Receptors on Chicken Erythrocytes for F42 Fimbriae of< i> Escherichia coli Isolated from Pigs (original) (raw)
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).
Identification and Purification of F11 Fimbriae on Avian Pathogenic Escherichia Coli
2000
A mannose-resistanthemagglutinating(MRHA)P or P-relatedfimbriae was purified from three E.coli isolates from chickenswith colisepticemia.The polyclonal antiserum against the fimbriae was prepared in rabbits. Examination of the purified fimbriae by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated a major fimbriae subunit of approximately 18 kDa. This band was also reacted with anti-FII serum on Western blotting. The antiserum against the avian P fimbriae strongly reacted with the major subunit of the homologousfimbriae and with FII fimbriae on Western blotting. The first 21 N-terminal amino acid sequence of the major fimbrial subunit of the avian P fimbriaewas identical to that of FII fimbriae. The adhesive properties of the avian P fimbriae was similar to that of FII fimbriae with regard to both MRHA of human erythrocytes and binding to the Gal-Gal class 11receptor. These results indicate that this fimbriae on avian E.coli isolates are closely related to FII fimbriae which are associated with E.coli isolatedfrom human urinarytract infection.
The F4 fimbrial antigen of Escherichia coli and its receptors
Veterinary Microbiology, 2000
F4 or K88 ®mbriae are long ®lamentous polymeric surface proteins of enterotoxigenic Escherichia coli (ETEC), consisting of so-called major (FaeG) and minor (FaeF, FaeH, FaeC, and probably FaeI) subunits. Several serotypes of F4 have been described, namely F4ab, F4ac, and F4ad. The F4 ®mbriae allow the microorganisms to adhere to F4-speci®c receptors present on brush borders of villous enterocytes and consequently to colonize the small intestine. Such ETEC infections are responsible for diarrhea and mortality in neonatal and recently weaned pigs. In this review emphasis is put on the morphology, genetic con®guration, and biosynthesis of F4 ®mbriae. Furthermore, the localization of the different a, b, c, and d epitopes, and the localization of the receptor binding site on the FaeG major subunit of F4 get ample attention. Subsequently, the F4-speci®c receptors are discussed. When the three variants of F4 (F4ab, F4ac, and F4ad) are considered, six porcine phenotypes can be distinguished with regard to the brush border adhesiveness: phenotype A binds all three variants, phenotype B binds F4ab and F4ac, phenotype C binds F4ab and F4ad, phenotype D binds F4ad, phenotype E binds none of the variants, and phenotype F binds F4ab. The following receptor model is described: receptor bcd is found in phenotype A pigs, receptor bc is found in phenotype A and B pigs, receptor d is found in phenotype C and D pigs, and receptor b is found in phenotype F pigs. Furthermore, the characterization of the different receptors is described in which the bcd receptor is proposed as collection of glycoproteins with molecular masses ranging from 45 to 70 kDa, the bc receptor as two glycoproteins with Veterinary Microbiology 71 (2000) 223±244
Identification and Purification of FU Fimbriae on Avian Pathogenic Escherichia coli
2000
A mannose-resistant hemagglutinating (MRHA) P or P-related fimbriae was purified from three Ecoli isolates from chickens with colisepticemia. The polycJonal antiserum against the fimbriae was prepared in rabbits. Examination of the purified fimbriae by SOS-polyacrylamide gel electrophoresis (SOS-PAGE) demonstrated a major fimbriae subunit of approximately 18 kOa. This band was also reacted with anti-FII serum on Western blotting. The antiserum against the avian P fimbriae strongly reacted with the major subunit of the homologous fimbriae and with Fil fimbriae on Western blotting. The first 21 N-terminal amino acid sequence of the major fimbrial subunit of the avian P fimbriae was identical to that of Fil fimbriae. The adhesive properties of the avian P fimbriae was similar to that of Fil fimbriae with regard to both MRHA ofhuman erythrocytes and binding to the Gal-Gal cJass II receptor. These results indicate that this fimbriae on avian Ecoli isolates are cJosely related to Fil fimb...
The F41 adhesin of enterotoxigenicEscherichia coli:inhibition of adhesion by monoclonal antibodies
Veterinary Quarterly, 1998
The anti-adhesive properties of 23 specific monoclonal antibodies (MAbs) against the F41 adhesive fimbrial antigen of enterotoxigenic Escherichia coli (ETEC) were studied in brush border adhesion inhibition tests and haemagglutination inhibition tests with four F41-positive E. coli strains and purified F41 antigen. These MAbs recognize five epitope clusters, F41-1 to F41-5. It was proven that these epitope clusters were located on the 29 kDa F41 major fimbrial subunits. All nine MAbs against epitope cluster 1 inhibited the adhesion of F41-positive strains to brush border preparations of calf and pig intestines and the haemagglutination of sheep and guinea pig erythrocytes by the F41-positive strains and purified F41 antigen. The fourteen MAbs against the other four epitope clusters showed very little to no blocking of adhesion and haemagglutination. The results indicate that the adhesion of F41 to intestinal epithelial cells is mediated by the same domain of the 29 kDa F41 major fimbrial subunit(s) as the adhesion of F41 to erythrocytes. Irrespective of their epitope specificity F41 MAbs protected infant mice against a challenge with F41-positive ETEC. MAbs against all epitope clusters partly protected piglets against challenge with F41-positive ETEC in a similar way. Therefore, we conclude that direct blocking of the receptor binding site located on the major fimbrial subunit is not the main mechanism how antibodies protect against ETEC infection.
