5'-TTGTGTACCCTTTTTGTTCAAG-3')-primer 2 (5'-CGCGGATCCGACCGGCGCTTCCAGTGCAG-3') and primer 3 (5'-CGCGGATCCGTTGTCAGGTTATTCGCTTTAGC-3')-primer 4 (5'-ATGTCTGGCTAACAGAAGAC-3'), were used to amplify DNA fragments of 951 bp and 712 bp (corresponding, respectively, to the 3' end of the espB gene and the 5' end of the espA gene and additional flanking regions from chromosomal DNA of strain O157Sakai) by PCR with Takara ExTaq (Takara Shuzo Co., Ltd.). The corresponding oligonucleotides contained restriction sites for the endonuclease BamHI (shown in bold in the primer sequence). This enzyme was used for ligation of both amplification products and subsequent cloning of the resulting fragment into the oriRR6k{gamma} lacZ{alpha} sacB plasmid pWM91 (30). The resulting recombinant suicide plasmid was introduced by conjugation into O157T or increased-adherence mutants and integrated into the chromosome. Further cultivation in the presence of 5% sucrose led to a second round of recombination and the excision of the plasmid with wild-type espADB genes. Bacteria were confirmed to harbor a mutated espADB allele by PCR using a combination of two primers, which are primers 1 and 4. The espADB mutants, O157T{Delta}espADB, C8-C8{Delta}espADB, and G1-C5{Delta}espADB, were confirmed to secrete Tir and the fusion protein of EspA and EspB into the culture medium. Furthermore, by using an agglutination assay with antiserum specific for O157, it was confirmed that there was no alteration in the length of the LPS of O157T{Delta}espADB, C8-C8{Delta}espADB, and G1-C5{Delta}espADB. When investigated by Northern blotting, the levels of mRNA for the espB gene (the transcript represents the mRNA of the fusion gene containing the 5' of espA and 3' of espB) and mRNA for the escC to escU genes in G1-C5{Delta}espADB or C8-C8{Delta}espADB were determined to have increased compared with that of O157T{Delta}espADB, which is consistent with the results in the parental strains (see Fig. 6A and B in Results and Discussion), showing that at least the two hallmarks of the parental G1-C5 and C8-C8 strains had been conserved throughout this construction.



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  		FIG. 6. Analysis of mRNAs generated from strains O157T, G1-C5, and C8-C8 by Northern blotting. Total RNA (2, 0.5, 0.125, or 0.3125 µg) prepared from each strain was subjected to Northern blot analysis. The name of the probe used to detect each mRNA is indicated in the lower right corner of the panel (see Materials and Methods for details on each probe). fim2 and fim3 are the names of the probes used to detect mRNAs from genes for fimbrial biosynthesis encoded by loci 2 and 3, respectively (19). As negative controls to show no contamination of chromosomal DNA, total RNA (2, 0.5, 0.125, or 0.3125 µg) prepared from O157T (WT) grown in 10 ml of LB at 30°C for 2 h was spotted on the top lane in each panel, because the levels of ler mRNA and espB mRNA from O157:H7 cells cultured in LB (L-broth) were reduced compared with those from cells cultured in DMEM (1). All RNA samples on each panel were prepared at the same time. Accordingly, the results for panel C or F were used as controls to ensure that the different levels of mRNA seen for the wild type and G1C5 in panels A and B were not due to degradation of the mRNA. The difference between increased-adherence mutants and the wild type is clearly recognized by comparing the 2-µg spots in panel B. Essentially thesame results were obtained in the independently prepared RNA. Additionally, when investigated by Northern blotting, the levels of mRNA for espB and mRNAs for escC to escU in G1-C5{Delta}espADB or C8-C8{Delta}espADB were increased as compared with those seen with O157T{Delta}espADB, confirming the results shown in Fig. 6A and B (see Materials and Methods).

