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Pectin-derived neoglycolipids: Tools for differentiation of Shiga toxin subtypes and inhibitors of Shiga toxin-mediated cellular injury
Carbohydrate Polymers 212 (2019) 323–333
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Pectin-derived neoglycolipids: Tools for differentiation of Shiga toxin subtypes and inhibitors of Shiga toxin-mediated cellular injury
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Gottfried Pohlentz, Daniel Steil, Dennis Rubin, Alexander Mellmann, Helge Karch, ⁎ Johannes Müthing Institute for Hygiene, University of Münster, Robert-Koch-Straße 41, D-48149 Münster, Germany
A R T I C LE I N FO
A B S T R A C T
Keywords: Citrus pectin EHEC Glycovesicles Neoglycolipids Shiga toxin STEC
Gut pathogenic enterohemorrhagic Escherichia coli (EHEC) release Shiga toxins (Stxs) as major virulence factors, which bind to globotriaosylceramide (Gb3Cer, Galα1-4 Galβ1-4Glcβ1-1Cer) on human target cells. The aim of this study was the production of neoglycolipids (neoGLs) using citrus pectin-derived oligosaccharides and their application as potential inhibitors of Stxs. The preparation of neoGLs starts with the reduction of the carboxylic acid group of the pectic poly(α1-4)GalUA core structure to the corresponding alcohol, followed by hydrolytic cleavage of resulting poly(α1-4)Gal into (α1-4)Galn oligosaccharides and their linkage to phosphatidylethanolamine (PE). Thin-layer chromatography overlay assays of the produced (α1-4)Galn-PE and corresponding Amadori (α1-4)Galn=PE neoGLs revealed distinguishable binding patterns for Stx1a, Stx2a, and Stx2e. Furthermore, prepared neoGLs protected Vero cells against the cytotoxic action of Stxs when applied as multivalent glycovesicles. The produced neoGLs are applicable for differentiation of Stx subtypes and represent a promising approach to combat infections of EHEC by blocking their major toxins.
1. Introduction Enterohemorrhagic Escherichia coli (EHEC), a certain subset of Shiga toxin (Stx)-producing E. coli (STEC), can colonize the human intestine and elicit fulminant enteropathies like hemorrhagic colitis (Karpman & Ståhl, 2014). Upon transfer into the circulation, released Stxs cause lifethreatening extraintestinal complications such as the hemolytic-uremic syndrome (HUS) and neurological deficiencies (Karch et al., 2012). Although some progress has been made in clinical practice (Davis, van de Kar, & Tarr, 2014), STEC still remains an important zoonotic issue worldwide due to lack of specific treatment for EHEC-associated HUS (Kampmeier, Berger, Mellmann, Karch, & Berger, 2018). Stxs are AB5 toxins (Bergan, Dyve Lingelem, Simm, Skotland, & Sandvig, 2012) and their B pentamers bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer) with Galα1-4 Galβ1-4Glcβ11Cer structure (Smith, Lord, Roberts, & Johannes, 2004). While the occurrence of STEC strains producing Stx1a and Stx2a, which represent the two major Stx subtypes being associated with severe human infections, correlates with the development of HUS, strains producing Stx2e have been isolated from patients with less severe disease (Friedrich et al., 2002; Karch et al., 2006). Stx1a and Stx2a preferentially bind to
Gb3Cer, whereas Stx2e exhibits some prevalence for globotetraosylceramide (Gb4Cer, Galβ3Galα4 Galβ4Glcβ1Cer), but also binds to Gb3Cer (Müthing et al., 2012; Steil et al., 2018). The Galα1-4 Gal-epitope is the key carbohydrate determinant, which is recognized by the B subunits of Stxs as a highly efficient receptor. When translocated to the blood stream, Stxs bind to surfaceexposed Gb3Cer particularly of human renal and cerebral microvascular endothelial cells (Legros, Dusny et al., 2017; Legros, Pohlentz et al., 2017; Legros, Pohlentz, Steil, & Müthing, 2018), followed by internalization and retrograde routing of the toxin to intracellular targets, where Stx exerts its cytotoxic effects (Johannes, 2017). Stxmediated damage of microvascular endothelial cells underlies the pathological changes in hemorrhagic colitis and HUS caused by EHEC (Bielaszewska & Karch, 2005). However, it is worth noting that Gb3Cerpositive and Stx-sensitive developing human erythrocytes have been recently identified as further Stx targets (Betz et al., 2016), a finding that might explain the occurrence of anemia observed in HUS patients (Zoja, Buelli, & Morigi, 2010). The rational design of soluble multivalent Stx inhibitors is a very real prospect to target the Gb3Cer-Stx recognition event, and a number of possible therapeutics designed to inhibit the actions of Stxs has been
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Corresponding author. E-mail addresses: [email protected] (G. Pohlentz), [email protected] (D. Steil), [email protected] (D. Rubin), [email protected] (A. Mellmann), [email protected] (H. Karch), [email protected] (J. Müthing). https://doi.org/10.1016/j.carbpol.2019.02.039 Received 15 November 2018; Received in revised form 4 February 2019; Accepted 12 February 2019 Available online 15 February 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.
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of 100 μL of a freshly prepared solution of sodium cyanoborohydride (10 mg/mL in chloroform/methanol (1/1, v/v) with 1% acetic acid) were successively added over a period of 16 h and incubated at 60 °C. The solvent was evaporated, the residue suspended in water and dialyzed, followed by lyophilization yielding 16.9 mg of a (α1-4)Galn-PEcontaining neoGL preparation.
developed (Melton-Celsa & O’Brien, 2014). Preparations of Gb3 derivatives with terminal Galα1-4 Gal-epitope via a chemical or chemoenzymatic synthesis warrant a lot of technical effort and a cost-intensive production line in particular on larger quantities required for therapeutic applications (Kamath et al., 2004). There is hence a need for preparing glycoconjugates with high binding affinity towards Stxs, which are easily accessible, producible at low costs and, most importantly, suitable for neutralizing Stx-mediated cellular injury. Citrus pectin is composed of D-galacturonic acid (GalUA) as the major constituent forming linear polymers, which have a backbone consisting of α1-4-linked GalUA monomers (Caffall & Mohnen, 2009). Citrus pectin has been found to exhibit marginal protection for human colonic cells against the cytotoxic action of Stxs, whereas pectic oligosaccharides exhibited a slight but significant protective effect when applied at high concentrations in cell cultures (Olano-Martin, Williams, Gibson, & Rastall, 2003). The aim of this study was to proceed in a way to produce pectin-derived neoglycolipids (neoGLs) with the potential to inhibit Stx-mediated cellular damage. To this end we chemically transformed pectin (α1-4)GalUA polymers into (α1-4)Galn oligosaccharides, which were then transferred onto a lipid anchor. We postulated that EHEC-released Stx1a, Stx2a, and Stx2e subtypes should exhibit different binding preferences to the prepared pectin-derived neoGLs and that lipid vesicles spiked with the produced neoGLs (glycovesicles) might act as multivalent inhibitors of the cytotoxic action of various Stx subtypes. Orally applied as a dietary supplement neoGLs, inserted in vesicles or encapsulated, e. g., in pectin-alginate particles to provide gastro-resistance (Gartziandia et al., 2018), should bind up free Stx(s) within the intestine so that the toxin(s) could not act either locally or systemically (Armstrong et al., 1995; Bengmark, 2003; MeltonCelsa & O’Brien, 2014).
2.3. Mass spectrometry of pectic oligosaccharides and pectin-derived neoGLs of (α1-4)Galn-PE type Pectin-derived (α1-4)Galn and (α1-4)GalUAn oligosaccharides as well as produced (α1-4)Galn-PE type neoGLs were structurally characterized by nano electrospray ionization mass spectrometry (nanoESI MS). To this end, oligosaccharides were dissolved in 50% methanol and neoGLs in chloroform/methanol (2/8, v/v). MS experiments were carried out on a SYNAPT G2-S mass spectrometer (Waters, Manchester, UK) equipped with a Z-spray source in the positive ion sensitivity mode. Typical source parameters were: source temperature: 80 °C, capillary voltage: 0.8 kV, sampling cone voltage: 20 V, and source offset voltage: 50 V. For low energy collision-induced dissociation (CID) experiments, precursor ions of interest were selected in the quadrupole analyzer and fragmented in the trap cell using a collision gas (Ar) flow rate of 2.0 mL/min and collision energies up to 50 eV (Elab). For MS analysis of overlay assay-detected Stx-binding (α1-4)Galn-PE type neoGLs (see 2.5. Thin-layer chromatography overlay assay detection of Stx-binding (α1-4)Galn-PE type neoGLs) the impregnation agent was removed with chloroform and the neoGLs were extracted from scraped silica gel with chloroform/methanol (1/1, v/v) following a previously published protocol (Meisen, Peter-Katalinić, & Müthing, 2004). The obtained residue was taken up in 20 μL of chloroform/methanol (2/8, v/v) and analysed by nanoESI MS as described above in this paragraph.
2. Material and methods 2.4. Stx1a, Stx2a, and Stx2e subtypes 2.1. Preparation of pectic (α1-4)Galn oligosaccharides (Krall & McFeeters, 1998; Taylor & Conrad, 1972)
Stx1a-containing supernatants from bacterial liquid cultures were produced with the EHEC wild-type strains of serotype O84:H- (7792/ 96), O118:H- (5028/97), and O145:H- (2074/97) (Müthing et al., 2012). Employed Stx2a-producing EHEC wild-type strains were those of serotype O26:H11 (356/97), O103:H2 (2636/97), and O157:H- (0296/ 96 and 3448/98); Stx2e-containing supernatants were from STEC wildtype strains of serotype ONT:H10 (24059/97) and O101:H9 (VUBEH60) (Müthing et al., 2012). The supernatants were used after sterile filtration in the overlay and cell culture assays.
An amount of 3 g of N-cyclohexyl-N’-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate was dissolved in 200 mL of deionized water and 100 mg of citrus low methylated pectin (ELICITYL, Crolles, France; GAT102, batch number E1004-09 EC8, degree of methylation < 5%, MW ˜150 kDa) in 20 mL of deionized water was added. A constant pH value of 4.75 was maintained with 0.1 M HCl for 2 h. A NaBH4 solution (20 g in 100 mL of deionized water) was added dropwise over 2 h keeping a constant pH of 7.0 by titration with 6 M HCl and the temperature between 25 °C and 35 °C. After dialysis, the glycosidic bonds of the polymer were hydrolyzed with 3.5 mM trifluoroacetic acid at 100 °C under reflux for 30 min. After chilling to 4 °C the hydrolysate was lyophilized, dissolved in water and remaining (α1-4)GalUA-containing oligosaccharides were separated from (α1-4)Galn oligosaccharides by anion exchange chromatography on a column packed with DEAE-Sepharose CL6B (GE Healthcare, Munich, Germany) in its acetate form with water as eluent. The (α1-4)Galn oligosaccharidecontaining eluate fractions were pooled and lyophilized. Yields of the (α1-4)Galn-mixture varied from 8 to 15 mg.
2.5. Thin-layer chromatography overlay assay detection of Stx-binding (α14)Galn-PE type neoGLs Four aliquots of neoGLs (see 2.2. Preparation of pectin-derived neoGLs with (α1-4)Galn oligosaccharides) were applied to a silica gel 60 precoated high-performance thin-layer chromatography (TLC) glass plate (HPTLC plates, size 10 cm x 10 cm, thickness 0.2 mm, no. 1.05633.0001; Merck, Darmstadt, Germany) and separated in the solvent chloroform/methanol/water (60/35/8, v/v/v). The TLC plate was cut into two strips: a small strip 1 (lane 1), which was used for orcinol staining, and a larger strip 2 (lanes 2, 3, and 4), which was used for the Stx overlay assays. After impregnation, strip 2 was cut into 3 parts, which were then subjected to the Stx1a, Stx2a, and Stx2e overlay assay, respectively (Müthing et al., 2012; Steil et al., 2018).
2.2. Preparation of pectin-derived neoGLs with (α1-4)Galn oligosaccharides Phosphatidylethanolamine (PE) was chosen as the lipid anchor for the preparation of neoGLs according to a procedure described by Pohlentz and Egge (1994).The amount of 10 mg of (α1-4)Galn oligosaccharides (see 2.1. Preparation of pectic (α1-4)Galn oligosaccharides) was suspended in 150 μL of dimethylsulfoxide and solubilized with 300 μL of methanol. This solution was mixed with 25 mg of 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (Sigma-Aldrich Chemie GmbH, Munich, Germany; cat. 37,161) dissolved in 2.5 mL of chloroform/methanol (2/1, v/v) and shaken for 2 h at 60 °C. Then 8 portions
2.6. Preparation of neoGL- and Gb3Cer-spiked glycovesicles Multilamellar vesicles were prepared by mixing the lipid constituents in organic solvents, followed by evaporation of the solvent and hydration in phosphate-buffered saline (PBS) as recently described (Steil et al., 2018). Briefly, a mixture of 1 mg of total lipid consisting of 0.4 mg of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC; Sigma324
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binding specificity towards these neoGLs using the TLC overlay assay (Fig. 2). The portrayal in Fig. 2A illustrates the rationale of the TLC overlay assay and Fig. 2B demonstrates binding of Stx1a, Stx2a, and Stx2e to the TLC-separated proposed (α1-4)Galn-PE neoGLs. The Stx subtypes gave characteristic binding patterns, which were markedly different from each other. Stx1a obviously “preferred” neoGLs with short oligosaccharide chains, whereas Stx2a exhibited preference for those with longer glycans as can be inferred from the TLC overlay assays. Stx2e showed a rather weak interaction with the produced neoGLs, but clearly “recognized” at least two neoGLs with opposing glycan chain lengths (Fig. 2B).
