Enterohemorrhagic Escherichia coli and a Fresh View on Shiga Toxin-Binding Glycosphingolipids of Primary Human Kidney and Colon Epithelial Cells and Their Toxin Susceptibility.

Enterohemorrhagic Escherichia coli (EHEC) are the human pathogenic subset of Shiga toxin (Stx)-producing E. coli (STEC). EHEC are responsible for severe colon infections associated with life-threatening extraintestinal complications such as the hemolytic-uremic syndrome (HUS) and neurological disturbances. Endothelial cells in various human organs are renowned targets of Stx, whereas the role of epithelial cells of colon and kidneys in the infection process has been and is still a matter of debate. This review shortly addresses the clinical impact of EHEC infections, novel aspects of vesicular package of Stx in the intestine and the blood stream as well as Stx-mediated extraintestinal complications and therapeutic options. Here follows a compilation of the Stx-binding glycosphingolipids (GSLs), globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) and their various lipoforms present in primary human kidney and colon epithelial cells and their distribution in lipid raft-analog membrane preparations. The last issues are the high and extremely low susceptibility of primary renal and colonic epithelial cells, respectively, suggesting a large resilience of the intestinal epithelium against the human-pathogenic Stx1a- and Stx2a-subtypes due to the low content of the high-affinity Stx-receptor Gb3Cer in colon epithelial cells. The review closes with a brief outlook on future challenges of Stx research.

Surface acoustic wave (SAW) real-time interaction analysis of influenza A virus hemagglutinins with sialylated neoglycolipids.

Real-time interaction analysis of H1 hemagglutinin from influenza A H1N1 (A/New York/18/2009) and H7 hemagglutinin from influenza A H7N7 (A/Netherlands/219/03) with sialylated neoglycolipids (neoGLs) was performed using the surface acoustic wave (SAW) technology. The produced neoGLs carried phosphatidylethanolamine (PE) as lipid anchor and terminally sialylated lactose (Lc2, Galß1-4Glc) or neolactotetraose (nLc4, Galß1-4GlcNAcß1-3Galß1-4Glc) harboring an N-acetylneuraminic acid (Neu5Ac). Using α2-6-sialylated neoGLs, H1 and H7 exhibited marginal attachment toward II6Neu5Ac-Lc2-PE, whereas Sambucus nigra lectin (SNL) exhibited strong binding and Maackia amurensis lectin (MAL) was negative in accordance with their known binding preference toward a distal Neu5Acα2-6Gal- and Neu5Acα2-3Gal-residue, respectively. H1 revealed significant binding toward IV6Neu5Ac-nLc4-PE when compared to weak interaction of H7, whereas SNL showed strong and MAL no attachment corresponding to their interaction specificities. Additional controls of MAL and SNL with α2-3-sialylated II3Neu5Ac-Lc2-PE and IV3Neu5Ac-nLc4-PE underscored the reliability of the SAW technology. Pre-exposure of model membranes spiked with α2-6-sialylated neoGLs to Vibrio cholerae neuraminidase substantially reduced the binding of the hemagglutinins and the SNL reference. Collectively, the SAW technology is capable of accurate measuring binding features of hemagglutinins toward neoGL-spiked lipid bilayers, which can be easily loaded to the functionalized biosensor gold surface thereby simulating biological membranes and suggesting promising clinical application for influenza virus research.

Primary Human Colon Epithelial Cells (pHCoEpiCs) Do Express the Shiga Toxin (Stx) Receptor Glycosphingolipids Gb3Cer and Gb4Cer and Are Largely Refractory but Not Resistant towards Stx.

