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EISEVIER FEMS Microbiology Letters 133 (1995) 35-39 Cloning and nucleotide sequence of a gene upstream of the eaeA gene of enterohemorrhagic Escherichia coZi 0157:H7 Shaohua Zhao a, Sharon E. Mitchell b, Jianghong Meng a, Michael P. Doyle a,*, Stephen Kresovich b ’ Centerfor Enhancement and Department qf’ FoodScience and Unilvrsity of Georgia, GrifJin, GA 30223, USA Unit, USDA/ARS, Unir ersity of Georgia, Griffin. GA 30223, Food Safety and Quality ’ Genetic Resources Technology. USA I August 1995; accepted 28 August 1995 Received Abstract A DNA segment located immediately upstream of the eaeA gene of enterohemorrhagic Escherichia coli 0157:H7 strain HAI was cloned and sequenced. This segment contained an open reading frame encoding a predicted protein of 156 amino acids. A database search identified similar open reading frames upstream of the eaeA gene in two other bacterial pathogens, i.e. enteropathogenic E. coli and Citrobacter freundii. The predicted amino acid sequence of the enterohemorrhagic E. coli protein shared 96.8% and 94.2% identity with the enteropathogenic E. coli and C. freundii sequences, respectively. Because the open reading frame is located within the locus of enterocyte effacement region of the E. coli chromosome, a ‘hot spot’ for insertion of virulence factor genes, and shares high sequence homology with attaching and effacing EPEC and C. freundii, this protein may be associated with pathogenicity of E. co/i 0157:H7. Keywords; Escherichia co/i 0 1S7:H7; Hemorrhagic colitis; Enterohemorrhagic 1. Introduction Enterohemorrhagic E~cherichia colt’ (EHEC) 0157:H7 is the most common cause of hemorrhagic colitis and hemolytic uremic syndrome [ 11. Although the precise mechanism of pathogenicity has not been fully elucidated, it is generally thought that E. coli 0157:H7 causes disease by adherence to the host cell membrane followed by production of one or more Shiga-like toxins (SLTs) [2,3]. The structure * Corresponding author. Tel.: + 1 (404) 228 7284; Fax: (404) 229 3216; E-mail: [email protected]. 037%1097/95/$09,50 0 SSDI 037X-1097(95)00330-4 1995 Federation of European + I Microbiological Escherichia co/i and function of SLT genes and their products have been extensively studied 141. The mechanism of bacterial adherence, however, is less clear, although some adherence factors have been identified [2,3]. Like the enteropathogenic E. coli (EPEC), E. coli 0157:H7 can colonize the intestine of humans and experimental animals, producing a characteristic attaching and effacing (AE) lesion. Two genes involved in the generation of these lesions have been characterized: eaeA encodes intimin, a protein required for intimate attachment [5] and eaeB encodes a secreted protein necessary for intimate attachment and transduction of signals to epithelial cells in EPEC [6]. The eaeB gene is located approx. 4.5 kb Societies. All rights reserved downstream from the 3’ end of the eaeA gene. Both eaeA and eaeB have been sequenced and characterized [6-81. Studies have shown that although eaeA is important for the production of lesions, this gene alone is insufficient to confer AE activity [9]. The function of eaeB of EHEC has not been fully elucidated. An approx. 35kb conserved region containing the earA, eaeB, and sep (secretion of E. coli protein) genes has been reported in both EHEC and EPEC [3]. This region, the locus of enterocyte effacement (LEE), is not present in normal flora E. coli, E. co/i K12 or enterotoxigenic E. cc&, but is present in other AE bacteria, including Citrobacter ,freundii biotype 4280, Hafnia alcei, and E. coli strain RDEC-I. In EPEC and EHEC, the LEE region is inserted into the E. coli K12 chromosome at approx. 