Aldrich, Steinheim, Germany; P6354), 0.2 mg of cholesterol (SigmaAldrich; C8667) and 0.4 mg of prepared (α1-4)Galn-PE type neoGLs or reference Gb3Cer was produced from these lipids dissolved in chloroform/methanol solutions. After evaporation of the organic solvents and hydration in 1 mL of PBS, extrusion of the suspension through a polycarbonate membrane resulted in small unilamellar glycovesicles spiked with (α1-4)Galn-PE type neoGLs or Gb3Cer (Steil et al., 2018). 2.7. Vero cell culture inhibition assay of Stx-mediated cytotoxicity The cytotoxic effects of Stxs towards Vero-B4 cells were determined using crystal violet as previously described (Kouzel et al., 2017; Steil et al., 2015). Briefly, aliquots of 4 × 103 cells were seeded in 100 μL medium per well and allowed to adhere for 24 h. Sterile-filtrated Stxcontaining bacterial supernatants, which were serially diluted in cell culture medium, were added in 100 μL volumes in parallel to 100 μL medium (control). After 1 h incubation, the solutions were removed and 200 μL of fresh medium were applied for 48 h. The cell viability was determined photometrically as previously described (Kouzel et al., 2017; Steil et al., 2015). The hypothesized inhibition of Stx-caused cytotoxicity by means of glycovesicles spiked with (α1-4)Galn-PE type neoGLs or reference gold standard Gb3Cer (see 2.6. Preparation of neoGL- and Gb3Cer-spiked glycovesicles) was analyzed in Vero-B4 cell cultures. For this purpose, the glycovesicle solutions were diluted 1:2 with Stx-containing medium or medium only (control) and incubated for 1 h. Afterwards, 100 μL of these solutions were added to the 100 μL cell cultures and incubated for 1 h. The applied amount of Gb3Cer and neoGLs was 20 μg/well, respectively, corresponding to a concentration of 100 μg/mL in the cell culture. The supernatants were discarded, followed by incubation of each well with 200 μL of fresh medium for 48 h. The cell viability was determined photometrically as described above in this paragraph. Results are presented as mean percentage values ± SD of four independent biological replicates related to untreated controls, which were set to 100% cell viability. Statistical data analysis of the inhibition assays was performed using R software (version 3.4.0). Analysis of variance (ANOVA) was used to test the difference between sample groups. Pairwise comparisons between groups were done using the Student’s t-test with corrections for multiple testing based on the Holm-Bonferroni method (Holm, 1979). The difference was considered significant at p < 0.001.
3.3. Structures of Stx1a-, Stx2a-, and Stx2e-binding (α1-4)Galn-PE type neoGLs The overlay-detected Stx-binding neoGLs were extracted from the silica gel and submitted to MS1 and MS2 analysis using the positive ion mode. During the linking reaction of the pectic oligosaccharides to the amino group of PE (see 2.2. Preparation of pectin-derived neoGLs with (α1-4)Galn oligosaccharides), side products of an Amadori rearrangement, so-called 1-amino-1-deoxy ketoses, are formed in addition to (α14)Galn-PE neoGLs, and Galn=PE denote the corresponding Amadori products. This second series of PE-containing species have minus 2 lower molecular masses than the corresponding Galn-PEs, which underwent the complete reductive amination reaction. 3.3.1. Stx1a-binding (α1-4)Galn-PE and (α1-4)Galn=PE neoGLs The ESI mass spectra obtained from TLC-separated Stx1a-positive neoGLs together with proposed structures are shown in Fig. 3A. Suggested structures were (α1-4)Galn-PE type neoGLs carrying two, three, four and six galactose. The neoGL with the Gal2 disaccharide was identified as Amadori rearrangement giving rise of a (α1-4)Gal2=PE structure deduced from the m/z values at 1010.66 and 1032.64, which correspond to the monosodiated [M + Na]+ and disodiated [M−H +2Na]+ ion adduct, respectively. Accordingly, threefold galactosylated neoGLs were identified with (α1-4)Gal3=PE and (α1-4)Gal3-PE structure evidenced by m/z values of 1172.72/1194.69 and 1174.71/ 1196.69 for the [M + Na]+/[M−H+2Na]+ pairs, respectively. Detected ions at m/z 1334.76/1356.74 and 1336.78/1358.77 correspond to the [M + Na]+/[M−H+2Na]+ adducts of (α1-4)Gal4=PE and (α14)Gal4-PE, respectively. The MS2 analysis of proposed Amadori products is exemplarily shown for Gal4= PE in supplementary Fig. S2 depicting the CID spectrum of Gal4=PE (Fig. S2 A) and the corresponding fragmentation scheme (Fig. S2B). The occurrence of Amadori rearrangement products exerting a substantial interaction activity towards Stxs is further addressed in the Discussion below. The presence of a sixfold galactosylated neoGL is indicated by m/z at 1660.89/1682.88 equivalent to (α1-4)Gal6-PE ionizing as [M + Na]+/[M−H+2Na]+ adducts as well. Proposed structures were further verified by CID measurements. The ESI MS2 analysis with the accompanying fragmentation scheme is exemplarily shown for (α1-4)Gal3-PE (Fig. 3B) representing a minor Stx1a-positive neoGL as concluded from the TLC analysis (see Fig. 2B).
3. Results 3.1. Production and purification of (α1-4)Galn oligosaccharides from citrus pectin To produce neoGLs harboring (α1-4)Galn oligosaccharides, low methylated citrus pectin (degree of methylation < 5%) with poly(α14)GalUA core structure was used as starting material (Fig. 1). After chemical reduction of the carboxyl to the corresponding alcohol groups, the glycosidic bonds between the saccharide units of the resulting (α14)Gal polymers were cleaved by acidic hydrolysis to yield (α1-4)Galn oligosaccharides. The ESI mass spectrum revealed presence of (α14)Galn oligosaccharides accompanied by non-reduced (α1-4)GalUAn in the hydrolysate (Fig. 1A). Anion exchange chromatography resulted in entire removal of (α1-4)GalUA units, and the ESI mass spectrum indicated a purified fraction of (α1-4)Galn oligosaccharides ranging from (α1-4)Gal2 to (α1-4)Gal6 as shown in Fig. 1B.
3.3.2. Stx2a-binding (α1-4)Galn-PE and (α1-4)Galn=PE neoGLs Following the procedure described above for MS analysis of Stx1apositive neoGLs (see 3.3.1. Stx1a-binding (α1-4)Galn-PE and (α14)Galn=PE neoGLs), a number of Stx2a-binding neoGLs with (α14)Galn-PE and (α1-4)Galn=PE structures was identified and the obtained ESI mass spectra are depicted in Fig. 4A. Structures recognized by Stx2a were (α1-4)Gal2=PE, (α1-4)Gal4=PE, (α1-4)Gal4-PE and (α1-4)Gal6-PE identical to attachment structures of Stx1a. However, the relative binding strengths, which may approximately correlate with color intensities of Stx-positive TLC bands, were markedly different (see insert of Fig. 4A) when compared to Stx1a (see insert of Fig. 3A). Interestingly, Stx2a did not adhere to (α1-4)Gal3=PE and (α1-4)Gal3-PE,
3.2. TLC overlay detection of Stx1a-, Stx2a-, and Stx2e-binding (α14)Galn-PE type neoGLs Chemical coupling of the (α1-4 Gal)n oligosaccharides to the amino group of PE was the final step for producing (α1-4)Galn-PE type neoGLs. The Stx1a, Stx2a, and Stx2e subtypes were then scrutinized for their 325
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Fig. 1. Flow sheet of the production, purification and structural proof of (α1-4)Galn oligosaccharides obtained from citrus pectin. (A) The carboxyl groups of poly(α1-4)GalUA were reduced to the corresponding alcohol, followed by chemical hydrolysis resulting in desired (α14)Galn oligosaccharides accompanied by oligosaccharides containing remained non-reduced (α1-4)GalUA. (B) Separation of (α14)Galn from (α1-4)GalUAn-harboring oligosaccharides was achieved by anion exchange chromatography. The ESI mass spectra were obtained using the positive ion mode. Asterisks indicate gas phase complexes of Galn. According to MS/MS analysis the numbered signals derive from carbodiimide (CDI) adducts of GalUA-containing oligosaccharides. The ions at m/z 608.31 (1) correspond to GalGalUA·CDI [M·CDI]+, those at m/z 806.31 (2) to Gal-GalUA2·CDI [M·CDI-H + Na]+ and those at m/z 968.38 (3) to Gal2-GalUA2·CDI [M·CDI-H + Na]+. They are either neutral or positively charged and thus escape anion exchange chromatography. In addition they give rise to intense signals that do not reflect their actual abundances in the oligosaccharide mixture.
Fig. 4B).
but exhibited strong binding to the neoGL with (α1-4)Gal5-PE structure, deducible from the [M + Na]+/[M−H+2Na]+ ion pair at m/z 1498.85/1520.82. The MS2 analysis of (α1-4)Gal4-PE and the corresponding fragmentation scheme are shown as an example for further structural characterization in Fig. 4B representing one of the major Stx2a-binding neoGLs according to the TLC overlay assay (see insert of
3.3.3. Stx2e-binding (α1-4)Galn-PE and (α1-4)Galn=PE neoGLs As demonstrated in Fig. 5A, the MS analysis of overlay positive (α14)Galn-PE and (α1-4)Galn=PE neoGLs revealed [M + Na]+/[M−H +2Na]+ ion pairs with m/z at 1010.66/1032.64 and 1660.90/1682.88 326
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3.5. Stx1a- and Stx2a-mediated cellular damage Stx1a- and Stx2a-caused cellular damage was investigated in vitro using serially diluted Stx-containing supernatants from liquid cultures of STEC strains to determine the approximate IC50 value for each subtype, i.e., the dilution of Stx-containing supernatant causing ˜50% growth inhibition. Stx2e was excluded, because this subtype did not exert any cytotoxic activity (not shown). Fig. 7 shows the concentration-dependent cytotoxic activity of Stx1a (Fig. 7A) and Stx2a (Fig. 7B). The dilutions employed for the subsequent Stx inhibition assays (see 3.6. Inhibition of Stx1a- and Stx2a-mediated cellular damage by (α14)Galn-PE-containing glycovesicles) are marked with arrows. 3.6. Inhibition of Stx1a- and Stx2a-mediated cellular damage by (α14)Galn-PE- and (α1-4)Galn=PE-containing glycovesicles Fig. 8A schematically depicts the attachment of Stx to Gb3Cer receptors on the cell surface and the adhesion of Stx to glycovesicles spiked with (α1-4)Galn-PE and (α1-4)Galn=PE neoGLs or Gb3Cer. For Stx1a inhibition experiments, Vero cells were treated first with Stx1a alone resulting in a survival rate of 29.3 ± 7.3% (Fig. 8B, +Stx1a, right side, control) in relation to untreated cells, which were set to 100.0 ± 12.3% viability (Fig. 8B, -Stx1a, left side, control). Incubation of the cells with Stx1a together with neoGL-spiked glycovesicles resulted in more than doubling of the survival rate indicated by 62.6 ± 9.0% cell viability (Fig. 8B, +Stx1a, right side, neoGLs). Treatment of the cells with Stx1a in the presence of Gb3Cer-doped glycovesicles (gold standard) increased the survival rate to 89.0 ± 9.1% (Fig. 8B, +Stx1a, right side, Gb3Cer). Exposure of the cells to neoGLs or Gb3Cer alone gave survival rates of 92.7 ± 9.5% (Fig. 8B, -Stx1a, left side, neoGLs) and 84.6 ± 11.1% (Fig. 8B, -Stx1a, left side, Gb3Cer), indicative for a marginal influence of the vesicles on the cell viability. Thus, neoGL-loaded glycovesicles raised more than twice the survival rate and hence protected cells from damage by Stx1a. Glycovesicles loaded with the native Gb3Cer receptor of the toxin exerted the highest efficacy by an almost threefold increase of cellular viability when co-exposed with Stx1a. Similarly, but less pronounced, treatment of cells with Stx2a alone lowered the cell viability to 39.1 ± 4.0% (Fig. 8C, +Stx2a, right side, control). Co-incubation of the cells with Stx2a and neoGL-loaded glycovesicles increased cell survival to 68.8 ± 8.1% (Fig. 8C, +Stx2a, right side, neoGLs) corresponding to an 1.75-fold improvement of survival. Co-exposure of Stx2a with Gb3Cer enhanced the viability to 88.7 ± 2.5% (Fig. 8C, +Stx2a, right side, Gb3Cer) equivalent to more than doubled cell survival. Again, neoGLs or Gb3Cer glycovesicles had per se no toxic effect (Fig. 8C, -Stx2a, left side). Collectively, glycovesicles doped with neoGLs improved cell survival 1.75-fold and protected cells significantly from Stx2a attack. The Gb3Cer gold standard caused somewhat higher protection by more than doubling the survival rate.
