| 1. |
- Quintana-Hayashi, Macarena P, et al.
(författare)
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Porcine intestinal glycosphingolipids recognized by Brachyspira hyodysenteriae
- 2023
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Ingår i: Microbial Pathogenesis. - : Elsevier BV. - 0882-4010. ; 175
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Tidskriftsartikel (refereegranskat)abstract
- Swine dysentery caused by Brachyspira hyodysenteriae is a disease present worldwide with an important economic impact on the farming business, resulting in an increased use of antibiotics. In the present study, we investigated the binding of B. hyodysenteriae to glycosphingolipids from porcine small intestinal epithelium in order to determine the glycosphingolipids involved in B. hyodysenteriae adhesion. Specific interactions between B. hyodysenteriae and two non-acid glycosphingolipids were obtained. These binding-active glycosphingolipids, were characterized by mass spectrometry as lactotetraosylceramide (Galβ3GlcNAcβ3Galβ4Glcβ1Cer) and the B5 glycosphingolipid (Galα3Galβ4GlcNAcβ3Galβ4Glcβ1Cer). Comparative binding studies using structurally related reference glycosphingolipids showed that B. hyodysenteriae binding to lactotetraosylceramide required an unsubstituted terminal Galβ3GlcNAc sequence, while for binding to the B5 pentaosylceramide the terminal Galα3Galβ4GlcNAc sequence is the minimum element recognized by the bacteria. Binding of Griffonia simplicifolia IB4 lectin to pig colon tissue sections from healthy control pig and B. hyodysenteriae infected pigs showed that in the healthy pigs the Galα3Gal epitope was mainly present in the lamina propria. In contrast, in four out of five pigs with swine dysentery there was an increased expression of Galα3Gal in the goblet cells and in the colonic crypts, where B. hyodysenteriae also was present. The one pig that had recovered by the time of necropsy had the Galα3Gal epitope only in the lamina propria. These data are consistent with a model where a transient increase in the carbohydrate sequence recognized by the bacteria occur in colonic mucins during B. hyodysenteriae infection, suggesting that the mucins may act as decoys contributing to clearance of the infection. These findings may lead to novel strategies for treatment of B. hyodysenteriae induced swine dysentery.
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| 2. |
- von Mentzer, Astrid, 1983, et al.
(författare)
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Colonization factor CS30 from enterotoxigenic Escherichia coli binds to sulfatide in human and porcine small intestine
- 2020
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Ingår i: Virulence. - : Informa UK Limited. - 2150-5594 .- 2150-5608. ; 11:1, s. 381-390
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Tidskriftsartikel (refereegranskat)abstract
- The ability to adhere via colonization factors to specific receptors located on the intestinal mucosa is a key virulence factor in enterotoxigenic Escherichia coli (ETEC) pathogenesis. Here, the potential glycosphingolipid receptors of the novel human ETEC colonization factor CS30 were examined by binding of CS30-expressing bacteria to glycosphingolipids on thin-layer chromatograms. We thereby found a highly specific binding of CS30-expressing bacteria to a fast-migrating acid glycosphingolipid of human and porcine small intestine, while no binding was obtained with a mutant ETEC strain unable to express CS30 fimbriae. The CS30 binding glycosphingolipid from human small intestine was isolated and characterized by mass spectrometry as sulfatide (SO3-3Gal beta 1Cer). Comparative binding studies using sulfatides with different ceramide compositions gave a preferential binding of CS30 to sulfatide with d18:1-h24:0 ceramide. This ceramide species of sulfatide was also isolated from human small intestine and characterized by mass spectrometry and antibody binding. These studies implicate sulfatide as candidate receptor for mediating attachment of CS30-fimbriated ETEC to human and porcine small intestinal cells. Our findings may be a basis for designing receptor saccharide analogues for inhibition of the intestinal adhesion of CS30-expressing E. coli.
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| 3. |
- Zalem, Dani, et al.
(författare)
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Biochemical and structural characterization of the novel sialic acid-binding site of Escherichia coli heat-labile enterotoxin LT-IIb.
