Characterization of Saa, a novel autoagglutinating adhesin produced by locus of enterocyte effacement-negative Shiga-toxigenic Escherichia coli strains that are virulent for humans

Abstract

The capacity of Shiga toxigenic Escherichia coli (STEC) to adhere to the intestinal mucosa undoubtedly contributes to pathogenesis of human disease. The majority of STEC strains isolated from severe cases produce attaching and effacing lesions on the intestinal mucosa, a property mediated by the locus of enterocyte effacement (LEE) pathogenicity island. This element is not essential for pathogenesis, as some cases of severe disease, including hemolytic uremic syndrome (HUS), are caused by LEE-negative STEC strains, but the mechanism whereby these adhere to the intestinal mucosa is not understood. We have isolated a gene from the megaplasmid of a LEE-negative O113:H21 STEC strain (98NK2) responsible for an outbreak of HUS, which encodes an auto-agglutinating adhesin designated Saa (STEC autoagglutinating adhesin). Introduction of saa cloned in pBC results in a 9.7-fold increase in adherence of E. coli JM109 to HEp-2 cells and a semilocalized adherence pattern. Mutagenesis of saa in 98NK2, or curing the wild-type strain of its megaplasmid, resulted in a significant reduction in adherence. Homologues of saa were found in several unrelated LEE-negative STEC serotypes, including O48:H21 (strain 94CR) and O91:H21 (strain B2F1), which were also isolated from patients with HUS. Saa exhibits a low degree of similarity (25% amino acid [aa] identity) with YadA of Yersinia enterocolitica and Eib, a recently described phage-encoded immunoglobulin binding protein from E. coli. Saa produced by 98NK2 is 516 aa long and includes four copies of a 37-aa direct repeat sequence. Interestingly, Saa produced by other STEC strains ranges in size from 460 to 534 aa as a consequence of variation in the number of repeats and/or other insertions or deletions immediately proximal to the repeat domain.

FIG. 1

Adherence of E. coli strains to HEp-2 cells (Giemsa stain). (A) STEC strain 98NK2; (B) E. coli DH1:pHC79; (C) E. coli DH1:pJCP561; (D) E. coli JM109:pJCP562.

FIG. 2

Map of the 98NK2 DNA insert of pJCP562 showing the location of the saa open reading frame (thick arrow) and its putative promoter (p) and transcription termination site (t). The regions with DNA sequence homology with portions of IS3 elements and pO157 (P) are shown beneath the map.

FIG. 3

Alignment of the deduced amino acid sequences of Saa proteins from various STEC strains was carried out using CLUSTAL W (37) with manual adjustment. The locations of the repeat sequences are indicated. -, absence of a residue. Residues which differ from the consensus sequence are shown in bold and are underlined. The numbers on the right indicate the position within the amino acid sequence for each protein. The arrow indicates the predicted signal peptidase cleavage site.

FIG. 4

Adherence of saa-positive STEC strains to HEp-2 cells. The indicated STEC strains were subjected to the quantitative HEp-2 adherence assay as described in Materials and Methods. Data are the mean ± standard error of quadruplicate assays (∗, significantly different from 98NK2 [P < 0.01]; ∗∗, significantly different from 98NK2 [P < 0.001]).

FIG. 5

Western blot analysis of STEC strains. STEC strains were grown in LB broth at 37°C, and lysates were separated by SDS-PAGE, blotted, and probed with anti-Saa antibody as described in Materials and Methods. The mobilities of protein size markers (in kilodaltons) are also shown.

FIG. 6

Electron micrographs of STEC strains after staining with anti-Saa and gold-labeled (20 nm) protein M conjugate. Bar, 0.5 μm.

FIG. 7

Western blot analysis of E. coli JM109 expressing cloned saa genes. (A) Lysates of JM109 carrying the indicated plasmids, or purified Saa, were separated by SDS-PAGE, blotted, and probed with anti-Saa antibody as described in Materials and Methods. The mobilities of protein size markers (in kilodaltons) are also shown. (B) A fresh lysate of E. coli JM109:pJCP563 was subjected to SDS-PAGE with or without boiling, blotted, and probed with anti-Saa antibody. The apparent size of immunoreactive species (arrows) was calculated with reference to the mobility of size markers.

FIG. 8

Adherence of E. coli JM109 expressing cloned saa genes to HEp-2 cells (Giemsa stain). (A) E. coli JM109:pBluescript; (B) JM109:pJCP563; (C) JM109:pJCP564; (D) JM109:pJCP565.

FIG. 9

Analysis of saa-negative derivative of 98NK2. (A) Western blot analysis of 98NK2 and 98NK2-S lysates using polyclonal anti-Saa antibody. (B) Adherence of 98NK2 and 98NK2-S to HEp-2 cells (Giemsa stain).