Fig. 2. Scheme of the Stx TLC overlay assay (A) and overlay detection of Stx1a-, Stx2a- and Stx2e-binding (α1-4)Galn-PE type neoGLs (B). After separation of the neoGLs, the cut TLC strips were overlaid with bacterial supernatants of Stx subtypes as indicated. Used variants were: Stx1a (O84:H-, 7792/96), Stx2a (O26:H11, 2574/97), and Stx2e (ONT:H10) (see also supplementary Fig. S1). The detection was performed with anti-Stx1 (for Stx1a) and anti-Stx2 antibody (for Stx2a and Stx2e), combined with alkaline phosphatase-conjugated secondary antibody and 5-bromo-4-chloro-3-indolylphosphate (BCIP) as the substrate. Applied amounts of neoGLs for orcinol staining and TLC overlay assays were 20 μg, respectively.
structures, respectively, which were assigned to (α1-4)Gal2=PE and (α1-4)Gal6-PE structures representing the dominant Stx2e receptors. In addition, although only weakly positive in the TLC overlay assay, (α14)Gal4=PE and (α1-4)Gal4-PE were detected as Stx2e ligands. The MS2 analysis of (α1-4)Gal2=PE together with the auxiliary fragmentation scheme are exemplarily shown in Fig. 5B for further structural characterization. 3.4. Differentiation of Stx subtypes based on specific binding patterns towards (α1-4)Galn-PE and (α1-4)Galn=PE neoGLs Fig. 6 comparatively shows the binding patterns of the three Stx subtypes towards the structurally characterized (α1-4)Galn-PE and (α14)Galn=PE neoGLs. Collectively, Stx1a prefers (α1-4)Galn-PEs/(α14)Galn=PEs with 2 ≤ n ≤ 4 and Stx2a (α1-4)Galn-PEs with n ≥ 4, whereas Stx2e showed a rather weak attachment strength to (α14)Galn-PEs/(α1-4)Galn=PEs with a slight preference for (α1-4)Gal6-PE that makes it different from Stx1a and Stx2a. These subtype-specific binding patterns were verified with further two Stx1a variants, three Stx2a variants and one Stx2e variant in comparison to those used so far in this study as shown in supplementary Fig. S1. The results indicate that the respective adhesion pattern towards (α1-4)Galn-PEs/(α14)Galn=PEs is not restricted to a certain variant of a defined Stx subtype, but holds true as a characteristic feature of each additionally probed Stx variant. In other words, the pectin-derived neoGLs are principally applicable for differentiation of the three investigated Stx subtypes.
4. Discussion Within the pharmaceutical industry pectins serve as an emergent source of raw material with potential health benefits. The different facets of pectic oligosaccharides both as prebiotic as well as therapeutic agent are currently extensively debated, especially their impact of certain human diseases (Babbar, Dejonghe, Gatti, Sforza, & Elst, 2016). Pectins meet many regulations easily regarding health applications and there is a body of evidence for the shift of pectin from its conventional role to progressive applications beyond food texture (Lara-Espinoza, Carvajal-Millán, Balandrán-Quintana, López-Franco, & Rascón-Chu, 2018; Nagash, Masoodi, Rather, Wani, & Gani, 2017). Native citrus pectin, harboring linear GalUA polymers consisting of α1-4-linked GalUA as major constituent (Caffall & Mohnen, 2009) have been previously shown to exert no positive effect, whereas pectin327
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Fig. 3. ESI mass spectra of Stx1a-binding (α1-4)Galn-PE and (α1-4)Galn=PE neoGLs (A) and the MS2 analysis of (α1-4)Gal3-PE (B). (A) Mass spectra were obtained from silica gel extracts of Stx1a-positive TLC bands as indicated in the insert (see also Fig. 2B). (B) The precursor ions at m/z 1174.72 were selected for CID analysis and their fragmentation provided the structural proof of the proposed (α1-4)Gal3-PE structure.
addition to the reductive amination products a second series of PEneoGLs was obtained with higher mobility on the TLC plate and substantial binding to Stx subtypes. These species are truly Amadori products for two reasons: First, since the formation of Schiff bases is reversible, thus, remaining ones should be destroyed during the dialysis where oligosaccharide educts are removed from the equilibrium.
derived oligosaccharides applied at high concentrations (10 mg/mL) slightly protected colonic cells from Stx (Olano-Martin et al., 2003). We went forward by conversion of the (α1-4)GalUAn-oligosaccharides to the corresponding (α1-4)Galn-oligosaccharides and transfer onto a PE lipid anchor. These procedures can be performed as easily as the ensuing linkage of the oligosaccharides to the amino group of PE. In 328
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Fig. 4. ESI mass spectra of Stx2a-binding (α1-4)Galn-PE and (α1-4)Galn=PE neoGLs (A) and the MS2 analysis of (α1-4)Gal4-PE (B). (A) Mass spectra were obtained from silica gel extracts of Stx2a-positive TLC bands as indicated in the insert (see also Fig. 2B). (B) The precursor ions at m/z 1336.77 were selected for CID analysis and their fragmentation provided the structural proof of the proposed (α1-4)Gal4-PE structure.
fragmentation schemes. Especially the fragment ion pairs H/I and R/S are highly diagnostic. Their respective differences in m/z of 42 match the loss of ketene, which should not be observed in fragment ion spectra of corresponding Schiff bases.
Second, the CID spectra derived from these compounds clearly corroborate the Amadori structures as exemplarily demonstrated for (α14)Gal2=PE and (α1-4)Gal4=PE in Fig. 5 and supplementary Fig. S2, respectively, showing their CID spectra and the accompanying 329
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Fig. 5. ESI mass spectra of Stx2e-binding (α1-4)Galn-PE and (α1-4)Galn=PE neoGLs (A) and the MS2 analysis of (α1-4)Gal2=PE (B). (A) Mass spectra were obtained from silica gel extracts of Stx2e-positive TLC bands as indicated in the insert (see also Fig. 2B). (B) The precursor ions at m/z 1010.66 were selected for CID analysis and their fragmentation provided the structural proof of the proposed (α1-4)Gal2=PE structure.
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Fig. 6. Stx subtype-specific binding to structurally characterized (α1-4)Galn-PE and (α1-4)Galn=PE neoGLs. The distinct recognition of (α1-4)Galn-PEs and (α14)Galn=PEs ranging from twofold (Gal2) to sixfold (Gal6) galactosylated neoGLs detected by means of TLC overlay assays allows for differentiation of Stx1a, Stx2a, and Stx2e (see Fig. 2 for methodological details).
Fig. 8. Scheme of the interaction of Stx with glycovesicles in the cell culture (A) and protective effect of glycovesicles against Stx1a (B) and Stx2a (C). (A) Illustration of the procedure employed for analyzing inhibitory effects of glycovesicles on Stxmediated cellular damage of Vero cells. (B and C) Cells were treated with Stx alone (+Stx1a, +Stx2a, control, right hand bars) or with Stx in the presence of neoGL- or Gb3Cer-spiked glycovesicles (+Stx1a, +Stx2a, right hand bars as indicated). In addition, cells were incubated without Stx in the absence of glycovesicles (-Stx1a, -Stx2a, control, left hand bars) or without Stx in the presence of neoGL- or Gb3Cer-doped glycovesicles (-Stx1a, -Stx2a, left hand bars as indicated). Significant differences between survival rates of cells exposed to Stx1a or Stx2a alone (control) and to Stx1a or Stx2a co-applied with neoGL- or Gb3Cer-loaded glycovesicles are marked with asterisks (***p < 0.001). Fig. 7. Stx1a- (A) and Stx2a-mediated cellular damage (B). Cells were exposed to decreasing dilutions of Stx1a- or Stx2a-containing supernatants corresponding to increasing Stx concentrations. Applied dilutions ranged from the lowest toxin concentration of 1:216 (65,536-fold) diluted to the highest concentration of 1:27 (128-fold) diluted bacterial cell culture supernatant in case of Stx1a (O145:H-, 2074/97) (A). Used Stx2a (O157:H-, 3448/98) supernatants ranged from the lowest concentration of 1:213 (8.192-fold) to 1:24 (16-fold) dilution (B). The arrows indicate 1:211-diluted Stx1a (A) and 1:25-diluted Stx2a supernatants (B), which were close to the IC50 value and employed for the ensuing inhibition assays (see Fig. 8).
Despite the altered proximal Gal configuration of the Amadori-rearranged (α1-4)Galn=PE neoGLs, which result in a slightly different glycan epitope when compared to the canonical (α1-4)Galn-PE neoGLs, the Amadori products exerted a substantial interaction activity towards the analyzed Stx1a, Stx2a, and Stx2e subtypes. This holds particularly true for the (α1-4)Gal2=PE neoGL, which represents the unique dimeric binding form of the produced Amadori rearrangements. Thus, the chemical modification of the Gal molecule at the reducing end of the 331
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2018).
Galα1-Gal disaccharide, which results in a severely altered glycan epitope in case of the disaccharide, is part of a key carbohydratebinding structure of Stxs when compared to the native cellular receptor Gb3Cer with Galα1-4 Galβ1-4Glcβ1-1Cer structure (Legros et al., 2018). The application of glycovesicles spiked with (α1-4)Galn-PEs and (α1-4)Galn=PEs revealed significant improvement of cell survival upon treatment with Stx1a or Stx2a. The attachment of Stx to the oligosaccharide-decorated glycovesicles is postulated to prevent Stx from binding to its Gb3Cer receptor on the cells and hence to protect cells from Stx. The applied amount of (α1-4)Galn-PEs/(α1-4)Galn=PEs and reference Gb3Cer was 20 μg/well, corresponding to a concentration of 100 μg/mL, respectively, in the cell culture. Thus, a significant toxin neutralization could be obtained with 100-fold lower concentrations of (α1-4)Galn-PEs/(α1-4)Galn=PEs by us compared to previously employed pectic oligosaccharides (Olano-Martin et al., 2003). In contrast to such “natural” products, a number of promising Stx receptor analogues, comprising the Gb3 trisaccharide linked to various scaffolds, has been developed (Kulkarni, Weiss, & Iyer, 2010). Such semi-synthetic toxin binders have been demonstrated in animal models of STEC disease to be effective, but showed no benefit in established HUS infections (MacConnachie & Todd, 2004). Compared to those sophisticated synthetic compounds, pectins represent a more easily accessible starting material being commercially available in almost unlimited quantity from natural resources at comparably low cost (Noreen et al., 2017). This makes pectin-derived (α1-4)Galn-oligosaccharides cost-efficient precursors for the preparation of any type of multivalent Stx receptor competitors. Moreover, pectin-derived (α1-4)Galn-PE type neoGLs work more efficiently than the pure oligosaccharides when exposed in clustered structures that mimic the natural display of lipid-linked glycans (Kulkarni et al., 2010). Importantly, antibiotic use in treatment of EHEC infections is controversial because of concerns about triggering HUS by increasing Stx release (Carnicelli et al., 2016; Wong et al., 2012). Since a causative treatment of EHEC infections is not available, innovative and cost-effective therapeutics for protection against Stxs are urgently needed (Kavaliauskiene, Dyve Lingelem, Skotland, & Sandvig, 2017; Melton-Celsa & O’Brien, 2014). In clinical practice the differentiation of Stx-subtypes is commonly performed by detection of stx genes using state-of-the-art real-time PCR analysis (Qin et al., 2015). The distinction, e.g., of stx1a and stx2a genes from the stx2e gene is important, because presence of stx2e may predict a milder disease with minimal risk of HUS (Friedrich et al., 2002). Additional and/or alternative serological assays are in use for indirect detection of Stx subtypes using more or less specific anti-Stx antibodies (Staples, Fang, Graham, Smith, & Jennison, 2017). However, discrepancies between PCR and serological assays have been described and the failure of several serological assays to differentiate between the various Stx1 and Stx2 subtypes may be of concern (Feng, Jinneman, Scheutz, & Monday, 2011). Unfortunately, PCR and serological assays do not provide information for receptor binding specificity or interaction strength, although binding to the receptor glycolipid(s) is the pivotal point for cellular attachment and internalization of the toxins (Johannes, 2017; Kavaliauskiene et al., 2017). An alternative to PCR and serological assays that operates on the protein level for subtyping of Stx1a, Stx2a, and Stx2e has been recently published by us based on real time interaction analysis that revealed characteristic binding kinetics of each subtype (Steil et al., 2018). Here we add a further alternative to PCR-based assays based on the different TLC adhesion patterns of Stx1a, Stx2a, and Stx2e towards (α1-4)Galn-PEs/(α1-4)Galn=PEs. Our vision is that neoGLs of (α1-4)Galn-PE type could serve as the basis for the development of an Stx-classifying microarray (Liu et al., 2012) or a test kit applicable as a diagnostic tool. Such a microarray or kit would work on the protein level and might be an alternative for commonly employed PCR and/or serological assays. Finally, neoGLs with the potential to neutralize Stx-mediated cellular damage might be applicable as a supportive measure to combat EHEC infections (Kampmeier et al.,
Funding This research was funded by grants from the German Federal Ministry of Education and Research (BMBF) of the German Center for Infection Research (DZIF, TTU 06.801) and InfectControl 2020 IRMRESS [grant number 03ZZ0805B] and conducted under the umbrella of the Interdisciplinary Center for Clinical Research (IZKF) Münster [grant number Müth2/021/15]. Conflicts of interest The authors disclose submitted patent ‘Oligosaccharide composition for binding to lectins’ (PCT/EP2016/082289 and international publication number WO 2017/109023 A1, inventors J.M. and G.P.). The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. Acknowledgments We thank Dagmar Mense, Nikola Skutta and Ralph Fischer for excellent technical assistance. The help with statistical analysis of Dr. Ivan Kouzel is gratefully acknowledged. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.carbpol.2019.02.039. References Armstrong, G. D., Rowe, P. C., Goodyer, P., Orrbine, E., Klassen, T. P., Welss, G., et al. (1995). A phase I study of chemically synthesized verotoxin (Shiga-like toxin) Pktrisaccharide receptors attached to chromosorb for preventing hemolytic-uremic syndrome. The Journal of Infectious Diseases, 171, 1042–1045. Babbar, N., Dejonghe, W., Gatti, M., Sforza, S., & Elst, K. (2016). Pectic oligosaccharides from agricultural by-products: Production, characterization and health benefits. Critical Reviews in Biotechnology, 36, 594–606. https://doi.org/10.3109/07388551. 2014.996732. Bengmark, S. (2003). Use of some pre-, pro- and synbiotics in critically ill patients. Best Practice & Research Clinical Gastroenterology, 17, 833–848. https://doi.org/10.1016/ S1521-6918(03)00073-8. Bergan, J., Dyve Lingelem, A. B., Simm, R., Skotland, T., & Sandvig, K. (2012). Shiga toxins. Toxicon, 60, 1085–1107. https://doi.org/10.1016/j.toxicon.2012.07.016. Betz, J., Dorn, I., Kouzel, I. U., Bauwens, A., Meisen, I., Kemper, B., et al. (2016). Shiga toxin of enterohaemorrhagic Escherichia coli directly injures developing human erythrocytes. Cellular Microbiology, 18, 339–348. https://doi.org/10.1111/cmi.12592. Bielaszewska, M., & Karch, H. (2005). Consequences of enterohaemorrhagic Escherichia coli infection for the vascular endothelium. Thrombosis and Haemostasis, 94, 312–318. https://doi.org/10.1160/TH05-04-0265. Caffall, K. H., & Mohnen, D. (2009). The structure, function, and biosynthesis of plant cell wall pectic polysaccharides. Carbohydrate Research, 344, 1879–1900. https://doi.org/ 10.1016/j.carres.2009.05.021. Carnicelli, D., Arfilli, V., Ricci, F., Velati, C., Tazzari, P. L., & Brigotti, M. (2016). The antibiotic polymyxin B impairs the interactions between Shiga toxins and human neutrophils. The Journal of Immunology, 196, 1177–1185. https://doi.org/10.4049/ jimmunol.1500671. Davis, T. K., van de Kar, N. C., & Tarr, P. I. (2014). Shiga toxin/verotoxin-producing Escherichia coli infections: Practical clinical perspectives. Microbiology Spectrum, 2. https://doi.org/10.1128/microbiolspec.EHEC-0025-2014 EHEC-0025-2014. Feng, P. C., Jinneman, K., Scheutz, F., & Monday, S. R. (2011). Specificity of PCR and serological assays in the detection of Escherichia coli Shiga toxin subtypes. Applied and Environmental Microbiology, 77, 6699–6702. https://doi.org/10.1128/AEM. 00370-11. Friedrich, A. W., Bielaszewska, M., Zhang, W. L., Pulz, M., Kuczius, T., Ammon, A., et al. (2002). Escherichia coli harboring Shiga toxin 2 gene variants: Frequency and association with clinical symptoms. The Journal of Infectious Diseases, 185, 74–84. https:// doi.org/10.1086/338115. Gartziandia, O., Lasa, A., Pedraz, J. L., Miranda, J., Portillo, M. P., Igartua, M., et al. (2018). Preparation and characterization of resveratrol loaded pectin/alginate blend gastro-resistant microparticles. Molecules, 23. https://doi.org/10.3390/ molecules23081886 pii: E1886. Holm, S. (1979). A simple sequentially rejective multiple test procedure. Scandinavian
