The primary virulence factors of many pathogenic bacteria are secreted protein toxins which bind to glycolipid receptors on host cell surfaces. The binding specificities of three such toxins for different glycolipids, mainly from the ganglioside series, were determined by surface plasmon resonance (SPR) using a liposome capture method. Unlike microtiter plate and thin layer chromatography overlay assays, the SPR/liposome methodology allows for real time analysis of toxin binding under conditions that mimic the natural cell surface venue of these interactions and without any requirement for labeling of toxin or receptor. Compared to conventional assays, the liposome technique showed more restricted oligosaccharide specificities for toxin binding. Cholera toxin demonstrated an absolute requirement for terminal galactose and internal sialic acid residues (as in G M1 ) with tolerance for substitution with a second internal sialic acid (as in G D1b ). Escherichia coli heat-labile enterotoxin bound to G M1 and tolerated removal or extension of the internal sialic acid residue (as in asialo-G M1 and G D1b , respectively) but not substitution of the terminal galactose of G M1 . Tetanus toxin showed a requirement for two internal sialic acid residues as in G D1b . Extension of terminal galactose with a single sialic acid was tolerated to some extent. The SPR analyses also yielded rate and affinity constants which are not attainable by conventional assays. Complex binding profiles were observed in that the association and dissociation rate constants varied with toxin:receptor ratios. The sub-nanomolar affinities of cholera toxin and heat-labile enterotoxin for liposome-anchored gangliosides were attributable largely to very slow dissociation rate constants. The SPR/liposome technology should have general applicability in the study of glycolipid-protein interactions and in the evaluation of reagents designed to interfere with these interactions.The protein toxins produced by many pathogenic bacteria are among the best characterized virulence factors. These toxins typically bind to oligosaccharide receptors on host cell surfaces (1). Many belong to the AB 5 family of toxins which are comprised of an enzymatically active and toxic A-subunit and five B-subunits which form the receptor binding portion of the molecule. In most instances the five B-subunits are identical and allow for pentameric attachment to the cell surface receptors. Crystal structures of five AB 5 toxins or their B-pentamers, three complexed with carbohydrate receptors, have been reported. These are cholera toxin (2, 3), Escherichia coli heatlabile toxin (4 -6), shiga toxin (7), shiga-like toxin (8), and pertussis toxin (9). However, this wealth of structural data has not answered all questions relating to the oligosaccharide-binding specificities of these molecules. For example, there is some controversy as to the nature of the functional receptor of LT. Although structurally very similar to CT, LT shows subtle differences in receptor binding specificity (1...

Despite the characterization of some Burkholderia cepacia complex exopolysaccharides (EPSs), little is known about the role of EPSs in the pathogenicity of B. cepacia complex organisms. We describe 2 Burkholderia cenocepacia (genomovar III) isolates obtained from a patient with cystic fibrosis (CF): the nonmucoid isolate C8963 and the mucoid isolate C9343. Both isolates had identical random amplified polymorphic DNA patterns. C9343 produced a capsule composed of the EPSs PS-I and PS-II, as well as alpha -1,6-glucan. These isolates exhibited several phenotypic differences: C8963 synthesized octanoyl-homoserine lactone and produced biofilms, but C9343 did not; in a mouse model of pulmonary infection, C8963 was cleared more rapidly than was C9343; and C9343 interacted poorly with macrophages and neutrophils, compared with C8963, suggesting that the C9343 capsule interfered with cell-surface interactions. Overproduction of EPS by C9343 resulted in a mucoid appearance and interfered with cell-surface interactions and clearance in an animal model. This mucoid colonial appearance could enhance the persistence and virulence of this important CF-related pathogen.

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The antigen binding fragment from an IgG2a kappa murine monoclonal antibody with specificity for alpha-(2-->8)-linked sialic acid polymers has been prepared and crystallized in the absence of hapten. Crystals were grown by vapor diffusion equilibrium with 16-18% polyethylene glycol 4000 solutions. The structure was solved by molecular replacement methods and refined to a conventional R factor of 0.164 for data to 2.8 A. The binding site is observed to display a shape and distribution of charges that is complementary to that of the predicted conformation of the oligosaccharide epitope. A thermodynamic description of ligand binding has been compiled for oligosaccharides ranging in length from 9 to 41 residues, and the data for the largest ligand has been used in a novel way to estimate the size of the antigen binding site. A model of antigen binding is presented that satisfies this thermodynamic data, as well as a previously reported requirement of conformational specificity of the oligosaccharide. X-ray crystallographic and thermodynamic evidence are consistent with a binding site that accommodates at least eight sialic acid residues.

Lipopolysaccharide (LPS) of the Pseudomonas aeruginosa serotype O5 wild-type strain PAO1 and derived rough-type mutant strains AK1401 and AK1012 was isolated by a modified phenol/chloroform/ petroleum-ether extraction method. Deoxycholate/PAGE of the LPS from the rough mutant AK1401 indicated two bands near the dye front with mobilities similar to those of the parent strain, indicating that both LPS contain a complete core and a species comprising a core and one repeating unit. Composition analysis of the LPS from strains PAO1 and AK1401 indicated that the complete core oligosaccharide was composed of D-glucose (four units), L-rhamnose (one unit), 2-amino-2-deoxy-D-galactose (one unit), L-glycero-D-manno-heptose (Hep; two units), 3-deoxy-D-manno-octulosonic acid (Kdo ; two units), L-alanine (one unit) and phosphate (three units). The glycan structure of the LPS was determined by onedimensional and two-dimensional (2D) NMR techniques in combination with MS-based methods on oligosaccharide samples obtained from the LPS by delipidation procedures. The locations of three phosphomonoester groups on the first heptose residue were established by a two-dimensional 31 P (ω 1 )-halffiltered COSY experiment on the reduced core oligosaccharide sample of the LPS from the wild-type strain. The presence of a 7-O-carbamoyl substituent was observed on the second heptose. The structure of the core region of the O-chain-deficient LPS from P. aeruginosa serotype O5 is as follows:A structural model is presented that is also representative of that for P. aeruginosa serotype O6 LPS. A revised structure for the serotype O6 mutant strain A28 is presented.Keywords : Pseudomonas aeruginosa; lipopolysaccharide; core oligosaccharide; structure ; NMR.Pseudomonas aeruginosa is an opportunistic pathogen that affects compromised individuals such as burn victims, and cystic fibrosis and cancer patients. Lipopolysaccharide (LPS) is considered to be one of the major virulence factors of P. aeruginosa [1,2]. As in most of gram-negative bacteria, it forms an essential part of the outer membrane and is the most immunoreactive surface antigen of the organism. LPS of P. aeruginosa shares theCorrespondence to E. Altman, Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A OR6, CanadaFax: ϩ1 613 941 1327. E-mail: eleonora.altman@nrc.ca Abbreviations. OS, oligosaccharide; LPS, lipopolysaccharide; Kdo, 3-deoxy-D-manno-octulosonic acid; HPAEC, high performance anion exchange chromatography; FAB, fast-atom bombardment, ES-MS, electrospray mass spectrometry; 1D, one-dimensional; 2D, two-dimensional ; HMQC, heteronuclear multiple-quantum coherence ; Hep, heptose.general architecture found in members of the family Enterobacteriaceae and is composed of three regions: a lipid A moiety; a core oligosaccharide, which can be subdivided into inner and outer core units; and an O-antigen polysaccharide. 20 major serotypes of P. aeruginosa have been described on the basis of the structural diversity of their O-antigens [3,4].It has...