82 min which is also the location of a large (approx. 70 kb) insert in the uropathogenic E. coli. This large insert or ‘pathogenicity island’ contains virulence factor genes in uropathogenic E. coli. The fact that the LEE of EHEC and EPEC is located at the same chromosomal site suggests that this region is a ‘hot spot’ for insertion of virulence factor genes. In the present study, we report the DNA sequence of the region immediately flanking the 5’ end of the EHEC eaeA gene, and compare the predicted amino acid sequence of an open reading frame (ORF) located within this region to those from similarly located ORFs in EPEC and C. ,freundii. 2. Materials and methods 2.1. Bacterial media and chemical reagents Luria broth (LB) and Luria plates were used for routine culturing of E. coli strains. All media were obtained from Difco, Detroit, MI. Ampicillin, kanamycin, nalidixic acid, 5-bromo-4-chloro-3-indolyl-b-galactoside (X-gal), and isopropyl-P-Dthiogalacto-pyranoside (IPTG) were purchased from Sigma Chemical Co., St. Louis, MO. 2.2. Bacterial strains and cectors our E. coli 0157:H7 strain HAI was maintained in laboratory. HA1 was derived from E. coli 0157:H7 strain 932. The parent strain was previously used for developing the monoclonal antibody (mAb) 4E8C12 that specifically reacts with a unique outer membrane protein (OMP) of E. coli 0157:H7 [ 101. E. coli 0157:H7 strain A10 was created in our laboratory by Tn phoA mutagenesis [I I] and did not express this unique OMP. E. coli strain JM 109 (Promega, Madison, WI) was used as the host cells for plasmid vector pUC 19 (Promega). 2.3. Construction screening of expression library and imrnuno- Genomic DNA of E. coli 0157:H7 strain HAI was prepared and purified by cesium chloride gradient centrifugation using the procedure described by Sambrook et al. [ 121. The chromosomal DNA of HA1 and plasmid DNA of pUC 19 were digested to completion with BamHI and EcoRI according to the manufacturer’s protocol (Gibco BRL, Gathersburg, MD). 50 ng of pUC 19 and I50 ng of E. coli 0157:H7 digested DNAs were mixed together and ligated by adding 5 units of T4 DNA ligase (Gibco BRL). The reaction mixture was incubated at 4°C overnight and recombinant plasmids were then transformed into E. coli JM 109. Cells were plated onto LB plates containing 50 pg ampicillin ml-‘, 25 ,ug IPTG ml ~~’ and 25 pg X-gal ml-‘. The white, ampicillin-resistant E. coli colonies were selected and immunoscreening was performed by the method described by Sambrook et al. [ 121 using mAb 4E8C I2 (detects an OMP-specific to E. coli 0157:H7) as the first antibody, and a goat anti-mouse IgG conjugated to alkaline phosphatase (Sigma, St. Louis, MO) as the second antibody. Western blots of proteins from one colony showing a strong positive result in the immunoblot assay (clone 6-F) were probed with mAb 4E8C 12. 2.4. Southern blot analysis Southern blot hybridization [ 121 was performed to determine if clone 6-F (positive in the immunoblot assay) contained the same DNA sequence as in the region flanking the TnphoA insertion site in the OMPmutant strain, AlO. Strain A10 does not express the protein detected by mAb 4E8C 12. Genomic DNAs from E. coli 0157:H7 strains HA 1 and S. Zhao rt 01. / FEMS Microbiology A10 were digested with EcoRV and probed purified insert DNA from clone 6-F. 2.5. DNA sequencing with und analysis Plasmid clone 6-F was sequenced directly by Taq cycle sequencing using fluorescence-based chain termination chemistry (Perkin Elmer/Applied Biosysterns, Foster City, CA) and an automatic DNA sequencer (Perkin Elmer/Applied Biosystems, model 373 A). Electropherograms were edited and DNA contigs were assembled with the aid of the computer program Sequencer 2.1 (Gene Codes Corp., Ann Arbor, MI). Additional analyses were performed using programs included in the GCG (Genetics Computer Group, Madison, WI) package [ 131. The entire 798-bp DNA sequence from 6-F and the deduced amino acid sequence from one complete open reading frame were used to search the combined data bases of the National Center for Biotechnology Information (Washington, D.C.) via the BLAST network service. The database searches employed the alignment algorithm of Altschul et al. [ 141. 