Glycosphingolipids Recognized by Acinetobacter baumannii.
ABSTRACT: Acinetobacter baumannii is an opportunistic bacterial pathogen associated with hospital-acquired infections, including pneumonia, meningitis, bacteremia, urinary tract infection, and wound infections. Recognition of host cell surface carbohydrates plays a crucial role in adhesion and enables microbes to colonize different host niches. Here the potential glycosphingolipid receptors of A. baumannii were examined by binding of 35S-labeled bacteria to glycosphingolipids on thin-layer chromatograms. Thereby a selective interaction with two non-acid glycosphingolipids of human and rabbit small intestine was found. The binding-active glycosphingolipids were isolated and, on the basis of mass spectrometry, identified as neolactotetraosylceramide (Gal?4GlcNAc?3Gal?4Glc?1Cer) and lactotetraosylceramide (Gal?3GlcNAc?3Gal?4Glc?1Cer). Further binding assays using reference glycosphingolipids showed that A. baumannii also bound to lactotriaosylceramide (GlcNAc?3Gal?4Glc?1Cer) demonstrating that GlcNAc was the basic element recognized. In addition, the bacteria occasionally bound to galactosylceramide, lactosylceramide with phytosphingosine and/or hydroxy fatty acids, isoglobotriaosylceramide, gangliotriaosylceramide, and gangliotetraosylceramide, in analogy with binding patterns that previously have been described for other bacteria classified as "lactosylceramide-binding". Finally, by isolation and characterization of glycosphingolipids from human skin, the presence of neolactotetraosylceramide was demonstrated in this A. baumannii target tissue.
Project description:Certain Helicobacter pylori strains adhere to the human gastric epithelium using the blood group antigen-binding adhesin (BabA). All BabA-expressing H. pylori strains bind to the blood group O determinants on type 1 core chains, i.e. to the Lewis b antigen (Fuc?2Gal?3(Fuc?4)GlcNAc; Le(b)) and the H type 1 determinant (Fuc?2Gal?3GlcNAc). Recently, BabA strains have been categorized into those recognizing only Le(b) and H type 1 determinants (designated specialist strains) and those that also bind to A and B type 1 determinants (designated generalist strains). Here, the structural requirements for carbohydrate recognition by generalist and specialist BabA were further explored by binding of these types of strains to a panel of different glycosphingolipids. Three glycosphingolipids recognized by both specialist and generalist BabA were isolated from the small intestine of a blood group O pig and characterized by mass spectrometry and proton NMR as H type 1 pentaglycosylceramide (Fuc?2Gal?3GlcNAc?3Gal?4Glc?1Cer), Globo H hexaglycosylceramide (Fuc?2Gal?3GalNAc?3Gal?4Gal?4Glc?1Cer), and a mixture of three complex glycosphingolipids (Fuc?2Gal?4GlcNAc?6(Fuc?2Gal?3GlcNAc?3)Gal?3GlcNAc?3Gal?4Glc?1Cer, Fuc?2Gal?3GlcNAc?6(Fuc?2Gal?3GlcNAc?3)Gal?3GlcNAc?3Gal?4Glc?1Cer, and Fuc?2Gal?4(Fuc?3)GlcNAc?6(Fuc?2Gal?3GlcNAc?3)Gal?3GlcNAc?3Gal?4Glc?1Cer). In addition to the binding of both strains to the Globo H hexaglycosylceramide, i.e. a blood group O determinant on a type 4 core chain, the generalist strain bound to the Globo A heptaglycosylceramide (GalNAc?3(Fuc?2)Gal?3GalNAc?3Gal?4Gal?4Glc?1Cer), i.e. a blood group A determinant on a type 4 core chain. The binding of BabA to the two sets of isoreceptors is due to conformational similarities of the terminal disaccharides of H type 1 and Globo H and of the terminal trisaccharides of A type 1 and Globo A.
