Application of thin‐layer chromatography/infrared matrix‐assisted laser desorption/ionization orthogonal time‐of‐flight mass spectrometry to structural analysis of bacteria‐binding glycosphingolipids selected by affinity detection

Müsken, Anne; Souady, Jamal; Dreisewerd, Klaus; Zhang, Wenlan; Distler, Ute; Peter‐Katalinić, Jasna; Miller‐Podraza, Halina; Karch, Helge; Müthing, Johannes

doi:10.1002/rcm.4480

Cited by 20 publications

(10 citation statements)

References 40 publications

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“…In light of these results, we evaluated the GSL composition of T24 and Vero cells. GSLs are cognate receptors of Stxs and the P fimbrial UPEC adhesin (48,49). We detected Gb3Cer and Gb4Cer in neutral GSL preparations of both cell lines.…”

Section: Molecular Epidemiology Of Ehec Strains Isolated From Hospitamentioning

confidence: 82%

Characterization of Urinary Tract Infection-Associated Shiga Toxin-Producing Escherichia coli

et al. 2014

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c Enterohemorrhagic Escherichia coli (EHEC), a subgroup of Shiga toxin (Stx)-producing E. coli (STEC), is a leading cause of diarrhea and hemolytic-uremic syndrome (HUS) in humans. However, urinary tract infections (UTIs) caused by this microorganism but not associated with diarrhea have occasionally been reported. We geno-and phenotypically characterized three EHEC isolates obtained from the urine of hospitalized patients suffering from UTIs. These isolates carried typical EHEC virulence markers and belonged to HUS-associated E. coli (HUSEC) clones, but they lacked virulence markers typical of uropathogenic E. coli. One isolate exhibited a localized adherence (LA)-like pattern on T24 urinary bladder epithelial cells. Since the glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) are well-known receptors for Stx but also for P fimbriae, a major virulence factor of extraintestinal pathogenic E. coli (ExPEC), the expression of Gb3Cer and Gb4Cer by T24 cells and in murine urinary bladder tissue was examined by thin-layer chromatography and mass spectrometry. We provide data indicating that Stxs released by the EHEC isolates bind to Gb3Cer and Gb4Cer isolated from T24 cells, which were susceptible to Stx. All three EHEC isolates expressed stx genes upon growth in urine. Two strains were able to cause UTI in a murine infection model and could not be outcompeted in urine in vitro by typical uropathogenic E. coli isolates. Our results indicate that despite the lack of ExPEC virulence markers, EHEC variants may exhibit in certain suitable hosts, e.g., in hospital patients, a uropathogenic potential. The contribution of EHEC virulence factors to uropathogenesis remains to be further investigated. Escherichia coli is one of the most versatile microorganisms that rapidly colonizes the gastrointestinal tract of newborns (1). Although E. coli usually represents an important commensal of the normal intestinal microbiota, other variants that are able to cause infections exist (1, 2). On the basis of their virulence properties and the site of infection, these bacteria are classified as intestinal pathogenic E. coli (IPEC), which are associated with diarrhea, and extraintestinal pathogenic E. coli (ExPEC), which cause infections beyond the intestinal tract (3). Enterohemorrhagic E. coli (EHEC) belongs to the IPEC group of bacteria and represents one of the main causative agents of diarrhea, hemorrhagic colitis (HC), and hemolytic-uremic syndrome (HUS) in humans (4). The characteristic main EHEC virulence markers include the locus of enterocyte effacement (LEE) pathogenicity island and Shiga toxin (Stx)-encoding bacteriophages. Additionally, other toxins, including the cytolethal distending toxin (CDT), EHEC hemolysin (Hly), serine protease EspP, subtilase cytotoxin, cyclomodulin Cif, and different siderophore systems, as well as adhesins can also contribute to EHEC pathogenesis (5). These EHEC virulence factors are absent from archetypal ExPEC strains.Similarly, the main ExPEC virulence fa...

