Evaluation of the Speed, Accuracy and Precision of the QuickMIC Rapid Antibiotic Susceptibility Testing Assay With Gram-Negative Bacteria in a Clinical Setting.

The rapidly changing landscape of antimicrobial resistance poses a challenge for empirical antibiotic therapy in severely ill patients and highlights the need for fast antibiotic susceptibility diagnostics to guide treatment. Traditional methods for antibiotic susceptibility testing (AST) of bacteria such as broth microdilution (BMD) or the disc diffusion method (DDM) are comparatively slow and show high variability. Rapid AST methods under development often trade speed for resolution, sometimes only measuring responses at a single antibiotic concentration. QuickMIC is a recently developed lab-on-a-chip system for rapid AST. Here we evaluate the performance of the QuickMIC method with regard to speed, precision and accuracy in comparison to traditional diagnostic methods. 151 blood cultures of clinical Gram-negative isolates with a high frequency of drug resistance were tested using the QuickMIC system and compared with BMD for 12 antibiotics. To investigate sample turnaround time and method functionality in a clinical setting, another 41 clinical blood culture samples were acquired from the Uppsala University Hospital and analyzed on site in the clinical laboratory with the QuickMIC system, and compared with DDM for 8 antibiotics routinely used in the clinical laboratory. The overall essential agreement between MIC values obtained by QuickMIC and BMD was 83.4%, with an average time to result of 3 h 2 min (SD: 24.8 min) for the QuickMIC method. For the clinical dataset, the categorical agreement between QuickMIC and DDM was 96.8%, whereas essential and categorical agreement against BMD was 91.0% and 96.7%, respectively, and the total turnaround time as compared to routine diagnostics was shown to be reduced by 40% (33 h vs. 55 h). Interexperiment variability was low (average SD: 44.6% from target MIC) compared to the acceptable standard of ±1 log2 unit (i.e. -50% to +100% deviation from target MIC) in BMD. We conclude that the QuickMIC method can provide rapid and accurate AST, and may be especially valuable in settings with high resistance rates, and for antibiotics where wildtype and antibiotic-resistant bacteria have MIC distributions that are close or overlapping.

Electron Beam-Induced Transformation in High-Density Amorphous Ices.

Amorphous ice is commonly used as a noncrystalline matrix for protecting sensitive biological samples in cryogenic electron microscopy (cryo-EM). The amorphization process of water is complex, and at least two amorphous states of different densities are known to exist, high- and low-density amorphous ices (HDA and LDA). These forms are considered to be the counterparts of two distinct liquid states, namely, high- and low-density liquid water. Herein, we investigate the HDA to LDA transition using electron diffraction and cryo-EM. The observed phase transition is induced by the impact of electrons, and we discuss two different mechanisms, namely, local heating and beam-induced motion of water molecules. The temperature increase is estimated by comparison with X-ray scattering experiments on identically prepared samples. Our results suggest that HDA, under the conditions used in our cryo-EM measurements, is locally heated above its glass-transition temperature.

Influence of sodium content on the thermal behavior of low vacancy Prussian white cathode material.

Rechargeable sodium-ion batteries are the most attractive substitutes for lithium-ion batteries in large-scale energy storage devices due to wide spread reserves and low-cost of sodium resources and the similarities between sodium and lithium chemistry. However, finding a suitable cathode material is still a hurdle to be overcome. To date, Prussian white (PW), NaxFe[Fe(CN)6]y·nH2O has stood out as one of the most promising Na-host materials due to its low cost, facile synthesis and competitive electrochemical capacity. Despite this, there are concerns that this material will thermally decompose at relatively low temperatures to form cyanogen gas, which is a safety hazard. Thus, low vacancy NaxFe[Fe(CN)6]y·nH2O (x = 1.5, 1, 0.5 and 0) has been synthesized, and the influence of x on its thermal behavior systematically investigated. It is demonstrated that the thermal decomposition temperature, water content and moisture sensitivity of the samples strongly depend on the sodium content. The sample with x = 1.5 is found to be the most thermally stable and has the highest water content under the same experimental conditions. In addition, the sodium-rich samples (x = 1.5, 1 and 0.5) have higher surface water than the sodium-deficient one (x = 0). The local structure for this sample is also very different to the sodium-rich ones. Our findings offer new insights into the profound implications of proper material handling and safer operating conditions for practical Na-ion batteries and may be extended to analogous systems.