332
Carbohydrate Polymers 212 (2019) 323–333
G. Pohlentz, et al.
prevention and treatment of haemolytic uraemic syndrome in Shiga toxin producing Escherichia coli infection. Current Opinion in Infectious Diseases, 17, 479–482. Meisen, I., Peter-Katalinić, J., & Müthing, J. (2004). Direct analysis of silica gel extracts from immunostained glycosphingolipids by nanoelectrospray ionization quadrupole time-of-flight mass spectrometry. Analytical Chemistry, 76, 2248–2255. https://doi. org/10.1021/ac035511t. Melton-Celsa, A. R., & O’Brien, A. D. (2014). New therapeutic developments against Shiga toxin-producing Escherichia coli. Microbiology Spectrum, 2. https://doi.org/10.1128/ microbiolspec.EHEC-0013-2013 EHEC-0013-2013. Müthing, J., Meisen, I., Zhang, W., Bielaszewska, M., Mormann, M., Bauerfeind, R., et al. (2012). Promiscuous Shiga toxin 2e and its intimate relationship to Forssman. Glycobiology, 22, 849–862. https://doi.org/10.1093/glycob/cws009. Nagash, F., Masoodi, F. A., Rather, S. A., Wani, S. M., & Gani, A. (2017). Emerging concepts in the neutraceutical and functional properties of pectin – a review. Carbohydrate Polymers, 168, 227–239. https://doi.org/10.1016/j.carbpol.2017.03. 058. Noreen, A., Nazli, Z. I., Akram, J., Rasul, I., Mansha, A., Yagoob, N., et al. (2017). Pectins functionalized biomaterials; A new viable approach for biomedical applications: A review. International Journal of Biological Macromolecules, 101, 254–272. https://doi. org/10.1016/j.ijbiomac.2017.03.029. Olano-Martin, E., Williams, M. R., Gibson, G. R., & Rastall, R. A. (2003). Pectins and pectic-oligosaccharides inhibit Escherichia coli O157:H7 Shiga toxin as directed towards the human colonic cell line HT29. FEMS Microbiology Letters, 218, 101–105. Pohlentz, G., & Egge, H. (1994). Neoglycolipids of 1-deoxy-1-phosphatidylethanolaminolactitol type: Synthesis, structure analysis, and use as probes for characterization of glycosyltransferases. Methods in Enzymology, 242, 127–145. Qin, X., Klein, E. J., Galanakis, E., Thomas, A. A., Stapp, J. R., Rich, S., et al. (2015). Realtime PCR assay for detection and differentiation of Shiga toxin-producing Escherichia coli from clinical samples. Journal of Clinical Microbiology, 53, 2148–2153. https:// doi.org/10.1128/JCM.00115-15. Smith, D. C., Lord, J. M., Roberts, L. M., & Johannes, L. (2004). Glycosphingolipids as toxin receptors. Seminars in Cell & Developmental Biology, 15, 397–408. https://doi. org/10.1016/j.semcdb.2004.03.005. Staples, M., Fang, N. X., Graham, R. M., Smith, H. V., & Jennison, A. V. (2017). Evaluation of SHIGA TOXIN QUIK CHEK and ImmunoCard STAT! EHEC as screening tools for the detection of Shiga toxin in fecal specimens. Diagnostic Microbiology and Infectious Disease, 87, 95–99. https://doi.org/10.1016/j.diagmicrobio.2016.03.011. Steil, D., Schepers, C. L., Pohlentz, G., Legros, N., Runde, J., Humpf, H. U., et al. (2015). Shiga toxin glycosphingolipid receptors of Vero-B4 kidney epithelial cells and their membrane microdomain lipid environment. Journal of Lipid Research, 56, 2322–2336. https://doi.org/10.1194/jlr.M063040. Steil, D., Pohlentz, G., Legros, N., Mormann, M., Mellmann, A., Karch, H., et al. (2018). Combining mass spectrometry, surface acoustic wave interaction analysis, and cell viability assays for characterization of Shiga toxin subtypes of pathogenic Escherichia coli bacteria. Analytical Chemistry, 90, 8989–8997. https://doi.org/10.1021/acs. analchem.8b01189. Taylor, R. L., & Conrad, H. E. (1972). Stoichiometric depolymerisation of polyuronides and glycosaminoglycorans to monosaccharides following reduction of their carbodiimide-activated carboxyl groups. Biochemistry, 11, 1383–1388. Wong, C. S., Mooney, J. C., Brandt, J. R., Staples, A. O., Jelacic, S., Boster, D. R., et al. (2012). Risk factors for the hemolytic-uremic syndrome in children infected with Escherichia coli O157:H7: A multivariable analysis. Clinical Infectious Diseases, 55, 33–41. https://doi.org/10.1093/cid/cis299. Zoja, C., Buelli, S., & Morigi, M. (2010). Shiga toxin-associated hemolytic uremic syndrome: Pathophysiology of endothelial dysfunction. Pediatric Nephrology, 25, 2231–2240. https://doi.org/10.1007/s00467-010-1522-1.
Journal of Statistics, 6, 65–70. Johannes, L. (2017). Shiga toxin – A model for glycolipid-dependent and lectin-driven endocytosis. Toxins, 9, 340. https://doi.org/10.3390/toxins9110340. Kamath, V. P., Yeske, R. E., Gregson, J. M., Ratcliffe, R. M., Fang, Y. R., & Palcic, M. M. (2004). Large-scale chemical and chemo-enzymatic synthesis of a spacer-containing Pk-trisaccharide. Carbohydrate Research, 339, 1141–1146. https://doi.org/10.1016/j. carres.2003.12.027. Kampmeier, S., Berger, M., Mellmann, A., Karch, H., & Berger, P. (2018). The 2011 German enterohemorrhagic Escherichia coli O104:H4 outbreak – The danger is still out there. Current Topics in Microbiology and Immunology. https://doi.org/10.1007/ 82_2018_107. Karch, H., Friedrich, A. W., Gerber, A., Zimmerhackl, L. B., Schmidt, M. A., & Bielaszewska, M. (2006). New aspects in the pathogenesis of enteropathic hemolytic uremic syndrome. Seminars in Thrombosis and Hemostasis, 32, 105–112. https://doi. org/10.1055/s-2006-939766. Karch, H., Denamur, E., Dobrindt, U., Finlay, B. B., Hengge, R., Johannes, L., et al. (2012). The enemy within us: Lessons from the 2011 European Escherichia coli O104:H4 outbreak. EMBO Molecular Medicine, 4, 841–848. https://doi.org/10.1002/emmm. 201201662. Karpman, D., & Ståhl, A. L. (2014). Enterohemorrhagic Escherichia coli pathogenesis and the host response. Microbiology Spectrum, 2. https://doi.org/10.1128/microbiolspec. EHEC-009-2013 EHEC-0009-2013. Kavaliauskiene, S., Dyve Lingelem, A. B., Skotland, T., & Sandvig, K. (2017). Protection against Shiga toxins. Toxins (Basel), 9. https://doi.org/10.3390/toxins9020044 pii:E44. Kouzel, I. U., Pohlentz, G., Schmitz, J. S., Steil, D., Humpf, H. U., Karch, H., et al. (2017). Shiga toxin glycosphingolipid receptors in human Caco-2 and HCT-8 colon epithelial cell lines. Toxins (Basel), 9. https://doi.org/10.3390/toxins9110338 pii: E338. Krall, S. M., & McFeeters, R. F. (1998). Pectin hydrolysis: Effect of temperature, degree of methylation, pH, and calcium on hydrolysis rates. Journal of Agricultural and Food Chemistry, 46, 1311–1315. https://doi.org/10.1021/jf970473y. Kulkarni, A. A., Weiss, A. A., & Iyer, S. S. (2010). Glycan-based high-affinity ligands for toxins and pathogen receptors. Medicinal Research Reviews, 30, 327–393. https://doi. org/10.1002/med.20196. Lara-Espinoza, C., Carvajal-Millán, E., Balandrán-Quintana, R., López-Franco, Y., & Rascón-Chu, A. (2018). Pectin and pectin-based composite materials: Beyond food texture. Molecules, 23. https://doi.org/10.3390/molecules23040942 pii: E942. Legros, N., Pohlentz, G., Steil, D., & Müthing, J. (2018). Shiga toxin-glycosphingolipid interaction: Status quo of research with focus on primary human brain and kidney endothelial cells. International Journal of Medical Microbiology, 308, 1073–1084. https://doi.org/10.1016/j.ijmm.2018.09.003. Legros, N., Dusny, S., Humpf, H. U., Pohlentz, G., Karch, H., & Müthing, J. (2017). Shiga toxin glycosphingolipid receptors and their lipid membrane ensemble in primary human blood-brain-barrier endothelial cells. Glycobiology, 27, 99–109. https://doi. org/10.1093/glycob/cww090. Legros, N., Pohlentz, G., Runde, J., Dusny, S., Humpf, H. U., Karch, H., et al. (2017). Colocalization of receptors for Shiga toxins with lipid rafts in primary human renal glomerular endothelial cells and influence of D-PDMP on synthesis and distribution of glycosphingolipid receptors. Glycobiology, 27, 947–965. https://doi.org/10.1093/ glycob/cwx048. Liu, Y., Childs, R. A., Palma, A. S., Campanero-Rhodes, M. A., Stoll, M. S., Chai, W., et al. (2012). Neoglycolipid-based oligosaccharide microassay system: Preparation of NGLs and their noncovalent immobilization on nitrocellulose-coated glass slides for microarray analyses. Methods in Molecular Biology, 808, 117–136. https://doi.org/10. 1007/978-1-61779-373-8_8. MacConnachie, A. A., & Todd, W. T. (2004). Potential therapeutic agents for the
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Pectin-derived neoglycolipids: Tools for differentiation of Shiga toxin subtypes and inhibitors of Shiga toxin-mediated cellular injury
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Gottfried Pohlentz, Daniel Steil, Dennis Rubin, Alexander Mellmann, Helge Karch, ⁎ Johannes Müthing Institute for Hygiene, University of Münster, Robert-Koch-Straße 41, D-48149 Münster, Germany
A R T I C LE I N FO
A B S T R A C T
Keywords: Citrus pectin EHEC Glycovesicles Neoglycolipids Shiga toxin STEC
Gut pathogenic enterohemorrhagic Escherichia coli (EHEC) release Shiga toxins (Stxs) as major virulence factors, which bind to globotriaosylceramide (Gb3Cer, Galα1-4 Galβ1-4Glcβ1-1Cer) on human target cells. The aim of this study was the production of neoglycolipids (neoGLs) using citrus pectin-derived oligosaccharides and their application as potential inhibitors of Stxs. The preparation of neoGLs starts with the reduction of the carboxylic acid group of the pectic poly(α1-4)GalUA core structure to the corresponding alcohol, followed by hydrolytic cleavage of resulting poly(α1-4)Gal into (α1-4)Galn oligosaccharides and their linkage to phosphatidylethanolamine (PE). Thin-layer chromatography overlay assays of the produced (α1-4)Galn-PE and corresponding Amadori (α1-4)Galn=PE neoGLs revealed distinguishable binding patterns for Stx1a, Stx2a, and Stx2e. Furthermore, prepared neoGLs protected Vero cells against the cytotoxic action of Stxs when applied as multivalent glycovesicles. The produced neoGLs are applicable for differentiation of Stx subtypes and represent a promising approach to combat infections of EHEC by blocking their major toxins.