Shiga toxin (Stx) is released by enterohemorrhagic Escherichia coli (EHEC) into the human intestinal lumen and transferred across the colon epithelium to the circulation. Stx-mediated damage of human kidney and brain endothelial cells and renal epithelial cells is a renowned feature, while the sensitivity of the human colon epithelium towards Stx and the decoration with the Stx receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer, Galα1-4Galß1-4Glcß1-1Cer) and globotetraosylceramide (Gb4Cer, GalNAcß1-3Galα1-4Galß1-4Glcß1-1Cer) is a matter of debate. Structural analysis of the globo-series GSLs of serum-free cultivated primary human colon epithelial cells (pHCoEpiCs) revealed Gb4Cer as the major neutral GSL with Cer (d18:1, C16:0), Cer (d18:1, C22:1/C22:0) and Cer (d18:1, C24:2/C24:1) accompanied by minor Gb3Cer with Cer (d18:1, C16:0) and Cer (d18:1, C24:1) as the dominant lipoforms. Gb3Cer and Gb4Cer co-distributed with cholesterol and sphingomyelin to detergent-resistant membranes (DRMs) used as microdomain analogs. Exposure to increasing Stx concentrations indicated only a slight cell-damaging effect at the highest toxin concentration of 1 µg/mL for Stx1a and Stx2a, whereas a significant effect was detected for Stx2e. Considerable Stx refractiveness of pHCoEpiCs that correlated with the rather low cellular content of the high-affinity Stx-receptor Gb3Cer renders the human colon epithelium questionable as a major target of Stx1a and Stx2a.

Blockade of Glycosphingolipid Synthesis Inhibits Cell Cycle and Spheroid Growth of Colon Cancer Cells In Vitro and Experimental Colon Cancer Incidence In Vivo.

Colorectal cancer (CRC) is one of the most frequently diagnosed cancers in humans. At early stages CRC is treated by surgery and at advanced stages combined with chemotherapy. We examined here the potential effect of glucosylceramide synthase (GCS)-inhibition on CRC biology. GCS is the rate-limiting enzyme in the glycosphingolipid (GSL)-biosynthesis pathway and overexpressed in many human tumors. We suppressed GSL-biosynthesis using the GCS inhibitor Genz-123346 (Genz), NB-DNJ (Miglustat) or by genetic targeting of the GCS-encoding gene UDP-glucose-ceramide-glucosyltransferase- (UGCG). GCS-inhibition or GSL-depletion led to a marked arrest of the cell cycle in Lovo cells. UGCG silencing strongly also inhibited tumor spheroid growth in Lovo cells and moderately in HCT116 cells. MS/MS analysis demonstrated markedly elevated levels of sphingomyelin (SM) and phosphatidylcholine (PC) that occurred in a Genz-concentration dependent manner. Ultrastructural analysis of Genz-treated cells indicated multi-lamellar lipid storage in vesicular compartments. In mice, Genz lowered the incidence of experimentally induced colorectal tumors and in particular the growth of colorectal adenomas. These results highlight the potential for GCS-based inhibition in the treatment of CRC.

Real-time interaction analysis of Shiga toxins and membrane microdomains of primary human brain microvascular endothelial cells.

Infections of the human intestinal tract with enterohemorrhagic Escherichia coli (EHEC) result in massive extraintestinal complications due to translocation of EHEC-released Shiga toxins (Stxs) from the gut into the circulation. Stx-mediated damage of the cerebral microvasculature raises serious brain dysfunction being the most frequent cause of acute mortality in patients suffering from severe EHEC infections. Stx2a and Stx2e are associated with heavy and mild course of infection, respectively. Stx2a preferentially binds to globotriaosylceramide (Gb3Cer, Galα1-4Galß1-4Glcß1-1Cer), while Stx2e prefers globotetraosylceramide (Gb4Cer, GalNAcß1-3Galα1-4Galß1-4Glcß1-1Cer). Both glycosphingolipids (GSLs) were detected in detergent-resistant membranes (DRMs) of primary human brain microvascular endothelial cells (pHBMECs) resembling microdomains of the plasma membrane. In this study, we show that Gb3Cer and Gb4Cer of pHBMECs with saturated C16:0, C22:0, and C24:0 fatty acids dominated in DRMs, corresponding to the liquid-ordered membrane phase, whereas lipoforms carrying unsaturated C24:1 and C24:2 fatty acids prevailed in the non-DRM fractions, which correspond to the liquid-disordered membrane phase. Similarly, a shift of the phospholipids from saturated lipoforms in the DRM to unsaturated species in the non-DRM fractions was observed. Real-time biomolecular interaction analysis using affinity-purified Stx2a and Stx2e, recorded with a surface acoustic wave (SAW) biosensor, evidenced high binding strength of both toxins toward DRMs and failure in interaction with non-DRMs. These results support the hypothesis of preferential binding of Stxs toward microdomains harboring GSL receptors carrying saturated fatty acids in their lipid anchors. Collectively, unraveling the precise mechanisms of Stx-microdomain interaction may help to develop antiadhesive compounds to combat Stx-mediated cellular injury.

