Glycohpids from mucosa scrapings of small intestine of neonatal and adult pigs were tested by the thin-layer chromatogram overlay assay for the binding of Escherichiu 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 NeuGca3GahW%$3Cer (NeuGcGM3), the two bands being due to heterogeneity of the ceramide. #en tested against various referenoe glycolipids, NeuAcGM3 was shown to be inactive. This ganghoside was dominating in adult pig. The apparent ~~~~rn~~ disappearance of N-glycolyl groups in glycofipids of intestinal mucosa may have a correspondence in prote~-links sequences as well and thus explain the resistance of adult pigs to infection with E. co& K99.Bacterial adhesion: E. co& K99; Pig intestine; Receptor; Glycohpid; Si~lyliactosyl~r~de; ~-Giycolylneu~mi~~c acidEscherichia culi strains possessing the K99 antigen cause diarrhea in neonatal calves, lambs and piglets [ 1,2], Two virulence factors have been identified, the ability to colonize the small intestine, and the production of enterotoxins that cause the diarrhea. The K99 antigen, which is invoived in the adherence of the bacteria to the brush border of epithelial cells, has been characterized as a fimbrial structure with a single repeating protein subunit. The purified antigen causes a mannose-insensitive agglutination of horse erythrocytes, but no agglutination of guinea-pig erythrocytes. The receptor on horse erythrocytes was identified as NeuGccu3Ga&?4Glc#Cer, based on mass spectrometry and NMR spectroscopy [3] and this was later confirmed f4]_ This info~atio~ was recently supplemented by inhibition of ag~utination of human erythrocytes using intact bacteria and a series of synthetic sialic acid derivatives [5]_ We were interested in knowing the nature of receptor substances in the target cells, the epithelial cells of the small intestine. Furthermore, the known resistance to infection among adult animals [ 1 J was of relevance to test for receptor characteristics.Glycolipids were therefore isolated from mucosa scrapings of piglets and adult pigs and these were tested for binding of E. coli K99 using the thin-layer chromatogram overlay assay [6]. Binding of bacteria to glycolipids coated in microtiter wells was also examined [7]. Only piglet mucosa contained a receptor-active glycofipid, which was isolated and shown to be identical with the earlier identified receptor of horse erythrocytes, namely NeuG~3Ga&34GlqYCer.The condensed representation of sugar chains follows the IUB-IUPAC JCBN recommendations [(1985) J. Biol. Chem. 262, 13-183.
MATERIALS AND METHODS2.1, Growth of bacteria and labeling E. colt strain K-12 Csooh-,ton~I,thr,leu-6,t;4i-1,supE44, faCy_p~'e was used as the host for plasmid pFK99, which ...