A set of oligonucleotides, primer yhiE deletion R (5'-TGTTTATGTGGTACCCATAATTGTG-3')-primer yhiE deletion F (5'-TGAGCGACATGGTACCCCTGGGTAT-3'), were used as described above to amplify DNA fragments of 5,059 bp (corresponding to the in-frame yhiE deletion gene and additional flanking regions from pIC132) by PCR. The corresponding oligonucleotides contained restriction sites for the endonuclease KpnI (shown in bold in the primer sequence). This enzyme was used for self-ligation of the amplification product. The resulting 2,139-bp ApaI-SacI fragment (containing the in-frame yhiE deletion) was subcloned into the plasmid pWM91 (30). The resulting recombinant suicide plasmid was introduced by conjugation into O157T or the C8-C8{Delta}espADB mutant with a deletion in the espADB operon and integrated into the chromosome. Further cultivation in the presence of 5% sucrose led to a second round of recombination and the excision of the plasmid with wild-type yhiE gene. Bacteria were confirmed to harbor a mutated yhiE allele by PCR using a combination of two primers, which are outside primers G1-C5 forward and G1-C5 reverse. The yhiE mutants O157T{Delta}yhiE and C8C8{Delta}espADB{Delta}yhiE were confirmed as described above to have no alteration in the length of the LPS.

A set of oligonucleotides, primer yhiF(O157F) (5'-ATATAAAGACGAACAATATC-3')-primer yhiF(O157R) (5'-CGGATGCACGGCGATATCCGT-3'), were used as described above to amplify DNA fragments of 2,894 bp (corresponding to the yhiF gene and additional flanking regions from chromosomal DNA of strain O157Sakai) by PCR. The amplified product was cloned into pGEM-T easy vector (Promega) to make the plasmid pIC194. A set of oligonucleotides, primer yhiF deletion R (5'-AAGAACATCGGTACCCTGGTAATT-3')-primer yhiF deletion F (5'-CAATGAGCTGGTACCCCATCAGCA-3'), were used as described above to amplify DNA fragments of 5,456 bp (corresponding to the in-frame yhiF deletion gene and the additional flanking regions from pIC194) by PCR. The corresponding oligonucleotides contained restriction sites for the endonuclease KpnI (shown in bold in the primer sequence). This enzyme was used for self-ligation of the amplification product. The resulting 2,536-bp ApaI-SacI fragment (containing the in-frame yhiF deletion) was subcloned into the plasmid pWM91 (30). The resulting recombinant suicide plasmid was introduced by conjugation into O157T and integrated into the chromosome. Further cultivation in the presence of 5% sucrose led to a second round of recombination and the excision of the plasmid with wild-type yhiF gene. Bacteria were confirmed to harbor a mutated yhiF allele by PCR using a combination of two primers, which are outside primers yhiF(O157F) and yhiF(O157R). The yhiF mutant O157T{Delta}yhiF was confirmed as described above to have no alteration in length of the LPS.

Mice. Female ICR mice 4 weeks of age were obtained from Japan SLC (Hamamatsu, Japan) and used for the experiments after a week of acclimatization. Mice were fed sterilized Charles River solid rodent chow (Oriental Yeast, Tokyo, Japan) and water and kept on stainless steel mesh to prevent them from feeding on their own feces. Infectious experiments were performed in an isolation chamber for animals (Toyoriko Co., Ltd., Tokyo, Japan) in the laboratory for animal experiments, in accordance with the standards for the care and use of laboratory animals of Gifu Pharmaceutical University.

Intragastrical inoculation of O157Sakai and its mutants. O157 strains stored at -80°C were seeded into 10 ml of nutrient broth and cultured statically for 15 h at 37°C. The cultures were diluted at 1:100 in the same medium and incubated for 4 h at 37°C. The bacteria in logarithmic growth were harvested by centrifugation for 20 min at 2,000 x g and resuspended into sterilized PBS. The bacterial concentration in the suspension was estimated from the optical density at 600 nm and adjusted to an appropriate concentration by dilution with PBS. Mice were starved of food for 8 h and then injected intraperitoneally with 25 mg of cimetidine/kg of body weight. Bacterial suspensions were inoculated intragastrically 15 min after the cimetidine treatment.

Number of bacteria in feces. Fresh feces of mice were suspended in PBS. To detect strain O157Sakai, the suspensions were plated on Sorbitor MacConkey agar (Nissui Pharmaceutical Co., Ltd.) supplemented with 20 µg of novobiocin/ml and 0.1 µg of cefixime/ml (NC-SMAC). To detect G1-C5 or {Delta}eae (41), the suspensions were plated on Sorbitor MacConkey agar supplemented with 50 µg of kanamycin/ml (KM-SMAC) or NC-SMAC. White colonies developed by incubating the plate for 24 h at 37°C were counted. In G1-C5 or {Delta}eae, the numbers of colonies on Sorbitor MacConkey agar supplemented with KM that were essentially the same as the number of colonies on NC-SMAC were adopted.

