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

Enterohemorrhagic (EHEC) are the human pathogenic subset of Shiga toxin (Stx)-producing (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.

Ingenious Action of Neuraminidase Recruiting Additional GM1 Cholera Toxin Receptors for Primary Human Colon Epithelial Cells.

For five decades it has been known that the pentamer of B subunits (choleragenoid) of the cholera toxin (CT) of binds with high preference to the ganglioside GM1 (IINeu5Ac-Gg4Cer). However, the exact structures of CT-binding GM1 lipoforms of primary human colon epithelial cells (pHCoEpiCs) have not yet been described in detail. The same holds true for generating further GM1 receptor molecules from higher sialylated gangliosides with a GM1 core through the neuraminidase of .

Sphingolipids control dermal fibroblast heterogeneity.

Human cells produce thousands of lipids that change during cell differentiation and can vary across individual cells of the same type. However, we are only starting to characterize the function of these cell-to-cell differences in lipid composition. Here, we measured the lipidomes and transcriptomes of individual human dermal fibroblasts by coupling high-resolution mass spectrometry imaging with single-cell transcriptomics.

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.

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 (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, Galα1-4Galβ1-4Glcβ1-1Cer) and globotetraosylceramide (Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer) 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.

Primary Human Renal Proximal Tubular Epithelial Cells (pHRPTEpiCs): Shiga Toxin (Stx) Glycosphingolipid Receptors, Stx Susceptibility, and Interaction with Membrane Microdomains.

Tubular epithelial cells of the human kidney are considered as targets of Shiga toxins (Stxs) in the Stx-mediated pathogenesis of hemolytic-uremic syndrome (HUS) caused by Stx-releasing enterohemorrhagic (EHEC). Analysis of Stx-binding glycosphingolipids (GSLs) of primary human renal proximal tubular epithelial cells (pHRPTEpiCs) yielded globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) with Cer (d18:1, C16:0), Cer (d18:1, C22:0), and Cer (d18:1, C24:1/C24:0) as the dominant lipoforms. Investigation of detergent-resistant membranes (DRMs) and nonDRMs, serving as equivalents for the liquid-ordered and liquid-disordered membrane phase, respectively, revealed the prevalence of Gb3Cer and Gb4Cer together with cholesterol and sphingomyelin in DRMs, suggesting lipid raft association.

Thin-Layer Chromatography in Structure and Recognition Studies of Shiga Toxin Glycosphingolipid Receptors.

Glycosphingolipids (GSLs) consist of a ceramide (Cer) lipid anchor, which is typically composed of the long-chain aminoalcohol sphingosine (d18:1) and a fatty acid (mostly C16-C24) and a sugar moiety harboring to a great extent one to five monosaccharides. GSLs of the globo-series are well-recognized receptors of Shiga toxins (Stxs) released by Stx-producing Escherichia coli (STEC). Receptors for the Stx subtypes Stx1a and Stx2a are globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), whereby Gb3Cer represents their high-affinity and Gb4Cer their low-affinity receptor.

Shiga Toxin (Stx)-Binding Glycosphingolipids of Primary Human Renal Cortical Epithelial Cells (pHRCEpiCs) and Stx-Mediated Cytotoxicity.

Human kidney epithelial cells are supposed to be directly involved in the pathogenesis of the hemolytic-uremic syndrome (HUS) caused by Shiga toxin (Stx)-producing enterohemorrhagic (EHEC). The characterization of the major and minor Stx-binding glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), respectively, of primary human renal cortical epithelial cells (pHRCEpiCs) revealed GSLs with Cer (d18:1, C16:0), Cer (d18:1, C22:0), and Cer (d18:1, C24:1/C24:0) as the dominant lipoforms. Using detergent-resistant membranes (DRMs) and non-DRMs, Gb3Cer and Gb4Cer prevailed in the DRM fractions, suggesting their association with microdomains in the liquid-ordered membrane phase.

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, Galβ1-4Glc) or neolactotetraose (nLc4, Galβ1-4GlcNAcβ1-3Galβ1-4Glc) 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.

Valid Presumption of Shiga Toxin-Mediated Damage of Developing Erythrocytes in EHEC-Associated Hemolytic Uremic Syndrome.

The global emergence of clinical diseases caused by enterohemorrhagic (EHEC) is an issue of great concern. EHEC release Shiga toxins (Stxs) as their key virulence factors, and investigations on the cell-damaging mechanisms toward target cells are inevitable for the development of novel mitigation strategies. Stx-mediated hemolytic uremic syndrome (HUS), characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute renal injury, is the most severe outcome of an EHEC infection.