1. Introduction Enterohemorrhagic Escherichia coli (EHEC), a certain subset of Shiga toxin (Stx)-producing E. coli (STEC), can colonize the human intestine and elicit fulminant enteropathies like hemorrhagic colitis (Karpman & Ståhl, 2014). Upon transfer into the circulation, released Stxs cause lifethreatening extraintestinal complications such as the hemolytic-uremic syndrome (HUS) and neurological deficiencies (Karch et al., 2012). Although some progress has been made in clinical practice (Davis, van de Kar, & Tarr, 2014), STEC still remains an important zoonotic issue worldwide due to lack of specific treatment for EHEC-associated HUS (Kampmeier, Berger, Mellmann, Karch, & Berger, 2018). Stxs are AB5 toxins (Bergan, Dyve Lingelem, Simm, Skotland, & Sandvig, 2012) and their B pentamers bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer) with Galα1-4 Galβ1-4Glcβ11Cer structure (Smith, Lord, Roberts, & Johannes, 2004). While the occurrence of STEC strains producing Stx1a and Stx2a, which represent the two major Stx subtypes being associated with severe human infections, correlates with the development of HUS, strains producing Stx2e have been isolated from patients with less severe disease (Friedrich et al., 2002; Karch et al., 2006). Stx1a and Stx2a preferentially bind to
Gb3Cer, whereas Stx2e exhibits some prevalence for globotetraosylceramide (Gb4Cer, Galβ3Galα4 Galβ4Glcβ1Cer), but also binds to Gb3Cer (Müthing et al., 2012; Steil et al., 2018). The Galα1-4 Gal-epitope is the key carbohydrate determinant, which is recognized by the B subunits of Stxs as a highly efficient receptor. When translocated to the blood stream, Stxs bind to surfaceexposed Gb3Cer particularly of human renal and cerebral microvascular endothelial cells (Legros, Dusny et al., 2017; Legros, Pohlentz et al., 2017; Legros, Pohlentz, Steil, & Müthing, 2018), followed by internalization and retrograde routing of the toxin to intracellular targets, where Stx exerts its cytotoxic effects (Johannes, 2017). Stxmediated damage of microvascular endothelial cells underlies the pathological changes in hemorrhagic colitis and HUS caused by EHEC (Bielaszewska & Karch, 2005). However, it is worth noting that Gb3Cerpositive and Stx-sensitive developing human erythrocytes have been recently identified as further Stx targets (Betz et al., 2016), a finding that might explain the occurrence of anemia observed in HUS patients (Zoja, Buelli, & Morigi, 2010). The rational design of soluble multivalent Stx inhibitors is a very real prospect to target the Gb3Cer-Stx recognition event, and a number of possible therapeutics designed to inhibit the actions of Stxs has been
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Corresponding author. E-mail addresses: [email protected] (G. Pohlentz), [email protected] (D. Steil), [email protected] (D. Rubin), [email protected] (A. Mellmann), [email protected] (H. Karch), [email protected] (J. Müthing). https://doi.org/10.1016/j.carbpol.2019.02.039 Received 15 November 2018; Received in revised form 4 February 2019; Accepted 12 February 2019 Available online 15 February 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.
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of 100 μL of a freshly prepared solution of sodium cyanoborohydride (10 mg/mL in chloroform/methanol (1/1, v/v) with 1% acetic acid) were successively added over a period of 16 h and incubated at 60 °C. The solvent was evaporated, the residue suspended in water and dialyzed, followed by lyophilization yielding 16.9 mg of a (α1-4)Galn-PEcontaining neoGL preparation.
developed (Melton-Celsa & O’Brien, 2014). Preparations of Gb3 derivatives with terminal Galα1-4 Gal-epitope via a chemical or chemoenzymatic synthesis warrant a lot of technical effort and a cost-intensive production line in particular on larger quantities required for therapeutic applications (Kamath et al., 2004). There is hence a need for preparing glycoconjugates with high binding affinity towards Stxs, which are easily accessible, producible at low costs and, most importantly, suitable for neutralizing Stx-mediated cellular injury. Citrus pectin is composed of D-galacturonic acid (GalUA) as the major constituent forming linear polymers, which have a backbone consisting of α1-4-linked GalUA monomers (Caffall & Mohnen, 2009). Citrus pectin has been found to exhibit marginal protection for human colonic cells against the cytotoxic action of Stxs, whereas pectic oligosaccharides exhibited a slight but significant protective effect when applied at high concentrations in cell cultures (Olano-Martin, Williams, Gibson, & Rastall, 2003). The aim of this study was to proceed in a way to produce pectin-derived neoglycolipids (neoGLs) with the potential to inhibit Stx-mediated cellular damage. To this end we chemically transformed pectin (α1-4)GalUA polymers into (α1-4)Galn oligosaccharides, which were then transferred onto a lipid anchor. We postulated that EHEC-released Stx1a, Stx2a, and Stx2e subtypes should exhibit different binding preferences to the prepared pectin-derived neoGLs and that lipid vesicles spiked with the produced neoGLs (glycovesicles) might act as multivalent inhibitors of the cytotoxic action of various Stx subtypes. Orally applied as a dietary supplement neoGLs, inserted in vesicles or encapsulated, e. g., in pectin-alginate particles to provide gastro-resistance (Gartziandia et al., 2018), should bind up free Stx(s) within the intestine so that the toxin(s) could not act either locally or systemically (Armstrong et al., 1995; Bengmark, 2003; MeltonCelsa & O’Brien, 2014).
2.3. Mass spectrometry of pectic oligosaccharides and pectin-derived neoGLs of (α1-4)Galn-PE type Pectin-derived (α1-4)Galn and (α1-4)GalUAn oligosaccharides as well as produced (α1-4)Galn-PE type neoGLs were structurally characterized by nano electrospray ionization mass spectrometry (nanoESI MS). To this end, oligosaccharides were dissolved in 50% methanol and neoGLs in chloroform/methanol (2/8, v/v). MS experiments were carried out on a SYNAPT G2-S mass spectrometer (Waters, Manchester, UK) equipped with a Z-spray source in the positive ion sensitivity mode. Typical source parameters were: source temperature: 80 °C, capillary voltage: 0.8 kV, sampling cone voltage: 20 V, and source offset voltage: 50 V. For low energy collision-induced dissociation (CID) experiments, precursor ions of interest were selected in the quadrupole analyzer and fragmented in the trap cell using a collision gas (Ar) flow rate of 2.0 mL/min and collision energies up to 50 eV (Elab). For MS analysis of overlay assay-detected Stx-binding (α1-4)Galn-PE type neoGLs (see 2.5. Thin-layer chromatography overlay assay detection of Stx-binding (α1-4)Galn-PE type neoGLs) the impregnation agent was removed with chloroform and the neoGLs were extracted from scraped silica gel with chloroform/methanol (1/1, v/v) following a previously published protocol (Meisen, Peter-Katalinić, & Müthing, 2004). The obtained residue was taken up in 20 μL of chloroform/methanol (2/8, v/v) and analysed by nanoESI MS as described above in this paragraph.
2. Material and methods 2.4. Stx1a, Stx2a, and Stx2e subtypes 2.1. Preparation of pectic (α1-4)Galn oligosaccharides (Krall & McFeeters, 1998; Taylor & Conrad, 1972)
Stx1a-containing supernatants from bacterial liquid cultures were produced with the EHEC wild-type strains of serotype O84:H- (7792/ 96), O118:H- (5028/97), and O145:H- (2074/97) (Müthing et al., 2012). Employed Stx2a-producing EHEC wild-type strains were those of serotype O26:H11 (356/97), O103:H2 (2636/97), and O157:H- (0296/ 96 and 3448/98); Stx2e-containing supernatants were from STEC wildtype strains of serotype ONT:H10 (24059/97) and O101:H9 (VUBEH60) (Müthing et al., 2012). The supernatants were used after sterile filtration in the overlay and cell culture assays.
An amount of 3 g of N-cyclohexyl-N’-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate was dissolved in 200 mL of deionized water and 100 mg of citrus low methylated pectin (ELICITYL, Crolles, France; GAT102, batch number E1004-09 EC8, degree of methylation < 5%, MW ˜150 kDa) in 20 mL of deionized water was added. A constant pH value of 4.75 was maintained with 0.1 M HCl for 2 h. A NaBH4 solution (20 g in 100 mL of deionized water) was added dropwise over 2 h keeping a constant pH of 7.0 by titration with 6 M HCl and the temperature between 25 °C and 35 °C. After dialysis, the glycosidic bonds of the polymer were hydrolyzed with 3.5 mM trifluoroacetic acid at 100 °C under reflux for 30 min. After chilling to 4 °C the hydrolysate was lyophilized, dissolved in water and remaining (α1-4)GalUA-containing oligosaccharides were separated from (α1-4)Galn oligosaccharides by anion exchange chromatography on a column packed with DEAE-Sepharose CL6B (GE Healthcare, Munich, Germany) in its acetate form with water as eluent. The (α1-4)Galn oligosaccharidecontaining eluate fractions were pooled and lyophilized. Yields of the (α1-4)Galn-mixture varied from 8 to 15 mg.
2.5. Thin-layer chromatography overlay assay detection of Stx-binding (α14)Galn-PE type neoGLs Four aliquots of neoGLs (see 2.2. Preparation of pectin-derived neoGLs with (α1-4)Galn oligosaccharides) were applied to a silica gel 60 precoated high-performance thin-layer chromatography (TLC) glass plate (HPTLC plates, size 10 cm x 10 cm, thickness 0.2 mm, no. 1.05633.0001; Merck, Darmstadt, Germany) and separated in the solvent chloroform/methanol/water (60/35/8, v/v/v). The TLC plate was cut into two strips: a small strip 1 (lane 1), which was used for orcinol staining, and a larger strip 2 (lanes 2, 3, and 4), which was used for the Stx overlay assays. After impregnation, strip 2 was cut into 3 parts, which were then subjected to the Stx1a, Stx2a, and Stx2e overlay assay, respectively (Müthing et al., 2012; Steil et al., 2018).
2.2. Preparation of pectin-derived neoGLs with (α1-4)Galn oligosaccharides Phosphatidylethanolamine (PE) was chosen as the lipid anchor for the preparation of neoGLs according to a procedure described by Pohlentz and Egge (1994).The amount of 10 mg of (α1-4)Galn oligosaccharides (see 2.1. Preparation of pectic (α1-4)Galn oligosaccharides) was suspended in 150 μL of dimethylsulfoxide and solubilized with 300 μL of methanol. This solution was mixed with 25 mg of 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (Sigma-Aldrich Chemie GmbH, Munich, Germany; cat. 37,161) dissolved in 2.5 mL of chloroform/methanol (2/1, v/v) and shaken for 2 h at 60 °C. Then 8 portions
2.6. Preparation of neoGL- and Gb3Cer-spiked glycovesicles Multilamellar vesicles were prepared by mixing the lipid constituents in organic solvents, followed by evaporation of the solvent and hydration in phosphate-buffered saline (PBS) as recently described (Steil et al., 2018). Briefly, a mixture of 1 mg of total lipid consisting of 0.4 mg of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC; Sigma324
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binding specificity towards these neoGLs using the TLC overlay assay (Fig. 2). The portrayal in Fig. 2A illustrates the rationale of the TLC overlay assay and Fig. 2B demonstrates binding of Stx1a, Stx2a, and Stx2e to the TLC-separated proposed (α1-4)Galn-PE neoGLs. The Stx subtypes gave characteristic binding patterns, which were markedly different from each other. Stx1a obviously “preferred” neoGLs with short oligosaccharide chains, whereas Stx2a exhibited preference for those with longer glycans as can be inferred from the TLC overlay assays. Stx2e showed a rather weak interaction with the produced neoGLs, but clearly “recognized” at least two neoGLs with opposing glycan chain lengths (Fig. 2B).