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Section: Molecular Epidemiology Of Ehec Strains Isolated From Hospitamentioning

confidence: 82%

Characterization of Urinary Tract Infection-Associated Shiga Toxin-Producing Escherichia coli

et al. 2014

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“…Such overlay techniques have been frequently applied to the characterization of GSLs as receptors for pathogens and toxins [121]. This type of GSL-profiling has also been combined with MS, such as MALDI for the structural characterization of pathogen receptors like those for Shiga toxins [122] or with IR-MALDI to determine neutral GSLs recognized by P-fimbriated E. coli bacteria [123]. TLC immuno-staining in combination with nano-ESI-MS and nano-ESI-MS/MS has been used to determine the structural profiles of Gb3Cer and Gb4Cer-binding Shiga toxin and antibodies [124].…”

Section: Identification Of Interacting Moleculesmentioning

confidence: 99%

Lipidomics of Glycosphingolipids

Farwanah

Kolter

2012

Metabolites

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Glycosphingolipids (GSLs) contain one or more sugars that are attached to a sphingolipid moiety, usually to a ceramide, but in rare cases also to a sphingoid base. A large structural heterogeneity results from differences in number, identity, linkage, and anomeric configuration of the carbohydrate residues, and also from structural differences within the hydrophobic part. GSLs form complex cell-type specific patterns, which change with the species, the cellular differentiation state, viral transformation, ontogenesis, and oncogenesis. Although GSL structures can be assigned to only a few series with a common carbohydrate core, their structural variety and the complex pattern are challenges for their elucidation and quantification by mass spectrometric techniques. We present a general overview of the application of lipidomics for GSL determination. This includes analytical procedures and instrumentation together with recent correlations of GSL molecular species with human diseases. Difficulties such as the structural complexity and the lack of standard substances for complex GSLs are discussed.

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“…Using the TLC overlay assay, combined with infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry (IR-MALDI–O-TOF-MS), Müsken et al (2010) carried out the structural characterization of globo-series neutral GSLs (microbial GSL-receptor), recognized by P-fimbriated Escherichia coli , the agent of upper urinary tract infections in humans. The method could be applicable for any GSL-binding non-pathogenic and/or pathogenic E. coli strain, or other microbes such as Helicobacter pylori or viruses.…”

Section: New Applications In the Mass Spectrometric Analysis Of Cerebmentioning

confidence: 99%

Structural Analysis of Fungal Cerebrosides

Barreto‐Bergter¹,

Sassaki²,

Souza³

2011

Front. Microbio.

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Of the ceramide monohexosides (CMHs), gluco- and galactosyl-ceramides are the main neutral glycosphingolipids expressed in fungal cells. Their structural determination is greatly dependent on the use of mass spectrometric techniques, including fast atom bombardment-mass spectrometry, electrospray ionization, and energy collision-induced dissociation mass spectrometry. Nuclear magnetic resonance has also been used successfully. Such a combination of techniques, combined with classical analytical separation, such as high-performance thin layer chromatography and column chromatography, has led to the structural elucidation of a great number of fungal CMHs. The structure of fungal CMH is conserved among fungal species and consists of a glucose or galactose residue attached to a ceramide moiety containing 9-methyl-4,8-sphingadienine with an amidic linkage to hydroxylated fatty acids, most commonly having 16 or 18 carbon atoms and unsaturation between C-3 and C-4. Along with their unique structural characteristics, fungal CMHs have a peculiar subcellular distribution and striking biological properties. Fungal cerebrosides were also characterized as antigenic molecules directly or indirectly involved in cell growth or differentiation in Schizophyllum commune, Cryptococcus neoformans, Pseudallescheria boydii, Candida albicans, Aspergillus nidulans, Aspergillus fumigatus, and Colletotrichum gloeosporioides. Besides classical techniques for cerebroside (CMH) analysis, we now describe new approaches, combining conventional thin layer chromatography and mass spectrometry, as well as emerging technologies for subcellular localization and distribution of glycosphingolipids by secondary ion mass spectrometry and imaging matrix-assisted laser desorption ionization time-of-flight.

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Application of thin‐layer chromatography/infrared matrix‐assisted laser desorption/ionization orthogonal time‐of‐flight mass spectrometry to structural analysis of bacteria‐binding glycosphingolipids selected by affinity detection

Cited by 20 publications

References 40 publications

Characterization of Urinary Tract Infection-Associated Shiga Toxin-Producing Escherichia coli

Characterization of Urinary Tract Infection-Associated Shiga Toxin-Producing Escherichia coli

Lipidomics of Glycosphingolipids

Structural Analysis of Fungal Cerebrosides

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