MALDI-2 Mass Spectrometry and Immunohistochemistry Imaging of Gb3Cer, Gb4Cer, and Further Glycosphingolipids in Human Colorectal Cancer Tissue.

The main cellular receptors of Shiga toxins (Stxs), the neutral glycosphingolipids (GSLs), globotriaosylceramide (Gb3Cer/CD77) and globotetraosylceramide (Gb4Cer), are significantly upregulated in about half of the human colorectal carcinomas (CRC) and in other cancers. Therefore, conjugates exploiting the Gb3Cer/Gb4Cer-binding B subunit of Stx (StxB) have attracted great interest for both diagnostic and adjuvant therapeutic interventions. Moreover, fucosylated GSLs were recognized as potential tumor-associated targets. One obstacle to a broader use of these receptor/ligand systems is that the contribution of specific GSLs to tumorigenesis, in particular, in the context of an altered lipid metabolism, is only poorly understood. A second is that also nondiseased organs (e.g., kidney) and blood vessels can express high levels of certain GSLs, not least Gb3Cer/Gb4Cer. Here, we used, in a proof-of-concept study, matrix-assisted laser desorption/ionization mass spectrometry imaging combined with laser-induced postionization (MALDI-2-MSI) to simultaneously visualize the distribution of several Gb3Cer/Gb4Cer lipoforms and those of related GSLs (e.g., Gb3Cer/Gb4Cer precursors and fucosylated GSLs) in tissue biopsies from three CRC patients. Using MALDI-2 and StxB-based immunofluorescence microscopy, Gb3Cer and Gb4Cer were mainly found in dedifferentiated tumor cell areas, tumor stroma, and tumor-infiltrating blood vessels. Notably, fucosylated GSL such as Fuc-(n)Lc4Cer generally showed a highly localized expression in dysplastic glands and indian file-like cells infiltrating adipose tissue. Our "molecular histology" approach could support stratifying patients for intratumoral GSL expression to identify an optimal therapeutic strategy. The improved chemical coverage by MALDI-2 can also help to improve our understanding of the molecular basis of tumor development and GSL metabolism.

How bacterial pathogens of the gastrointestinal tract use the mucosal glyco-code to harness mucus and microbiota: New ways to study an ancient bag of tricks.

During the last decades, the flourishing scientific field of molecular pathogenesis brought groundbreaking knowledge of the mechanisms of pathogenicity and the underlying bacterial virulence factors to cause infectious diseases. However, a major paradigm shift is currently occurring after it became increasingly evident that bacterial-host and host-host cell interactions including immune responses orchestrated by defined virulence factors are not the sole drivers of infectious disease development. Strong evidence has been collected that information and nutrient flow within complex microbial communities, as well as to and from host cells and matrices are equally important for successful infection. This particularly holds true for gastrointestinal (GI) pathogens and the GI microbiota interacting and communicating with each other as well as with the host GI mucus and mucosa. Gut-adapted pathogens appear to have developed powerful and specific strategies to interact with human GI mucus including the microbiota for nutrient acquisition, mucosal adhesion, inter-species communication and traversing the mucus barrier. This review covers the existing evidence on these topics and explores the mutual dynamics of host GI mucus, the mucosal habitat and incoming acute and chronic pathogens during GI infections. A particular focus is placed on the role of carbohydrates in diverse mucosal interaction, communication and competition processes. Novel techniques to analyze and synthesize mucus-derived carbohydrates and to generate mucus mimetics are introduced. Finally, open questions and future objectives for pathogen - host GI mucus research will be discussed.

