Transfer region of pO113 from enterohemorrhagic Escherichia coli: similarity with R64 and identification of a novel plasmid-encoded autotransporter, EpeA

Abstract

Enterohemorrhagic Escherichia coli (EHEC) is a prominent, food-borne cause of diarrhea, bloody diarrhea, and the hemolytic uremic syndrome in industrialized countries. Most strains of EHEC carry the locus for enterocyte effacement (LEE) pathogenicity island, but a proportion of isolates from patients with severe disease do not carry LEE and very little is known about virulence factors in these organisms. LEE-negative strains of EHEC typically express Shiga toxin 2 and carry a large plasmid that encodes the production of EHEC hemolysin. In this study, we determined the nucleotide sequence of the transfer region of pO113, the large hemolysin plasmid from LEE-negative EHEC O113:H21 (EH41). This 63.9-kb region showed a high degree of similarity with the transfer region of R64, and pO113 was capable of self-transmission at low frequencies. Unlike R64 and the related dot/icm system of Legionella pneumophila, however, pO113 was unable to mobilize RSF1010. In addition, the pO113 transfer region encoded a novel high-molecular-weight serine protease autotransporter of Enterobacteriaceae (SPATE) protein, termed EpeA. Like other SPATEs, EpeA exhibited protease activity and mucinase activity, but expression was not associated with a cytopathic effect on epithelial cells. Analysis of a second high-molecular-weight secreted protein revealed that pO113 also encodes EspP, a cytopathic SPATE identified previously in EHEC O157:H7. The nucleotide sequences encoding the predicted beta-domains of espP and epeA were identical and also shared significant homology with a third SPATE protein, EspI. Both espP and epeA were detected in several LEE-negative clinical isolates of EHEC and thus may contribute to the pathogenesis of this subset of EHEC.

FIG. 1.

Schematic representation of the 63.9-kb transfer region of pO113 and comparison with R64. Grey arrows represent ORFs shared by pO113 and R64. White arrows represent ORFs unique to pO113, and black arrows indicate ORFs present in R64 and not pO113. Arrows indicate the direction of gene transcription. The scale shown is for pO113, and the R64 gene names are provided below the corresponding ORFs. The dotted line represents the absence of an equivalent R64 DNA sequence.

FIG. 2.

Schematic representation of a 9.1-kb BamHI fragment of pO113 containing epeA. The white arrow represents epeA (4,080 bp), and the dark arrows represent other putative ORFs encoded by this fragment. Cleavage positions for KpnI, SacI, BamHI, and XhoI are indicated, and the direction of lacZ transcription from pCR-Script is shown. Structure of EpeA is indicated below the 9.1-kb fragment and shows the N- and C-terminal cleavage sites, serine protease, and P-loop motifs and their corresponding amino acid sequences and positions. The predicted molecular mass of each domain is indicated.

FIG. 3.

SDS-PAGE gel showing the dominant high-molecular-mass proteins, EpeA and EspP, in secreted protein fractions from LEE-negative strains of EHEC. Lane 1, EHEC O157:H7 (EDL933) (LEE positive); lane 2, EHEC O113:H21 (EH41); lane 3, EHEC O113:H21 (EH53); lane 4, EHEC O113:H21 (EH71); lane 5, EHEC O116:H21 (EH42); lane 6, EHEC O130:H11 (EH43); lane 7, EHEC O1:H7 (EH69). Bacteria were grown to mid-log phase, and supernatant proteins were precipitated with TCA and resolved by an SDS-12.5% PAGE gel. Arrows indicate protein bands corresponding to EpeA and EspP. Arrowheads indicate LEE-encoded proteins.

FIG. 4.

Secreted proteins isolated from EHEC O113:H21 (EH41) (lane 1), EH41epeA (lane 2), E. coli XL1-Blue(p13g) (lane 3), and E. coli XL1-Blue(p13gXho) (lane 4). Bacterial strains were grown to mid-log phase, and supernatant proteins were precipitated with TCA and resolved on an SDS-12.5% PAGE gel. The open arrowhead indicates EpeA, and the closed arrowhead indicates EspP. The asterisk indicates a breakdown product of EpeA.

FIG. 5.

(A) Immunoblot demonstrating cleavage of swine pepsin A by concentrated supernatant proteins derived from EH41 (lane 2), EH41epeA (lane 3), E. coli XL1-Blue(p13g) (lane 4), and E. coli XL1-Blue(p13gXho) (lane 5). The arrow indicates uncleaved pepsin, which is also shown for reference in lane 1. (B) Gelatinase zymogram analysis of concentrated supernatant proteins derived from EH41 (lane 2), EH41epeA (lane 3), E. coli XL1-Blue(p13g) (lane 4), and E. coli XL1-Blue(p13gXho) (lane 5). Lane 1 shows activity of secreted proteases from D. nodosus as a positive control. (C) Mucinolytic activity of EpeA. Agar containing 0.5% bovine submaxillary mucin was inoculated with 5 μg of supernatant proteins derived from E. coli XL1-Blue(p13g) (i) and E. coli XL1-Blue(p13gXho) (ii) per ml. Agar was stained with 0.1% amido black.

FIG. 6.

Effect of temperature and pH on EpeA secretion. Western blot analysis of EpeA in whole-cell protein extracts (a lanes) and precipitated supernatant proteins (b lanes) from EH41 cultures grown to an OD₆₀₀ of 1.0 at 24, 30, 37, and 39°C (A) and pH 4.0, 6.0, and 8.0 (B).

FIG. 7.

Southern blot analysis of EcoRI-digested genomic DNA derived from EH41c (lane 1) and EH41 (lane 2). The membrane was hybridized with DIG-labeled beta probe specific for the β-domain of epeA.