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

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.

[1]  C. Guilleminault,et al.  Outbreaks of enterohaemorrhagic Escherichia coli O157:H7 infection by two different genotype strains in Japan, 1996 , 1996 .

[2]  Nicole T. Perna,et al.  Molecular Evolution of a Pathogenicity Island from Enterohemorrhagic Escherichia coli O157:H7 , 1998, Infection and Immunity.

[3]  T. Baldwin,et al.  Transcriptional regulation of bioluminesence genes from Vibrio fischeri , 1995, Molecular microbiology.

[4]  J. Wells,et al.  Hemorrhagic colitis associated with a rare Escherichia coli serotype. , 1983, The New England journal of medicine.

[5]  T. Chakraborty,et al.  EspE, a novel secreted protein of attaching and effacing bacteria, is directly translocated into infected host cells, where it appears as a tyrosine‐phosphorylated 90 kDa protein , 1998, Molecular microbiology.

[6]  E. C. Teixeira,et al.  Comparison of the genomes of two Xanthomonas pathogens with differing host specificities , 2002, Nature.

[7]  F. Blattner,et al.  Identification and Characterization of lpfABCC′DE, a Fimbrial Operon of Enterohemorrhagic Escherichia coli O157:H7 , 2002, Infection and Immunity.

[8]  Tetsuya Hayashi,et al.  toxB Gene on pO157 of Enterohemorrhagic EscherichiacoliO157:H7 Is Required for Full Epithelial Cell Adherence Phenotype , 2001, Infection and Immunity.

[9]  M. McKee,et al.  Enterohemorrhagic Escherichia coli O157:H7 requires intimin to colonize the gnotobiotic pig intestine and to adhere to HEp-2 cells , 1995, Infection and immunity.

[10]  P. Williams,et al.  Actin accumulation at sites of bacterial adhesion to tissue culture cells: basis of a new diagnostic test for enteropathogenic and enterohemorrhagic Escherichia coli , 1989, Infection and immunity.

[11]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[12]  Y. Nagai,et al.  Genome and virulence determinants of high virulence community-acquired MRSA , 2002, The Lancet.

[13]  C. Sasakawa,et al.  Bicarbonate Ion Stimulates the Expression of Locus of Enterocyte Effacement-Encoded Genes in Enterohemorrhagic Escherichia coli O157:H7 , 2002, Infection and Immunity.

[14]  M. Pallen,et al.  A novel EspA‐associated surface organelle of enteropathogenic Escherichia coli involved in protein translocation into epithelial cells , 1998, The EMBO journal.

[15]  V. Sperandio,et al.  The Locus of Enterocyte Effacement (LEE)-Encoded Regulator Controls Expression of Both LEE- and Non-LEE-Encoded Virulence Factors in Enteropathogenic and EnterohemorrhagicEscherichia coli , 2000, Infection and Immunity.

[16]  R. Tauxe,et al.  The epidemiology of infections caused by Escherichia coli O157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome. , 1991, Epidemiologic reviews.

[17]  V. de Lorenzo,et al.  Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria , 1990, Journal of bacteriology.

[18]  M. Rohde,et al.  Initial binding of Shiga toxin‐producing Escherichia coli to host cells and subsequent induction of actin rearrangements depend on filamentous EspA‐containing surface appendages , 1998, Molecular microbiology.

[19]  I. Rosenshine,et al.  Hierarchy in the expression of the locus of enterocyte effacement genes of enteropathogenic Escherichia coli , 1999, Molecular microbiology.

[20]  W. Huse,et al.  Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties. , 1988, Nucleic acids research.

[21]  Y. Inagaki,et al.  Outbreaks of enterohaemorrhagic Escherichia colt O157:H7 infection by two different genotype strains in Japan, 1996 , 1996, The Lancet.

