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 (original) (raw)
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Porcine intestinal glycosphingolipids recognized by F6-fimbriated enterotoxigenic Escherichia coli
Microbial Pathogenesis, 2014
One important virulence factor of enterotoxigenic Escherichia coli is their ability to adhere via fimbrial adhesins to specific receptors located on the intestinal mucosa. Here, the potential glycosphingolipid receptors of enterotoxigenic F6-fimbriated E. coli were examined by binding of purified F6 fimbriae, and F6-expressing bacteria, to glycosphingolipids on thin-layer chromatograms. When intestinal mucosal non-acid glycosphingolipids from single pigs were assayed for F6 binding capacity, a selective interaction with two glycosphingolipids was observed. The binding-active glycosphingolipids were isolated and characterized as lactotriaosylceramide (GlcNAcb3Galb4Glcb1Cer) and lactotetraosylceramide (Galb3GlcNAcb3Galb4Glcb1Cer). Further binding assays using a panel of reference glycosphingolipids showed a specific interaction between the F6 fimbriae and a number of neolacto core chain (Galb4GlcNAc) glycosphingolipids. In addition, an occasional binding of the F6 fimbriae to sulfatide, galactosylceramide, lactosylceramide with phytosphingosine and/or hydroxy fatty acids, isoglobotriaosylceramide, gangliotriaosylceramide, and gangliotetraosylceramide was obtained. From the results we conclude that lactotriaosylceramide and lactotetraosylceramide are major porcine intestinal receptors for F6-fimbriated E. coli.
The Journal of Biochemistry, 1991
Non-acid glycosphingolipid expression was studied in the large intestines from four individuals with the A]Le(a-b +), BLe(a-b +), and OLe(a-b +) blood group phenotypes. In the A, Le(a-b +) case, specimens were taken from the ascending and sigmoid parts of the large intestine in order to compare the expression of glycolipids in the proximal and distal regions of the intestine. In one blood group OLe(a-b +) individual, epithelial cells were isolated from the residual stroma to compare the glycolipid compositions in these two tissue compartments. GlcCer, GalCer, LacCer, Gb 3 Cer, and Gb,Cer were the major compounds in all three individuals, as shown by mass spectrometry, proton NMR spectroscopy, and degradation studies. The Le°-5 glycolipid was the major complex blood group glycolipid in all individuals, except in the proximal ascending part of the large intestine of the AjLefa-b +) case, in which the Le b-6 glycolipid was predominant. There were trace amounts of blood group ABH glycolipids, in agreement with the ABO blood group phenotypes of the donors, Lewis antigens with more than six sugar residues in the carbohydrate chain, and blood group X and Y glycolipid antigens. The epithelial cells were dominated by monoglycosylceramides and the Le a-5 glycolipid, while only trace amounts of di-, tri-, and tetraglycosylceramide structures were present. No reactivity was seen in the epithelial cell fraction with Galal-4Gal specific Escherichia coli, anti-P\ or anti-P antibodies, indicating the absence of the glycolipid-borne Galal-4Gal sequence in human large intestinal epithelial cells.
PLoS ONE, 2011
Enterotoxigenic F4-fimbriated Escherichia coli is associated with diarrheal disease in neonatal and postweaning pigs. The F4 fimbriae mediate attachment of the bacteria to the pig intestinal epithelium, enabling an efficient delivery of diarrheainducing enterotoxins to the target epithelial cells. There are three variants of F4 fimbriae designated F4ab, F4ac and F4ad, respectively, having different antigenic and adhesive properties. In the present study, the binding of isolated F4ab, F4ac and F4ad fimbriae, and F4ab/ac/ad-fimbriated E. coli, to glycosphingolipids from erythrocytes and from porcine small intestinal epithelium was examined, in order to get a comprehensive view of the F4-binding glycosphingolipids involved in F4mediated hemagglutination and adhesion to the epithelial cells of porcine intestine. Specific interactions between the F4ab, F4ac and F4ad fimbriae and both acid and non-acid glycosphingolipids were obtained, and after isolation of binding-active glycosphingolipids and characterization by mass spectrometry and proton NMR, distinct carbohydrate binding patterns were defined for each fimbrial subtype. Two novel glycosphingolipids were isolated from chicken erythrocytes, and characterized as GalNAca3GalNAcß3Galß4Glcß1Cer and GalNAca3GalNAcß3Galß4GlcNAcß3Galß4Glcß1Cer. These two compounds, and lactosylceramide (Galß4Glcß1Cer) with phytosphingosine and hydroxy fatty acid, were recognized by all three variants of F4 fimbriae. No binding of the F4ad fimbriae or F4ad-fimbriated E. coli to the porcine intestinal glycosphingolipids occurred. However, for F4ab and F4ac two distinct binding patterns were observed. The F4ac fimbriae and the F4ac-expressing E. coli selectively bound to galactosylceramide (Galß1Cer) with sphingosine and hydroxy 24:0 fatty acid, while the porcine intestinal glycosphingolipids recognized by F4ab fimbriae and the F4ab-fimbriated bacteria were characterized as galactosylceramide, sulfatide (SO 3-3Galß1Cer), sulf-lactosylceramide (SO 3-3Galß4Glcß1Cer), and globotriaosylceramide (Gala4Galß4Glcß1Cer) with phytosphingosine and hydroxy 24:0 fatty acid. Finally, the F4ad fimbriae and the F4ad-fimbriated E. coli, but not the F4ab or F4ac subtypes, bound to reference gangliotriaosylceramide (GalNAcß4Galß4Glcß1Cer), gangliotetraosylceramide (Galß3GalNAcß4Galß4Glcß1Cer), isoglobotriaosylceramide (Gala3-Galß4Glcß1Cer), and neolactotetraosylceramide (Galß4GlcNAcß3Galß4Glcß1Cer).