Aldrich, Steinheim, Germany; P6354), 0.2 mg of cholesterol (SigmaAldrich; C8667) and 0.4 mg of prepared (α1-4)Galn-PE type neoGLs or reference Gb3Cer was produced from these lipids dissolved in chloroform/methanol solutions. After evaporation of the organic solvents and hydration in 1 mL of PBS, extrusion of the suspension through a polycarbonate membrane resulted in small unilamellar glycovesicles spiked with (α1-4)Galn-PE type neoGLs or Gb3Cer (Steil et al., 2018). 2.7. Vero cell culture inhibition assay of Stx-mediated cytotoxicity The cytotoxic effects of Stxs towards Vero-B4 cells were determined using crystal violet as previously described (Kouzel et al., 2017; Steil et al., 2015). Briefly, aliquots of 4 × 103 cells were seeded in 100 μL medium per well and allowed to adhere for 24 h. Sterile-filtrated Stxcontaining bacterial supernatants, which were serially diluted in cell culture medium, were added in 100 μL volumes in parallel to 100 μL medium (control). After 1 h incubation, the solutions were removed and 200 μL of fresh medium were applied for 48 h. The cell viability was determined photometrically as previously described (Kouzel et al., 2017; Steil et al., 2015). The hypothesized inhibition of Stx-caused cytotoxicity by means of glycovesicles spiked with (α1-4)Galn-PE type neoGLs or reference gold standard Gb3Cer (see 2.6. Preparation of neoGL- and Gb3Cer-spiked glycovesicles) was analyzed in Vero-B4 cell cultures. For this purpose, the glycovesicle solutions were diluted 1:2 with Stx-containing medium or medium only (control) and incubated for 1 h. Afterwards, 100 μL of these solutions were added to the 100 μL cell cultures and incubated for 1 h. The applied amount of Gb3Cer and neoGLs was 20 μg/well, respectively, corresponding to a concentration of 100 μg/mL in the cell culture. The supernatants were discarded, followed by incubation of each well with 200 μL of fresh medium for 48 h. The cell viability was determined photometrically as described above in this paragraph. Results are presented as mean percentage values ± SD of four independent biological replicates related to untreated controls, which were set to 100% cell viability. Statistical data analysis of the inhibition assays was performed using R software (version 3.4.0). Analysis of variance (ANOVA) was used to test the difference between sample groups. Pairwise comparisons between groups were done using the Student’s t-test with corrections for multiple testing based on the Holm-Bonferroni method (Holm, 1979). The difference was considered significant at p < 0.001.
3.3. Structures of Stx1a-, Stx2a-, and Stx2e-binding (α1-4)Galn-PE type neoGLs The overlay-detected Stx-binding neoGLs were extracted from the silica gel and submitted to MS1 and MS2 analysis using the positive ion mode. During the linking reaction of the pectic oligosaccharides to the amino group of PE (see 2.2. Preparation of pectin-derived neoGLs with (α1-4)Galn oligosaccharides), side products of an Amadori rearrangement, so-called 1-amino-1-deoxy ketoses, are formed in addition to (α14)Galn-PE neoGLs, and Galn=PE denote the corresponding Amadori products. This second series of PE-containing species have minus 2 lower molecular masses than the corresponding Galn-PEs, which underwent the complete reductive amination reaction. 3.3.1. Stx1a-binding (α1-4)Galn-PE and (α1-4)Galn=PE neoGLs The ESI mass spectra obtained from TLC-separated Stx1a-positive neoGLs together with proposed structures are shown in Fig. 3A. Suggested structures were (α1-4)Galn-PE type neoGLs carrying two, three, four and six galactose. The neoGL with the Gal2 disaccharide was identified as Amadori rearrangement giving rise of a (α1-4)Gal2=PE structure deduced from the m/z values at 1010.66 and 1032.64, which correspond to the monosodiated [M + Na]+ and disodiated [M−H +2Na]+ ion adduct, respectively. Accordingly, threefold galactosylated neoGLs were identified with (α1-4)Gal3=PE and (α1-4)Gal3-PE structure evidenced by m/z values of 1172.72/1194.69 and 1174.71/ 1196.69 for the [M + Na]+/[M−H+2Na]+ pairs, respectively. Detected ions at m/z 1334.76/1356.74 and 1336.78/1358.77 correspond to the [M + Na]+/[M−H+2Na]+ adducts of (α1-4)Gal4=PE and (α14)Gal4-PE, respectively. The MS2 analysis of proposed Amadori products is exemplarily shown for Gal4= PE in supplementary Fig. S2 depicting the CID spectrum of Gal4=PE (Fig. S2 A) and the corresponding fragmentation scheme (Fig. S2B). The occurrence of Amadori rearrangement products exerting a substantial interaction activity towards Stxs is further addressed in the Discussion below. The presence of a sixfold galactosylated neoGL is indicated by m/z at 1660.89/1682.88 equivalent to (α1-4)Gal6-PE ionizing as [M + Na]+/[M−H+2Na]+ adducts as well. Proposed structures were further verified by CID measurements. The ESI MS2 analysis with the accompanying fragmentation scheme is exemplarily shown for (α1-4)Gal3-PE (Fig. 3B) representing a minor Stx1a-positive neoGL as concluded from the TLC analysis (see Fig. 2B).
3. Results 3.1. Production and purification of (α1-4)Galn oligosaccharides from citrus pectin To produce neoGLs harboring (α1-4)Galn oligosaccharides, low methylated citrus pectin (degree of methylation < 5%) with poly(α14)GalUA core structure was used as starting material (Fig. 1). After chemical reduction of the carboxyl to the corresponding alcohol groups, the glycosidic bonds between the saccharide units of the resulting (α14)Gal polymers were cleaved by acidic hydrolysis to yield (α1-4)Galn oligosaccharides. The ESI mass spectrum revealed presence of (α14)Galn oligosaccharides accompanied by non-reduced (α1-4)GalUAn in the hydrolysate (Fig. 1A). Anion exchange chromatography resulted in entire removal of (α1-4)GalUA units, and the ESI mass spectrum indicated a purified fraction of (α1-4)Galn oligosaccharides ranging from (α1-4)Gal2 to (α1-4)Gal6 as shown in Fig. 1B.
3.3.2. Stx2a-binding (α1-4)Galn-PE and (α1-4)Galn=PE neoGLs Following the procedure described above for MS analysis of Stx1apositive neoGLs (see 3.3.1. Stx1a-binding (α1-4)Galn-PE and (α14)Galn=PE neoGLs), a number of Stx2a-binding neoGLs with (α14)Galn-PE and (α1-4)Galn=PE structures was identified and the obtained ESI mass spectra are depicted in Fig. 4A. Structures recognized by Stx2a were (α1-4)Gal2=PE, (α1-4)Gal4=PE, (α1-4)Gal4-PE and (α1-4)Gal6-PE identical to attachment structures of Stx1a. However, the relative binding strengths, which may approximately correlate with color intensities of Stx-positive TLC bands, were markedly different (see insert of Fig. 4A) when compared to Stx1a (see insert of Fig. 3A). Interestingly, Stx2a did not adhere to (α1-4)Gal3=PE and (α1-4)Gal3-PE,
3.2. TLC overlay detection of Stx1a-, Stx2a-, and Stx2e-binding (α14)Galn-PE type neoGLs Chemical coupling of the (α1-4 Gal)n oligosaccharides to the amino group of PE was the final step for producing (α1-4)Galn-PE type neoGLs. The Stx1a, Stx2a, and Stx2e subtypes were then scrutinized for their 325
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Fig. 1. Flow sheet of the production, purification and structural proof of (α1-4)Galn oligosaccharides obtained from citrus pectin. (A) The carboxyl groups of poly(α1-4)GalUA were reduced to the corresponding alcohol, followed by chemical hydrolysis resulting in desired (α14)Galn oligosaccharides accompanied by oligosaccharides containing remained non-reduced (α1-4)GalUA. (B) Separation of (α14)Galn from (α1-4)GalUAn-harboring oligosaccharides was achieved by anion exchange chromatography. The ESI mass spectra were obtained using the positive ion mode. Asterisks indicate gas phase complexes of Galn. According to MS/MS analysis the numbered signals derive from carbodiimide (CDI) adducts of GalUA-containing oligosaccharides. The ions at m/z 608.31 (1) correspond to GalGalUA·CDI [M·CDI]+, those at m/z 806.31 (2) to Gal-GalUA2·CDI [M·CDI-H + Na]+ and those at m/z 968.38 (3) to Gal2-GalUA2·CDI [M·CDI-H + Na]+. They are either neutral or positively charged and thus escape anion exchange chromatography. In addition they give rise to intense signals that do not reflect their actual abundances in the oligosaccharide mixture.
Fig. 4B).
but exhibited strong binding to the neoGL with (α1-4)Gal5-PE structure, deducible from the [M + Na]+/[M−H+2Na]+ ion pair at m/z 1498.85/1520.82. The MS2 analysis of (α1-4)Gal4-PE and the corresponding fragmentation scheme are shown as an example for further structural characterization in Fig. 4B representing one of the major Stx2a-binding neoGLs according to the TLC overlay assay (see insert of
3.3.3. Stx2e-binding (α1-4)Galn-PE and (α1-4)Galn=PE neoGLs As demonstrated in Fig. 5A, the MS analysis of overlay positive (α14)Galn-PE and (α1-4)Galn=PE neoGLs revealed [M + Na]+/[M−H +2Na]+ ion pairs with m/z at 1010.66/1032.64 and 1660.90/1682.88 326
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3.5. Stx1a- and Stx2a-mediated cellular damage Stx1a- and Stx2a-caused cellular damage was investigated in vitro using serially diluted Stx-containing supernatants from liquid cultures of STEC strains to determine the approximate IC50 value for each subtype, i.e., the dilution of Stx-containing supernatant causing ˜50% growth inhibition. Stx2e was excluded, because this subtype did not exert any cytotoxic activity (not shown). Fig. 7 shows the concentration-dependent cytotoxic activity of Stx1a (Fig. 7A) and Stx2a (Fig. 7B). The dilutions employed for the subsequent Stx inhibition assays (see 3.6. Inhibition of Stx1a- and Stx2a-mediated cellular damage by (α14)Galn-PE-containing glycovesicles) are marked with arrows. 3.6. Inhibition of Stx1a- and Stx2a-mediated cellular damage by (α14)Galn-PE- and (α1-4)Galn=PE-containing glycovesicles Fig. 8A schematically depicts the attachment of Stx to Gb3Cer receptors on the cell surface and the adhesion of Stx to glycovesicles spiked with (α1-4)Galn-PE and (α1-4)Galn=PE neoGLs or Gb3Cer. For Stx1a inhibition experiments, Vero cells were treated first with Stx1a alone resulting in a survival rate of 29.3 ± 7.3% (Fig. 8B, +Stx1a, right side, control) in relation to untreated cells, which were set to 100.0 ± 12.3% viability (Fig. 8B, -Stx1a, left side, control). Incubation of the cells with Stx1a together with neoGL-spiked glycovesicles resulted in more than doubling of the survival rate indicated by 62.6 ± 9.0% cell viability (Fig. 8B, +Stx1a, right side, neoGLs). Treatment of the cells with Stx1a in the presence of Gb3Cer-doped glycovesicles (gold standard) increased the survival rate to 89.0 ± 9.1% (Fig. 8B, +Stx1a, right side, Gb3Cer). Exposure of the cells to neoGLs or Gb3Cer alone gave survival rates of 92.7 ± 9.5% (Fig. 8B, -Stx1a, left side, neoGLs) and 84.6 ± 11.1% (Fig. 8B, -Stx1a, left side, Gb3Cer), indicative for a marginal influence of the vesicles on the cell viability. Thus, neoGL-loaded glycovesicles raised more than twice the survival rate and hence protected cells from damage by Stx1a. Glycovesicles loaded with the native Gb3Cer receptor of the toxin exerted the highest efficacy by an almost threefold increase of cellular viability when co-exposed with Stx1a. Similarly, but less pronounced, treatment of cells with Stx2a alone lowered the cell viability to 39.1 ± 4.0% (Fig. 8C, +Stx2a, right side, control). Co-incubation of the cells with Stx2a and neoGL-loaded glycovesicles increased cell survival to 68.8 ± 8.1% (Fig. 8C, +Stx2a, right side, neoGLs) corresponding to an 1.75-fold improvement of survival. Co-exposure of Stx2a with Gb3Cer enhanced the viability to 88.7 ± 2.5% (Fig. 8C, +Stx2a, right side, Gb3Cer) equivalent to more than doubled cell survival. Again, neoGLs or Gb3Cer glycovesicles had per se no toxic effect (Fig. 8C, -Stx2a, left side). Collectively, glycovesicles doped with neoGLs improved cell survival 1.75-fold and protected cells significantly from Stx2a attack. The Gb3Cer gold standard caused somewhat higher protection by more than doubling the survival rate.
