Diarrheal disease caused by enterotoxigenic Escherichia coli expressing the K88 (F4) fimbrial adhesin (K88 ETEC) is a significant source of mortality and morbidity among newborn and weaned piglets. K88 fimbrial adhesins are filamentous surface appendages whose lectin (carbohydrate-binding) activity allows K88 ETEC to attach to specific glycoconjugates (receptors) on porcine intestinal epithelial cells. There are three variants of K88 adhesin (K88ab, K88ac, and K88ad), which possess different, yet related, carbohydrate-binding specificities. We used porcine serum transferrin (pSTf) and purified glycosphingolipids (GSL) to begin to define the minimal recognition sequence for K88 adhesin variants. We found that K88ab adhesin binds with high affinity to pSTf (dissociation constant, 75 M), while neither K88ac nor K88ad adhesin recognizes pSTf. Degradation of the N-glycan on pSTf by extensive metaperiodate treatment abolished its interaction with the K88ab adhesin, indicating that the K88ab adhesin binds to the single N-glycan found on pSTf. Using exoglycosidase digestion of the pSTf glycan, we demonstrated that K88ab adhesin recognizes N-acetylglucosamine (GlcNAc) residues in the core of the N-glycan on pSTf. All three K88 variants were found to bind preferentially to GSL containing a ␤-linked N-acetylhexosamine (HexNAc), either GlcNAc or N-acetylgalactosamine, in the terminal position or, alternatively, in the penultimate position with galactose in the terminal position. Considering the results from pSTf and GSL binding studies together, we propose that the minimal recognition sequence for the K88 adhesin variants contains a ␤-linked HexNAc. In addition, the presence of a terminal galactose ␤-linked to this HexNAc residue enhances K88 adhesin binding.Diarrheal disease caused by enterotoxigenic Escherichia coli expressing the K88 (F4) fimbrial adhesin (K88 ETEC) is a significant source of mortality and morbidity among newborn and weaned piglets (9,25,38,41,59). K88 fimbrial adhesins are filamentous surface appendages whose lectin (carbohydratebinding) activity allows K88 ETEC to attach to specific glycoconjugates (receptors) on porcine intestinal epithelial cells (28). Attachment of bacteria to their receptors on intestinal epithelial cells is an essential step in the colonization of the small intestine by ETEC.Three serologically distinguishable variants of K88 adhesin (K88ab, K88ac, and K88ad) have been identified (21, 40). Each variant consists of a conserved antigenic region shared by all three variants, designated a, and variant-specific antigenic regions, designated b, c, and d for K88ab, K88ac, and K88ad, respectively (13, 29). The antigenic differences among the three K88 variants can be ascribed exclusively to a small number of nucleotide changes in the major fimbrial subunit gene, resulting in the amino acid substitutions that distinguish the three K88 variants. Several researchers have investigated the molecular interaction of K88 fimbrial adhesin variants with erythrocytes, intestinal mucus, and intestina...

Calf diarrhea due to infection by enterotoxigenic Escherichia coli was treated by administration of glycoprotein glycans derived from bovine plasma. The glycan moieties of the nonimmunoglobulin fraction of plasma mimicked the oligosaccharide moiety of intestinal receptors recognized by K99 pili. These glycoprotein glycans inhibited adhesion of E. coli K99+ ST' to erythrocyte glycoconjugates in vitro, and they protected colostrum-deprived newborn calves against lethal doses of enterotoxigenic E. coli (1010 bacteria). Adhesion of bacteria to the intestines (duodenum, jejunum, and ileum) was significantly reduced (by 2 orders of magnitude) in treated calves.

In this study we show that the adhesion to mucus of the enterotoxigenic Escherichia coli strains responsible for diarrhea in calves involves a bacterium-mucin recognition phenomenon in which the bacterial pii and specific mucus receptors carried by the glycoproteins (2,000 to 400 kilodalton) play a major role. An adhesion maximum was observed at a pH of less than 6 (4.75 to 5.25). The sialic acids and galactose appeared to be at least partly responsible for the attachment of K99 pili, whereas F41 pili preferentially recognized desialylated receptors. The attachment of different strains of E. coli characterized by the presence of the three main pili, K99, F41, and FY, known to be responsible for the binding of enterotoxigenic E. coli to the intestinal epithelium of the calf, was studied using Scatchard and Hill analyses. The attachment mechanism of bacteria carrying K99 pili showed positive cooperativity. FY and F41 pili recognized independent receptor sites, the first on sialylated mucus and the second on sialidase-treated mucus. Moreover, F41 pili were found to bind to native mucus according to a negative cooperativity phenomenon. Finally, the recognition sites carried by bacterial pilins may be saturated by some animal glycoprotein glycans which are therefore adhesion inhibitors.

Putative receptors of Escherichia coli K88 fimbriae are either tightly membrane bound or an integral part of membranes. Thus, proteins associated with piglet small intestinal mucosae were solubilized by a detergent (deoxycholate). A 74-kDa glycoprotein (GP74) purified from enterocyte and brush border membrane preparations was specifically detected in vitro by K88ab fimbriae. GP74 was recognized only in the mucosae of phenotypically adhesive animals. Metaperiodate treatment abolished the recognition, indicating that K88ab fimbriae-GP74 binding required the carbohydrate moiety. This glycoprotein belongs to the transferrin family and differed from the serum transferrin of the same adhesive-phenotype piglets. Unlike intestinal transferrin, serum transferrin was recognized independently of the adhesion phenotype. The glycan moieties of intestinal and serum transferrins differed in their molar compositions. Transferrin GP74 contained one monosialylated and monofucosylated glycan chain of the N-acetyllactosamine type. Intestinal holotransferrin exhibited pI values of 5.2, 5.3, 5.5, and 5.6, whereas serum holotransferrin pI values ranged between 5.4 and 6.2. Since mucosal transferrin was found intimately entrapped on membranes, we hypothesize that a K88ab fimbriaetransferrin-cell transferrin receptor complex might allow the bacteria to adhere to specific sites of the mucosa.

Evidence for the existence of two phenotypes of piglets born to experimental herds was obtained based on the susceptibility of intestinal brush borders to adhesion of K99-positive Escherichia cofi. The enterocytes of the K99-receptive piglets displayed a characteristic sialoglycolipid pattern, with a higher content of the monosialoglycolipids I13NeuGc-LacCer (GM~Gc), IV3NeuGc-nLcOse4Cer (SPGGc) and IV3NeuAc-nLcOse4Cer (SPG) and the oligosialogangliosides IV3NeuAc,I13NeuAc-GgOse,Cer (GDla), I13(NeuAc),-GgOse,Cer (GD2), I13(NeuAc)z-GgOse4Cer (GD1 b) and IV3NeuAc,I13(NeuAc)2-GgOse4Cer (GTlb) when compared to the gangliosides of non-receptive piglets. The gangliosides from enterocytes of the non-receptive piglets were mainly the monosialogangliosides 113 NeuAc-GgOse,Cer (GM2) and I13NeuAc-LacCer (GM3), only traces of the other sialoglycolipids being detected. Adhesion of 4C-labelled K99-positive E. cofi cells to the piglet small intestinal sialoglycolipids, as tested by the thin-layer chromatogram overlay assay, revealed that the receptive enterocyte membrane was richer in glycolipids containing K99 receptor structures than the non-receptive enterocyte.Adhesion of K99-positive E. cofi correlated with the degree of sialylation of the brush border glycolipids.