Escherichia coli K88 receptor expression in intestine of disease-susceptible weaned pigs
Veterinary Microbiology, 1999
A challenge trial was carried out in which Escherichia coli O157 K88ac was administered to a litter of weaned pigs and the development of the disease monitored over a five-day experimental period. The eight animals in the trial were assigned to two groups depending on whether they exhibited disease symptoms. Six pigs developed diarrhoea and two appeared unaffected; these were designated as the test (or K88-susceptible) group and the control (or K88-resistant) group, respectively. The animals were euthanised and the intestine was removed and sections processed for brush border membrane vesicle preparation. Microscopic and biochemical assays were undertaken on tissue samples from each animal and a strong correlation was observed between the expression of a glycoprotein receptor complex associated with the brush border membrane and the development of disease symptoms. Further investigation revealed the presence of an analogous glycoprotein complex in the K88-resistant group which did not bind the K88-fimbriae antigen. These results suggest that genetic differences in the glycosyl moieties of the receptor complex provide the basis for disease susceptibility to K88-positive E. coli.
Infection and Immunity, 1999
In this study, we identified a receptor for the K88ad fimbrial adhesin of Escherichia coli in neutral glycosphingolipid preparations from intestinal epithelial cells of K88ad-adhesive pigs, which was absent in preparations from K88ad-nonadhesive pigs. Neither K88ab nor K88ac adhesin variants bound to this neutral glycosphingolipid. Because this receptor is an intestinal glycosphingolipid that binds K88ad adhesin, it has been designated IGLad. Carbohydrate compositional analysis of a partially purified preparation of IGLad identified galactose, glucose, andN-acetylglucosamine in a ratio of 1.5:1.0:0.5 as the major monosaccharides. Preliminary characterization experiments using lectins showed that IGLad contains the terminal glycanic structure Galβ1-4GlcNAc. Removal of terminal β-linked galactose residues from IGLad decreased the recognition of IGLad by the K88ad adhesin, indicating that terminal β-linked galactose is an essential component of the K88ad adhesin recognition site on IGL...
Structural requirements for the glycolipid receptor of human uropathogenic Escherichia coli
Molecular Microbiology, 1995
The binding of uropathogenic Bscberichia coli to the globo series of glycolipids via P pili is a critical step in the infectious process that is mediated by a humanspecific PapG adhesin. Three classes of PapG adhesins exist with different binding specificities to Galii4Galcontaining glycotipids. The structural basis for PapG recognition of the human glycolipid receptor globoside was investigated by using soluble saccharide analogues as inhibitors of bacterial haemagglutination. The minimum binding epitope was confirmed as the Galu4Gal moiety, but parts of the GalNAcjt and giucose residues, which flank the Gala4Gal in globoside (GbO4). were also shown to be important for strong binding. Furthermore, the same five hydroxyl groups of GalM4Gat In globotriasyl ceramide that were recognized by a previously characterized PapG variant were also recognized by the human-specific PapG in binding the GbO4 that dominates In the human kidney. Saccharide analogues that blocked haemagglutination also blocked the adherence of human uropathogenic E. coli to human kidney sections. Knowledge of the molecular details of the PapG-GbO^ interaction will make it possible to design antiadherence therapeutics.