High-throughput continuous rotation electron diffraction data acquisition via software automation.

Single-crystal electron diffraction (SCED) is emerging as an effective technique to determine and refine the structures of unknown nano-sized crystals. In this work, the implementation of the continuous rotation electron diffraction (cRED) method for high-throughput data collection is described. This is achieved through dedicated software that controls the transmission electron microscope and the camera. Crystal tracking can be performed by defocusing every nth diffraction pattern while the crystal rotates, which addresses the problem of the crystal moving out of view of the selected area aperture during rotation. This has greatly increased the number of successful experiments with larger rotation ranges and turned cRED data collection into a high-throughput method. The experimental parameters are logged, and input files for data processing software are written automatically. This reduces the risk of human error, and makes data collection more reproducible and accessible for novice and irregular users. In addition, it is demonstrated how data from the recently developed serial electron diffraction technique can be used to supplement the cRED data collection by automatic screening for suitable crystals using a deep convolutional neural network that can identify promising crystals through the corresponding diffraction data. The screening routine and cRED data collection are demonstrated using a sample of the zeolite mordenite, and the quality of the cRED data is assessed on the basis of the refined crystal structure.

Hydrogenation-Induced Structure and Property Changes in the Rare-Earth Metal Gallide NdGa: Evolution of a [GaH]2- Polyanion Containing Peierls-like Ga-H Chains.

The hydride NdGaH1+x (x ≈ 0.66) and its deuterized analogue were obtained by sintering the Zintl phase NdGa with the CrB structure in a hydrogen atmosphere at pressures of 10-20 bar and temperatures near 300 °C. The system NdGa/NdGaH1+x exhibits reversible H storage capability. H uptake and release were investigated by kinetic absorption measurements and thermal desorption mass spectroscopy, which showed a maximum H concentration corresponding to "NdGaH2" (0.93 wt % H) and a two-step desorption process, respectively. The crystal structure of NdGaH1+x was characterized by neutron diffraction (P21/m, a = 4.1103(7), b = 4.1662(7), c = 6.464(1) Å, ß = 108.61(1)° Z = 2). H incorporates in NdGa by occupying two distinct positions, H1 and H2. H1 is coordinated in a tetrahedral fashion by Nd atoms. The H2 position displays flexible occupancy, and H2 atoms attain a trigonal bipyramidal coordination by centering a triangle of Nd atoms and bridging two Ga atoms. The phase stability and electronic structure of NdGaH1+x were analyzed by first-principles DFT calculations. NdGaH1H2 (NdGaH2) may be expressed as Nd(3+)(H1(-))[GaH2](2-). The two-dimensional polyanion [GaH](2-) features linear -H-Ga-H-Ga- chains with alternating short (1.8 Å) and long (2.4 Å) Ga-H distances, which resembles a Peierls distortion. H2 deficiency (x < 1) results in the fragmentation of chains. For x = 0.66 arrangements with five-atom moieties, Ga-H-Ga-H-Ga are energetically most favorable. From magnetic measurements, the Curie-Weiss constant and effective magnetic moment of NdGaH1.66 were obtained. The former indicates antiferromagnetic interactions, and the latter attains a value of ∼3.6 µB, which is typical for compounds containing Nd(3+) ions.

Electrochemical fabrication and characterization of Cu/Cu2O multi-layered micro and nanorods in Li-ion batteries.