PapG subtype-specific binding characteristics of Escherichia coli towards globo-series glycosphingolipids of human kidney and bladder uroepithelial cells.

Uropathogenic Escherichia coli (UPEC) are the primary cause of urinary tract infections (UTIs) in humans. P-fimbriae are key players for bacterial adherence to the uroepithelium through the Galα1-4Gal-binding PapG adhesin. The three identified classes I, II and III of PapG are supposed to adhere differently to host cell glycosphingolipids (GSLs) of the uroepithelial tract harboring a distal or internal Galα1-4Gal sequence. In this study, GSL binding characteristics were obtained in a nonradioactive adhesion assay using biotinylated E. coli UTI and urine isolates combined with enzyme-linked NeutrAvidin for detection. Initial experiments with reference globotriaosylceramide (Gb3Cer, Galα1-4Galß1-4Glcß1-1Cer), globotetraosylceramide (Gb4Cer, GalNAcß1-3Galα1-4Galß1-4Glcß1-1Cer) and Forssman GSL (GalNAcα1-3GalNAcß1-3Galα1-4Galß1-4Glcß1-1Cer) revealed balanced adhesion toward the three GSLs for PapG I-mediated attachment. In contrast, E. coli carrying PapG II or PapG III increasingly adhered to growing oligosaccharide chain lengths of Gb3Cer, Gb4Cer and Forssman GSL. Binding studies with GSLs from human A498 kidney and human T24 bladder epithelial cells, both being negative for the Forssman GSL, revealed the less abundant Gb4Cer vs. Gb3Cer as the prevalent receptor in A498 cells of E. coli expressing PapG II or PapG III. On the other hand, T24 cells exhibited a higher relative content of Gb4Cer vs. Gb3Cer alongside dominant binding of PapG II- or PapG III-harboring E. coli toward Gb4Cer and vastly lowered attachment to minor Gb3Cer. Further studies on PapG-mediated interaction with cell surface-exposed GSLs will improve our knowledge on the molecular mechanisms of P-fimbriae-mediated adhesion and may contribute to the development of antiadhesion therapeutics to combat UTIs.

Renal globotriaosylceramide facilitates tubular albumin absorption and its inhibition protects against acute kidney injury.

To elucidate the physiologic function of renal globotriaosylceramide (Gb3/CD77), which up-to-date has been associated exclusively with Shiga toxin binding, we have analyzed renal function in Gb3-deficient mice. Gb3 synthase KO (Gb3S-/-) mice displayed an increased renal albumin and low molecular weight protein excretion compared to WT. Gb3 localized at the brush border and within vesicular structures in WT proximal tubules and has now been shown to be closely associated with the receptor complex megalin/cubilin and with albumin uptake. In two clinically relevant mouse models of acute kidney injury caused by myoglobin as seen in rhabdomyolysis and the aminoglycoside gentamicin, Gb3S-/- mice showed a preserved renal function and morphology, compared to WT. Pharmacologic inhibition of glucosylceramide-based glycosphingolipids, including Gb3, in WT mice corroborated the results of genetically Gb3-deficient mice. In conclusion, our data significantly advance the current knowledge on the physiologic and pathophysiologic role of Gb3 in proximal tubules, showing an involvement in the reabsorption of filtered albumin, myoglobin and the aminoglycoside gentamicin.

Membrane assembly of Shiga toxin glycosphingolipid receptors and toxin refractiveness of MDCK II epithelial cells.

