Formation of moiré interlayer excitons in space and time.

Moiré superlattices in atomically thin van der Waals heterostructures hold great promise for extended control of electronic and valleytronic lifetimes1-7, the confinement of excitons in artificial moiré lattices8-13 and the formation of exotic quantum phases14-18. Such moiré-induced emergent phenomena are particularly strong for interlayer excitons, where the hole and the electron are localized in different layers of the heterostructure19,20. To exploit the full potential of correlated moiré and exciton physics, a thorough understanding of the ultrafast interlayer exciton formation process and the real-space wavefunction confinement is indispensable. Here we show that femtosecond photoemission momentum microscopy provides quantitative access to these key properties of the moiré interlayer excitons. First, we elucidate that interlayer excitons are dominantly formed through femtosecond exciton-phonon scattering and subsequent charge transfer at the interlayer-hybridized Σ valleys. Second, we show that interlayer excitons exhibit a momentum fingerprint that is a direct hallmark of the superlattice moiré modification. Third, we reconstruct the wavefunction distribution of the electronic part of the exciton and compare the size with the real-space moiré superlattice. Our work provides direct access to interlayer exciton formation dynamics in space and time and reveals opportunities to study correlated moiré and exciton physics for the future realization of exotic quantum phases of matter.

Far-from-Equilibrium Electron-Phonon Interactions in Optically Excited Graphene.

Comprehending far-from-equilibrium many-body interactions is one of the major goals of current ultrafast condensed matter physics research. Here, a particularly interesting but barely understood situation occurs during a strong optical excitation, where the electron and phonon systems can be significantly perturbed and the quasiparticle distributions cannot be described with equilibrium functions. In this work, we use time- and angle-resolved photoelectron spectroscopy to study such far-from-equilibrium many-body interactions for the prototypical material graphene. In accordance with theoretical simulations, we find remarkable transient renormalizations of the quasiparticle self-energy caused by the photoinduced nonequilibrium conditions. These observations can be understood by ultrafast scatterings between nonequilibrium electrons and strongly coupled optical phonons, which signify the crucial role of ultrafast nonequilibrium dynamics on many-body interactions. Our results advance the understanding of many-body physics in extreme conditions, which is important for any endeavor to optically manipulate or create non-equilibrium states of matter.

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.

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.

Advanced Methods for MALDI-MS Imaging of the Chemical Communication in Microbial Communities.

Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) is increasingly used to visualize the chemical communication between microorganisms. However, to fully exploit the potential of this label-free technique, crucial methodological advances are still needed. In particular, with current microbial MALDI-MSI methods chemical coverage is strongly limited to well ionizing compounds and a safe MSI-compatible inactivation of microbial viability and quenching of metabolism is not possible. Here, we introduce a membrane-based culturing workflow that enables a rapid MSI-compatible steam inactivation of pathogens and generation of a flat surface. We equipped precision mass spectrometers with laser-postionization (MALDI-2) modules to increase the analytical sensitivity by up to several orders of magnitude. In this way, for example 39 different 2-alkylquinolones with differential expression patterns and a similar number of glycerophospholipids were simultaneously visualized from single cultures of Pseudomonas aeruginosa at about 50 µm resolution. To visualize the metabolic exchange between competing microorganisms, we challenged commensal Escherichia coli MG1655 and virulence factor-depleted E. coli C600 strains with enteropathogenic Shiga-toxin negative E. coli O26:H11, and Staphylococcus aureus with antagonistic P. aeruginosa. Insight into the three-dimensional organization of a biofilm of the probiotic E. coli Nissle 1917 at 15 µm pixel size was obtained after developing an embedding/cryosectioning protocol. Our advanced protocols could help to substantially increase the application range of microbial MS imaging.

Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury.

Outer membrane vesicles (OMVs) are important tools in bacterial virulence but their role in the pathogenesis of infections caused by enterohemorrhagic Escherichia coli (EHEC) O157, the leading cause of life-threatening hemolytic uremic syndrome, is poorly understood. Using proteomics, electron and confocal laser scanning microscopy, immunoblotting, and bioassays, we investigated OMVs secreted by EHEC O157 clinical isolates for virulence factors cargoes, interactions with pathogenetically relevant human cells, and mechanisms of cell injury. We demonstrate that O157 OMVs carry a cocktail of key virulence factors of EHEC O157 including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, and flagellin. The toxins are internalized by cells via dynamin-dependent endocytosis of OMVs and differentially separate from vesicles during intracellular trafficking. Stx2a and CdtV-B, the DNase-like CdtV subunit, separate from OMVs in early endosomes. Stx2a is trafficked, in association with its receptor globotriaosylceramide within detergent-resistant membranes, to the Golgi complex and the endoplasmic reticulum from where the catalytic Stx2a A1 fragment is translocated to the cytosol. CdtV-B is, after its retrograde transport to the endoplasmic reticulum, translocated to the nucleus to reach DNA. CdtV-A and CdtV-C subunits remain OMV-associated and are sorted with OMVs to lysosomes. EHEC hemolysin separates from OMVs in lysosomes and targets mitochondria. The OMV-delivered CdtV-B causes cellular DNA damage, which activates DNA damage responses leading to G2 cell cycle arrest. The arrested cells ultimately die of apoptosis induced by Stx2a and CdtV via caspase-9 activation. By demonstrating that naturally secreted EHEC O157 OMVs carry and deliver into cells a cocktail of biologically active virulence factors, thereby causing cell death, and by performing first comprehensive analysis of intracellular trafficking of OMVs and OMV-delivered virulence factors, we provide new insights into the pathogenesis of EHEC O157 infections. Our data have implications for considering O157 OMVs as vaccine candidates.

