Suri S. Iyer

Carbohydrate-based label-free detection of Escherichia coli ORN 178 using electrochemical impedance spectroscopy.

A label-free biosensor for Escherichia coli (E. coli) ORN 178 based on faradaic electrochemical impedance spectroscopy (EIS) was developed. α-Mannoside or β-galactoside was immobilized on a gold disk electrode using a self-assembled monolayer (SAM) via a spacer terminated in a thiol functionality. Impedance measurements (Nyquist plot) showed shifts due to the binding of E. coli ORN 178, which is specific for α-mannoside. No significant change in impedance was observed for E. coli ORN 208, which does not bind to α-mannoside. With increasing concentrations of E. coli ORN 178, electron-transfer resistance (R(et)) increases before the sensor is saturated. After the Nyquist plot of E. coli/mixed SAM/gold electrode was modeled, a linear relationship between normalized R(et) and the logarithmic value of E. coli concentrations was found in a range of bacterial concentration from 10(2) to 10(3) CFU/mL. The combination of robust carbohydrate ligands with EIS provides a label-free, sensitive, specific, user-friendly, robust, and portable biosensing system that could potentially be used in a point-of-care or continuous environmental monitoring setting.

Glycan-based high-affinity ligands for toxins and pathogen receptors

Glycans decorate over 95% of the mammalian cell surface in the form of glycolipids and glycoproteins. Several toxins and pathogens bind to these glycans to enter the cells. Understanding the fundamentals of the complex interplay between microbial pathogens and their glycan receptors at the molecular level could lead to the development of novel therapeutics and diagnostics. Using Shiga toxin and influenza virus as examples, we describe the complex biological interface between host glycans and these infectious agents, and recent strategies to develop glycan‐based high‐affinity ligands. These molecules are expected to ultimately be incorporated into diagnostics and therapeutics, and can be used as probes to study important biological processes. Additionally, by focusing on the specific glycans that microbial pathogens target, we can begin to decipher the “glycocode” and how these glycans participate in normal and aberrant cellular communication.

Glycan Encapsulated Gold Nanoparticles Selectively Inhibit Shiga Toxins 1 and 2

Shiga toxins (Stx) released by Escherichia coli O157:H7 and Shigella dysentriae cause life-threatening conditions that include hemolytic uremic syndrome (HUS), kidney failure, and neurological complications. Cellular entry is mediated by the B-subunit of the AB(5) toxin, which recognizes cell surface glycolipids present in lipid raft-like structures. We developed gold glyconanoparticles that present a multivalent display similar to the cell surface glycolipids to compete for these toxins. These highly soluble glyconanoparticles were nontoxic to the Vero monkey kidney cell line and protected Vero cells from Stx-mediated toxicity in a dose-dependent manner. The inhibition is highly dependent on the structure and density of the glycans; selective inhibition of Stx1 and the more clinically relevant Stx2 was achieved. Interestingly, natural variants of Stx2, Stx2c, and Stx2d possessing minimal amino acid variation in the receptor binding site of the B-subunit or changes in the A-subunit were not neutralized by either the Stx1- or Stx2-specific gold glyconanoparticles. Our results suggest that tailored glyconanoparticles that mimic the natural display of glycans in lipid rafts could serve as potential therapeutics for Stx1 and Stx2. However, a few amino acid changes in emerging Stx2 variants can change receptor specificity, and further research is needed to develop receptor mimics for the emerging variants of Stx2.

Factors Affecting Protein―Glycan Specificity: Effect of Spacers and Incubation Time

Glycan binding: We monitored the binding of synthetic glycans to influenza hemagglutinin by using ELISA and surface plasmon resonance, thereby demonstrating that the glycans presentation influences binding dramatically. Also, the binding observed in static systems was very different from that in dynamic fluid systems. These studies suggest that binding specificities are dependent on glycan structure, valency, presentation, and assay conditions.

Biotinylated Bi‐ and Tetra‐antennary Glycoconjugates for Escherichia coli Detection

Glycans cover the surface of all mammalian cells. Several toxins and pathogens use these glycans to bind and infect the cell. Using a versatile modular synthetic strategy, we have developed biotinylated bi‐ and tetraantennary glycoconjugates to capture and detect E. coli and compared the capturing ability of these molecules to commercial polyclonal antibodies. Magnetic beads were coated with biotinylated glycoconjugate or antibody, and these beads were used to capture, isolate, and quantify bacterial recovery by using a luminescence assay. The glycoconjugate‐coated magnetic beads outperformed antibody‐coated magnetic beads in sensitivity and selectivity when compared under identical experimental conditions. Glycoconjugates could capture Escherichia coli from stagnant water, and the ability of a panel of glycoconjugates to capture a selection of pathogenic bacteria was also evaluated. To the best of our knowledge, this study represents the first comprehensive study that compares synthetic glycoconjugates and antibodies for E. coli detection. The glycoconjugates are also very stable and inexpensive. The results presented here are expected to lead to an increased interest in developing glycoconjugate‐based high affinity reagents for diagnostics.

Identification and characterization of the carbohydrate ligands recognized by pertussis toxin via a glycan microarray and surface plasmon resonance.