Project description:The FORS histo-blood group system is the most recently discovered carbohydrate-based human blood group system. FORS is a rare blood group system, and most individuals have naturally occurring anti-FORS1 antibodies in plasma. Screening for anti-FORS1 antibodies is often done by hemagglutination assays using FORS1-expressing sheep erythrocytes, since FORS1-positive human erythrocytes are most often not available. Here, we have characterized the non-acid glycosphingolipids from sheep erythrocytes and isolated subfractions, with mass spectrometry, binding of antibodies and lectins, and by enzymatic hydrolysis. This demonstrated the presence of Forssman and Galili pentaosylceramides, and a Galili heptaosylceramide. Two complex glycosphingolipids recognized by human anti-FORS1 antibodies were characterized as a Forssman neolacto hybrid hexaosylceramide (GalNAc?3GalNAc?3Gal?4GlcNAc?3Gal?4Glc?1Cer) and a Forssman Galili hybrid heptaosylceramide (GalNAc?3GalNAc?3Gal?3Gal?4GlcNAc?3Gal?4Glc?1Cer). These are novel glycosphingolipid structures, and to our knowledge, the first case of an elongated Galili antigen. Thus, the anti-Forssman antibodies in human serum bind not only to the classical Forssman pentaosylceramide (GalNAc?3GalNAc?3Gal?4Gal?4Glc?1Cer), but also when the GalNAc?3GalNAc?3 sequence is presented on a neolacto core chain and even on a Galili carbohydrate sequence.
Project description:Carbohydrate-carbohydrate interactions (CCIs) are of central importance for several biological processes. However, the ultra-weak nature of CCIs generates difficulties in studying this interaction, thus only little is known about CCIs. Here we present a highly sensitive equilibrium-fluctuation-analysis of single liposome binding events to supported lipid bilayers (SLBs) based on total internal reflection fluorescence (TIRF) microscopy that allows us to determine apparent kinetic rate constants of CCIs. The liposomes and SLBs both contained natural Le(x) glycosphingolipids (Gal?4(Fuc?3)GlcNAc?3Gal?4Glc?1Cer), which were employed to mimic cell-cell contacts. The kinetic parameters of the self-interaction between Le(x)-containing liposomes and SLBs were measured and found to be modulated by bivalent cations. Even more interestingly, upon addition of cholesterol, the strength of the CCIs increases, suggesting that this interaction is strongly influenced by a cholesterol-dependent presentation and/or spatial organization of glycosphingolipids in cell membranes.
Project description:OBJECTIVE:To investigate the expression of glycosphingolipids in serum and tissue from patients with cholangiocarcinoma compared with healthy controls. METHODS:Nanospray ionization-linear ion trap mass spectrometry (NSI-MSn) was used to demonstrate the comparative structural glycomics of glycosphingolipids in serum from patients with cholangiocarcinoma (n=15), compared with healthy controls (n?=?15). GM2 expression in cholangiocarcinoma tissues (n?=?60) was evaluated by immunohistochemistry. RESULTS:Eleven glycosphingolipids were detected by NSI-MSn: CMH (ceramide monohexose), Lac-Cer (galactose (Gal)?1-4 glucose (Glc)?1-1'-ceramide), Gb3 (Gal?1-4Gal?1-4Glc?1-1'-ceramide), Gb4/Lc4 (N-acetylgalactosamine (GalNAc)?1-3Gal?1-4Gal?1-4Glc?1-1'-ceramide/Gal?1-4 N-acetylglucosamine (GlcNAc)?1-3Gal?1-4Glc?1-1'-ceramide), GM3 (N-acetylneuraminic acid (NeuAc)2-3Gal?1-4Glc?1-1'-ceramide), GM2 (GalNAc?1-4[NeuAc2-3]Gal?1-4Glc?1-1'-ceramide), GM1 (Gal?1-3GalNAc?1-4[NeuAc2-3]Gal?1-4Glc?1-1'-ceramide), hFA (hydroxylated fatty acid)-CMH, hFA-Lac-Cer, hFA-Gb3, and hFA-GM3. Lac-Cer was the most abundant structure among the lactosides and globosides (normal, 24.40%?±?0.11%; tumor, 24.61%?±?2.10%), while GM3 predominated among the gangliosides (normal, 29.14%?±?1.31%; tumor, 30.53%?±?4.04%). The two glycosphingolipids that significantly differed between healthy controls and patients with cholangiocarcinoma were Gb3 and GM2. High expression of GM2 was associated with vascular invasion in tissue from patients with cholangiocarcinoma. CONCLUSIONS:Altered expression of glycosphingolipids in tissue and serum from patients with cholangiocarcinoma may contribute to tumor growth and progression. The ganglioside GM2, which significantly increased in the serum of patients with cholangiocarcinoma, represents a promising target as a biomarker for cholangiocarcinoma.