- 2016
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Ingår i: The Biochemical journal. - 1470-8728. ; 473:21, s. 3923-3936
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Tidskriftsartikel (refereegranskat)abstract
- The structurally related AB5-type heat-labile enterotoxins of Escherichia coli and Vibrio cholerae are classified into two major types. The type I group includes cholera toxin (CT) and E. coli LT-I, whereas the type II subfamily comprises LT-IIa, LT-IIb and LT-IIc. The carbohydrate-binding specificities of LT-IIa, LT-IIb and LT-IIc are distinctive from those of cholera toxin and E. coli LT-I. Whereas CT and LT-I bind primarily to the GM1 ganglioside, LT-IIa binds to gangliosides GD1a, GD1b and GM1, LT-IIb binds to the GD1a and GT1b gangliosides, and LT-IIc binds to GM1, GM2, GM3 and GD1a. These previous studies of the binding properties of type II B-subunits have been focused on ganglio core chain gangliosides. To further define the carbohydrate binding specificity of LT-IIb B-subunits, we have investigated its binding to a collection of gangliosides and non-acid glycosphingolipids with different core chains. A high-affinity binding of LT-IIb B-subunits to gangliosides with a neolacto core chain, such as Neu5Gcα3- and Neu5Acα3-neolactohexaosylceramide, and Neu5Gcα3- and Neu5Acα3-neolactooctaosylceramide was detected. An LT-IIb-binding ganglioside was isolated from human small intestine and characterized as Neu5Acα3-neolactohexaosylceramide. The crystal structure of the B-subunit of LT-IIb with the pentasaccharide moiety of Neu5Acα3-neolactotetraosylceramide (Neu5Ac-nLT: Neu5Acα3Galβ4GlcNAcβ3Galβ4Glc) was determined providing the first information for a sialic-binding site in this subfamily, with clear differences from that of CT and LT-I.
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| 4. |
- Zalem, Dani, et al.
(författare)
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Characterization of the ganglioside recognition profile of Escherichia coli heat-labile enterotoxin LT-IIc
- 2022
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 32:5, s. 391-403
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Tidskriftsartikel (refereegranskat)abstract
- The heat-labile enterotoxins of Escherichia coli and cholera toxin of Vibrio cholerae are related in structure and function. Each of these oligomeric toxins is comprised of one A polypeptide and five B polypeptides. The B-subunits bind to gangliosides, which are followed by uptake into the intoxicated cell and activation of the host's adenylate cyclase by the A-subunits. There are two antigenically distinct groups of these toxins. Group I includes cholera toxin and type I heat-labile enterotoxin of E. coli; group II contains the type II heat-labile enterotoxins of E. coli. Three variants of type II toxins, designated LT-IIa, LT-IIb and LT-IIc have been described. Earlier studies revealed the crystalline structure of LT-IIb. Herein the carbohydrate binding specificity of LT-IIc B-subunits was investigated by glycosphingolipid binding studies on thin-layer chromatograms and in microtiter wells. Binding studies using a large variety of glycosphingolipids showed that LT-IIc binds with high affinity to gangliosides with a terminal Neu5Ac alpha 3Gal or Neu5Gc alpha 3Gal, e.g. the gangliosides GM3, GD1a and Neu5Ac alpha 3-/Neu5Gc alpha 3--neolactotetraosylceramide and Neu5Ac alpha 3-/Neu5Gc alpha 3-neolactohexaosylceramide. The crystal structure of LT-IIc B-subunits alone and with bound LSTd/sialyl-lacto-N-neotetraose d pentasaccharide uncovered the molecular basis of the ganglioside recognition. These studies revealed common and unique functional structures of the type II family of heat-labile enterotoxins.
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| 5. |
- Zalem, Dani
(författare)
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Structural analyses of carbohydrate receptors for enterotoxins and adhesins of enterotoxigenic Escherichia coli
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Carbohydrate-binding proteins expressed by microbes are key determinants in initiating and sustaining infections that account for millions of deaths each year. This thesis fo- cused on proteins integral to infections instigated by enterotoxigenic Escherichia coli (ETEC); estimated as the largest bacterial cause of diarrhea in the world with hundreds of millions of cases each year. ETEC infections are mediated by two primary carbohy- drate-binding proteins; 1) Colonization factors (CF), which facilitate host cell attach- ment, and 2) Enterotoxins, which penetrate host cells to induce a potentially lethal diar- rheal response. By employing biochemical techniques, such as chromatogram binding assays, mass spectrometry and NMR, we dissected the precise mechanisms fundamental for the interactions of ETEC carbohydrate-binding proteins. In the presented papers, the novel colonization factor CS30, and the enterotoxins LT- IIb, and LT-IIc where investigated. Our findings identified the sulfatide glycosphin- golipid as the principal receptor for CS30 and emphasized the significance of the car- bohydrate-presenting lipid moiety in binding. The diarrhea-inducing toxins, LT-IIb and LT-IIc, demonstrated distinct binding specificity to sialic acid presenting glycosphin- golipids, and the presence of such receptors were confirmed in the human intestine. Lastly, structural studies detailed the atomic framework of these binding interactions and quantified the binding affinities. By revealing the specific carbohydrate interactions underpinning both adhesion and toxin action, our study uncovers the intricate processes governing pathogenic infection mechanisms, which may inform the design of next-generation anti-bacterial therapeu- tics, vaccines and diagnostical tools.
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