Receptor Structure for F1C Fimbriae of Uropathogenic Escherichia coli
Infection and Immunity, 2000
F1C fimbriae are correlated with uropathogenic Escherichia coli strains. Although F1C fimbriae mediate binding to kidney tubular cells, their receptor is not known. In this paper, we demonstrate for the first time specific carbohydrate residues as receptor structure for F1C-fimbria-expressing E. coli. The binding of the F1C fimbriated recombinant E. coli strain HB101(pPIL110-54) and purified F1C fimbriae to reference glycolipids of different carbohydrate compositions was evaluated by using thin-layer chromatography (TLC) overlay and solid-phase binding assays. TLC fimbrial overlay analysis revealed the binding ability of purified F1C fimbriae only to glucosylceramide (GlcCer), 1-linked galactosylceramide 2 (GalCer2) with nonhydroxy fatty acids, lactosylceramide, globotriaosylceramide, paragloboside (nLc 4 Cer), lactotriaosylceramide, gangliotriaosylceramide (asialo-GM 2 [GgO 3 Cer]) and gangliotetraosylceramide (asialo-GM 1 [GgO 4 Cer]). The binding of purified F1C fimbriae as well as F1C fimbriated recombinant E. coli strain HB101(pPIL110-54) was optimal to microtiter plates coated with asialo-GM 2 (GgO 3 Cer). The bacterial interaction with asialo-GM 1 (GgO 4 Cer) and asialo-GM 2 (GgO 3 Cer) was strongly inhibited only by disaccharide GalNAc1-4Gal linked to bovine serum albumin. We observed no binding to globotetraosylceramide or Forssman antigen (Gb 5 Cer) glycosphingolipids or to sialic-acid-containing gangliosides. It was demonstrated that the presence of a GalCer or GlcCer residue alone is not sufficient for optimal binding, and additional carbohydrate residues are required for high-affinity adherence. Indeed, the binding efficiency of F1C fimbriated recombinant bacteria increased by 19-fold when disaccharide sequence GalNAc1-4Gal is linked to glucosylceramide as in asialo-GM 2 (GgO 3 Cer). Thus, it is suggested that the disaccharide sequence GalNAc1-4Gal of asialo-GM 2 (GgO 3 Cer) which is positioned internally in asialo-GM 1 (GgO 4 Cer) is the high-affinity binding epitope for the F1C fimbriae of uropathogenic E. coli.
Biochemical and serological characterization of Escherichia coli fimbrial antigen F1652
FEMS Microbiology Letters, 1992
Mannose-rcsistant hemagglutinating fimbrial antigen F!65 is produced by L~'cherichia col(strains associated with septicemia in piglets anti calves. A fimbrial component with an M~ of 17 21~) as determined by SDS-PAGE was purified to homogeneity from FI65-positive E. col(strain 4787 of serogroup O115. This fimbrial component of F!65 antigen was named F165,. Separation procedures included fast protein liquid chromatography with a Superose 12 column followed by ultracentrifugation and 0.15 M ethanolamine buffer (pH 10.5) dissociation. Upon removal of ethanolaminc, the fimbrial component rcassociated into fimbriae. Amino acid composition analysis indicated that the fimbrial component molecule comprised 158 amino acid residues of
Receptor-specific binding of purified F4 to isolated villi
Veterinary Microbiology, 1999
Different procedures for preparing and purifying F4ac fimbriae of the enterotoxigenic Escherichia coli strain GIS 26 (O149:K91:F4ac LT + Sta + STb + ) were performed and the purity and yield of F4ac were compared. Fimbriae were prepared by either mechanical shearing or heatshock treatment of concentrated bacterial suspensions (10 11 bacteria/ml). The mechanical shearing procedure resulted in approximately 1.7 mg fimbriae (i.e. 74.4% of the isolated protein) and 0.6 mg (25.6%) contaminating proteins per 10 12 bacteria, whereas the yield of fimbriae following heatshock treatment was lower (0.3 mg per 10 12 bacteria, i.e. 26.2%) and the relative contamination higher (1.0 mg per 10 12 bacteria, i.e. 73.8%). A further purification consisted of either anion exchange chromatography (AEC) or electro-elution from SDS-polyacrylamide gels. The electroelution procedure was performed under reducing and denaturing conditions, so that purified FaeG subunits, the major subunit of F4, were finally obtained. The binding activity of fimbriae, nonpurified as well as purified, and FaeG to F4-specific receptors on isolated intestinal villi was assessed in an inhibition adhesion assay. Native fimbriae as well as major subunits were able to bind to the receptors, and the specificity of the binding was demonstrated by blockage with F4ac-specific MAb. # (W. Van den Broeck) 0378-1135/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 -1 1 3 5 ( 9 9 ) 0 0 0 7 6 -0
Veterinary Microbiology, 2000
The adhesin F18ac puri®ed on Sepharose CL 4B column chromatography and SDS-PAGE stained with Coomassie Blue and Western blotting using speci®c anti-F18ac serum presented one band of %17 kDa. Gold immunolabeling revealed that the adhesin F18ac has a ®mbrial structure on the bacterial surface. The ®rst 27 amino acid residues of the N-terminal portion of the adhesin F18ac, showed 92.5% homology (25 amino acids) with the F107 (F18ab) ®mbriae. # 2000 Published by Elsevier Science B.V.