Electrodes composed of freestanding nano- and microrods composed of stacked layers of copper and cuprous oxide have been fabricated using a straightforward one-step template-assisted pulsed galvanostatic electrodeposition approach. The approach provided precise control of the thickness of each individual layer of the high-aspect-ratio rods as was verified by SEM, EDS, XRD, TEM and EELS measurements. Rods with diameters of 80, 200 and 1000 nm were deposited and the influence of the template pore size on the structure and electrochemical performance of the conversion reaction based electrodes in lithium-ion batteries was investigated. The multi-layered Cu2O/Cu nano- and microrod electrodes exhibited a potential window of more than 2 V, which was ascribed to the presence of a distribution of Cu2O (and Cu, respectively) nanoparticles with different sizes and redox potentials. As approximately the same areal capacity was obtained independent of the diameter of the multi-layered rods the results demonstrate the presence of an electroactive Cu2O layer with a thickness defined by the time domain of the measurements. It is also demonstrated that while the areal capacity of the electrodes decreased dramatically when the scan rate was increased from 0.1 to 2 mV s(-1), the capacity remained practically constant when the scan rate was further increased to 100 mV s(-1). This behaviour can be explained by assuming that the capacity is limited by the lithium ion diffusion rate though the Cu2O layer generated during the oxidation step. The electrochemical performance of present type of 3-D multi-layered rods provides new insights into the lithiation and delithiation reactions taking place for conversion reaction materials such as Cu2O.

The alpha1,3GalT knockout/alpha1,2FucT transgenic pig does not appear to have an advantage over the alpha1,3GalT knockout pig with respect to glycolipid reactivity with human serum antibodies.

BACKGROUND: The human H-transferase (α2FucT) was introduced in Gal-negative pigs to produce pig organs not only free from Gal-antigens, but also in which the uncapped N-acetyllactosamine precursor had been transformed into non-xenogenic blood group H type 2 compounds. This work is the first descriptive analysis of glycolipids from the GalT-KO/FucT-TG pig. The aim was to investigate the cell membrane antigens in GalT-KO/FucT-TG tissues to explore its efficacy as an organ donor. Also, detailed knowledge on the correlation between the cellular glycosyltransferase configuration and the resulting carbohydrate phenotype expression is valuable from a basic glycobiological perspective. METHODS: Neutral and acidic glycolipids from GalT-KO/FucT-TG small intestine were compared with glycolipids from two wildtype and two GalT-KO pig intestines. Glycolipid reactivity was tested on thin layer chromatography plates using chemical reagents, antibodies, lectins, and human serum. Structural characterization of neutral glycolipids was performed by LC-ESI/MS and proton NMR spectroscopy. RESULTS: Characterization of the glycolipid expression in GalT-KO/FucT-TG intestine showed absence of Gal antigens and decreased/unchanged levels of the N-acetyllactosamine precursor and the blood group H type 2 expression, when compared with the wildtype. The reactivity of human serum antibodies to GalT-KO/FucT-TG derived glycolipids was similar or slightly elevated when compared with GalT-KO glycolipids. Results from LC-ESI/MS and proton NMR spectroscopy revealed no established neutral xenogenic antigens in the GalT-KO/FucT-TG pig, and could thus not explain the immunologic reactivity to human serum antibodies. The antibody binding to acidic glycolipids is most likely to be explained by the abundance of N-glycolylneuraminic acid epitopes in pig tissues. Six neutral complex biantennary glycolipids with blood group H type 1, 2, Lewis(x) and Lewis(y) determinants were found, of which three were identified in this work for the first time. One of these was a nonaglycosylceramide with blood group H type 2 and lactosyl determinants linked to a lactotetraosyl core, and the other two were decaglycosylceramides with blood group H type 1 and H type 2 determinants linked to a neolactotetraosyl core, and Lewis(x) and blood group H type 1 determinants on a lactotetraosyl core, respectively. CONCLUSIONS: Lipid-linked carbohydrate antigens in the GalT-KO/FucT-TG pig intestine showed no or minor qualitative difference when compared with GalT-KO pigs. The GalT-KO/FucT-TG pig did not appear to have an advantage over the GalT-KO pig with respect to reactivity with human antibodies from a xenotransplantation perspective.

Structural complexity of non-acid glycosphingolipids in human embryonic stem cells grown under feeder-free conditions.