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 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.

Probing electron-hole Coulomb correlations in the exciton landscape of a twisted semiconductor heterostructure.

In two-dimensional semiconductors, cooperative and correlated interactions determine the material's excitonic properties and can even lead to the creation of correlated states of matter. Here, we study the fundamental two-particle correlated exciton state formed by the Coulomb interaction between single-particle holes and electrons. We find that the ultrafast transfer of an exciton's hole across a type II band-aligned semiconductor heterostructure leads to an unexpected sub-200-femtosecond upshift of the single-particle energy of the electron being photoemitted from the two-particle exciton state. While energy relaxation usually leads to an energetic downshift of the spectroscopic signature, we show that this upshift is a clear fingerprint of the correlated interaction of the electron and hole parts of the exciton. In this way, time-resolved photoelectron spectroscopy is straightforwardly established as a powerful method to access electron-hole correlations and cooperative behavior in quantum materials. Our work highlights this capability and motivates the future study of optically inaccessible correlated excitonic and electronic states of matter.

Disentangling the multiorbital contributions of excitons by photoemission exciton tomography.

Excitons are realizations of a correlated many-particle wave function, specifically consisting of electrons and holes in an entangled state. Excitons occur widely in semiconductors and are dominant excitations in semiconducting organic and low-dimensional quantum materials. To efficiently harness the strong optical response and high tuneability of excitons in optoelectronics and in energy-transformation processes, access to the full wavefunction of the entangled state is critical, but has so far not been feasible. Here, we show how time-resolved photoemission momentum microscopy can be used to gain access to the entangled wavefunction and to unravel the exciton's multiorbital electron and hole contributions. For the prototypical organic semiconductor buckminsterfullerene (C60), we exemplify the capabilities of exciton tomography and achieve unprecedented access to key properties of the entangled exciton state including localization, charge-transfer character, and ultrafast exciton formation and relaxation dynamics.

Ultrafast element-resolved magneto-optics using a fiber-laser-driven extreme ultraviolet light source.

We present a novel setup to measure the transverse magneto-optical Kerr effect in the extreme ultraviolet spectral range based on a fiber laser amplifier system with a repetition rate between 100 and 300 kHz, which we use to measure element-resolved demagnetization dynamics. The setup is equipped with a strong electromagnet and a cryostat, allowing measurements between 10 and 420 K using magnetic fields up to 0.86 T. The performance of our setup is demonstrated by a set of temperature- and time-dependent magnetization measurements with elemental resolution.

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.

Band-structure-dependent demagnetization in the Heusler alloy Co2Mn(1-x)FexSi.

We investigate the ultrafast demagnetization for two Heusler alloys (Co2Mn(1-x)FexSi) with a different lineup of the minority band gap and the Fermi level. Even though electronic spin-flip transitions are partially blocked by the band gap in one compound, the respective magnetization dynamics, as measured by the time-resolved Kerr effect, are remarkably similar. Based on a dynamical model that includes momentum and spin-dependent carrier scattering, we show that the magnetization dynamics are dominated by hole spin-flip processes, which are not influenced by the gap.

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.

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. Though both lines exhibited the Stx receptor globotriaosylceramide, RNAseq revealed strikingly different transcriptomic responses to an Stx challenge. Using RNAi to silence factors involved in ACHN cells' Stx response, the greatest protection occurred when silencing RAB5A and TRAPPC6B, two host factors that we newly link to Stx trafficking. Silencing these factors alongside YKT6 fully prevented the cytotoxic Stx effect. Overall, our approach reveals novel subcellular targets for potential therapies against Stx-mediated kidney failure.

Time-resolved momentum microscopy with a 1 MHz high-harmonic extreme ultraviolet beamline.

Recent progress in laser-based high-repetition rate extreme ultraviolet (EUV) light sources and multidimensional photoelectron spectroscopy enables the build-up of a new generation of time-resolved photoemission experiments. Here, we present a setup for time-resolved momentum microscopy driven by a 1 MHz fs EUV table-top light source optimized for the generation of 26.5 eV photons. The setup provides simultaneous access to the temporal evolution of the photoelectron's kinetic energy and in-plane momentum. We discuss opportunities and limitations of our new experiment based on a series of static and time-resolved measurements on graphene.

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 Galß1-4Glcß1-1Cer) 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.