Shiga toxins (Stxs) are the major virulence factors of Stx-producing Escherichia coli (STEC), which cause hemorrhagic colitis and severe extraintestinal complications due to injury of renal endothelial cells, resulting in kidney failure. Since kidney epithelial cells are suggested additional targets for Stxs, we analyzed Madin-Darby canine kidney (MDCK) II epithelial cells for presence of Stx-binding glycosphingolipids (GSLs), determined their distribution to detergent-resistant membranes (DRMs), and ascertained the lipid composition of DRM and non-DRM preparations. Globotriaosylceramide and globotetraosylceramide, known as receptors for Stx1a, Stx2a, and Stx2e, and Forssman GSL as a specific receptor for Stx2e, were found to cooccur with SM and cholesterol in DRMs of MDCK II cells, which was shown using TLC overlay assay detection combined with mass spectrometry. The various lipoforms of GSLs were found to mainly harbor ceramide moieties composed of sphingosine (d18:1) and C24:1/C24:0 or C16:0 FA. The cells were highly refractory toward Stx1a, Stx2a, and Stx2e, most likely due to the absence of Stx-binding GSLs in the apical plasma membrane determined by immunofluorescence confocal laser scanning microscopy. The results suggest that the cellular content of Stx receptor GSLs and their biochemical detection in DRM preparations alone are inadequate to predict cellular sensitivity toward Stxs.

Combining Mass Spectrometry, Surface Acoustic Wave Interaction Analysis, and Cell Viability Assays for Characterization of Shiga Toxin Subtypes of Pathogenic Escherichia coli Bacteria.

Shiga toxin (Stx)-producing Escherichia coli (STEC) and enterohemorrhagic E. coli (EHEC) as a human pathogenic subgroup of STEC are characterized by releasing Stx AB5-toxin as the major virulence factor. Worldwide disseminated EHEC strains cause sporadic infections and outbreaks in the human population and swine pathogenic STEC strains represent greatly feared pathogens in pig breeding and fattening plants. Among the various Stx subtypes, Stx1a and Stx2a are of eminent clinical importance in human infections being associated with life-threatening hemorrhagic colitis and hemolytic uremic syndrome, whereas Stx2e subtype is associated with porcine edema disease with a generalized fatal outcome for the animals. Binding toward the glycosphingolipid globotriaosylceramide (Gb3Cer) is a common feature of all Stx subtypes analyzed so far. Here, we report on the development of a matched strategy combining (i) miniaturized one-step affinity purification of native Stx subtypes from culture supernatant of bacterial wild-type strains using Gb3-functionalized magnetic beads, (ii) structural analysis and identification of Stx holotoxins by electrospray ionization ion mobility mass spectrometry (ESI MS), (iii) functional Stx-receptor real-time interaction analysis employing the surface acoustic wave (SAW) technology, and (iv) Vero cell culture assays for determining Stx-caused cytotoxic effects. Structural investigations revealed diagnostic tryptic peptide ions for purified Stx1a, Stx2a, and Stx2e, respectively, and functional analysis resulted in characteristic binding kinetics of each Stx subtype. Cytotoxicity studies revealed differing toxin-mediated cell damage ranked with Stx1a > Stx2a > Stx2e. Collectively, this matched procedure represents a promising clinical application for the characterization of life-endangering Stx subtypes at the protein level.

Shiga toxin-glycosphingolipid interaction: Status quo of research with focus on primary human brain and kidney endothelial cells.

Shiga toxin (Stx)-mediated injury of the kidneys and the brain represent the major extraintestinal complications in humans upon infection by enterohemorrhagic Escherichia coli (EHEC). Damage of renal and cerebral endothelial cells is the key event in the pathogenesis of the life-threatening hemolytic uremic syndrome (HUS). Stxs are AB5 toxins and the B-pentamers of the two clinically important Stx subtypes Stx1a and Stx2a preferentially bind to the glycosphingolipid globotriaosylceramide (Gb3Cer, Galα4Galß4Glcß1Cer) and to less extent to globotetraosylceramide (Gb4Cer, GalNAcß3Galα4Galß4Glcß1), which are expected to reside in lipid rafts in the plasma membrane of the human endothelium. This review summarizes the current knowledge on the Stx glycosphingolipid receptors and their lipid membrane ensemble in primary human brain microvascular endothelial cells (pHBMECs) and primary human renal glomerular endothelial cells (pHRGECs). Increasing knowledge on the precise initial molecular mechanisms by which Stxs interact with cellular targets will help to develop specific therapeutics and/or preventive measures to combat EHEC-caused diseases.

Shiga toxin glycosphingolipid receptors and their lipid membrane ensemble in primary human blood-brain barrier endothelial cells.

