Identification of a novel fimbrial gene cluster related to long polar fimbriae in locus of enterocyte effacement-negative strains of enterohemorrhagic Escherichia coli

Abstract

Enterohemorrhagic Escherichia coli (EHEC) is a food-borne cause of bloody diarrhea and the hemolytic-uremic syndrome (HUS) in humans. Most strains of EHEC belong to a group of bacterial pathogens that cause distinctive lesions on the host intestine termed attaching-and-effacing (A/E) lesions. A/E strains of EHEC, including the predominant serotype, O157:H7, are responsible for the majority of HUS outbreaks worldwide. However, several serotypes of EHEC are not A/E pathogens because they lack the locus of enterocyte effacement (LEE) pathogenicity island. Nevertheless, such strains have been associated with sporadic cases and small outbreaks of hemorrhagic colitis and HUS. Of these LEE-negative organisms, O113:H21 is one of the most commonly isolated EHEC serotypes in many regions. Clinical isolates of LEE-negative EHEC typically express Shiga toxin 2 and carry an approximately 90-kb plasmid that encodes EHEC hemolysin, but in the absence of LEE, little is known about the way in which these pathogens colonize the host intestine. In this study we describe the identification of a novel fimbrial gene cluster related to long polar fimbriae in EHEC O113:H21. This chromosomal region comprises four open reading frames, lpfA to lfpD, and has the same location in the EHEC O113:H21 genome as O island 154 in the prototype EHEC O157:H7 strain, EDL933. In a survey of EHEC of other serotypes, homologues of lpfA(O113) were found in 26 of 28 LEE-negative and 8 of 11 non-O157:H7 LEE-positive EHEC strains. Deletion of the putative major fimbrial subunit gene, lpfA, from EHEC O113:H21 resulted in decreased adherence of this strain to epithelial cells, suggesting that lpf(O113) may function as an adhesin in LEE-negative isolates of EHEC.

FIG. 1.

(A) Genetic organization of the lpf gene clusters from strains of E. coli and S. enterica serovar Typhimurium showing the approximate sizes and number of ORFs of the gene clusters and the E. coli K-12 gene adjacent to the chromosomal insertion site. (B) Analysis of the region upstream from the lpf_O113 gene cluster. Putative −10 and −35 promoter sites are underlined, and a putative ribosome binding site is shaded. The amino acid translation is shown above the nucleotide sequence. A potential transcription start point is in bold type and uppercase, and the coding region of lpfA is in bold type. The promoter region was analyzed with the NNPP program for prokaryotes.

FIG. 2.

Amino acid alignment of the predicted lpfA_O113 product and related fimbrial proteins. The alignment was performed with CLUSTAL software, and amino acid numbers are on the right. Gaps are indicated by a dashed line, and conserved amino acids are shaded in black. Cysteine residues (Cys39 and Cys79) predicted to form a disulfide bridge are indicated by an asterisk. The invariant glycine (Gly176) and aromatic (Tyr188) residues predicted to comprise a chaperone-binding domain are also indicated (#).

FIG. 3.

Adherence of derivatives of E. coli ORN103 and EHEC O113:H21 (EH41) to Chinese hamster ovary cells (CHO-K1). (A) E. coli ORN103(pWSK:lpf); (B) E. coli ORN103(pWSK29); (C) parent strain EHEC O113:H21 (EH41); (D) EH41lpf−. Arrows indicate adherent bacteria.

FIG. 4.

Nucleotide sequence of the regions flanking different lpf sites adjacent to pstS. Conserved nucleotides are shaded in black, and two nucleotides unique to EHEC O157: H7 (EDL933) are shaded in grey.