P. T. J. Willemsen

Characterization of the antigenic and adhesive properties of FaeG, the major subunit of K88 fimbriae

The two K88 serotypes, K88ab and K88ac, differ in terms of antigenic and adhesive properties. The structural determinants of the serotype‐specific epitopes and the identity of the amino acid residues involved in fimbriae‐receptor interaction were studied by the construction and analysis of K88 hybrid proteins in which various parts of the K88ab and K88ac fimbrial subunit FaeG were exchanged, and by in vitro mutagenesis of non‐conserved amino acid residues. Using a set of monoclonal antibodies, several regions or amino acid residues involved in the formation of serotype‐specific antigenic determinants were located. The haemagglutinating activity of the hybrid and mutant proteins revealed several amino acid residues involved in the formation of the receptor binding site. A clear correlation was found between the receptor binding site and the serotype‐specific antigenic determinants.

Receptor-active glycolipids of epithelial cells of the small intestine of young and adult pigs in relation to susceptibility to infection with Escherichia coli K99.

Glycolipids from mucosa scrapings of small intestine of neonatal and adult pigs were tested by the thin‐layer chromatogram overlay assay for the binding of Escherichia coli K99. There was practically no binding to acid or non‐acid glycolipids of adult pig, known to be resistant to infection with this bacterium. However, piglets, which are susceptible to infection, showed a clear binding to a doublet band in the acid glycolipid fraction. The receptor‐active glycolipid was isolated and shown by mass spectrometry, NMR spectroscopy and degradation methods to be NeuGcα‐3Galß4GlcßCer (NeuGc‐GM3), the two bands being due to heterogeneity of the ceramide. When tested against various reference glycolipids, NeuAc‐GM3 was shown to be inactive. This ganglioside was dominating in adult pig. The apparent developmental disappearance of N‐glycolyl groups in glycolipids of intestinal mucosa may have a correspondence in protein‐linked sequences as well and thus explain the resistance of adult pigs to infection with E. coli K99.

Age and serotype dependent binding of K88 fimbriae to porcine intestinal receptors

The porcine small intestine contains several polypeptides that could function as receptors for K88-positive Escherichia coli. The mucus fraction contained three proteins with molecular weights of 25, 35 and 60 kDa respectively, which showed a high affinity for K88-positive E. coli cells, whereas brush borders contained a 16 kDa protein and a set of proteins ranging from 40-70 kDa. Depending on the K88 serotype tested, differences in binding to these proteins were observed. In particular, E. coli cells carrying K88ad fimbriae exhibited only a rather weak binding to mucus proteins. The influence of age of the pig on the presence of K88 receptors was also investigated. One-week-old and 35-days-old post-weaning piglets were shown to contain K88 receptors in their mucus while these receptors were hardly detectable in the mucus of 6-month-old pigs. The presence of receptors in the brush border fraction was shown to be independent of age. The binding of K88 fimbriae to mucus proteins was blocked using a lectin of Euonymus europeaus which specifically recognizes the Gal alpha(1-3)Gal sequence, indicating that this disaccharide forms a significant part of the receptor structure.

Localization and function of FanH and FanG, minor components of K99 fimbriae of enterotoxigenic Escherichia coli

Specific antisera against FanG and against FanH were prepared by immunization with hybrid Cro-LacZ-FanG and Cro-LacZ-FanH proteins, respectively. Immunoblotting with these antisera revealed the presence of FanG and FanH as minor components in purified K99 fimbriae. Mutations were constructed in fanG and fanH and cells defective in FanG or FanH were characterized by ELISA, immunoblotting, adhesion assays and electron microscopy. A minicell experiment showed that the mutations in fanG or fanH had no effect on the expression of the other K99-specific proteins. Cells defective in FanG produced no fimbriae and did not agglutinate horse erythrocytes, but cell-free heat-shock preparations of these cells still bound the K99 glycolipid receptor. Cells defective in FanH produced 1-2% of the K99 fimbriae as compared with wild-type K99 producing cells. These mutant fimbriae appeared to be shorter but were still capable of binding the K99 glycolipid receptor. Apparently, FanG and FanH are not required for binding the K99 receptor. These results and analysis of K99 mutants by immunoblotting using a specific antiserum against another K99 minor component, FanF, indicated that the combinations FanF/FanG and FanF/FanH are required for the initiation and elongation (length determination) of K99 fimbriae formation, respectively.