Shiga toxin (Stx)-mediated injury to microvascular endothelial cells in the brain significantly contributes to the pathogenesis of the hemolytic-uremic syndrome caused by enterohemorrhagic Escherichia coli (EHEC). Stxs are AB5 toxins and the B-pentamers of the two major Stx subtypes Stx1a and Stx2a preferentially bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer) expressed by human endothelial cells. Here we report on comprehensive structural analysis of the different lipoforms of Gb3Cer (Galα4Galß4Glcß1Cer) and globotetraosylceramide (Gb4Cer, GalNAcß3Galα4Galß4Glcß1Cer, the less effective Stx receptor) of primary human brain microvascular endothelial cells and their association with lipid rafts. Detergent-resistant membranes (DRMs), obtained by sucrose density gradient ultracentrifugation, were used as lipid raft-analogous microdomains of the liquid-ordered phase and nonDRM fractions were employed as equivalents for the liquid-disordered phase of cell membranes. Structures of the prevalent lipoforms of Gb3Cer and Gb4Cer were those with Cer (d18:1, C16:0), Cer (d18:1, C22:0) and Cer (d18:1, C24:1/C24:0) determined by electrospray ionization mass spectrometry that was combined with thin-layer chromatography immunodetection using anti-Gb3Cer and anti-Gb4Cer antibodies as well as Stx1a and Stx2a subtypes. Association of Stx receptor GSLs was determined by co-localization with lipid raft-specific membrane protein flotillin-2 and canonical lipid raft marker sphingomyelin with Cer (d18:1, C16:0) and Cer (d18:1, C24:1/C24:0) in the liquid-ordered phase, whereas lyso-phosphatidylcholine was detectable exclusively in the liquid-disordered phase. Defining the precise microdomain structures of primary endothelial cells may help to unravel the initial mechanisms by which Stxs interact with their target cells and will help to develop novel preventive and therapeutic measures for EHEC-mediated diseases.

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.

Damage of human renal glomerular endothelial cells (HRGECs) of the kidney represents the linchpin in the pathogenesis of the hemolytic uremic syndrome caused by Shiga toxins of enterohemorrhagic Escherichia coli (EHEC). We performed a comprehensive structural analysis of the Stx-receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer, Galα4Galß4Glcß1Cer) and globotetraosylceramide (Gb4Cer, GalNAcß3Galα4Galß4Glcß1Cer) and their distribution in lipid raft analog detergent-resistant membranes (DRMs) and nonDRMs prepared from primary HRGECs. Predominant receptor lipoforms were Gb3Cer and Gb4Cer with Cer (d18:1, C16:0), Cer (d18:1, C22:0) and Cer (d18:1, C24:1/C24:0). Stx-receptor GSLs co-distribute with sphingomyelin (SM) and cholesterol as well as flotillin-2 in DRMs, representing the liquid-ordered membrane phase and indicating lipid raft association. Lyso-phosphatidylcholine (lyso-PC) was identified as a nonDRM marker phospholipid of the liquid-disordered membrane phase. Exposure of primary HRGECs to the ceramide analogon d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) reduced total Gb3Cer and Gb4Cer content, roughly calculated from two biological replicates, down to half and quarter of its primordial content, respectively, but strengthened their prevalence and cholesterol preponderance in DRMs. At the same time, the distribution of PC, SM and lyso-PC to subcellular membrane fractions remained unaffected by D-PDMP treatment. Defining the GSL composition and precise microdomain structures of primary HRGECs may help to develop novel therapeutic options to combat life-threatening EHEC infections.

Shiga toxin of enterohaemorrhagic Escherichia coli directly injures developing human erythrocytes.