Fig. 2. Scheme of the Stx TLC overlay assay (A) and overlay detection of Stx1a-, Stx2a- and Stx2e-binding (α1-4)Galn-PE type neoGLs (B). After separation of the neoGLs, the cut TLC strips were overlaid with bacterial supernatants of Stx subtypes as indicated. Used variants were: Stx1a (O84:H-, 7792/96), Stx2a (O26:H11, 2574/97), and Stx2e (ONT:H10) (see also supplementary Fig. S1). The detection was performed with anti-Stx1 (for Stx1a) and anti-Stx2 antibody (for Stx2a and Stx2e), combined with alkaline phosphatase-conjugated secondary antibody and 5-bromo-4-chloro-3-indolylphosphate (BCIP) as the substrate. Applied amounts of neoGLs for orcinol staining and TLC overlay assays were 20 μg, respectively.
structures, respectively, which were assigned to (α1-4)Gal2=PE and (α1-4)Gal6-PE structures representing the dominant Stx2e receptors. In addition, although only weakly positive in the TLC overlay assay, (α14)Gal4=PE and (α1-4)Gal4-PE were detected as Stx2e ligands. The MS2 analysis of (α1-4)Gal2=PE together with the auxiliary fragmentation scheme are exemplarily shown in Fig. 5B for further structural characterization. 3.4. Differentiation of Stx subtypes based on specific binding patterns towards (α1-4)Galn-PE and (α1-4)Galn=PE neoGLs Fig. 6 comparatively shows the binding patterns of the three Stx subtypes towards the structurally characterized (α1-4)Galn-PE and (α14)Galn=PE neoGLs. Collectively, Stx1a prefers (α1-4)Galn-PEs/(α14)Galn=PEs with 2 ≤ n ≤ 4 and Stx2a (α1-4)Galn-PEs with n ≥ 4, whereas Stx2e showed a rather weak attachment strength to (α14)Galn-PEs/(α1-4)Galn=PEs with a slight preference for (α1-4)Gal6-PE that makes it different from Stx1a and Stx2a. These subtype-specific binding patterns were verified with further two Stx1a variants, three Stx2a variants and one Stx2e variant in comparison to those used so far in this study as shown in supplementary Fig. S1. The results indicate that the respective adhesion pattern towards (α1-4)Galn-PEs/(α14)Galn=PEs is not restricted to a certain variant of a defined Stx subtype, but holds true as a characteristic feature of each additionally probed Stx variant. In other words, the pectin-derived neoGLs are principally applicable for differentiation of the three investigated Stx subtypes.
4. Discussion Within the pharmaceutical industry pectins serve as an emergent source of raw material with potential health benefits. The different facets of pectic oligosaccharides both as prebiotic as well as therapeutic agent are currently extensively debated, especially their impact of certain human diseases (Babbar, Dejonghe, Gatti, Sforza, & Elst, 2016). Pectins meet many regulations easily regarding health applications and there is a body of evidence for the shift of pectin from its conventional role to progressive applications beyond food texture (Lara-Espinoza, Carvajal-Millán, Balandrán-Quintana, López-Franco, & Rascón-Chu, 2018; Nagash, Masoodi, Rather, Wani, & Gani, 2017). Native citrus pectin, harboring linear GalUA polymers consisting of α1-4-linked GalUA as major constituent (Caffall & Mohnen, 2009) have been previously shown to exert no positive effect, whereas pectin327
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Fig. 3. ESI mass spectra of Stx1a-binding (α1-4)Galn-PE and (α1-4)Galn=PE neoGLs (A) and the MS2 analysis of (α1-4)Gal3-PE (B). (A) Mass spectra were obtained from silica gel extracts of Stx1a-positive TLC bands as indicated in the insert (see also Fig. 2B). (B) The precursor ions at m/z 1174.72 were selected for CID analysis and their fragmentation provided the structural proof of the proposed (α1-4)Gal3-PE structure.
addition to the reductive amination products a second series of PEneoGLs was obtained with higher mobility on the TLC plate and substantial binding to Stx subtypes. These species are truly Amadori products for two reasons: First, since the formation of Schiff bases is reversible, thus, remaining ones should be destroyed during the dialysis where oligosaccharide educts are removed from the equilibrium.
derived oligosaccharides applied at high concentrations (10 mg/mL) slightly protected colonic cells from Stx (Olano-Martin et al., 2003). We went forward by conversion of the (α1-4)GalUAn-oligosaccharides to the corresponding (α1-4)Galn-oligosaccharides and transfer onto a PE lipid anchor. These procedures can be performed as easily as the ensuing linkage of the oligosaccharides to the amino group of PE. In 328
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Fig. 4. ESI mass spectra of Stx2a-binding (α1-4)Galn-PE and (α1-4)Galn=PE neoGLs (A) and the MS2 analysis of (α1-4)Gal4-PE (B). (A) Mass spectra were obtained from silica gel extracts of Stx2a-positive TLC bands as indicated in the insert (see also Fig. 2B). (B) The precursor ions at m/z 1336.77 were selected for CID analysis and their fragmentation provided the structural proof of the proposed (α1-4)Gal4-PE structure.
fragmentation schemes. Especially the fragment ion pairs H/I and R/S are highly diagnostic. Their respective differences in m/z of 42 match the loss of ketene, which should not be observed in fragment ion spectra of corresponding Schiff bases.
Second, the CID spectra derived from these compounds clearly corroborate the Amadori structures as exemplarily demonstrated for (α14)Gal2=PE and (α1-4)Gal4=PE in Fig. 5 and supplementary Fig. S2, respectively, showing their CID spectra and the accompanying 329
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Fig. 5. ESI mass spectra of Stx2e-binding (α1-4)Galn-PE and (α1-4)Galn=PE neoGLs (A) and the MS2 analysis of (α1-4)Gal2=PE (B). (A) Mass spectra were obtained from silica gel extracts of Stx2e-positive TLC bands as indicated in the insert (see also Fig. 2B). (B) The precursor ions at m/z 1010.66 were selected for CID analysis and their fragmentation provided the structural proof of the proposed (α1-4)Gal2=PE structure.
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Fig. 6. Stx subtype-specific binding to structurally characterized (α1-4)Galn-PE and (α1-4)Galn=PE neoGLs. The distinct recognition of (α1-4)Galn-PEs and (α14)Galn=PEs ranging from twofold (Gal2) to sixfold (Gal6) galactosylated neoGLs detected by means of TLC overlay assays allows for differentiation of Stx1a, Stx2a, and Stx2e (see Fig. 2 for methodological details).
Fig. 8. Scheme of the interaction of Stx with glycovesicles in the cell culture (A) and protective effect of glycovesicles against Stx1a (B) and Stx2a (C). (A) Illustration of the procedure employed for analyzing inhibitory effects of glycovesicles on Stxmediated cellular damage of Vero cells. (B and C) Cells were treated with Stx alone (+Stx1a, +Stx2a, control, right hand bars) or with Stx in the presence of neoGL- or Gb3Cer-spiked glycovesicles (+Stx1a, +Stx2a, right hand bars as indicated). In addition, cells were incubated without Stx in the absence of glycovesicles (-Stx1a, -Stx2a, control, left hand bars) or without Stx in the presence of neoGL- or Gb3Cer-doped glycovesicles (-Stx1a, -Stx2a, left hand bars as indicated). Significant differences between survival rates of cells exposed to Stx1a or Stx2a alone (control) and to Stx1a or Stx2a co-applied with neoGL- or Gb3Cer-loaded glycovesicles are marked with asterisks (***p < 0.001). Fig. 7. Stx1a- (A) and Stx2a-mediated cellular damage (B). Cells were exposed to decreasing dilutions of Stx1a- or Stx2a-containing supernatants corresponding to increasing Stx concentrations. Applied dilutions ranged from the lowest toxin concentration of 1:216 (65,536-fold) diluted to the highest concentration of 1:27 (128-fold) diluted bacterial cell culture supernatant in case of Stx1a (O145:H-, 2074/97) (A). Used Stx2a (O157:H-, 3448/98) supernatants ranged from the lowest concentration of 1:213 (8.192-fold) to 1:24 (16-fold) dilution (B). The arrows indicate 1:211-diluted Stx1a (A) and 1:25-diluted Stx2a supernatants (B), which were close to the IC50 value and employed for the ensuing inhibition assays (see Fig. 8).
Despite the altered proximal Gal configuration of the Amadori-rearranged (α1-4)Galn=PE neoGLs, which result in a slightly different glycan epitope when compared to the canonical (α1-4)Galn-PE neoGLs, the Amadori products exerted a substantial interaction activity towards the analyzed Stx1a, Stx2a, and Stx2e subtypes. This holds particularly true for the (α1-4)Gal2=PE neoGL, which represents the unique dimeric binding form of the produced Amadori rearrangements. Thus, the chemical modification of the Gal molecule at the reducing end of the 331
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2018).
Galα1-Gal disaccharide, which results in a severely altered glycan epitope in case of the disaccharide, is part of a key carbohydratebinding structure of Stxs when compared to the native cellular receptor Gb3Cer with Galα1-4 Galβ1-4Glcβ1-1Cer structure (Legros et al., 2018). The application of glycovesicles spiked with (α1-4)Galn-PEs and (α1-4)Galn=PEs revealed significant improvement of cell survival upon treatment with Stx1a or Stx2a. The attachment of Stx to the oligosaccharide-decorated glycovesicles is postulated to prevent Stx from binding to its Gb3Cer receptor on the cells and hence to protect cells from Stx. The applied amount of (α1-4)Galn-PEs/(α1-4)Galn=PEs and reference Gb3Cer was 20 μg/well, corresponding to a concentration of 100 μg/mL, respectively, in the cell culture. Thus, a significant toxin neutralization could be obtained with 100-fold lower concentrations of (α1-4)Galn-PEs/(α1-4)Galn=PEs by us compared to previously employed pectic oligosaccharides (Olano-Martin et al., 2003). In contrast to such “natural” products, a number of promising Stx receptor analogues, comprising the Gb3 trisaccharide linked to various scaffolds, has been developed (Kulkarni, Weiss, & Iyer, 2010). Such semi-synthetic toxin binders have been demonstrated in animal models of STEC disease to be effective, but showed no benefit in established HUS infections (MacConnachie & Todd, 2004). Compared to those sophisticated synthetic compounds, pectins represent a more easily accessible starting material being commercially available in almost unlimited quantity from natural resources at comparably low cost (Noreen et al., 2017). This makes pectin-derived (α1-4)Galn-oligosaccharides cost-efficient precursors for the preparation of any type of multivalent Stx receptor competitors. Moreover, pectin-derived (α1-4)Galn-PE type neoGLs work more efficiently than the pure oligosaccharides when exposed in clustered structures that mimic the natural display of lipid-linked glycans (Kulkarni et al., 2010). Importantly, antibiotic use in treatment of EHEC infections is controversial because of concerns about triggering HUS by increasing Stx release (Carnicelli et al., 2016; Wong et al., 2012). Since a causative treatment of EHEC infections is not available, innovative and cost-effective therapeutics for protection against Stxs are urgently needed (Kavaliauskiene, Dyve Lingelem, Skotland, & Sandvig, 2017; Melton-Celsa & O’Brien, 2014). In clinical practice the differentiation of Stx-subtypes is commonly performed by detection of stx genes using state-of-the-art real-time PCR analysis (Qin et al., 2015). The distinction, e.g., of stx1a and stx2a genes from the stx2e gene is important, because presence of stx2e may predict a milder disease with minimal risk of HUS (Friedrich et al., 2002). Additional and/or alternative serological assays are in use for indirect detection of Stx subtypes using more or less specific anti-Stx antibodies (Staples, Fang, Graham, Smith, & Jennison, 2017). However, discrepancies between PCR and serological assays have been described and the failure of several serological assays to differentiate between the various Stx1 and Stx2 subtypes may be of concern (Feng, Jinneman, Scheutz, & Monday, 2011). Unfortunately, PCR and serological assays do not provide information for receptor binding specificity or interaction strength, although binding to the receptor glycolipid(s) is the pivotal point for cellular attachment and internalization of the toxins (Johannes, 2017; Kavaliauskiene et al., 2017). An alternative to PCR and serological assays that operates on the protein level for subtyping of Stx1a, Stx2a, and Stx2e has been recently published by us based on real time interaction analysis that revealed characteristic binding kinetics of each subtype (Steil et al., 2018). Here we add a further alternative to PCR-based assays based on the different TLC adhesion patterns of Stx1a, Stx2a, and Stx2e towards (α1-4)Galn-PEs/(α1-4)Galn=PEs. Our vision is that neoGLs of (α1-4)Galn-PE type could serve as the basis for the development of an Stx-classifying microarray (Liu et al., 2012) or a test kit applicable as a diagnostic tool. Such a microarray or kit would work on the protein level and might be an alternative for commonly employed PCR and/or serological assays. Finally, neoGLs with the potential to neutralize Stx-mediated cellular damage might be applicable as a supportive measure to combat EHEC infections (Kampmeier et al.,
Funding This research was funded by grants from the German Federal Ministry of Education and Research (BMBF) of the German Center for Infection Research (DZIF, TTU 06.801) and InfectControl 2020 IRMRESS [grant number 03ZZ0805B] and conducted under the umbrella of the Interdisciplinary Center for Clinical Research (IZKF) Münster [grant number Müth2/021/15]. Conflicts of interest The authors disclose submitted patent ‘Oligosaccharide composition for binding to lectins’ (PCT/EP2016/082289 and international publication number WO 2017/109023 A1, inventors J.M. and G.P.). The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. Acknowledgments We thank Dagmar Mense, Nikola Skutta and Ralph Fischer for excellent technical assistance. The help with statistical analysis of Dr. Ivan Kouzel is gratefully acknowledged. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.carbpol.2019.02.039. References Armstrong, G. D., Rowe, P. C., Goodyer, P., Orrbine, E., Klassen, T. P., Welss, G., et al. (1995). A phase I study of chemically synthesized verotoxin (Shiga-like toxin) Pktrisaccharide receptors attached to chromosorb for preventing hemolytic-uremic syndrome. The Journal of Infectious Diseases, 171, 1042–1045. Babbar, N., Dejonghe, W., Gatti, M., Sforza, S., & Elst, K. (2016). Pectic oligosaccharides from agricultural by-products: Production, characterization and health benefits. Critical Reviews in Biotechnology, 36, 594–606. https://doi.org/10.3109/07388551. 2014.996732. Bengmark, S. (2003). Use of some pre-, pro- and synbiotics in critically ill patients. Best Practice & Research Clinical Gastroenterology, 17, 833–848. https://doi.org/10.1016/ S1521-6918(03)00073-8. Bergan, J., Dyve Lingelem, A. B., Simm, R., Skotland, T., & Sandvig, K. (2012). Shiga toxins. Toxicon, 60, 1085–1107. https://doi.org/10.1016/j.toxicon.2012.07.016. Betz, J., Dorn, I., Kouzel, I. U., Bauwens, A., Meisen, I., Kemper, B., et al. (2016). Shiga toxin of enterohaemorrhagic Escherichia coli directly injures developing human erythrocytes. Cellular Microbiology, 18, 339–348. https://doi.org/10.1111/cmi.12592. Bielaszewska, M., & Karch, H. (2005). Consequences of enterohaemorrhagic Escherichia coli infection for the vascular endothelium. Thrombosis and Haemostasis, 94, 312–318. https://doi.org/10.1160/TH05-04-0265. Caffall, K. H., & Mohnen, D. (2009). The structure, function, and biosynthesis of plant cell wall pectic polysaccharides. Carbohydrate Research, 344, 1879–1900. https://doi.org/ 10.1016/j.carres.2009.05.021. Carnicelli, D., Arfilli, V., Ricci, F., Velati, C., Tazzari, P. L., & Brigotti, M. (2016). The antibiotic polymyxin B impairs the interactions between Shiga toxins and human neutrophils. The Journal of Immunology, 196, 1177–1185. https://doi.org/10.4049/ jimmunol.1500671. Davis, T. K., van de Kar, N. C., & Tarr, P. I. (2014). Shiga toxin/verotoxin-producing Escherichia coli infections: Practical clinical perspectives. Microbiology Spectrum, 2. https://doi.org/10.1128/microbiolspec.EHEC-0025-2014 EHEC-0025-2014. Feng, P. C., Jinneman, K., Scheutz, F., & Monday, S. R. (2011). Specificity of PCR and serological assays in the detection of Escherichia coli Shiga toxin subtypes. Applied and Environmental Microbiology, 77, 6699–6702. https://doi.org/10.1128/AEM. 00370-11. Friedrich, A. W., Bielaszewska, M., Zhang, W. L., Pulz, M., Kuczius, T., Ammon, A., et al. (2002). Escherichia coli harboring Shiga toxin 2 gene variants: Frequency and association with clinical symptoms. The Journal of Infectious Diseases, 185, 74–84. https:// doi.org/10.1086/338115. Gartziandia, O., Lasa, A., Pedraz, J. L., Miranda, J., Portillo, M. P., Igartua, M., et al. (2018). Preparation and characterization of resveratrol loaded pectin/alginate blend gastro-resistant microparticles. Molecules, 23. https://doi.org/10.3390/ molecules23081886 pii: E1886. Holm, S. (1979). A simple sequentially rejective multiple test procedure. Scandinavian
