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.

Ingenious Action of Vibrio cholerae 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 Vibrio cholerae binds with high preference to the ganglioside GM1 (II3Neu5Ac-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 V. cholerae. To avoid the artificial incorporation of exogenous gangliosides from animal serum harboring GM1 and higher sialylated ganglio-series gangliosides, pHCoEpiCs were cultured in serum-free medium. Thin-layer chromatography overlay binding assays using a choleragenoid combined with electrospray ionization mass spectrometry revealed GM1 lipoforms with sphingosine (d18:1) as the sole sphingoid base linked to C14:0, C16:0, C18:0 or C20:0 fatty acyl chains forming the ceramide (Cer) moieties of the main choleragenoid-binding GM1 species. Desialylation of GD1a (IV3Neu5Ac,II3Neu5Ac-Gg4Cer) and GT1b (IV3Neu5Ac,II3(Neu5Ac)2-Gg4Cer) of pHCoEpiCs by V. cholerae neuraminidase was observed. GD1a-derived GM1 species with stable sphingosine (d18:1) and saturated fatty acyl chains varying in chain length from C16:0 up to C22:0 could be identified, indicating the ingenious interplay between CT and the neuraminidase of V. cholerae recruiting additional GM1 receptors of pHCoEpiCs.

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. We found that the cell-to-cell variations of specific lipid metabolic pathways contribute to the establishment of cell states involved in the organization of skin architecture. Sphingolipid composition is shown to define fibroblast subpopulations, with sphingolipid metabolic rewiring driving cell-state transitions. Therefore, cell-to-cell lipid heterogeneity affects the determination of cell states, adding a new regulatory component to the self-organization of multicellular systems.

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.

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 Escherichia coli (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. Stx1a and Stx2a exerted strong cellular damage with half-maximal cytotoxic doses (CD50) of 1.31 × 102 pg/mL and 1.66 × 103 pg/mL, respectively, indicating one order of magnitude higher cellular cytotoxicity of Stx1a. Surface acoustic wave (SAW) real-time interaction analysis using biosensor surfaces coated with DRM or nonDRM fractions gave stronger binding capability of Stx1a versus Stx2a that correlated with the lower cytotoxicity of Stx2a. Our study underlines the substantial role of proximal tubular epithelial cells of the human kidney being associated with the development of Stx-mediated HUS at least for Stx1a, while the impact of Stx2a remains somewhat ambiguous.

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 Escherichia coli (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. A preference of Gb3Cer and Gb4Cer endowed with C24:0 fatty acid accompanied by minor monounsaturated C24:1-harboring counterparts was observed in DRMs, whereas the C24:1 fatty acid increased in relation to the saturated equivalents in non-DRMs. A shift of the dominant phospholipid phosphatidylcholine with saturated fatty acids in the DRM to unsaturated species in the non-DRM fractions correlated with the GSL distribution. Cytotoxicity assays gave a moderate susceptibility of pHRCEpiCs to the Stx1a and Stx2a subtypes when compared to highly sensitive Vero-B4 cells. The results indicate that presence of Stx-binding GSLs per se and preferred occurrence in microdomains do not necessarily lead to a high cellular susceptibility towards Stx.

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. In addition to Gb3Cer and Gb4Cer, Gb5Cer and Forssman GSL are further receptors of the Stx2e subtype rendering Stx2e unique among the various Stx subtypes. Thin-layer chromatography (TLC) is a convenient and ubiquitously employed method for analyzing GSL mixtures of unknown composition. In particular, TLC immunochemical overlay detection allows for sensitive identification of Stx-binding GSLs in complex mixtures directly on the TLC plate. For this purpose, specific anti-GSL antibodies or Stxs themselves in conjunction with anti-Stx antibodies can be used. The described protocols of antibody-mediated detection of TLC-separated globo-series GSLs and corresponding identification of Stx-binding globo-series GSLs will provide detailed advice for successful GSL analysis and particularly highlight the power of the TLC overlay technique.

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.

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 Escherichia coli (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. Hemolytic anemia during HUS is defined as the loss of erythrocytes by mechanical disruption when passing through narrowed microvessels. The formation of thrombi in the microvasculature is considered an indirect effect of Stx-mediated injury mainly of the renal microvascular endothelial cells, resulting in obstructions of vessels. In this review, we summarize and discuss recent data providing evidence that HUS-associated hemolytic anemia may arise not only from intravascular rupture of erythrocytes, but also from the extravascular impairment of erythropoiesis, the development of red blood cells in the bone marrow, via direct Stx-mediated damage of maturing erythrocytes, leading to "non-hemolytic" anemia.

Modeling Native EHEC Outer Membrane Vesicles by Creating Synthetic Surrogates.

Enterohemorrhagic Escherichia coli (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. Thus, it is difficult to determine to which extent a single virulence factor in nOMVs, such as Stx, contributes to the molecular pathogenesis of EHEC. To reduce this complexity, we successfully developed a protocol for the preparation of synthetic OMVs (sOMVs) with a defined lipid composition resembling the E. coli outer membrane and loaded with specific proteins, i.e., bovine serum albumin (BSA) as a proxy for functional Stx2a. Using BSA for parameter evaluation, we found that (1) functional sOMVs can be prepared at room temperature instead of potentially detrimental higher temperatures (e.g., 45 °C), (2) a 1:10 ratio of protein to lipid, i.e., 100 µg protein with 1 mg of lipid mixture, yields homogenously sized sOMVs, and (3) long-term storage for up to one year at 4 °C is possible without losing structural integrity. Accordingly, we reproducibly generated Stx2a-loaded sOMVs with an average diameter of 132.4 ± 9.6 nm that preserve Stx2a's injuring activity, as determined by cytotoxicity assays with Vero cells. Overall, we successfully created sOMVs and loaded them with an EHEC toxin, which opens the door for future studies on the degree of virulence associated with individual toxins from EHEC and other bacterial pathogens.

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.

Structural Insights into Escherichia coli 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 Escherichia coli (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, Galα1-4Galß1-4Glcß1-1Cer) and globotetraosylceramide (Gb4Cer, GalNAcß1-3Galα1-4Galß1-4Glcß1-1Cer). 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. Immunochemical and mass spectrometric analysis revealed various Gb3Cer and Gb4Cer lipoforms as the dominant Stx-binding GSLs in both LLC-PK1 and PK-15 cells. A dihexosylceramide with proposed Galα1-4Gal-sequence (Gal2Cer) was detected in PK-15 cells, whereas LLC-PK1 cells lacked this compound. Both cell lines were susceptible towards Stx2e with LLC-PK1 representing an extremely Stx2e-sensitive cell line. Gb3-PE and Gb4-PE applied as glycovesicles significantly reduced the cytotoxic activity of Stx2e towards LLC-PK1 cells, whereas only Gb4-PE exhibited some protection against Stx2e for PK-15 cells. This is the first report identifying Stx2e receptors of porcine kidney epithelial cells and providing first data on their Stx2e-mediated damage suggesting possible involvement in the edema disease.