Identification of an aggregative adhesion fimbria (AAF) type III-encoding operon in enteroaggregative Escherichia coli as a sensitive probe for detecting the AAF-encoding operon family

Abstract

Enteroaggregative Escherichia coli (EAEC) is recognized as an emerging cause of diarrhea in children and adults worldwide, and recent studies have implicated EAEC in persistent diarrhea in patients infected with human immunodeficiency virus (HIV). In this study, we identified aggregative adhesion fimbria type III (AAF-III) in isolate 55989, a typical EAEC strain. Analysis of the sequence of the plasmid-borne agg-3 gene cluster encoding AAF-III showed this cluster to be closely related to the agg and aaf operons and to the afa operons carried by diffusely adherent pathogenic E. coli. We investigated the adhesion properties of a collection of 25 EAEC strains isolated from HIV-infected patients presenting with persistent diarrhea. We found that a minority of strains (36%) carried sequences similar to those of the agg and aaf operons, which encode AAF-I and AAF-II, respectively. We developed PCR assays specific for the agg-3 operon. In our collection, the frequency of AAF-III strains was similar (12%) to that of AAF-I strains (16%) but higher than that of AAF-II isolates (0%). Differences between EAEC strains in terms of the virulence factors present render detection of these strains difficult with the available DNA probes. Based on comparison of the agg, aaf, and agg-3 operons, we defined an AAF probe internal to the adhesion gene clusters and demonstrated that it was efficient for the identification of EAEC strains. We investigated 32 EAEC isolates, of which only 34.4% were detected with the classical CVD432 probe (detecting pAA virulence plasmids) whereas 65.6% were detected with the AAF probe.

FIG. 1.

Photomicrographs of HeLa cells infected with AAF-III-producing strains: the wild-type strain 55989 (A) and the recombinant HB101(pILL1268) (B). Bacteria displayed an aggregative pattern of adhesion to the cells and, to a lesser extent, to glass coverslips. (C) HB101 (negative control).

FIG. 2.

Structure of the agg-3 region. (A) Physical map of the pILL1268 insert. Restriction fragments hybridizing with the aggR, aspU, and CVD432 probes are indicated. (B) Genetic organization of the 12,012-bp sequenced region. The locations and directions of transcription of the ORFs described in this study are indicated by arrows. The truncated IS1414 element (IS1414∗) is indicated by a box. The region used as the AAF probe is also shown. Abbreviations: B, BamHI; S, SalI.

FIG. 3.

Electron micrographs of AAF-III-producing E. coli strain preparations negatively stained with 1% aqueous ammonium molybdate and shadowed with platinum. (A) Strain 55989; (B) detail of the region framed in panel A; (C) AAEC185(pILL1268). The wild-type 55989 isolate produces both flagella and long, flexible fimbriae, which are involved in the cohesion of bacterial aggregates, as indicated by arrows in panel B. AAEC185(pILL1268) produces fimbriae similar to those observed in 55989. These fimbriae are designated AAF-III. Bars, 0.5 μm.

FIG. 4.

Transmission electron micrographs of HeLa cells infected with E. coli strains expressing the agg-3 operon. (a) Strain 55989; (b) HB101 carrying the recombinant plasmid pILL1268. Bars, 1 μm. (Insets) Intracellular bacteria. Bars, 0.5 μm.

FIG. 5.

Sequence alignment of adhesins encoded by the agg-3, agg, and aaf operons. Gaps, indicated by dashes, have been inserted to optimize the alignment. Asterisks and points represent identical and similar residues, respectively, identified with the CLUSTAL W program. Numbers correspond to amino acid positions in the protein encoded by the agg-3 gene cluster. The predicted translational start site of Agg-3A is indicated by +1.