Microbiology, 1994
The porcine brush border glycoproteins of 210 and 240kDa, recognized by Escherichia coli K88ac f imbriae, contained O-linked oligosaccharides. The carbohydrate moieties were analysed by deglycosylation, lectin-binding and agglutination assays. Neuraminidase susceptibility of the 210 kDa receptor suggested that a sialoglycoprotein may act as receptor for the K88ac fimbriae. In contrast, K88ac-binding to the 210 and 240 kDa glycoproteins totally disappeared after fucosidase treatment, indicating the critical role of fucosyl residues at the receptor sites. Among the oligosaccharides extracted from these O-glycoproteins, K88ac fimbriae showed affinity for neutral sugar chains while sialylated species were not recognized. Our data suggest a possible role of the polypeptide backbone in the definition of receptor sites. Specific agglutination b y K88ac-fimbriated E. coli of the erythrocytes of the hamster Mesocricetus auratus was inhibited by the anti-T peanut lectin and the lectins of Datura stramonium, Aleuria aurantia and Maackia amurensis. Hence, w e propose that Galpl-3GalNAc-and Fucal-2Gal~l-3/4GIcNAc-are the main sequences mediating K88ac f imbrial binding. These structures were not detected in the non-adhesive piglet brush borders characterized b y a high carbohydrate content. Additional oligosaccharides probably masked the underlying receptor structures.
Fimbria-specific antibodies detach Escherichia coli from human cells
Infection and Immunity
Antibodies obtained from rabbits immunized with purified adhesion-mediating fimbriae of Escherichia coli SS142 were specific for the fimbriae of the homologous strain; they did not cross-react with isolated fimbriae of three different E. coli strains or with protein extracts from nine other adhesive E. coli strains. The antibodies inhibited adhesion to Intestine 407 tissue culture cell monolayers and hemagglutinating activity of E. coli SS142 but not of several other E. coli strains. The antibodies were able not only to prevent but also to reverse the adhesion of E. coli SS142 to Intestine 407 cells or human erythrocytes. Analysis of the kinetics of inhibition suggested that the antibodies did not competitively inhibit adhesion. Such antibodies can be useful for distinguishing different mechanistic steps of bacterial adhesion. Their ability to reverse bacterial adhesion in vitro may be of clinical relevance.
2003
The F17-G adhesin at the tip of flexible F17 fimbriae of enterotoxigenic Escherichia coli mediates binding to N -acetylb b b b -D -glucosamine-presenting receptors on the microvilli of the intestinal epithelium of ruminants. We report the 1.7 Å resolution crystal structure of the lectin domain of F17-G, both free and in complex with N -acetylglucosamine. The monosaccharide is bound on the side of the ellipsoid-shaped protein in a conserved site around which all natural variations of F17-G are clustered. A model is proposed for the interaction between F17-fimbriated E. coli and microvilli with enhanced affinity compared with the binding constant we determined for F17-G binding to N -acetylglucosamine (0.85 mM ----1 ). Unexpectedly, the F17-G structure reveals that the lectin domains of the F17-G, PapGII and FimH fimbrial adhesins all share the immunoglobulin-like fold of the structural components (pilins) of their fimbriae, despite lack of any sequence identity. Fold comparisons with pilin and chaperone structures of the chaperone/usher pathway highlight the central role of the C-terminal b b b
Infection and Immunity, 2002
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 μM), 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 K8...
Molecular Microbiology, 2004
The F17-G adhesin at the tip of flexible F17 fimbriae of enterotoxigenic Escherichia coli mediates binding to N-acetyl-β-d-glucosamine-presenting receptors on the microvilli of the intestinal epithelium of ruminants. We report the 1.7 Å resolution crystal structure of the lectin domain of F17-G, both free and in complex with N-acetylglucosamine. The monosaccharide is bound on the side of the ellipsoid-shaped protein in a conserved site around which all natural variations of F17-G are clustered. A model is proposed for the interaction between F17-fimbriated E. coli and microvilli with enhanced affinity compared with the binding constant we determined for F17-G binding to N-acetylglucosamine (0.85 mM−1). Unexpectedly, the F17-G structure reveals that the lectin domains of the F17-G, PapGII and FimH fimbrial adhesins all share the immunoglobulin-like fold of the structural components (pilins) of their fimbriae, despite lack of any sequence identity. Fold comparisons with pilin and chaperone structures of the chaperone/usher pathway highlight the central role of the C-terminal β-strand G of the immunoglobulin-like fold and provides new insights into pilus assembly, function and adhesion.