Project description:The binding of cholera toxin to the ganglioside GM1 as the initial step in the process leading to diarrhea is nowadays textbook knowledge. In contrast, the knowledge about the mechanisms for attachment of Vibrio cholerae bacterial cells to the intestinal epithelium is limited. In order to clarify this issue, a large number of glycosphingolipid mixtures were screened for binding of El Tor V. cholerae. Several specific interactions with minor complex non-acid glycosphingolipids were thereby detected. After isolation of binding-active glycosphingolipids, characterization by mass spectrometry and proton NMR, and comparative binding studies, three distinct glycosphingolipid binding patterns were defined. Firstly, V. cholerae bound to complex lacto/neolacto glycosphingolipids with the GlcNAc?3Gal?4GlcNAc sequence as the minimal binding epitope. Secondly, glycosphingolipids with a terminal Gal?3Gal?3Gal moiety were recognized, and the third specificity was the binding to lactosylceramide and related compounds. V. cholerae binding to lacto/neolacto glycosphingolipids, and to the other classes of binding-active compounds, remained after deletion of the chitin binding protein GbpA. Thus, the binding of V. cholerae to chitin and to lacto/neolacto containing glycosphingolipids represents two separate binding specificities.
Project description:Shiga toxin (Stx) producing Escherichia coli (STEC) cause the edema disease in pigs by releasing the swine-pathogenic Stx2e subtype as the key virulence factor. Stx2e targets endothelial cells of animal organs including the kidney harboring the Stx receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer, Gal?1-4Gal?1-4Glc?1-1Cer) and globotetraosylceramide (Gb4Cer, GalNAc?1-3Gal?1-4Gal?1-4Glc?1-1Cer). Since the involvement of renal epithelial cells in the edema disease is unknown, in this study, we analyzed the porcine kidney epithelial cell lines, LLC-PK1 and PK-15, regarding the presence of Stx-binding GSLs, their sensitivity towards Stx2e, and the inhibitory potential of Gb3- and Gb4-neoglycolipids, carrying phosphatidylethanolamine (PE) as the lipid anchor, towards Stx2e. Immunochemical and mass spectrometric analysis revealed various Gb3Cer and Gb4Cer lipoforms as the dominant Stx-binding GSLs in both LLC-PK1 and PK-15 cells. A dihexosylceramide with proposed Gal?1-4Gal-sequence (Gal2Cer) was detected in PK-15 cells, whereas LLC-PK1 cells lacked this compound. Both cell lines were susceptible towards Stx2e with LLC-PK1 representing an extremely Stx2e-sensitive cell line. Gb3-PE and Gb4-PE applied as glycovesicles significantly reduced the cytotoxic activity of Stx2e towards LLC-PK1 cells, whereas only Gb4-PE exhibited some protection against Stx2e for PK-15 cells. This is the first report identifying Stx2e receptors of porcine kidney epithelial cells and providing first data on their Stx2e-mediated damage suggesting possible involvement in the edema disease.
