Identification and characterization of the locus for diffuse adherence, which encodes a novel afimbrial adhesin found in atypical enteropathogenic Escherichia coli

Abstract

The O26 serogroup of enteropathogenic Escherichia coli (EPEC) is one of the serogroups most frequently implicated in infant diarrhea and is also common among enterohemorrhagic E. coli (EHEC) strains. The most common O26 strains belong to EPEC/EHEC serotype O26:H11 and are generally Shiga toxin (Stx) positive. Stx-negative E. coli strains that are negative for the EPEC EAF plasmid and bundle-forming pilus (Bfp) are classified as atypical EPEC. Here, we report a novel adhesin present in an stx-negative bfpA-negative atypical EPEC O26:H11 strain isolated from an infant with diarrhea. A cloned 15-kb genomic region from this strain, designated the locus for diffuse adherence (lda), confers diffuse adherence on HEp-2 cells when expressed in E. coli K-12. Sequence analysis of lda revealed a G+C content of 46.8% and 15 open reading frames sharing homology with the E. coli K88 fae and CS31A clp fimbrial operons. The lda region is part of a putative 26-kb genomic island inserted into the proP gene of the E. coli chromosome. Hybridization studies have demonstrated the prevalence of the minor structural subunit gene, ldaH, across E. coli serogroups O5, O26, O111, and O145. A second plasmid-encoded factor that contributed to the Hep-2 adherence of this strain was also identified but was not characterized. Null mutations that abolish adherence to HEp-2 cells can be restored by plasmid complementation. Antiserum raised against the major structural subunit, LdaG, recognizes a 25-kDa protein from crude heat-extracted protein preparations and inhibits the adherence of the E. coli DH5alpha lda(+) clone to HEp-2 cells. Electron microscopy revealed a nonfimbrial structure surrounding the bacterial cell.

FIG. 1.

HEp-2 adherence assay. (A) E. coli 22 wild type. (B) E. coli DH5α(pV-B-6). (C) E. coli DH5α(pV-B-6-Tn).

FIG. 2.

Comparison of predicted amino acid sequence of LdaH with E. coli K88 fimbrial subunit FaeH. Identical residues in both proteins are indicated by asterisks, and conserved residues are indicated by dots (:, strongly similar; ., weakly similar). The alignment was created by CLUSTAL W.

FIG. 3.

Maps of lda and K88 fae loci (drawn to scale). The locations and directions of genes are indicated by arrows, and the point of Tn5 insertion in pV-B-6-Tn is shown by an inverted triangle. A line scale drawn below indicates the size in kb. The shading patterns within the arrows indicate the predicted functions of the proteins as indicated.

FIG. 4.

Chromosomal location of LDA island. Wide arrows indicate the locations and directions of ORFs and genes. The interrupted proP gene is represented as a discontinuous filled arrow. The small arrows indicate the locations of PCR primers. The region encompassing the cosmid clone is represented by a thin horizontal line. Double bold vertical lines indicate gaps in the sequence.

FIG. 5.

HEp-2 adherence assay. (A) E. coli LDA1. (B) E. coli XL-10(pIJ22). (C) E. coli LDA3 mutant. (D) E. coli LDA3(pIJ20) complement.

FIG. 6.

Plasmid profiles. M, 1-kb DNA ladder markers (Invitrogen); lane 1, E. coli 22; lane 2, E. coli LDA3. The arrow indicates the location of pIJ22-1. The asterisk in lane 2 identifies pIJ30, used to cure pIJ22-1.

FIG. 7.

Detection of LdaG. (A) SDS-PAGE of whole-cell lysates (lanes 1 and 2) and heat-extracted proteins (lanes 3 and 4) from E. coli 22 (lanes 1 and 3) and E. coli LDA1 (lanes 2 and 4). The arrow indicates the location of LdaG (lanes 1 and 3). (B) Western blot analysis of LdaG. E. coli 22 (lane 1), E. coli LDA3 (lane 2), and E. coli DH5α(pIJ20) (lane 3) are shown.

FIG. 8.

Antibody inhibition of HEp-2 adherence. (A) E. coli DH5α(pV-B-6) incubated with preimmune serum (1:50 dilution). (B) E. coli DH5α(pV-B-6) incubated with α-LdaG antiserum (1:50 dilution). (C) E. coli 22 incubated with α-LdaG antiserum (1:5 dilution).

FIG. 9.

Immunogold labeling and negative staining of LDA. (A and B) E. coli 22. The bars represent 200 nm. (C) E. coli 22. The bar represents 50 nm. (D) E. coli LDA1. The bar represents 200 nm. (E) E. coli XL10(pIJ20). The bar represents 200 nm.