The detection limit was 102 CFU/g of feces.


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RESULTS AND DISCUSSION
 
Isolation of elevated-adherence mutants of strain O157Sakai. Previously collected (41) mini-Tn5Km2 insertion mutants (2,000) were screened for their capacity to adhere to Caco-2 cell monolayers. The Caco-2 cells were infected for 1.5 h, and after the nonadherent bacteria had been washed out, the infected cells were further incubated in fresh DMEM-glycerol for another 2.5 h to allow the adherent bacteria to develop MC visible by Giemsa staining (see Materials and Methods). By these methods, eight mutants (designated B12-D5, C8-C8, G1-C5, G1-E7, G1-G8, G3-A4, G3-F6, and G5-B6) were selected as reproducibly exhibiting an ability to form MC at least 3.5 times greater in number than those of the wild type. (As described in Materials and Methods, O157T was used as the wild type throughout this study.) The resulting eight mutants were further characterized in terms of growth rate and pattern of adherence to Caco-2 cells. No substantial differences in growth in DMEM-glycerol at 37°C (data not shown) or ability to form MC onto Caco-2 cells at 4 h postinfection were observed (Fig. 1).



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  		FIG. 1. Adherence phenotypes of mutants. Microscopic fields having representative MC were chosen. The bacteria grown in DMEM-glycerol for 2 h were used to infect Caco-2 cell monolayers. The infected monolayers were incubated for 1.5 h and washed five times with PBS. After another 2.5 h of incubation, the monolayers were again washed three times, fixed with methanol, and stained with Giemsa's solution to visualize the adherent bacterial colonies.

Sites of mini-Tn5Km2. To directly estimate the sites of the transposon in the eight mutants, the sequences of the miniTn5Km2-flanked DNA were determined as described in Materials and Methods. The sites of mini-Tn5Km2 on the chromosome were then estimated on the basis of sequence homology with the sequence of strain O157Sakai in the DNAdatabase (18) (http://genome.gen-info.osaka-u.ac.jp/bacteria/o157/index.html). As shown in Fig. 2, B12-D5 possessed mini-Tn5Km2 in the wzy gene within the 14-kb O157 O-antigen gene cluster of O157Sakai (44). The C8-C8 mutant had mini-Tn5Km2 in the yhiF gene, which was highly (99%) homologous to the DNA sequence of the E. coli K-12 yhiF gene, while the sites of the remaining six mutants (G1-C5, G1-E7, G1-G8, G3-A4, G3-F6, and G5-B6) were mapped to the yhiE gene, which was highly (99%) homologous to the E. coli K-12 yhiE gene (Fig. 2). On collecting the mini-Tn5Km2 insertion mutants, B12-D5 and C8-C8 were derived from separate conjugation experiments used for insertion mutagenesis with mini-Tn5Km2 into the O157T chromosome, while G1-C5, G1-E7, G1-G8, G3-A4, G3-F6, and G5-B6 were from another conjugation experiment. Since the latter six mutants had insertions into similar sites in the yhiE gene, they might have emerged from a single insertion event. G1-C5 and G1-E7 were thus further investigated as representative yhiE insertion mutants in this study.



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  		FIG. 2. Genetic organization of mini-Tn5Km2-insertion genes and the flanking regions in O157:H7. Insertion sites of all increased-adherence mutants are shown.

Expression of LPS. Since out of all eight mutants, only the B12-D5 mutant had the insertion in a gene whose function was previously defined, we first examined B12-D5 for the ability to be agglutinated and for LPS profiles on immunoblotting with polyclonal O157-specific antiserum. As shown in Fig. 3, B12-D5, but not C8-C8 and G1-C5, lost the wild-type O157 LPS, and B12-D5 was unable to be agglutinated with the antiserum at all (data not shown). Since previous studies indicated that the loss of the O-side chain of LPS from EHEC promoted bacterial adherence to cultured epithelial cells (5, 7), the increased adherence of B12-D5 to Caco-2 cells could be also due to the change in the LPS structure. In this context, B12-D5 was excluded from subsequent experiments.