Due to their pluripotency and growth capability, there are great expectations for human embryonic stem cells, both as a resource for functional studies of early human development and as a renewable source of cells for use in regenerative medicine and transplantation. However, to bring human embryonic stem cells into clinical applications, their cell surface antigen expression and its chemical structural complexity have to be defined. In the present study, total non-acid glycosphingolipid fractions were isolated from two human embryonic stem cell lines (SA121 and SA181) originating from leftover in vitro fertilized human embryos, using large amounts of starting material (1 × 10(9) cells/cell line). The total non-acid glycosphingolipid fractions were characterized by antibody and lectin binding, mass spectrometry, and proton NMR. In addition to the globo-series and type 1 core chain glycosphingolipids previously described in human embryonic stem cells, a number of type 2 core chain glycosphingolipids (neo-lactotetraosylceramide, the H type 2 pentaosylceramide, the Le(x) pentaosylceramide, and the Le(y) hexaosylceramide) were identified as well as the blood group A type 1 hexaosylceramide. Finally, the mono-, di-, and triglycosylceramides were characterized as galactosylceramide, glucosylceramide, lactosylceramide, galabiaosylceramide, globotriaosylceramide, and lactotriaosylceramide. Thus, the glycan diversity of human embryonic stem cells, including cell surface immune determinants, is more complex than previously appreciated.

The repertoire of glycosphingolipids recognized by Vibrio cholerae.

The binding of cholera toxin to the ganglioside GM1 as the initial step in the process leading to diarrhea is nowadays textbook knowledge. In contrast, the knowledge about the mechanisms for attachment of Vibrio cholerae bacterial cells to the intestinal epithelium is limited. In order to clarify this issue, a large number of glycosphingolipid mixtures were screened for binding of El Tor V. cholerae. Several specific interactions with minor complex non-acid glycosphingolipids were thereby detected. After isolation of binding-active glycosphingolipids, characterization by mass spectrometry and proton NMR, and comparative binding studies, three distinct glycosphingolipid binding patterns were defined. Firstly, V. cholerae bound to complex lacto/neolacto glycosphingolipids with the GlcNAcß3Galß4GlcNAc sequence as the minimal binding epitope. Secondly, glycosphingolipids with a terminal Galα3Galα3Gal moiety were recognized, and the third specificity was the binding to lactosylceramide and related compounds. V. cholerae binding to lacto/neolacto glycosphingolipids, and to the other classes of binding-active compounds, remained after deletion of the chitin binding protein GbpA. Thus, the binding of V. cholerae to chitin and to lacto/neolacto containing glycosphingolipids represents two separate binding specificities.

Forssman expression on human erythrocytes: biochemical and genetic evidence of a new histo-blood group system.

In analogy with histo-blood group A antigen, Forssman (Fs) antigen terminates with α3-N-acetylgalactosamine and can be used by pathogens as a host receptor in many mammals. However, primates including humans lack Fs synthase activity and have naturally occurring Fs antibodies in plasma. We investigated individuals with the enigmatic ABO subgroup A(pae) and found them to be homozygous for common O alleles. Their erythrocytes had no A antigens but instead expressed Fs glycolipids. The unexpected Fs antigen was confirmed in structural, serologic, and flow-cytometric studies. The Fs synthase gene, GBGT1, in A(pae) individuals encoded an arginine to glutamine change at residue 296. Gln296 is present in lower mammals, whereas Arg296 was found in 6 other primates, > 250 blood donors and A(pae) family relatives without the A(pae) phenotype. Transfection experiments and molecular modeling showed that Agr296Gln reactivates the human Fs synthase. Uropathogenic E coli containing prsG-adhesin-encoding plasmids agglutinated A(pae) but not group O cells, suggesting biologic implications. Predictive tests for intravascular hemolysis with crossmatch-incompatible sera indicated complement-mediated destruction of Fs-positive erythrocytes. Taken together, we provide the first conclusive description of Fs expression in normal human hematopoietic tissue and the basis of a new histo-blood group system in man, FORS.

Redefinition of the carbohydrate binding specificity of Helicobacter pylori BabA adhesin.