[22]  T. R. Ward,et al.  Iha: a Novel Escherichia coli O157:H7 Adherence-Conferring Molecule Encoded on a Recently Acquired Chromosomal Island of Conserved Structure , 2000, Infection and Immunity.

[23]  G. Dougan,et al.  Enteropathogenic and enterohaemorrhagic Escherichia coli : more subversive elements , 1998, Molecular microbiology.

[24]  J. Kvittingen.  [Enteropathogenic Escherichia coli]. , 1966, Tidsskrift for den Norske laegeforening : tidsskrift for praktisk medicin, ny raekke.

[25]  P. Sherman,et al.  Polysaccharide side chains are not required for attaching and effacing adhesion of Escherichia coli O157:H7 , 1996, Infection and immunity.

[26]  S. Dowell,et al.  Role of the Escherichia coli O157:H7 O side chain in adherence and analysis of an rfb locus , 1996, Infection and immunity.

[27]  M. Hattori,et al.  Complete nucleotide sequences of 93-kb and 3.3-kb plasmids of an enterohemorrhagic Escherichia coli O157:H7 derived from Sakai outbreak. , 1998, DNA research : an international journal for rapid publication of reports on genes and genomes.

[28]  B. Finlay,et al.  Enteropathogenic E. coli (EPEC) Transfers Its Receptor for Intimate Adherence into Mammalian Cells , 1997, Cell.

[29]  M. Bott,et al.  In vitro binding of the response regulator CitB and of its carboxy-terminal domain to A + T-rich DNA target sequences in the control region of the divergent citC and citS operons of Klebsiella pneumoniae. , 1997, Journal of molecular biology.

[30]  V. Sperandio,et al.  The Per regulon of enteropathogenic Escherichia coli : identification of a regulatory cascade and a novel transcriptional activator, the locus of enterocyte effacement (LEE)‐encoded regulator (Ler) , 1999, Molecular microbiology.

[31]  J. Leong,et al.  The Tir‐binding region of enterohaemorrhagic Escherichia coli intimin is sufficient to trigger actin condensation after bacterial‐induced host cell signalling , 1999, Molecular microbiology.

[32]  P. Reeves,et al.  Organization of Escherichia coli O157 O Antigen Gene Cluster and Identification of Its Specific Genes , 1998, Infection and Immunity.

[33]  O. White,et al.  Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. , 2002, Environmental microbiology.

[34]  S. Yokoyama,et al.  Adhesion and Colonization of Enterohemorrhagic Escherichia coli O157:H7 in Cecum of Mice , 2003, Microbiology and immunology.

[35]  P. Hitchcock,et al.  Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels , 1983, Journal of bacteriology.

[36]  M. Hattori,et al.  Complete genome sequence of enterohemorrhagic Escherichia coli O157:H7 and genomic comparison with a laboratory strain K-12. , 2001, DNA research : an international journal for rapid publication of reports on genes and genomes.

[37]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[38]  N. Fairweather,et al.  Versatile low-copy-number plasmid vectors for cloning in Escherichia coli. , 1982, Gene.

[39]  O. White,et al.  Genome sequence of the dissimilatory metal ion–reducing bacterium Shewanella oneidensis , 2002, Nature Biotechnology.

[40]  D. Swerdlow,et al.  Escherichia coli O157:H7 and the hemolytic-uremic syndrome. , 1995, The New England journal of medicine.

[41]  V. de Lorenzo,et al.  Analysis and construction of stable phenotypes in gram-negative bacteria with Tn5- and Tn10-derived minitransposons. , 1994, Methods in enzymology.

[42]  B. Wanner,et al.  Conditionally replicative and conjugative plasmids carrying lacZ alpha for cloning, mutagenesis, and allele replacement in bacteria. , 1996, Plasmid.

[43]  M. McKee,et al.  Investigation of enterohemorrhagic Escherichia coli O157:H7 adherence characteristics and invasion potential reveals a new attachment pattern shared by intestinal E. coli , 1995, Infection and immunity.