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  		FIG. 3. Expression of LPS of the increased-adherence mutants. Proteinase K-treated bacterial cell lysates (whole) derived from equal amounts of wild-type O157T (lane 1), B12-D5 (lane 2), G1-C5 (lane 3), and C8-C8 (lane 4) were resolved by SDS-12% PAGE, transferred to nitrocellulose membrane, and probed with polyclonal rabbit antisera specific to O157.

Characterization of yhiE and yhiF genes. To ensure that the increased-adhesion phenotype of G1-C5, G1-E7, and C8-C8 resulted from disruption of either the yhiE or the yhiF gene, we cloned yhiE or yhiF in a plasmid vector and introduced the resulting plasmids into their corresponding insertion mutants. When the resultant strains were investigated for the ability to adhere to Caco-2 cells, they showed levels of adherence capacity almost equal to that of the wild type, suggesting that the disruption of the two genes is involved in enhancement of the adherence capacity (Fig. 4). To confirm this, we constructed yhiE and yhiF in-frame deletion mutants, which we designated O157T{Delta}yhiE and O157T{Delta}yhiF (see Materials and Methods). When the O157T{Delta}yhiE or O157T{Delta}yhiF mutant was investigated for the ability to adhere to Caco-2 cells, it showed a significantly improved adherence capacity similar to those of G1-C5 or C8-C8, respectively (Fig. 4).



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  		FIG. 4. (A) Complementation of the enhancement in the adherence of G1-C5 (O157T having an insertion in yhiE) or C8-C8 (O157T having an insertion in yhiF) by yhiE (pIC132) or yhiF (pIC133), respectively. The number of MC on Caco-2 cells infected with bacteria and visualized as described in Fig. 1 was scored as the sum of 20 microscopic fields. The 20 fields were chosen concentrically from the center of a cover glass. The data shown are the means and standard deviations of three representative experiments. Essentially the same results were obtained with G1-E7. Plasmid pIC134 has the yhiF gene oriented in the opposite direction to pIC133 (see Materials and Methods). pIC134 also complements the adherence of C8-C8 but only to a moderate extent. (B) Comparison of adherence properties of the isogenic mutants (O157T{Delta}yhiE and O157T{Delta}yhiF) and the transposon mutants. The data were obtained as described for panel A.

Since the inactivation of either yhiE or yhiF in strain O157Sakai resulted in an increase in the capacity to adhere to Caco-2 cells, yhiE and yhiF are presumed to encode some negative factors affecting the bacterial adherence. The yhiE and yhiF genes encode 175 and 176 amino acids, respectively, and the amino acids of YhiF share 23% identity with those of YhiE, the highest similarity (except for the 99% similarity determined for YhiF of E. coli K-12) among all deduced proteins, according to a search of the database (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). Although the biological functions of YhiE and YhiF in O157:H7 have not yet been established, both of them showed homology with some transcriptional regulators when analyzed by BLAST search: YhiE showed homology with part of the two-component-system regulatory protein encoded by the XAC2168 gene in Xanthomonas axonopodis pv. citri or with the C-terminal part of the LuxR-like transcriptional regulator encoded by the PP2587 gene in Pseudomonas putida (8, 33); YhiF showed homology with the C-terminal part of the DNA-binding response regulator encoded by the SO4157 gene in Shewanella oneidensis or the C-terminal part of the hypothetical protein similar to response regulators of the two-component regulatory protein encoded by the MW2313 gene in Staphylococcus aureus (3, 20). In addition, a conserved-domain search (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) (2) led to the hypothesis that the C-terminal portion of YhiE is homologous to the C-terminal consensus sequence of members of the CitB response regulator family, containing a CheY-like receiver domain and a helix-turn-helix DNA-binding domain (31). Members of the family include an ECs4400 gene-encoded putative regulator and an ECs5326-encoded two-component transcriptional regulator (18). The conserved-domain search also led to the hypothesis that the C-terminal portion of YhiF possesses a helix-turn-helix motif involved in DNA binding that is homologous to that of the LuxR family of transcriptional regulators and that the overall portion shows weak homology to members of the CitB family of proteins (38). Therefore, it is possible that YhiE and YhiF serve as negative regulators for the expression of gene(s) involved in O157 adherence.