Modeling Native EHEC Outer Membrane Vesicles by Creating Synthetic Surrogates.

Enterohemorrhagic (EHEC) is a zoonotic pathogen responsible for life-threating diseases such as hemolytic uremic syndrome. While its major virulence factor, the Shiga toxin (Stx), is known to exert its cytotoxic effect on various endothelial and epithelial cells when in its free, soluble form, Stx was also recently found to be associated with EHEC outer membrane vesicles (OMVs). However, depending on the strain background, other toxins can also be associated with native OMVs (nOMVs), and nOMVs are also made up of immunomodulatory agents such as lipopolysaccharides and flagellin.

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.

RAB5A and TRAPPC6B are novel targets for Shiga toxin 2a inactivation in kidney epithelial cells.

The cardinal virulence factor of human-pathogenic enterohaemorrhagic Escherichia coli (EHEC) is Shiga toxin (Stx), which causes severe extraintestinal complications including kidney failure by damaging renal endothelial cells. In EHEC pathogenesis, the disturbance of the kidney epithelium by Stx becomes increasingly recognised, but how this exactly occurs is unknown. To explore this molecularly, we investigated the Stx receptor content and transcriptomic profile of two human renal epithelial cell lines: highly Stx-sensitive ACHN cells and largely Stx-insensitive Caki-2 cells.

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.

Structural Insights into Shiga Toxin (Stx) Glycosphingolipid Receptors of Porcine Renal Epithelial Cells and Inhibition of Stx-Mediated Cellular Injury Using Neoglycolipid-Spiked Glycovesicles.

Shiga toxin (Stx) producing (STEC) cause the edema disease in pigs by releasing the swine-pathogenic Stx2e subtype as the key virulence factor. Stx2e targets endothelial cells of animal organs including the kidney harboring the Stx receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer, Galα1-4Galβ1-4Glcβ1-1Cer) and globotetraosylceramide (Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer). Since the involvement of renal epithelial cells in the edema disease is unknown, in this study, we analyzed the porcine kidney epithelial cell lines, LLC-PK1 and PK-15, regarding the presence of Stx-binding GSLs, their sensitivity towards Stx2e, and the inhibitory potential of Gb3- and Gb4-neoglycolipids, carrying phosphatidylethanolamine (PE) as the lipid anchor, towards Stx2e.

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.

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.

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.

Pectin-derived neoglycolipids: Tools for differentiation of Shiga toxin subtypes and inhibitors of Shiga toxin-mediated cellular injury.

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)Gal oligosaccharides and their linkage to phosphatidylethanolamine (PE).

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 AB toxins and the B-pentamers of the two clinically important Stx subtypes Stx1a and Stx2a preferentially bind to the glycosphingolipid globotriaosylceramide (Gb3Cer, Galα4Galβ4Glcβ1Cer) and to less extent to globotetraosylceramide (Gb4Cer, GalNAcβ3Galα4Galβ4Glcβ1), which are expected to reside in lipid rafts in the plasma membrane of the human endothelium.

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 AB-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.

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 (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.

Shiga Toxin Glycosphingolipid Receptors in Human Caco-2 and HCT-8 Colon Epithelial Cell Lines.

Shiga toxins (Stxs) released by enterohemorrhagic (EHEC) into the human colon are the causative agents for fatal outcome of EHEC infections. Colon epithelial Caco-2 and HCT-8 cells are widely used for investigating Stx-mediated intestinal cytotoxicity. Only limited data are available regarding precise structures of their Stx receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), and association.

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, Galα4Galβ4Glcβ1Cer) and globotetraosylceramide (Gb4Cer, GalNAcβ3Galα4Galβ4Glcβ1Cer) 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).

A Topographical Atlas of Shiga Toxin 2e Receptor Distribution in the Tissues of Weaned Piglets.

Shiga toxin (Stx) 2e of Stx-producing (STEC) is the primary virulence factor in the development of pig edema disease shortly after weaning. Stx2e binds to the globo-series glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer, Galα1-4Galβ1-4Glcβ1-1Cer) and globotetraosylceramide (Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer), the latter acting as the preferential Stx2e receptor. We determined Stx receptor profiles of 25 different tissues of a male and a female weaned piglet using immunochemical solid phase binding assays combined with mass spectrometry.