globotetraose and globotriose

Many human pathogens initiate disease by utilizing their microbial adhesin proteins to attach to glycoconjugates on host cell mucosal surfaces. Soluble oligosaccharides of identical or similar structure to these naturally occurring ligands can both prevent bacterial attachment as well as mediate the release of attached bacteria. Since it has not been possible to isolate large quantities of these compounds, we have developed enzyme-based technologies to synthesize several relevant human oligosaccharides. Using cloned bacterial glycosyltransferases, we can synthesize several hundred grams of these oligosaccharides at a time. The availability of these large quantities will allow these compounds to be tested as anti-adhesive pharmaceutical agents as well as lead to expanded practical applications.

Topics: Bacteria; Biotechnology; Carbohydrate Sequence; Gene Expression; Genetic Vectors; Globosides; Glycosyltransferases; Humans; Molecular Sequence Data; Oligosaccharides; Recombinant Proteins; Trisaccharides

Shiga-like toxin 2 (Stx2) is one of the most important virulence factors in enterohaemorrhagic Escherichia coli (E. coli) strains such as O157H7. Subtypes of Stx2 are diverse with respect to their sequence, toxicity, and distribution. The most diverse Stx2 subtype, Stx2f, is difficult to detect immunologically, but is becoming more frequently associated with human illness.. A purification regimen was developed for the purification of Stx2f involving cation exchange, hydrophobic interaction, anion exchange, and gel filtration. The molecular weight of Stx2f B-subunit was approximately 5 kDa, which appeared significantly smaller than that of Stx2a (6 kDa) on a SDS-PAGE gel, although the size of the A subunit was similar to Stx2a (30 kDa). Stx2f was shown to be active in both cell-free and cell-based assays. The 50% cytotoxic dose in Vero cells was 3.4 or 1.7 pg (depending on the assay conditions), about 3-5 times higher than the archetypical Stx2a, while the activity of Stx2f and Stx2a in a cell-free rabbit reticulocyte system was similar. Stx2f bound to both globotriose-lipopolysaccharide (Gb3-LPS) and globotetraose-LPS (Gb4-LPS, mimics for globotriaosylceramide and globotetraosylceramide, respectively), but its ability to bind Gb4-LPS was much stronger than Stx2a. Stx2f was also much more stable at low pH and high temperature compared to Stx2a, suggesting the toxin itself may survive harsher food preparation practices.. Here, we detail the purification, biochemical properties, and toxicity of Stx2f, from an E. coli strain isolated from a feral pigeon. Information obtained in this study will be valuable for characterizing Stx2f and explaining the differences of Stx2a and Stx2f in host specificity and cytotoxicity.

Topics: Ammonium Sulfate; Animals; Cell-Free System; Chlorocebus aethiops; Chromatography, Gel; Chromatography, Ion Exchange; Enterohemorrhagic Escherichia coli; Enzyme-Linked Immunosorbent Assay; Globosides; Hot Temperature; Hydrogen-Ion Concentration; Lipopolysaccharides; Protein Binding; Rabbits; Recombinant Proteins; Shiga Toxin 2; Trisaccharides; Vero Cells

Large amounts of globotriose (Galalpha-4Galbeta-4Glc) are shown to be produced by the high cell density culture of an Escherichia coli strain over-expressing the Neisseria meningitidis lgtC gene for alpha-1,4-Gal transferase. The strain which was devoid of both alpha and beta galactosidase activity was fed with glycerol as the energy and carbon source and with lactose as precursor for globotriose synthesis. After complete exhaustion of lactose, globotriose could serve as an alternative acceptor for LgtC and the formation of a series of polygalactosylated compounds was observed. The system was extended to the synthesis of globotetraose (GalNAcbeta-3Galalpha-4Galbeta-4Glc) by overexpressing two additional genes: lgtD from Haemophilus influenzae Rd which encodes a beta-1,3-GalNAc transferase and wbpP from Pseudomonas aeruginosa which encodes a UDP-GalNAc C4 epimerase. Globotetraose could also be produced from exogenous globotriose which was shown to be actively taken up by the cells.

Topics: Bacterial Proteins; Escherichia coli; Globosides; Trisaccharides