Certain Helicobacter pylori strains adhere to the human gastric epithelium using the blood group antigen-binding adhesin (BabA). All BabA-expressing H. pylori strains bind to the blood group O determinants on type 1 core chains, i.e. to the Lewis b antigen (Fucα2Galß3(Fucα4)GlcNAc; Le(b)) and the H type 1 determinant (Fucα2Galß3GlcNAc). Recently, BabA strains have been categorized into those recognizing only Le(b) and H type 1 determinants (designated specialist strains) and those that also bind to A and B type 1 determinants (designated generalist strains). Here, the structural requirements for carbohydrate recognition by generalist and specialist BabA were further explored by binding of these types of strains to a panel of different glycosphingolipids. Three glycosphingolipids recognized by both specialist and generalist BabA were isolated from the small intestine of a blood group O pig and characterized by mass spectrometry and proton NMR as H type 1 pentaglycosylceramide (Fucα2Galß3GlcNAcß3Galß4Glcß1Cer), Globo H hexaglycosylceramide (Fucα2Galß3GalNAcß3Galα4Galß4Glcß1Cer), and a mixture of three complex glycosphingolipids (Fucα2Galß4GlcNAcß6(Fucα2Galß3GlcNAcß3)Galß3GlcNAcß3Galß4Glcß1Cer, Fucα2Galß3GlcNAcß6(Fucα2Galß3GlcNAcß3)Galß3GlcNAcß3Galß4Glcß1Cer, and Fucα2Galß4(Fucα3)GlcNAcß6(Fucα2Galß3GlcNAcß3)Galß3GlcNAcß3Galß4Glcß1Cer). In addition to the binding of both strains to the Globo H hexaglycosylceramide, i.e. a blood group O determinant on a type 4 core chain, the generalist strain bound to the Globo A heptaglycosylceramide (GalNAcα3(Fucα2)Galß3GalNAcß3Galα4Galß4Glcß1Cer), i.e. a blood group A determinant on a type 4 core chain. The binding of BabA to the two sets of isoreceptors is due to conformational similarities of the terminal disaccharides of H type 1 and Globo H and of the terminal trisaccharides of A type 1 and Globo A.

Detailed O-glycomics of the Muc2 mucin from colon of wild-type, core 1- and core 3-transferase-deficient mice highlights differences compared with human MUC2.

The heavily O-glycosylated mucin MUC2 constitutes the major protein in the mucosal layer that acts as a physical barrier protecting the epithelial layer in the colon. In this study, Muc2 was purified from mucosal scrapings from the colon of wild-type (WT) mice, core 3 transferase knockout (C3Gnt(-/-)) mice and intestinal epithelial cell-specific core 1 knockout (IEC C1Galt1(-/-)) mice. The Muc2 O-glycans were released by reductive ß-elimination and analyzed with liquid chromatography-mass spectrometry in the negative-ion mode. Muc2 from the distal colon of WT and C3Gnt(-/-) knockout mice carried a mixture of core 1- or core 2-type glycans, whereas Muc2 from IEC C1Galt1(-/-) mice carried highly sialylated core 3- and core 4-type glycans. A large portion of NeuAc in all mouse models was positioned on disialylated N-acetyllactosamine units, an epitope not reported on human colonic MUC2. Mass spectra and proton NMR spectroscopy revealed an abundant NeuAc linked to internally positioned N-acetylglucosamine on colonic murine Muc2, which also differs markedly from human MUC2. Our results highlight that murine colonic Muc2 O-glycosylation is substantially different from human MUC2, which could be one explanation for the different commensal microbiota of these two species.

Erythrocyte and porcine intestinal glycosphingolipids recognized by F4 fimbriae of enterotoxigenic Escherichia coli.