Project description:Shiga toxin (Stx) 2e of Stx-producing Escherichia coli (STEC) is the primary virulence factor in the development of pig edema disease shortly after weaning. Stx2e binds to the globo-series glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer, Gal?1-4Gal?1-4Glc?1-1Cer) and globotetraosylceramide (Gb4Cer, GalNAc?1-3Gal?1-4Gal?1-4Glc?1-1Cer), the latter acting as the preferential Stx2e receptor. We determined Stx receptor profiles of 25 different tissues of a male and a female weaned piglet using immunochemical solid phase binding assays combined with mass spectrometry. All probed tissues harbored GSL receptors, ranging from high (category I) over moderate (category II) to low content (category III). Examples of Gb4Cer expression in category I tissues are small intestinal ileum, kidney pelvis and whole blood, followed by colon, small intestinal duodenum and jejunum belonging to category II, and kidney cortex, cerebrum and cerebellum as members of category III organs holding true for both genders. Dominant Gb3Cer and Gb4Cer lipoforms were those with ceramides carrying constant sphingosine (d18:1) and a variable C16:0, C22:0 or C24:1/C24:0 fatty acid. From the mapping data, we created a topographical atlas for Stx2e receptors in piglet tissues and organs, which might be helpful to further investigations on the molecular and cellular mechanisms that underlie infections of Stx2e-producing STEC in pigs and their zoonotic potential for humans.
Project description:Helicobacter suis colonizes the stomach of most pigs and is the most prevalent non-Helicobacter pylori Helicobacter species found in the human stomach. In the human host, H. suis contributes to the development of chronic gastritis, peptic ulcer disease and MALT lymphoma, whereas in pigs it is associated with gastritis, decreased growth and ulcers. Here, we demonstrate that the level of H. pylori and H. suis binding to human and pig gastric mucins varies between individuals with species dependent specificity. The binding optimum of H. pylori is at neutral pH whereas that of H. suis has an acidic pH optimum, and the mucins that H. pylori bind to are different than those that H. suis bind to. Mass spectrometric analysis of mucin O-glycans from the porcine mucin showed that individual variation in binding is reflected by a difference in glycosylation; of 109 oligosaccharide structures identified, only 14 were present in all examined samples. H. suis binding to mucins correlated with glycans containing sulfate, sialic acid and terminal galactose. Among the glycolipids present in pig stomach, binding to lactotetraosylceramide (Gal?3GlcNAc?3Gal?4Glc?1Cer) was identified, and adhesion to Gal?3GlcNAc?3Gal?4Glc at both acidic and neutral pH was confirmed using other glycoconjugates. Together with that H. suis bound to DNA (used as a proxy for acidic charge), we conclude that H. suis has two binding modes: one to glycans terminating with Gal?3GlcNAc, and one to negatively charged structures. Identification of the glycan structures H. suis interacts with can contribute to development of therapeutic strategies alternative to antibiotics.
Project description:Carbohydrate recognition by bovine serum conglutinin has been investigated by inhibition and direct binding assays using glycoproteins and polysaccharides from Saccharomyces cerevisiae (baker's yeast), and neoglycolipids derived from N-acetylglucosamine oligomers, mannobiose and human milk oligosaccharides. The results clearly show that conglutinin is a lectin which binds terminal N-acetylglucosamine, mannose and fucose residues as found in chitobiose (GlcNAc beta 1-4GlcNAc), mannobiose (Man alpha 1-3Man) and lacto-N-fucopentaose II [Fuc alpha 1-4(Gal beta 1-3)GlcNAc beta 1-3Gal beta 1-4Glc] respectively.
Project description: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 diarrhea-inducing 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 F4-mediated 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 GalNAc?3GalNAcß3Galß4Glcß1Cer and GalNAc?3GalNAcß3Galß4GlcNAcß3Galß4Glcß1Cer. 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 (Gal?4Galß4Glcß1Cer) 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 (Gal?3Galß4Glcß1Cer), and neolactotetraosylceramide (Galß4GlcNAcß3Galß4Glcß1Cer).