The diseases caused by Shiga and cholera toxins account for the loss of millions of lives each year. Both belong to the clinically significant subset of bacterial AB5 toxins consisting of an enzymatically active A subunit that gains entry to susceptible mammalian cells after oligosaccharide recognition by the B5 homopentamer. Therapies might target the obligatory oligosaccharide-toxin recognition event, but the low intrinsic affinity of carbohydrate-protein interactions hampers the development of low-molecular-weight inhibitors. The toxins circumvent low affinity by binding simultaneously to five or more cell-surface carbohydrates. Here we demonstrate the use of the crystal structure of the B5 subunit of Escherichia coli O157:H7 Shiga-like toxin I (SLT-I) in complex with an analogue of its carbohydrate receptor to design an oligovalent, water-soluble carbohydrate ligand (named STARFISH), with subnanomolar inhibitory activity. The in vitro inhibitory activity is 1-10-million-fold higher than that of univalent ligands and is by far the highest molar activity of any inhibitor yet reported for Shiga-like toxins I and II. Crystallography of the STARFISH/Shiga-like toxin I complex explains this activity. Two trisaccharide receptors at the tips of each of five spacer arms simultaneously engage all five B subunits of two toxin molecules.

Shiga-like toxin I (SLT-I) is a virulence factor of Escherichia coli strains that cause disease in humans. Like other members of the Shiga toxin family, it consists of an enzymatic (A) subunit and five copies of a binding subunit (the B-pentamer). The B-pentamer binds to a specific glycolipid, globotriaosylceramide (Gb3), on the surface of target cells and thereby plays a crucial role in the entry of the toxin. Here we present the crystal structure at 2.8 A resolution of the SLT-I B-pentamer complexed with an analogue of the Gb3 trisaccharide. The structure reveals a surprising density of binding sites, with three trisaccharide molecules bound to each B-subunit monomer of 69 residues. All 15 trisaccharides bind to one side of the B-pentamer, providing further evidence that this side faces the cell membrane. The structural model is consistent with data from site-directed mutagenesis and binding of carbohydrate analogues, and allows the rational design of therapeutic Gb3 analogues that block the attachment of toxin to cells.

The structure provides insight into the pathogenic mechanisms of pertussis toxin and the evolution of bacterial toxins. Knowledge of the tertiary structure of the active site forms a rational basis for elimination of catalytic activity in recombinant molecules for vaccine use.

Prebiotic oligosaccharides are thought to provide beneficial effects in the gastrointestinal tract of humans and animals by stimulating growth of selected members of the intestinal microflora. Another means by which prebiotic oligosaccharides may confer health benefits is via their antiadhesive activity. Specifically, these oligosaccharides may directly inhibit infections by enteric pathogens due to their ability to act as structural mimics of the pathogen binding sites that coat the surface of gastrointestinal epithelial cells. In this study, the ability of commercial prebiotics to inhibit attachment of microcolony-forming enteropathogenic Escherichia coli (EPEC) was investigated. The adherence of EPEC strain E2348/69 on HEp-2 and Caco-2 cells, in the presence of fructooligosaccharides, inulin, galactooligosaccharides (GOS), lactulose, and raffinose was determined by cultural enumeration and microscopy. Purified GOS exhibited the greatest adherence inhibition on both HEp-2 and Caco-2 cells, reducing the adherence of EPEC by 65 and 70%, respectively. In addition, the average number of bacteria per microcolony was significantly reduced from 14 to 4 when GOS was present. Adherence inhibition by GOS was dose dependent, reaching a maximum at 16 mg/ml. When GOS was added to adhered EPEC cells, no displacement was observed. The expression of BfpA, a bundle-forming-pilus protein involved in localized adherence, was not affected by GOS, indicating that adherence inhibition was not due to the absence of this adherence factor. In addition, GOS did not affect autoaggregation. These observations suggest that some prebiotic oligosaccharides may have antiadhesive activity and directly inhibit the adherence of pathogens to the host epithelial cell surface.Prebiotic oligosaccharides are defined as nondigestible food ingredients that provide beneficial effects to the host by stimulating the growth of selected microbial members of the gastrointestinal tract (16). Among the colonic bacteria capable of metabolizing prebiotic oligosaccharides and whose growth is stimulated are species of Lactobacillus and Bifidobacterium. The presence of these bacteria in the gastrointestinal tract has long been associated with various health benefits (53). For example, lactobacilli and bifidobacteria, by virtue of their ability to produce organic acids and other antagonistic agents, may directly inhibit opportunistic pathogens (18,48,58). Also, enrichment of lactobacilli and bifidobacteria by prebiotics may indirectly result in the displacement of less-desirable members of the competing microflora (14, 17).Recently, another mechanism by which prebiotics might interfere with and inhibit infectious bacteria has been proposed (5,17,22,26). Specifically, this model is based on the observation that certain exogenous oligosaccharides structurally resemble the receptor sites coating the intestinal epithelial cells to which intestinal pathogens recognize and adhere (31). Accordingly, these oligosaccharides may act as molecular receptor decoys or antiadhesi...