332
Carbohydrate Polymers 212 (2019) 323–333
G. Pohlentz, et al.
prevention and treatment of haemolytic uraemic syndrome in Shiga toxin producing Escherichia coli infection. Current Opinion in Infectious Diseases, 17, 479–482. Meisen, I., Peter-Katalinić, J., & Müthing, J. (2004). Direct analysis of silica gel extracts from immunostained glycosphingolipids by nanoelectrospray ionization quadrupole time-of-flight mass spectrometry. Analytical Chemistry, 76, 2248–2255. https://doi. org/10.1021/ac035511t. Melton-Celsa, A. R., & O’Brien, A. D. (2014). New therapeutic developments against Shiga toxin-producing Escherichia coli. Microbiology Spectrum, 2. https://doi.org/10.1128/ microbiolspec.EHEC-0013-2013 EHEC-0013-2013. Müthing, J., Meisen, I., Zhang, W., Bielaszewska, M., Mormann, M., Bauerfeind, R., et al. (2012). Promiscuous Shiga toxin 2e and its intimate relationship to Forssman. Glycobiology, 22, 849–862. https://doi.org/10.1093/glycob/cws009. Nagash, F., Masoodi, F. A., Rather, S. A., Wani, S. M., & Gani, A. (2017). Emerging concepts in the neutraceutical and functional properties of pectin – a review. Carbohydrate Polymers, 168, 227–239. https://doi.org/10.1016/j.carbpol.2017.03. 058. Noreen, A., Nazli, Z. I., Akram, J., Rasul, I., Mansha, A., Yagoob, N., et al. (2017). Pectins functionalized biomaterials; A new viable approach for biomedical applications: A review. International Journal of Biological Macromolecules, 101, 254–272. https://doi. org/10.1016/j.ijbiomac.2017.03.029. Olano-Martin, E., Williams, M. R., Gibson, G. R., & Rastall, R. A. (2003). Pectins and pectic-oligosaccharides inhibit Escherichia coli O157:H7 Shiga toxin as directed towards the human colonic cell line HT29. FEMS Microbiology Letters, 218, 101–105. Pohlentz, G., & Egge, H. (1994). Neoglycolipids of 1-deoxy-1-phosphatidylethanolaminolactitol type: Synthesis, structure analysis, and use as probes for characterization of glycosyltransferases. Methods in Enzymology, 242, 127–145. Qin, X., Klein, E. J., Galanakis, E., Thomas, A. A., Stapp, J. R., Rich, S., et al. (2015). Realtime PCR assay for detection and differentiation of Shiga toxin-producing Escherichia coli from clinical samples. Journal of Clinical Microbiology, 53, 2148–2153. https:// doi.org/10.1128/JCM.00115-15. Smith, D. C., Lord, J. M., Roberts, L. M., & Johannes, L. (2004). Glycosphingolipids as toxin receptors. Seminars in Cell & Developmental Biology, 15, 397–408. https://doi. org/10.1016/j.semcdb.2004.03.005. Staples, M., Fang, N. X., Graham, R. M., Smith, H. V., & Jennison, A. V. (2017). Evaluation of SHIGA TOXIN QUIK CHEK and ImmunoCard STAT! EHEC as screening tools for the detection of Shiga toxin in fecal specimens. Diagnostic Microbiology and Infectious Disease, 87, 95–99. https://doi.org/10.1016/j.diagmicrobio.2016.03.011. Steil, D., Schepers, C. L., Pohlentz, G., Legros, N., Runde, J., Humpf, H. U., et al. (2015). Shiga toxin glycosphingolipid receptors of Vero-B4 kidney epithelial cells and their membrane microdomain lipid environment. Journal of Lipid Research, 56, 2322–2336. https://doi.org/10.1194/jlr.M063040. Steil, D., Pohlentz, G., Legros, N., Mormann, M., Mellmann, A., Karch, H., et al. (2018). Combining mass spectrometry, surface acoustic wave interaction analysis, and cell viability assays for characterization of Shiga toxin subtypes of pathogenic Escherichia coli bacteria. Analytical Chemistry, 90, 8989–8997. https://doi.org/10.1021/acs. analchem.8b01189. Taylor, R. L., & Conrad, H. E. (1972). Stoichiometric depolymerisation of polyuronides and glycosaminoglycorans to monosaccharides following reduction of their carbodiimide-activated carboxyl groups. Biochemistry, 11, 1383–1388. Wong, C. S., Mooney, J. C., Brandt, J. R., Staples, A. O., Jelacic, S., Boster, D. R., et al. (2012). Risk factors for the hemolytic-uremic syndrome in children infected with Escherichia coli O157:H7: A multivariable analysis. Clinical Infectious Diseases, 55, 33–41. https://doi.org/10.1093/cid/cis299. Zoja, C., Buelli, S., & Morigi, M. (2010). Shiga toxin-associated hemolytic uremic syndrome: Pathophysiology of endothelial dysfunction. Pediatric Nephrology, 25, 2231–2240. https://doi.org/10.1007/s00467-010-1522-1.
Journal of Statistics, 6, 65–70. Johannes, L. (2017). Shiga toxin – A model for glycolipid-dependent and lectin-driven endocytosis. Toxins, 9, 340. https://doi.org/10.3390/toxins9110340. Kamath, V. P., Yeske, R. E., Gregson, J. M., Ratcliffe, R. M., Fang, Y. R., & Palcic, M. M. (2004). Large-scale chemical and chemo-enzymatic synthesis of a spacer-containing Pk-trisaccharide. Carbohydrate Research, 339, 1141–1146. https://doi.org/10.1016/j. carres.2003.12.027. Kampmeier, S., Berger, M., Mellmann, A., Karch, H., & Berger, P. (2018). The 2011 German enterohemorrhagic Escherichia coli O104:H4 outbreak – The danger is still out there. Current Topics in Microbiology and Immunology. https://doi.org/10.1007/ 82_2018_107. Karch, H., Friedrich, A. W., Gerber, A., Zimmerhackl, L. B., Schmidt, M. A., & Bielaszewska, M. (2006). New aspects in the pathogenesis of enteropathic hemolytic uremic syndrome. Seminars in Thrombosis and Hemostasis, 32, 105–112. https://doi. org/10.1055/s-2006-939766. Karch, H., Denamur, E., Dobrindt, U., Finlay, B. B., Hengge, R., Johannes, L., et al. (2012). The enemy within us: Lessons from the 2011 European Escherichia coli O104:H4 outbreak. EMBO Molecular Medicine, 4, 841–848. https://doi.org/10.1002/emmm. 201201662. Karpman, D., & Ståhl, A. L. (2014). Enterohemorrhagic Escherichia coli pathogenesis and the host response. Microbiology Spectrum, 2. https://doi.org/10.1128/microbiolspec. EHEC-009-2013 EHEC-0009-2013. Kavaliauskiene, S., Dyve Lingelem, A. B., Skotland, T., & Sandvig, K. (2017). Protection against Shiga toxins. Toxins (Basel), 9. https://doi.org/10.3390/toxins9020044 pii:E44. Kouzel, I. U., Pohlentz, G., Schmitz, J. S., Steil, D., Humpf, H. U., Karch, H., et al. (2017). Shiga toxin glycosphingolipid receptors in human Caco-2 and HCT-8 colon epithelial cell lines. Toxins (Basel), 9. https://doi.org/10.3390/toxins9110338 pii: E338. Krall, S. M., & McFeeters, R. F. (1998). Pectin hydrolysis: Effect of temperature, degree of methylation, pH, and calcium on hydrolysis rates. Journal of Agricultural and Food Chemistry, 46, 1311–1315. https://doi.org/10.1021/jf970473y. Kulkarni, A. A., Weiss, A. A., & Iyer, S. S. (2010). Glycan-based high-affinity ligands for toxins and pathogen receptors. Medicinal Research Reviews, 30, 327–393. https://doi. org/10.1002/med.20196. Lara-Espinoza, C., Carvajal-Millán, E., Balandrán-Quintana, R., López-Franco, Y., & Rascón-Chu, A. (2018). Pectin and pectin-based composite materials: Beyond food texture. Molecules, 23. https://doi.org/10.3390/molecules23040942 pii: E942. Legros, N., Pohlentz, G., Steil, D., & Müthing, J. (2018). Shiga toxin-glycosphingolipid interaction: Status quo of research with focus on primary human brain and kidney endothelial cells. International Journal of Medical Microbiology, 308, 1073–1084. https://doi.org/10.1016/j.ijmm.2018.09.003. Legros, N., Dusny, S., Humpf, H. U., Pohlentz, G., Karch, H., & Müthing, J. (2017). Shiga toxin glycosphingolipid receptors and their lipid membrane ensemble in primary human blood-brain-barrier endothelial cells. Glycobiology, 27, 99–109. https://doi. org/10.1093/glycob/cww090. Legros, N., Pohlentz, G., Runde, J., Dusny, S., Humpf, H. U., Karch, H., et al. (2017). Colocalization of receptors for Shiga toxins with lipid rafts in primary human renal glomerular endothelial cells and influence of D-PDMP on synthesis and distribution of glycosphingolipid receptors. Glycobiology, 27, 947–965. https://doi.org/10.1093/ glycob/cwx048. Liu, Y., Childs, R. A., Palma, A. S., Campanero-Rhodes, M. A., Stoll, M. S., Chai, W., et al. (2012). Neoglycolipid-based oligosaccharide microassay system: Preparation of NGLs and their noncovalent immobilization on nitrocellulose-coated glass slides for microarray analyses. Methods in Molecular Biology, 808, 117–136. https://doi.org/10. 1007/978-1-61779-373-8_8. MacConnachie, A. A., & Todd, W. T. (2004). Potential therapeutic agents for the
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