Journal of General Microbiology, 1990
Binding of purified K99 fimbriae to cryostat sections of pig small intestine was detected. Binding sites were located in the mucus layer, but not in the submucosal connective tissue. High-M, mucin glycopeptides from pig small intestine were found to bind to KW-fimbriated enterotoxigenic Escherjchiu coli, in contrast to non-fimbriated cells. Sialic acid specificity of K99 fimbriae was demonstrated by the significant reduction in binding upon desialylation of mucin glycopeptides. The binding was saturable and the dissociation constant was estimated to be 6 x lo-' M. Fimbriated bacteria were calculated to possess 2.3 x lo3 binding sites per cell. Methods Bacterial cells. E. coli strain B117 (08 : K85ab, K99 : H-), kindly supplied by Dr J. A
FEMS Microbiology Letters, 1989
We examined the effects of a soluble receptorlike complex carbohydrate (an oligomannosidetype glycopeptide) on adherence of a type 1 fimbriated rabbit enteropathogen, Escherichia coli strain RDEC-1. Oligomannoside-type glycopeptide, but not a non-glycosylated peptide mixture used as control, inhibited adherence of type 1 fimbriated RDEC-1 to both rabbit ileal brush borders and guinea pig erythrocytes in vitro. In contrast, during RDEC-1 infection of rabbits the receptor-like oligomannoside did not affect the presence and severity of diarrhea, fecal shedding of organisms, luminal colonization by RDEC-1, or enteroadherence of organisms.
Transplantation, 2007
To avoid hyperacute rejection of xeno-organs, ␣1,3-galactosyltransferase knockout (GalT-KO) pigs have been produced. Gal␣1,3Gal determinant elimination may expose cryptic carbohydrate antigens and/or generate new antigens. This is the first biochemical study of carbohydrate antigens in GalT-KO pig organs. Methods. Neutral and acidic glycolipids were isolated from small intestine and pancreas of two GalT-KO and one wild-type (WT) pig. Glycolipid immune reactivity was tested on thin-layer chromatograms. Small intestine neutral glycolipids were separated by high-performance liquid chromatography and selected fractions were analyzed by proton nuclear magnetic resonance spectroscopy. Total gangliosides were quantified on thin-layer chromatograms and in microtiter wells. Results. Using Gal␣1,3nLc4 glycolipid reference, total Gal␣1,3Gal glycolipid antigens in the WT animal was estimated at about 30 g (small intestine) and 3 g (pancreas) per gram of dry tissue. Gal␣1,3Gal determinants were not detected in GalT-KO tissues at a detection limit of less than 0.25% (small intestine) and 0.5% (pancreas) of the WT tissues. Isoglobotriaosylceramide (iGb3) was absent but trace amounts of Fuc-iGb3 was found in both GalT-KO and WT pig small intestine. Blood group H type 2 core saccharide compounds were increased in GalT-KO pancreas. Total amount of gangliosides was decreased in GalT-KO tissues. The ␣1,3-galactosyltransferase acceptor, N-acetyllactosamine determinant, was not increased in GalT-KO tissues. Human serum antibodies reacted with WT organ Gal␣1,3Gal antigens and gangliosides, of which the ganglioside reactivity remained in GalT-KO tissues. Conclusions. Knockout of porcine ␣1,3-galactosyltransferase gene results in elimination of Gal␣1,3Gal-terminated glycolipid compounds. GalT-KO genetic modification did not produce new compensatory glycolipid compounds reactive with human serum antibodies.
1998
Three antigenic variants of the K88 fimbrial adhesin exist in nature, K88ab, K88ac, and K88ad. Enterotoxigenic Escherichia coli (ETEC) strains that produce these fimbriae cause life-threatening diarrhea in some but not all young pigs. The susceptibility of pigs to these organisms has been correlated with the adherence of bacteria to isolated enterocyte brush borders. Whether that correlation holds for multiple K88 variants and over a broad genetic base of pigs is unknown and was the impetus for this study. We also desired to examine the correlation of the expression of a porcine intestinal brush border mucin-type glycoprotein (IMTGP) which binds K88ab and K88ac with the susceptibility of piglets to K88 ؉ ETEC. Of 31 neonatal gnotobiotic pigs inoculated with K88ab ؉ or K88ac ؉ ETEC, 13 developed severe diarrhea, became dehydrated, and died or became moribund. Another pig became severely lethargic but not dehydrated. In vitro brush border adherence analysis was not possible for 10 of the severely ill pigs due to colonization by challenge strains. However, of the 17 pigs that did not become severely ill, 8 (47%) had brush borders that supported the adherence of K88ab ؉ and K88ac ؉ bacteria in vitro, suggesting a poor correlation between in vitro brush border adherence and piglet susceptibility to K88 ؉ ETEC. By contrast, the expression of IMTGP was highly correlated with susceptibility to K88 ؉ ETEC. Of the 12 pigs that produced IMTGP, 11 developed severe diarrhea. The other pig that produced IMTGP became lethargic but not severely diarrheic. Only 2 of 18 pigs that did not produce IMTGP became severely diarrheic. Colonizing bacteria were observed in histologic sections of intestines from all pigs that expressed IMTGP except for the one that did not develop severe diarrhea. However, colonizing bacteria were observed in histologic sections from only one pig that did not produce IMTGP. The bacterial concentration in the jejuna and ilea of pigs expressing IMTGP was significantly greater (P < 0.005) than that in pigs not expressing IMTGP. These observations suggest the IMTGP is a biologically relevant receptor for K88ab ؉ and K88ac ؉ E. coli or a correlate for expression for such a receptor.