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Characterization of AfaE adhesins produced by extraintestinal and intestinal human Escherichia coli isolates: PCR assays for detection of Afa adhesins that do or do not recognize Dr blood group antigens.

ABSTRACT: Operons of the afa family are expressed by pathogenic Escherichia coli strains associated with intestinal and extraintestinal infections in humans and animals. The recently demonstrated heterogeneity of these operons (L. Lalioui, M. Jouve, P. Gounon, and C. Le Bouguénec, Infect. Immun. 67:5048-5059, 1999) was used to develop a new PCR assay for detecting all the operons of the afa family with a single genetic tool. This PCR approach was validated by investigating three collections of human E. coli isolates originating from the stools of infants with diarrhea (88 strains), the urine of patients with pyelonephritis (97 strains), and the blood of cancer patients (115 strains). The results obtained with this single test and those previously obtained with several PCR assays were closely correlated. The AfaE adhesins encoded by the afa operons are variable, particularly with respect to the primary sequence encoded by the afaE gene. The receptor binding specificities have not been determined for all of these adhesins; some recognize the Dr blood group antigen (Afa/Dr(+) adhesins) on the human decay-accelerating factor (DAF) as a receptor, and others (Afa/Dr(-) adhesins) do not. Thus, the afa operons detected in this study were characterized by subtyping the afaE gene using specific PCRs. In addition, the DAF-binding capacities of as-yet-uncharacterized AfaE adhesins were tested by various cellular approaches. The afaE8 subtype (Afa/Dr(-) adhesin) was found to predominate in afa-positive isolates from sepsis patients (75%); it was frequent in afa-positive pyelonephritis E. coli (55.5%) and absent from diarrhea-associated strains. In contrast, Afa/Dr(+) strains (regardless of the afaE subtype) were associated with both diarrhea (100%) and extraintestinal infections (44 and 25% in afa-positive pyelonephritis and sepsis strains, respectively). These data suggest that there is an association between the subtype of AfaE adhesin and the physiological site of the infection caused by afa-positive strains.

SUBMITTER: Le Bouguenec C

PROVIDER: S-EPMC88018 | biostudies-literature | 2001 May

REPOSITORIES: biostudies-literature

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Characterization of AfaE adhesins produced by extraintestinal and intestinal human Escherichia coli isolates: PCR assays for detection of Afa adhesins that do or do not recognize Dr blood group antigens.

Le Bouguénec C C Lalioui L L du Merle L L Jouve M M Courcoux P P Bouzari S S Selvarangan R R Nowicki B J BJ Germani Y Y Andremont A A Gounon P P Garcia M I MI

Journal of clinical microbiology 20010501 5

Operons of the afa family are expressed by pathogenic Escherichia coli strains associated with intestinal and extraintestinal infections in humans and animals. The recently demonstrated heterogeneity of these operons (L. Lalioui, M. Jouve, P. Gounon, and C. Le Bouguénec, Infect. Immun. 67:5048-5059, 1999) was used to develop a new PCR assay for detecting all the operons of the afa family with a single genetic tool. This PCR approach was validated by investigating three collections of human E. co ...[more]

PMID: 11325983

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Project description:Over the last few years, dramatic increases in our knowledge about diffusely adhering Escherichia coli (DAEC) pathogenesis have taken place. The typical class of DAEC includes E. coli strains harboring AfaE-I, AfaE-II, AfaE-III, AfaE-V, Dr, Dr-II, F1845, and NFA-I adhesins (Afa/Dr DAEC); these strains (i) have an identical genetic organization and (ii) allow binding to human decay-accelerating factor (DAF) (Afa/Dr(DAF) subclass) or carcinoembryonic antigen (CEA) (Afa/Dr(CEA) subclass). The atypical class of DAEC includes two subclasses of strains; the atypical subclass 1 includes E. coli strains that express AfaE-VII, AfaE-VIII, AAF-I, AAF-II, and AAF-III adhesins, which (i) have an identical genetic organization and (ii) do not bind to human DAF, and the atypical subclass 2 includes E. coli strains that harbor Afa/Dr adhesins or others adhesins promoting diffuse adhesion, together with pathogenicity islands such as the LEE pathogenicity island (DA-EPEC). In this review, the focus is on Afa/Dr DAEC strains that have been found to be associated with urinary tract infections and with enteric infection. The review aims to provide a broad overview and update of the virulence aspects of these intriguing pathogens. Epidemiological studies, diagnostic techniques, characteristic molecular features of Afa/Dr operons, and the respective role of Afa/Dr adhesins and invasins in pathogenesis are described. Following the recognition of membrane-bound receptors, including type IV collagen, DAF, CEACAM1, CEA, and CEACAM6, by Afa/Dr adhesins, activation of signal transduction pathways leads to structural and functional injuries at brush border and junctional domains and to proinflammatory responses in polarized intestinal cells. In addition, uropathogenic Afa/Dr DAEC strains, following recognition of beta(1) integrin as a receptor, enter epithelial cells by a zipper-like, raft- and microtubule-dependent mechanism. Finally, the presence of other, unknown virulence factors and the way that an Afa/Dr DAEC strain emerges from the human intestinal microbiota as a "silent pathogen" are discussed.

Project description:BACKGROUND: Angiogenesis has been recently described as a novel component of inflammatory bowel disease pathogenesis. The level of vascular endothelial growth factor (VEGF) has been found increased in Crohn's disease and ulcerative colitis mucosa. To question whether a pro-inflammatory Escherichia coli could regulate the expression of VEGF in human intestinal epithelial cells, we examine the response of cultured human colonic T84 cells to infection by E. coli strain C1845 that belongs to the typical Afa/Dr diffusely adhering E. coli family (Afa/Dr DAEC). METHODOLOGY: VEGF mRNA expression was examined by Northern blotting and q-PCR. VEGF protein levels were assayed by ELISA and its bioactivity was analysed in endothelial cells. The bacterial factor involved in VEGF induction was identified using recombinant E. coli expressing Dr adhesin, purified Dr adhesin and lipopolysaccharide. The signaling pathway activated for the up-regulation of VEGF was identified using a blocking monoclonal anti-DAF antibody, Western blot analysis and specific pharmacological inhibitors. PRINCIPAL FINDINGS: C1845 bacteria induce the production of VEGF protein which is bioactive. VEGF is induced by adhering C1845 in both a time- and bacteria concentration-dependent manner. This phenomenon is not cell line dependent since we reproduced this observation in intestinal LS174, Caco2/TC7 and INT407 cells. Up-regulation of VEGF production requires: (1) the interaction of the bacterial F1845 adhesin with the brush border-associated decay accelerating factor (DAF, CD55) acting as a bacterial receptor, and (2) the activation of a Src protein kinase upstream of the activation of the Erk and Akt signaling pathways. CONCLUSIONS: Results demonstrate that a Afa/Dr DAEC strain induces an adhesin-dependent activation of DAF signaling that leads to the up-regulation of bioactive VEGF in cultured human intestinal cells. Thus, these results suggest a link between an entero-adherent, pro-inflammatory E. coli strain and angiogenesis which appeared recently as a novel component of IBD pathogenesis.

Dataset Information

Characterization of AfaE adhesins produced by extraintestinal and intestinal human Escherichia coli isolates: PCR assays for detection of Afa adhesins that do or do not recognize Dr blood group antigens.

Publications

Characterization of AfaE adhesins produced by extraintestinal and intestinal human Escherichia coli isolates: PCR assays for detection of Afa adhesins that do or do not recognize Dr blood group antigens.

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