Enterotoxigenic F4-fimbriated Escherichia coli is associated with diarrheal disease in neonatal and postweaning pigs. The F4 fimbriae mediate attachment of the bacteria to the pig intestinal epithelium, enabling an efficient delivery of diarrhea-inducing enterotoxins to the target epithelial cells. There are three variants of F4 fimbriae designated F4ab, F4ac and F4ad, respectively, having different antigenic and adhesive properties. In the present study, the binding of isolated F4ab, F4ac and F4ad fimbriae, and F4ab/ac/ad-fimbriated E. coli, to glycosphingolipids from erythrocytes and from porcine small intestinal epithelium was examined, in order to get a comprehensive view of the F4-binding glycosphingolipids involved in F4-mediated hemagglutination and adhesion to the epithelial cells of porcine intestine. Specific interactions between the F4ab, F4ac and F4ad fimbriae and both acid and non-acid glycosphingolipids were obtained, and after isolation of binding-active glycosphingolipids and characterization by mass spectrometry and proton NMR, distinct carbohydrate binding patterns were defined for each fimbrial subtype. Two novel glycosphingolipids were isolated from chicken erythrocytes, and characterized as GalNAcα3GalNAcß3Galß4Glcß1Cer and GalNAcα3GalNAcß3Galß4GlcNAcß3Galß4Glcß1Cer. These two compounds, and lactosylceramide (Galß4Glcß1Cer) with phytosphingosine and hydroxy fatty acid, were recognized by all three variants of F4 fimbriae. No binding of the F4ad fimbriae or F4ad-fimbriated E. coli to the porcine intestinal glycosphingolipids occurred. However, for F4ab and F4ac two distinct binding patterns were observed. The F4ac fimbriae and the F4ac-expressing E. coli selectively bound to galactosylceramide (Galß1Cer) with sphingosine and hydroxy 24:0 fatty acid, while the porcine intestinal glycosphingolipids recognized by F4ab fimbriae and the F4ab-fimbriated bacteria were characterized as galactosylceramide, sulfatide (SO(3)-3Galß1Cer), sulf-lactosylceramide (SO(3)-3Galß4Glcß1Cer), and globotriaosylceramide (Galα4Galß4Glcß1Cer) with phytosphingosine and hydroxy 24:0 fatty acid. Finally, the F4ad fimbriae and the F4ad-fimbriated E. coli, but not the F4ab or F4ac subtypes, bound to reference gangliotriaosylceramide (GalNAcß4Galß4Glcß1Cer), gangliotetraosylceramide (Galß3GalNAcß4Galß4Glcß1Cer), isoglobotriaosylceramide (Galα3Galß4Glcß1Cer), and neolactotetraosylceramide (Galß4GlcNAcß3Galß4Glcß1Cer).

Pichia pastoris-produced mucin-type fusion proteins with multivalent O-glycan substitution as targeting molecules for mannose-specific receptors of the immune system.

Mannose-binding proteins like the macrophage mannose receptor (MR), the dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) and mannose-binding lectin (MBL) play crucial roles in both innate and adaptive immune responses. Immunoglobulin fusion proteins of the P-selectin glycoprotein ligand-1 (PSGL-1/mIgG(2b)) carrying mostly O-glycans and, as a control, the α1-acid glycoprotein (AGP/mIgG(2b)) carrying mainly N-linked glycans were stably expressed in the yeast Pichia pastoris. Pichia pastoris-produced PSGL-1/mIgG(2b) was shown to carry O-glycans that mediated strong binding to mannose-specific lectins in a lectin array and were susceptible to cleavage by α-mannosidases including an α1,2- but not an α1,6-mannosidase. Electrospray ionization ion-trap mass spectrometry confirmed the presence of O-glycans containing up to nine hexoses with the penta- and hexasaccharides being the predominant ones. α1,2- and α1,3-linked, but not α1,6-linked, mannose residues were detected by (1)H-nuclear magnetic resonance spectroscopy confirming the results of the mannosidase cleavage. The apparent equilibrium dissociation constants for binding of PNGase F-treated mannosylated PSGL-1/mIgG(2b) to MR, DC-SIGN and MBL were shown by surface plasmon resonance to be 126, 56 and 16 nM, respectively. In conclusion, PSGL-1/mIgG(2b) expressed in P. pastoris carried O-glycans mainly comprised of α-linked mannoses and with up to nine residues. It bound mannose-specific receptors with high apparent affinity and may become a potent targeting molecule for these receptors in vivo.

The structural basis of blood group A-related glycolipids in an A3 red cell phenotype and a potential explanation to a serological phenomenon.