Binding of pertussis toxin (PTx) was examined by a glycan microarray; 53 positive hits fell into four general groups. One group represents sialylated biantennary compounds with an N-glycan core terminating in alpha2-6-linked sialic acid. The second group consists of multiantennary compounds with a canonical N-glycan core, but lacking terminal sialic acids, which represents a departure from the previous understanding of PTx binding to N-glycans. The third group consists of Neu5Acalpha2-3(lactose or N-acetyllactosamine) forms that lack the branched mannose core found in N-glycans; thus, their presentation is more similar to that of O-linked glycans and glycolipids. The fourth group of compounds consists of Neu5Acalpha2-8Neu5Acalpha2-8Neu5Ac, which is seen in the c series gangliosides and some N-glycans. Quantitative analysis by surface plasmon resonance of the relative affinities of PTx for terminal Neu5Acalpha2-3 versus Neu5Acalpha2-6, as well as the affinities for the trisaccharide Neu5Acalpha2-8Neu5Acalpha2-8Neu5Ac versus disaccharide, revealed identical global affinities, even though the amount of bound glycan varied by 4-5-fold. These studies suggest that the conformational space occupied by a glycan can play an important role in binding, independent of affinity. Characterization of N-terminal and C-terminal binding sites on the S2 and S3 subunits by mutational analysis revealed that binding to all sialylated compounds was mediated by the C-terminal binding sites, and binding to nonsialylated N-linked glycans is mediated by the N-terminal sites present on both the S2 and S3 subunits. A detailed understanding of the glycans recognized by pertussis toxin is essential to understanding which cells are targeted in clinical disease.

Comparison of Binding Platforms Yields Insights into Receptor Binding Differences between Shiga Toxins 1 and 2

Protein-glycan interactions are typically very weak, and avid binding is achieved when proteins express multiple glycan binding sites. Shiga toxin (Stx) uses glycan receptors to enter cells. Stx has five identical binding subunits, each with three nonidentical glycan binding sites. Previous studies examined binding to biantennary glycans expressing Pk trisaccharide mimics immobilized on streptavidin, resulting in display of four trisaccharides per streptavidin face. Stx1 preferred the Pk trisaccharide of its native receptor, globotriaosylceramide (Gb3), while the more potent and clinically relevant variant, Stx2, preferred the Pk trisaccharide with the terminal galactose replaced with N-acetylgalactosamine (NHAc-Pk). In the present study, binding of Stxs to Pk analogues was examined using two experimental platforms, ELISA and surface plasmon resonance (SPR). ELISA was more sensitive than SPR. Sensitivity in the ELISA was due to high streptavidin density, suggesting that avid binding may require engagement of more than four trisaccharides. Selectivity for the Pk analogues was maintained in both experimental platforms. Glycan preference was mapped to binding site 2, since reciprocal mutation of a single amino acid (asparagine 32 of Stx1 B-subunit/serine 31 of Stx2 B-subunit) reversed binding preference. However, native Stx1 bound well to plates loaded with a 50:50 mixture of Pk-NHAc-Pk, while Stx2 bound less efficiently, suggesting that one of the Stx1 binding sites may only engage Pk, while another may tolerate either Pk or NHAc-Pk. Varying glycan structure and density across different in vitro binding platforms revealed important differences in receptor binding properties between Stx1 and Stx2.

Detection of Carbohydrate Binding Proteins Using Magnetic Relaxation Switches

We have developed a simple, rapid, and sensitive carbohydrate-based magnetic relaxation switch assay for the detection of carbohydrate binding proteins. This technique was used to detect lectins and toxins that are known to bind to specific carbohydrates. Lectins that bind to the same carbohydrate displayed differential aggregation profiles because of differences in the structure and number of binding sites of the lectins. We demonstrated that selectivity and sensitivity can be enhanced using two different recognition elements. We have also demonstrated that magnetic relaxation switch assays can be used to detect toxins in a complex medium such as stool and environmental samples.

Design and synthesis of hyaluronan-mimetic gemini disaccharides

An efficient strategy has been designed for the preparation of synthetic mimics of hyaluronan (HA, 1) and its dimerized (Gemini) disaccharides (2a,b) via n-pentenyl glycoside formation. Construction of the target molecules was achieved through a combination of protection/deprotection protocols, imidate glycosylation methodology followed by ozonolysis, and reductive amination. These tailored synthetic mimics could act as versatile building blocks with therapeutic applications in tissue engineering, treatment of cancer and as drug delivery agents.

Glycan based Detection and Drug Susceptibility of Influenza Virus

We have developed a panel of synthetic glycans as receptor mimics for the specific capture of influenza viruses. The glycans were printed onto commercial glass slides using a free amine at the end of a spacer to generate a small focused microarray. The microarray was evaluated for its ability to capture three different strains of influenza A virus, two H1N1, A/Brisbane/59/2007 and A/Solomon Islands/3/2006 and one H3N2, A/Aichi/2/1968. We observed an excellent detection ability with some compounds exhibiting clinically relevant (101 plaque forming units) limit of detection. We also tested the drug susceptibility of current antivirals, Zanamivir and Ostelamivir using this microarray and could determine antiviral resistance for these strains.