Haemolytic anaemia is one of the characteristics of life-threatening extraintestinal complications in humans during infection with enterohaemorrhagic Escherichia coli (EHEC). Shiga toxins (Stxs) of EHEC preferentially damage microvascular endothelial cells of the kidney and the brain, whereby occluded small blood vessels may elicit anaemia through mechanical erythrocyte disruption. Here we show for the first time that Stx2a, the major virulence factor of EHEC, is also capable of direct targeting developing human erythrocytes. We employed an ex vivo erythropoiesis model using mobilized CD34(+) haematopoietic stem/progenitor cells from human blood and monitored expression of Stx receptors and Stx2a-mediated cellular injury of developing erythrocytes. CD34(+) haematopoietic stem/progenitor cells were negative for Stx2a receptors and resistant towards the toxin. Expression of Stx2a-binding glycosphingolipids and toxin sensitivity was apparent immediately after initiation of erythropoietic differentiation, peaked for basophilic and polychromatic erythroblast stages and declined during maturation into orthochromatic erythroblasts and reticulocytes, which became highly refractory to Stx2a. The observed Stx-mediated toxicity towards erythroblasts during the course of erythropoiesis might contribute, although speculative at this stage of research, to the anaemia caused by Stx-producing pathogens.

On-Tissue Phospholipase C Digestion for Enhanced MALDI-MS Imaging of Neutral Glycosphingolipids.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can be used to simultaneously visualize the lateral distribution of different lipid classes in tissue sections, but the applicability of the method to real-life samples is often limited by ion suppression effects. In particular, the presence of abundant phosphatidylcholines (PCs) can reduce the ion yields for all other lipid species in positive ion mode measurements. Here, we used on-tissue treatment with buffer-free phospholipase C (PLC) to near-quantitatively degrade PCs in fresh-frozen tissue sections. The ion signal intensities of mono-, di-, and oligohexosylceramides were enhanced by up to 10-fold. In addition, visualization of Shiga toxin receptor globotriaosylceramide (Gb3Cer) in the kidneys of wild-type and α-galactosidase A-knockout (Fabry) mice was possible at about ten micrometer resolution. Importantly, the PLC treatment did not decrease the high lateral resolution of the MS imaging analysis.

Physico-chemical characteristics and primary structure of an affinity-purified α-D-galactose-specific, jacalin-related lectin from the latex of mulberry (Morus indica).

An α-D-galactose specific lectin belonging to the family of jacalin-related lectins (JRL) has been purified by affinity chromatography on cross-linked guar-gum. Mass spectrometric data revealed that the protein harbors two chains like all the members of galactose-specific jacalin-related lectins (gJRL). De novo sequencing of proteolytic peptides demonstrated that the heavier chain consists of 133 amino acids and the lighter chain comprises of 21 or 24 amino acids. The heavier chain contains one N-glycosylation site (Asn47) occupied with either pauci-mannose type [GlcNAc2(Fuc)Man3(Xyl)] or complex type [GlcNAc2(Fuc)Man3(Xyl)GlcNAc(Fuc)Gal] N-glycans. Circular dichroism spectroscopy indicated that the secondary structure of the lectin is predominantly made up of ß-sheets, and differential scanning calorimetry revealed a thermal denaturation temperature of 77.6 °C. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assays on MCF-7 and MDCK cells showed that the lectin is highly cytotoxic towards both cell lines when dosed at micromolar concentrations, suggesting that it may play a role in the defense mechanism of the plant.

Shiga toxin glycosphingolipid receptors of Vero-B4 kidney epithelial cells and their membrane microdomain lipid environment.

Shiga toxins (Stxs) are produced by enterohemorrhagic Escherichia coli (EHEC), which cause human infections with an often fatal outcome. Vero cell lines, derived from African green monkey kidney, represent the gold standard for determining the cytotoxic effects of Stxs. Despite their global use, knowledge about the exact structures of the Stx receptor glycosphingolipids (GSLs) and their assembly in lipid rafts is poor. Here we present a comprehensive structural analysis of Stx receptor GSLs and their distribution to detergent-resistant membranes (DRMs), which were prepared from Vero-B4 cells and used as lipid raft equivalents. We identified globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) as the GSL receptors for Stx1a, Stx2a, and Stx2e subtypes using TLC overlay detection combined with MS. The uncommon Stx receptor, globopentaosylceramide (Gb5Cer, Galß3GalNAcß3Galα4Galß4Glcß1Cer), which was specifically recognized (in addition to Gb3Cer and Gb4Cer) by Stx2e, was fully structurally characterized. Lipoforms of Stx receptor GSLs were found to mainly harbor ceramide moieties composed of sphingosine (d18:1) and C24:0/C24:1 or C16:0 fatty acid. Moreover, co-occurrence with lipid raft markers, SM and cholesterol, in DRMs suggested GSL association with membrane microdomains. This study provides the basis for further exploring the functional impact of lipid raft-associated Stx receptors for toxin-mediated injury of Vero-B4 cells.