Activity of the type III secretion system. If yhiE and yhiF encode negative regulators, some positive factors must be activated in the mutants which include disruptions in either of the genes. Since secretion of EspA, EspB, and Tir from EHEC is positively associated with adherence to epithelial cells, the secretion into the medium from C8-C8, G1-C5, or G1-E7 was analyzed by silver-staining SDS-PAGE or immunoblotting with anti-EspB or anti-Tir-antibody. As shown in Fig. 5, the level of proteins secreted from the insertion mutants was increased compared with that from the wild type. Though the increase of EspB and Tir in the whole bacterial lysates, meanwhile, was apparently marginal compared with that in the wild type, total production levels for the secreted proteins were clearly increased. The same was also essentially true of O157T{Delta}yhiE and O157T{Delta}yhiF, whose levels of protein secretion into the medium were increased compared to the levels of the wild type (data not shown). In this context, it is possible that type III secretion contributes to the increased-adherence phenotype in the yhiE or yhiF gene mutant.



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  		FIG. 5. Levels of secretion and expression of EspA, EspB, and Tir of C8-C8 and G1-C5 mutants. Trichloroacetic acid-precipitated culture supernatants derived from equal amounts of wild-type O157T (lane 1), G1-C5 (lane 2), G1-E7 (lane 3), C8-C8 (lane 4), and B9-F9 (lane 5) were resolved by SDS-12% PAGE and stained with silver (A) or immunoblotted with specific antibodies (B). The B9-F9 mutant has an insertion of mini-Tn5Km2 in the escC, and the type III secretion system is inactivated (41).

Disruption of yhiE or yhiF affects LEE-encoded genes at the mRNA level. Using RNA dot blotting with DNA probes specific for each of the genes to assess whether disruption of the yhiE or yhiF gene would promote type III secretion at the transcriptional level, levels of mRNAs in G1-C5 and C8-C8 were investigated together with that of the wild type. As shown in Fig. 6A, the levels of espB mRNA in G1-C5 and C8-C8 were increased compared with those in the wild type. Since the espB gene forms an operon (espADB operon) together with the espA and espD genes (4, 42), it was most likely that the production of EspA and EspD would also be activated at the mRNA level. Similarly, the levels of the mRNAs containing from escC to escU, which encode parts of the type III secretion system, were also increased compared with the wild-type levels (compare each 2 µg of spots in Fig. 6B). Thus, these results suggest that the presence of functional yhiE and yhiF genes in strain O157Sakai somehow negatively regulates the expression of genes coding for type III secretion.

Meanwhile, expression of the espADB operon and other operons encoding the type III system in the LEE have been shown to be increased by stimulation such as with NaHCO3 via activation of the ler (LEE-encoded regulator) gene (1, 14, 16, 29, 34). Therefore, to investigate whether YhiE or YhiF also controls type III secretion via Ler, the levels of ler mRNA in G1-C5 and C8-C8 were examined by RNA dot blotting with a DNA probe specific for ler. As shown in Fig. 6C, the levels of ler mRNA in the two strains were similar to those of the wild type, suggesting that the regulation by YhiE or YhiF is not mediated by the ler gene.

No effect on expression of other putative adherence-associated gene clusters is seen in the absence of YhiE or YhiF. The complete genomic sequence of strain O157Sakai has indicated the presence of 14 putative fimbrial biosynthesis gene clusters, scattered along the chromosomal DNA (19, 43). Hence, using RNA dot blotting, we examined whether disruption of the yhiE or yhiF gene would affect the expression of the genes in the 13 loci (except for locus 7) (see Materials and Methods), while locus 7 was not examined due to the incomplete set of the genes required for fimbrial biosynthesis. As shown in Fig. 6, the level of mRNA for fimbrial locus 2 or 3 in C8-C8 was slightly increased compared with that of the wild type; however, the amounts of mRNA for the remaining loci in G1-C5 and C8-C8 were the same as the wild-type amounts (data not shown). In addition, the level of iha mRNA, which was previously indicated to encode a fimbrial adherence-associated protein (40), was not altered in G1-C5 or C8-C8. Therefore, to investigate whether or not the increased-adherence phenotype of G1-C5 and C8-C8 is caused by some putative adherent factors including those encoded in fimbrial loci 2 and 3, but not the LEE, a large in-frame deletion was introduced into espADB in the wild type, G1-C5, and C8-C8 and the resulting mutants were designated O157T{Delta}espADB, G1-C5{Delta}espADB, and C8-C8{Delta}espADB, respectively. When G1-C5{Delta}espADB, C8-C8{Delta}espADB, and O157T{Delta}espADB were investigated for the ability to adhere to Caco-2 cells, all the adherence capacities were greatly decreased to a low level similar to those of the type III secretion-deficient mutants (A5-E4 and B9-F9), which had been constructed in the previously study by Tatsuno et al. (41). Thus, we could not find any novel factors such as that of BFP of EPEC, which still retains its adherence capacity even in the type III defective mutant (data not shown). However these results do not completely rule out the involvement of the factors including the fimbrial loci 2 and 3 in adherence, since they might act in concert with the type III system.