2.6. Nucleotide sequence and accession number The DNA sequence data reported in this article have been submitted to GenBank database and assigned the accession number U323 12. 1 Letters 133 ClYY5l 35-39 31 3. Results and discussion Several thousand colonies from the E. coli 0157:H7 library were screened in the immunoblot assay. One colony containing a plasmid with an approx. 800-bp insert, 6-F, consistently reacted with mAb 4E8C12. This antibody detects an OMP unique to serotype 0157:H7 and a few SLT-producing serotypes of E. coli [lo]. Western blots, however, revealed no reaction with mAb 4E8C12 despite the fact that protein samples from clone 6-F were prepared using several different methods. Southern blot analysis was performed to determine if clone 6-F contained the same gene interrupted by Tn phoA in the OMP- mutant strain, A 10. The insert DNA from clone 6-F was purified and hybridized to EcoRV-digested genomic DNAs from E. coli 0157:H7 strains HA1 (OMP+) and A10 (OMP-1. In strain AlO, the EcoRV fragment containing TnphoA was approx. 9.4 kb [ 111. Since EcoRV does not cut within TnphoA, this fragment includes the complete transposon plus approx. 2 kb of flanking sequence. Therefore, if clone 6-F contained DNA sequences homologous to those flanking the transposon insertion site in strain AlO, a 9.4-kb fragment should have been detected for EcoRV-digested A 10 DNA and an approx. 2-kb fragment should have been detected for similarly digested 120 38 S. Zhao et al. / FEMS Microbiology Letters 133 f 19951 35-39 1 100 KPAEKIGnxISFNmRLCSFAIDEIWIS IsD?4iDEYmIYGVCGKFPT DNSNPALBIL NANLWFAENG GPYLCYEAGA QSLLLALRPP EH!x MSSRSELLLE ______*--- --_____s__ __________ EPGC _____----fJ R_-----V-S __________ -A--______ _--_A---- __________ --p------__~~______ __________ ~-p------- ----__R--- ----_-_s__ __________ CBFR ________-E ~------~-~ ___^_.____ -v-------156 101 NEmwvKSMBNLYLVLHNQ GITLENmmK IEBISSSDNK HYmGR’ BHBC LmJATPBKLE ___A_-____ __________ ___.---___ __________ -_____--_______ EPF.C ----______ ______N_____-.___ CBQR ---v-_____ _--.______ ___.---.-- Fig. 2. Alignment of the predicted amino acid sequences encoded by ORFs located upstream of the eaeA gene in: E.wherichia coli 0157:H7 strain HAI (EHEC), EPEC E2348/69 ([17]; EMBL/GeneBank/DDBJ Nucleotide Sequence Data Libraries accession no. M3405 I) and Cirrobacterfreundii strain 4280 (CBFR) ([15]; accession no. Ll 1691). Dashes represent sequence identity. wild-type DNA (strain HAl). Results from the Southern analysis revealed, however, that insert DNA from clone 6-F hybridized to a 3.5-kb EcoRV fragment in both HAI and A10 (data not shown). We, therefore, concluded that clone 6-F did not contain the gene encoding the unique OMP and that the positive reaction with mAb 4E8C12 was due to non-specific binding. The complete DNA sequence of the clone 6-F insert (798 bp) is shown in Fig. 1. This clone contained 110 bp of the 5’ region of the EHEC eaeA gene (encoding 36 N-terminal amino acids of the intimin protein), and 688 bp of upstream DNA sequence. The sequence upstream of the eaeA gene included an open reading frame potentially encoding a 156-amino acid protein (Fig. 1). Results from