Host protein binding and adhesive properties of H6 and H7 flagella of attaching and effacing Escherichia coli

Abstract

It had been suggested that the flagella of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) might contribute to host colonization. In this study, we set out to investigate the adhesive properties of H7 and H6 flagella. We studied the abilities of EHEC EDL933 (O157:H7) and EPEC E2348/69 (O127:H6) flagella to bind to bovine mucus, host proteins such as mucins, and extracellular matrix proteins. Through several approaches, we found that H6 and H7 flagella and their flagellin monomers bind to mucins I and II and to freshly isolated bovine mucus. A genetic approach showed that EHEC and EPEC fliC deletion mutants were significantly less adherent to bovine intestinal tissue than the parental wild-type strains. In addition, we found that EPEC bacteria and H6 flagella, but not EHEC, bound largely, in a dose-dependent manner, to collagen and to a lesser extent to laminin and fibronectin. We also report that EHEC O157:H7 strains agglutinate rabbit red blood cells via their flagella, a heretofore unknown phenotype in this pathogroup. Collectively, our data demonstrate that the H6 and H7 flagella possess adhesive properties, particularly the ability to bind mucins, that may contribute to colonization of mucosal surfaces.

FIG. 1.

Purification of EHEC H7 flagella. A transmission electron micrograph of EPEC E2348/69 (O127:H6) (A) and EHEC EDL933 (O157:H7) (B) grown in 1% tryptone expressing flagella (arrows) is shown. (C) Transmission electron micrograph of purified flagella from EDL933. (D) SDS-PAGE analysis of purified H7 and H6 in 16% SDS-PAGE gel. Both flagella are composed of an approximately 60-kDa subunit. Immunogold labeling of EPEC H6 (E) and EHEC H7 (F) flagella is shown. The inset is a high magnification of the decorated flagella. (G) Western blot analysis of whole bacterial cells by use of antibodies against H6 or H7 flagella demonstrating the specificity of antibodies utilized. Molecular mass markers (in kDa) are indicated on the left.

FIG. 2.

Binding of H6 and H7 flagella and flagellin monomers to mucins I and II. Different concentrations of mucins I and II were immobilized onto nitrocellulose membranes and then incubated with purified H7 or H6 flagellum filaments (A) or flagellin monomers (B). Note the dose-dependent binding of flagella and flagellins to mucins.

FIG. 3.

AEEC flagella mediate binding to cow intestinal mucus. (A) H7 and H6 flagellins binding to 10-fold serial dilutions of crude bovine mucus immobilized onto nitrocellulose membranes. (B) Quantification of bacteria adherent to bovine intestinal tissue, demonstrating the difference in adherence between the wild-type strains and their respective aflagellate fliC mutants. The results shown represent the averages for three separate experiments. *, P < 0.05.

FIG. 4.

Demonstration of binding of H7 flagella to mucins and bovine mucus. Purified H7 flagella were incubated with mucins I (A) and II (B) and bovine mucus (C) immobilized onto glass coverslips. Flagella were stained by immunofluorescence using primary antibodies against flagella from chicken and secondary goat anti-chicken antibodies conjugated to Alexa Fluor 594 (red). The control slide was precoated with albumin only (D).

FIG. 5.

Molecular exclusion chromatography of mucin I and H7 flagella. Elution patterns of mucin I (A) and H7 flagella (B) are shown. (C) SDS-PAGE Coomassie staining of protein peaks obtained from the elution of mucin I (lane a), mucin II (lane b), H6 flagella (lane c), and H7 flagella (lane d). (D) Protein elution pattern after interaction of H7 flagella and mucin I. Note the presence of two peaks, where peak 1 is mucin I bound to H7 flagella (0.2 mg/ml) and peak 2 is only mucin I. (E) SDS-PAGE Coomassie staining of peak 1 (lane 1) and peak 2 (lane 2). (F) SDS-PAGE Coomassie staining of mucin I bound to H7 flagella (0.5 mg/ml) (lane 1). Note that all mucin was bound by flagella. M, mass standards (kDa). An asterisk indicates mucin I, and an arrowhead indicates H7 flagella.

FIG. 6.

Dose-dependent binding of purified H6 and H7 flagella to ECM proteins. Purified H6 (▪) and H7 (▴) flagella (1 ng/well) were immobilized onto 96-well plates and incubated with 10-fold dilutions of collagen (A), laminin (B), fibronectin (C), and vitronectin (D). Binding was quantified by ELISA at an absorbance of 405 nm. The data are a representative of three identical experiments performed in quadruplicate. H6 flagella showed affinity for collagen, laminin, and fibronectin, whereas H7 did not have an affinity for any of these ECM proteins tested. For panels E and F, immobilized flagellins were reacted with collagen or laminin. The binding was detected with anti-collagen (α-collagen) and anti-laminin (α-laminin) antibodies. Note the strong affinity of H6 flagellin for collagen. Molecular mass markers (in kDa) are indicated on the left.

FIG. 7.

HA assays. (A) EHEC EDL933 (O157:H7) hemagglutinates rabbit RBC (positive control). For inhibition of HA, whole bacteria were incubated with rabbit RBC, followed by the addition of twofold serial dilutions of anti-H7, anti-O157, or anti-H6 antibodies. (B) HA mediated by purified H7 and not H6 flagella. (C) Inhibition of HA by mucins I and II. Twofold serial dilutions of mucins were incubated with rabbit RBC and 1 HA unit of purified H7 flagella. A dose-dependent inhibition of H7-mediated HA by mucins was observed. For all HA assays, wells containing RBC with PBS were used as negative controls.