Furthermore, we created an additional yhiE-yhiF double-knockout mutant (C8C8{Delta}espADB{Delta}yhiE) from C8-C8{Delta}espADB and investigated its adherence capacity with respect to factor(s) independent of the type III system. The results showed that the mutant, like the parental C8-C8{Delta}espADB strain, still failed to adhere to Caco-2 cells (data not shown).

Thus, the results of the series of experiments suggested that the increased-adherence phenotype of G1-C5 and C8-C8 is not due to activation of any other genes involved in the bacterial adherence than LEE-associated genes.

Long-term fecal shedding of the increased-adherence mutant in mice. Recently, Mori and his coworkers have found that SPF-ICR mice can be used for estimating the colonization of strain O157:H7, including strain O157Sakai, onto the lower intestine (31a). In the study, the fecal shedding of O157:H7 was observed in ICR mice for up to 5 weeks, while the fecal shedding periods of some type III-deficient mutants were greatly shortened compared with those of wild-type O157:H7 (31a). Hence, ICR mice were inoculated with 1011 CFU/kg of body weight of wild-type, G1-C5, or {Delta}eae (intimin-deficient mutant) bacteria, and the number of bacteria shed into feces was examined at 3, 7, and 14 days after the inoculation (see Materials and Methods). On day 3, wild-type and G1-C5 strains were detected in feces in numbers of around 105 CFU/g of feces, whereas the number of {Delta}eae mutants shed into feces was below the detectable level (less than 102 CFU/g of feces) (Fig. 7). Importantly, though the number of wild-type bacteria in feces had gradually declined by day 14, the number of G1-C5 bacteria in feces retained a level similar to that seen on day 3 (Fig. 7), suggesting that the absence of yhiE in O157Sakai enhances colonization onto mouse intestine.



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  		FIG. 7. Time course of fecal shedding of strain O157Sakai and its mutants. Mice were inoculated with 1011 CFU of O157Sakai (wild type), G1-C5, or {Delta}eae (nonpolar mutant)/kg of body weight. Broken lines show detection limits. When the data under the detection limit were set at 2, the mean and the range for the set of data for O157Sakai, G1-C5, and {Delta}eae were 2.9 ± 0.3, 4.9 ± 0.7, and 2.0 ± 0.0, respectively, on day 14. A statistical analysis (t test) was used to determine the difference between the wild type and the G1-C5 mutant. *, P < 0.01 for the mutant versus the wild type.

In summary, the yhiE and yhiF genes found in strain O157Sakai take part in modulating levels of expression of LEE genes. Though the mechanisms of control of LEE expression by YhiE and YhiF are still to be elucidated, they appear to serve as a negative regulator of LEE-encoded gene expression at the RNA level. Thus, our findings provide further evidence supporting the notion that LEE-encoded genes in EHEC play a central role in bacteria colonizing on intestinal epithelium. Furthermore, the isolation of increased-adherence mutants of O157Sakai in the present study may facilitate further study of the regulatory system for the adherence of EHEC to host intestinal epithelium under both in vitro and in vivo conditions.


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ACKNOWLEDGMENTS
 
This research was supported by grant number 13670263 from the Ministry of Education, Science and Culture of the Japanese government.

We thank Chizu Sasako for technical assistance. We are grateful to Naresh Verma and Reiko Akakura for helpful discussions.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Bacteriology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Phone: 81-3-5449-5252. Fax: 81-3-5449-5405. E-mail: sasakawa{at}ims.u-tokyo.ac.jp. Back

Editor: A. D. O'Brien


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Infection and Immunity, May 2003, p. 2598-2606, Vol. 71, No. 5
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.5.2598-2606.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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