Direct acute tubular damage contributes to Shigatoxin-mediated kidney failure.

The pathogenesis and therapy of Shigatoxin 2 (Stx2)-mediated kidney failure remain controversial. Our aim was to test whether, during an infection with Stx2-producing E. coli (STEC), Stx2 exerts direct effects on renal tubular epithelium and thereby possibly contributes to acute renal failure. Mice represent a suitable model because they, like humans, express the Stx2-receptor Gb3 in the tubular epithelium but, in contrast to humans, not in glomerular endothelia, and are thus free of glomerular thrombotic microangiopathy (TMA). In wild-type mice, Stx2 caused acute tubular dysfunction with consequent electrolyte disturbance, which was most likely the cause of death. Tubule-specific depletion of Gb3 protected the mice from acute renal failure. In vitro, Stx2 induced secretion of proinflammatory cytokines and apoptosis in human tubular epithelial cells, thus implicating a direct effect of Stx2 on the tubular epithelium. To correlate these results to human disease, kidney biopsies and outcome were analysed in patients with Stx2-associated kidney failure (n = 11, aged 22-44 years). The majority of kidney biopsies showed different stages of an ongoing TMA; however, no glomerular complement activation could be demonstrated. All biopsies, including those without TMA, showed severe acute tubular damage. Due to these findings, patients were treated with supportive therapy without complement-inhibiting antibodies (eculizumab) or immunoadsorption. Despite the severity of the initial disease [creatinine 6.34 (1.31-17.60) mg/dl, lactate dehydrogenase 1944 (753-2792) U/l, platelets 33 (19-124)/nl and haemoglobin 6.2 (5.2-7.8) g/dl; median (range)], all patients were discharged after 33 (range 19-43) days with no neurological symptoms and no dialysis requirement [creatinine 1.39 (range 0.84-2.86) mg/dl]. The creatinine decreased further to 0.90 (range 0.66-1.27) mg/dl after 24 months. Based on these data, one may surmise that acute tubular damage represents a separate pathophysiological mechanism, importantly contributing to Stx2-mediated acute kidney failure. Specifically in young adults, an excellent outcome can be achieved by supportive therapy only.

Characterization of freeze-fractured epithelial plasma membranes on nanometer scale with ToF-SIMS.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used to characterize the freeze-fracturing process of human epithelial PANC-1 and UROtsa cells. For this purpose, phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and phosphatidylserine standard samples were investigated to find specific signals with both high specificity and signal intensity. The results were used to investigate single cells of subconfluent cell layers prepared with a special silicon wafer sandwich preparation technique. This freeze-fracturing technique strips cell membranes off the cells, isolating them on opposing silicon wafer substrates. Criteria were found for defining regions with stripped off cell membranes and, on the opposing wafer, complementary regions with the remaining cells. Measured ethanolamine/choline and serine/choline ratios in these regions clearly showed that in the freeze-fracturing process, the lipid bilayer of the plasma membrane is split along its central zone. Accordingly, only the outer lipid monolayer is stripped off the cell, while the inner lipid monolayer remains attached to the cell on the opposing wafer, thus allowing detailed analysis of a single lipid monolayer. Furthermore, it could be shown that using different washing procedures did not influence the transmembrane lipid distribution. Under optimized preparation conditions, it became feasible to detect lipids with a lateral resolution of approximately 100 nm. The data indicate that ToF-SIMS would be a very useful technique to study with very high lateral resolution changes in lipid composition caused, for example, by lipid storage diseases or pharmaceuticals that interfere with the lipid metabolism.