Glycolipids from the red cells of a rare blood group A subgroup individual, expressing the blood group A(3) phenotype with the classical mixed-field agglutination phenomenon, A(2(539G>A))/O(1) genotype, and an unusual blood group A glycolipid profile, were submitted to a comprehensive biochemical and structural analysis. To determine the nature of blood group A glycolipids in this A(3) phenotype, structural determination was carried out with complementary techniques including proton nuclear magnetic resonance (1D and 2D), mass spectrometry (MS) (nano-electrospray ionization/quadrupole time-of-flight and tandem mass spectrometry) and thin layer chromatography with immunostaining detection. As expected, total blood group A structures were of low abundance, but contrary to expectations extended-A type 2 and A type 3 glycolipids were more dominant than A hexaglycosylceramides based on type 2 chain (A-6-2 glycolipids), which normally is the major A glycolipid. Several para-Forssman (GalNAcß3GbO(4)) structures, including extended forms, were identified but surmised not to contribute to the classic mixed-field agglutination of the A(3) phenotype. It is proposed that the low level of A antigen combined with an absence of extended branched glycolipids may be the factor determining the mixed-field agglutination phenomenon in this individual.

The GD1a glycan is a cellular receptor for adenoviruses causing epidemic keratoconjunctivitis.

Adenovirus type 37 (Ad37) is a leading cause of epidemic keratoconjunctivitis (EKC), a severe and highly contagious ocular disease. Whereas most other adenoviruses infect cells by engaging CD46 or the coxsackie and adenovirus receptor (CAR), Ad37 binds previously unknown sialic acid-containing cell surface molecules. By glycan array screening, we show here that the receptor-recognizing knob domain of the Ad37 fiber protein specifically binds a branched hexasaccharide that is present in the GD1a ganglioside and that features two terminal sialic acids. Soluble GD1a glycan and GD1a-binding antibodies efficiently prevented Ad37 virions from binding and infecting corneal cells. Unexpectedly, the receptor is constituted by one or more glycoproteins containing the GD1a glycan motif rather than the ganglioside itself, as shown by binding, infection and flow cytometry experiments. Molecular modeling, nuclear magnetic resonance and X-ray crystallography reveal that the two terminal sialic acids dock into two of three previously established sialic acid-binding sites in the trimeric Ad37 knob. Surface plasmon resonance analysis shows that the knob-GD1a glycan interaction has high affinity. Our findings therefore form a basis for the design and development of sialic acid-containing antiviral drugs for topical treatment of EKC.

Carbohydrate binding specificities and crystal structure of the cholera toxin-like B-subunit from Citrobacter freundii.

Enterotoxigenic Escherichia coli and Vibrio cholerae are well known causative agents of severe diarrheal diseases. Both pathogens produce AB(5) toxins, with one enzymatically active A-subunit and a pentamer of receptor-binding B-subunits. The primary receptor for both B-subunits is the GM1 ganglioside (Galbeta3GalNAcbeta4(NeuAcalpha3)Galbeta4GlcbetaCer), but the B-subunits from porcine isolates of E. coli also bind neolacto-(Galbeta4GlcNAcbeta-)terminated glycoconjugates and the B-subunits from human isolates of E. coli (hLTB) have affinity for blood group A type 2-(GalNAcalpha3(Fucalpha2)Galbeta4GlcNAcbeta-)terminated glycoconjugates. A B-subunit with 73% sequence identity to the B-subunits of cholera toxin and the heat-labile toxin of E. coli is produced by certain strains of enteropathogenic E. coli and by Citrobacter freundii. This C. freundii B-subunit (CFXB) has now been expressed in V. cholerae, and isolated in high yields. Glycosphingolipid binding studies show that CFXB binds to the GM1 ganglioside with high affinity. In addition, CFXB has high affinity for both neolacto-terminated and blood group A type 2-terminated glycoconjugates. The crystal structure of the pentameric arrangement of C. freundii B-subunits display high structural similarity with related proteins from E. coli and V. cholerae and oligosaccharide binding sites can be identified on the protein surface. Small changes in the 88-95 loop connecting the GM1 and blood group A binding sites explains the minor changes in affinity seen for these two ligands. However, the enhanced affinity of CFXB for neolacto-terminated structures can be sought in the Lys34Tyr substitution affording additional hydrogen bond interactions between the tyrosyl side chain and the GlcNAcbeta3Galb4Glcbeta1 segment of neolactotetraosylceramide via bridging water molecules.

Structural characterization of alpha1,3-galactosyltransferase knockout pig heart and kidney glycolipids and their reactivity with human and baboon antibodies.

