Identification and characterization of mouse small intestine mucosal receptors for Escherichia coli K-12(K88ab)

Abstract

Adhesion of 3H-labeled Escherichia coli K-12(K88ab) to CID-I mouse small intestine mucus and brush border preparations, immobilized on polystyrene, was studied. E. coli K12(K88ab) was shown to adhere readily to either crude mucus or brush border preparations, but not to bovine serum albumin. In contrast, the nearly isogenic E. coli K-12 strain, i.e., lacking the K88ab plasmid, did not bind well to either mucus, brush borders, or bovine serum albumin. The adhesion of E. coli K-12(K88ab) to both mucus and brush borders required pilus expression (i.e., growth at temperatures greater than 18°C) and was inhibited by pretreatment of either mucus or brush borders with trypsin, pronase, or sodium metaperiodate and by the presence of D-galactosamine. Crude mucus was fractionated by gel filtration, and the proteins in receptor-containing fractions were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Separated proteins were Western blotted to nitrocellulose. Adhesion of 35SO4-labeled E. coli K-12(K88ab) and 35S04-labeled E. coli K-12 to Western blots followed by autoradiography revealed two E. coli K-12(K88ab)-specific mucus receptor proteins (57 and 64 kilodaltons). Brush borders contained the same two receptor proteins present in mucus and an additional 91-kilodalton receptor protein. (Gyles) (K88ab) strain represents a strain of E. coli K-12 (Gyles) which has received the K88ab plasmid.Hereafter these strains will be referred to as E. coli K-12 and E. coli K-12(K88ab). Mucus preparations. Crude mucus was prepared from the small intestines of 5to 8-week-old CD-1 mice (Charles River Breeding Laboratories, Inc., Wilmington, Mass.) as previously described (21). Twenty-four hours before use the mice were deprived of food and given sterile water containing 0.5% (wt/wt) streptomycin sulfate. The following day the animals (usually four to six) were sacrificed, and the small intestines were removed and placed in sterile petri dishes containing HEPES (N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid)-Hanks buffer (pH 7.4). The individual intestines were pooled and cut into 2to 3-cm lengths. Any feces and partially digested food present were expressed from each section with a rubber spatula. The sections were than transferred to a second set of petri dishes containing HEPES-Hanks buffer (pH 7.4) and split open with a scalpel. The split sections were agitated to remove any remaining debris and transferred to a third set of petri dishes. Each section was then gently scraped with a rubber spatula to remove the mucus layer covering the mucosal surface.After the intestinal sections were discarded, the mucosal scrapings were centrifuged at 27,000 x g for 15 min to remove particulate and cellular material. The resulting supernatant, hereafter called crude mucus, was then analyzed for protein content (22) and employed in adhesion assays.Preparation of brush borders. Intestinal cells from the mouse small intestine were prepared by the method of Weiser (42). Twenty-four hours before use the mice were deprived of foo...

In vitro inhibition of adhesion of enterotoxigenic Escherichia coli K88 to piglet intestinal mucus by egg-yolk antibodies

“…to adhere to immobilised crude mucus has been previously described in di¡erent species of animals [17,25]. The K88 adhesin exists in at least three antigenic forms, i.e., K88ab, K88ac, and K88ad, which appear to have adhesive speci¢city for brush border membranes isolated from pigs of di¡erent phenotype [2,3,26,27].…”

Characterization and purification of porcine small intestinal mucus receptor for Escherichia coli K88ac fimbrial adhesin

“…Earlier, Sellwood et al [19] and Anderson et al [22] reported that glycoproteins are involved in the attachment of K88 ¢mbriae to intestinal epithelial cells. Laux et al [12] have also shown that the receptors to K88 ¢mbriae present in mouse small intestinal mucus are glycoproteins, which may contain D-galactosamine or D-galactosamine-like residues.…”

“…The adhesion of radioactively labeled E. coli cells to the intestinal mucus of 14-day-old piglets was studied by using a modi¢cation of the methods of Laux et al [12]. All adhesion assays were performed in triplicate in multiwell polystyrene tissue culture plates (Linbro, £at bottom, 1.6 cm : Nunc, polysorp).…”

“…The presence of both high-and low-a¤nity receptors for the K88 adhesin was ¢rst detected on adhesive brush borders by the use of kinetic binding studies [11]. Since then, putative K88 adhesin receptors have been identi¢ed in both intestinal brush border [12,13] and mucus [7,12,14] preparations. These putative receptors have been reported as being either glycoproteins [7,8,12,13] or glycolipids [14].…”

“…Westerman et al [15] reported that the K88ac variant is the most common, if not the only, variant of K88 adhesin found in pathogenic E. coli isolates from the major pork-producing region of the US. Most of the studies have been concerned with the characterization of receptors of K88ab ¢mbriae [6,7,8,12,14] and K88ac ¢mbriae receptors on intestinal brush border cells [13,16,17]. Few detailed studies on the K88ac ¢mbrial receptor in intestinal mucus are available.…”