the BLAST search indicated that this putative protein has significant homology to predicted proteins, also located upstream of eueA, in two pathogenic bacteria, EPEC and C. ,freundii. EPEC produce diarrhea in humans, whereas C. freundii is the causative agent of transmissible murine colonic hyperplasia [ 151. The amino acid sequence alignment of the predicted protein in EHEC, EPEC and C. freundii is shown in Fig. 2. The protein sequence of EHEC shared 96.8% and 94.2% identity with the sequences from EPEC and C. freundii, respectively. Three out of a total of five amino acid substitutions between EHEC and EPEC occurred within the first 18 Nterminal residues of the predicted protein, whereas the pattern of amino acid substitution between EHEC and C. freundii was more uniform. Recent studies have revealed that the eaeA, eaeB, and spa (cfm) genes are all located within an approx. 35-kb region that is present in EPEC and E. coli 0157:H7 [3]. This so-called LEE region is not present in normal flora E. coli, E. coli K- 12, or enterotoxigenic E. coli but is present in other AE bacteria, including C. freundii biotype 4280, H. alvei, and E. coli strain RDEC-1 that causes diarrhea in rabbits, and is thought to be a hot spot for insertion of virulence factor genes in the E. co/i chromosome. Lai and Donnenberg [ 161 reported that genes located between eueA and eaeB as well as downstream of eaeB are required for attaching and effacing activity. Characterization of genes in the LEE region should help clarify the biochemical mechanisms involved in the pathogenicity of E. coli 0157:H7. We propose that the open reading frame immediately upstream of the eueA gene might encode a virulence factor and participate in the formation of AE lesions. Expression and mutation analyses of this gene are presently underway, and these studies may determine if genes located upstream of eueA are important in E. coli 0157:H7 pathogenesis. Acknowledgements This study was supported in part by USDA Special Grant 93-COOP-2-959 I. References [I] Doyle. M.P. (199 1) Escherichia coli 0 157:H7 and its significance in foods. Int. J. Food Microbial. 12, 289-301. [2] Tesh, V.L. and O’Brien, A.D. (1992) Adherence and colonization mechanisms of enteropathogenic and enterohemorrhagic Escherichia cd. Microb. Pathog. 12, 245-254. [3] Kaper, J.B. (1994) Molecular pathogenesis of enteropathogenic Escherichia co/i. In: Molecular Genetics of Bacterial Pathogenesis (Miller, V.L., Kaper, J.B., Portnoy, D.A. and Isberg, R.R., Ed%), pp. 173- 195. American Society for Microbiology, Washington, D.C. S. Zhao et nl. / FEMS Microbiology [4] O’Brien, A., Tesh, V., Donohue-Rolfe, A., Jackson, M., Olsnes, S., Sandvig, K., Lindberg, A. and Keusch, G.T. (1992) Shiga toxin: biochemistry, genetics mode of action and role in pathogenesis. Curr. Top. Microbial. Immunol. 180, 65-94. [5] Donnenberg. MS, Tzipori, S., McKee, M.L.. O’Brien, A.D., Ahoy, J. and Kaper, J.B. (1993) The role of the eae gene of enterohemorrhagic Escherichia coli in intimate attachment in vitro and in a porcine model. J. Clin. Invest. 92, 14181224. [6] Donnenberg, MS, Yu, J. and Kaper, J.B. (1993) A second chromosomai gene necessary for intimate attachment of enteropathogenic Escherichicl cob to epithelial cells. J. Bacteriol. 175, 4670-4680. [7] Beebakhee. G., Louie, M., De Azavedo, J. and Brunton. J. (1992) Cloning and nucleotide sequence of the eae gene homologue from enterohemorrhagic Escherichia co/i. FEMS Microbial. Lett. 91, 63-68. [8] Yu. J. and Kaper, J.B. (1992) Cloning and characterization of the eae gene of enterohemorrhagic Escherichia co/i 0157:H7. Mol. Microbial. 6, 41 I-417. [9] Lottie, M.. 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