BACKGROUND: alpha1,3-galactosyltranferase knockout (GalT-KO) pigs have been established to avoid hyperacute rejection in GalT-KO pig-to-human xenotransplantation. GalT-KO pig heart and kidney glycolipids were studied focusing on elimination of Gal-antigens and whether novel antigens would appear. Non-human primates are used as pre-clinical transplantation experimental models. Therefore, sera from baboons transplanted with GalT-KO hearts were compared with human serum regarding reactivity with pig glycolipids. METHODS: Neutral and acidic glycolipids were isolated from GalT-KO and WT pig hearts and kidneys. Glycolipid immune reactivity was tested on TLC plates using human affinity-purified anti-Gal Ig, anti-blood group monoclonal antibodies, lectins, and human serum as well as baboon serum collected before and after GalT-KO pig heart transplantations. Selected glycolipid fractions, isolated by HPLC, were structurally characterized by mass spectrometry and proton NMR spectroscopy. RESULTS: GalT-KO heart and kidney lacked alpha3Gal-terminated glycolipids completely. Levels of uncapped N-acetyllactosamine precursor compounds, blood group H type 2 core chain compounds, the P1 antigen and the x(2) antigen were increased. Human serum antibodies reacted with Gal-antigens and N-glycolylneuraminic acid (NeuGc) in WT organs of which only the NeuGc reactivity remained in the GalT-KO tissues. A clear difference in reactivity between baboon and human antibodies with pig glycolipids was found. This was most pronounced for acidic, not yet identified, compounds in GalT-KO organs which were less abundant or lacking in the corresponding WT tissues. CONCLUSIONS: GalT-KO pig heart and kidney completely lacked Gal glycolipid antigens whilst glycolipids synthesized by competing pathways were increased. Baboon and human serum antibodies showed a different reactivity pattern to pig glycolipid antigens indicating that non-human primates have limitations as a human pre-clinical model for immune rejection studies.

No direct binding of the heat-labile enterotoxin of Escherichia coli to E. coli lipopolysaccharides.

A novel carbohydrate binding site recognizing blood group A and B determinants in a hybrid of cholera toxin and Escherichia coli heat-labile enterotoxin B-subunits (termed LCTBK) has previously been described, and also the native heat-labile enterotoxin bind to some extent to blood group A/B terminated glycoconjugates. The blood group antigen binding site is located at the interface of the B-subunits. Interestingly, the same area of the B-subunits has been proposed to be involved in binding of the heat-labile enterotoxin to lipopolysaccharides on the bacterial cell surface. Binding of the toxin to lipopolysaccharides does not affect the GM1 binding capacity. The present study aimed at characterizing the relationship between the blood group A/B antigen binding site and the lipopolysaccharide binding site. However, no binding of the B-subunits to E. coli lipopolysaccharides in microtiter wells or on thin-layer chromatograms was obtained. Incubation with lipopolysaccharides did not affect the binding of the B-subunits of heat-labile enterotoxin of human isolates to blood group A-carrying glycosphingolipids, indicating that the blood group antigen site is not involved in LPS binding. However, the saccharide competition experiments showed that GM1 binding reduced the affinity for blood group A determinants and vice versa, suggesting that a concurrent occupancy of the two binding sites does not occur. The latter finding is related to a connection between the blood group antigen binding site and the GM1 binding site through residues interacting with both ligands.

Relapsing fever Borrelia binds to neolacto glycans and mediates rosetting of human erythrocytes.

A hallmark of acute relapsing fever borreliosis is severe bacteremia. Some Borrelia species, such as B. duttonii and B. crocidurae, associate with erythrocytes and induce aggregation recognized as erythrocyte rosetting. Erythrocyte rosettes contribute to disease severity by increased tissue invasiveness (such as invasion of CNS and encephalitis), hemorrhaging, and reduced blood flow in affected microcapillaries. Here we report that relapsing fever Borrelia binds to neolacto (Galbeta4GlcNAcbeta3Galbeta4Glcbeta1)-carrying glycoconjugates that are present on human erythrocytes. This interaction is of low affinity but is compensated for by the multivalency of neo-lacto-oligosaccharides on the erythrocyte cell surface. Hence, the protein-carbohydrate interaction is dependent on multivalent neolacto-glycans to mediate binding.