The presence of a fucose utilization operon in the Streptococcus pneumoniae genome and its established importance in virulence indicates a reliance of this bacterium on the harvesting of host fucose-containing glycans. The identities of these glycans, however, and how they are harvested is presently unknown. The biochemical and high resolution x-ray crystallographic analysis of two family 98 glycoside hydrolases (GH98s) from distinctive forms of the fucose utilization operon that originate from different S. pneumoniae strains reveal that one enzyme, the predominant type among pneumococcal isolates, has a unique endo-␤-galactosidase activity on the Lewis Y antigen. Altered active site topography in the other species of GH98 enzyme tune its endo-␤-galactosidase activity to the blood group A and B antigens. Despite their different specificities, these enzymes, and by extension all family 98 glycoside hydrolases, use an inverting catalytic mechanism. Many bacterial and viral pathogens exploit host carbohydrate antigens for adherence as a precursor to colonization or infection. However, this is the first evidence of bacterial endoglycosidase enzymes that are known to play a role in virulence and are specific for distinct host carbohydrate antigens. The strain-specific distribution of two distinct types of GH98 enzymes further suggests that S. pneumoniae strains may specialize to exploit host-specific antigens that vary from host to host, a factor that may feature in whether a strain is capable of colonizing a host or establishing an invasive infection.Streptococcus pneumoniae asymptomatically colonizes the nasopharynx of 10 -40% of people, but given the appropriate opportunity, it can become an extremely aggressive pathogen (1-3). This bacterium causes millions of deaths annually (1), is acquiring antibiotic resistance (4), and shows a disturbing and lethal synergy with the Influenza virus (5). The ability of S. pneumoniae to cause invasive disease is increasingly being linked with the capacity of this bacterium to attack and process the glycans present in host tissues (see Ref. 6 for a review). Indeed, large scale screening of pneumococcal virulence factors has revealed a large complement of genes devoted to complex carbohydrate metabolism that contribute to pneumococcal virulence (7-9). Recent elegant studies have focused on showing how a group of three exo-glycosidases sequentially trim complex human N-glycans (10, 11). These enzymes, however, only make up a fraction of the 39 glycosidases predicted to be in the pneumococcal genome (TIGR4 strain); at least 18 of these 39 are required for full virulence of the bacterium (7). Despite the growing appreciation for the role of carbohydrate metabolism in pneumococcal virulence and the possibility of targeting such metabolic pathways with small molecule therapeutic compounds, the bulk of the carbohydrate-active proteins of S. pneumoniae remain unexamined. As such, we presently have a relatively superficial but growing appreciation for the array of host glycans that S. pn...

We have previously described a bacterial system for the conversion of globotriaose (Gb3) into globotetraose (Gb4) by a metabolically engineered Escherichia coli strain expressing the Haemophilus influenzae lgtD gene encoding b1,3-N-acetylgalactosaminyltransferase [Antoine, T., Bosso, C., Heyraud, A. Samain, E. (2005) Large scale in vivo synthesis of globotriose and globotetraose by high cell density culture of metabolically engineered Escherichia coli. Biochimie 87,[197][198][199][200][201][202][203]. Here, we found that LgtD has an additional b1,3-galactosyltransferase activity which allows our bacterial system to be extended to the synthesis of the carbohydrate portion of globopentaosylceramide (Galb-3GalNAcb-3Gala-4Galb-4Glc) which reacts with the monoclonal antibody defining the stage-specific embryonic antigen-3. In vitro assays confirmed that LgtD had both b1,3-GalT and b1,3-GalNAcT activities and showed that differences in the affinity for Gb3 and Gb4 explain the specific and exclusive formation of globopentaose.

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The Gram negative bacterium Pseudomonas aeruginosa (PA) is an opportunistic bacterium that causes severe and chronic infection of immune-depressed patients. It has the ability to form a biofilm that gives a selective advantage to the bacteria with respect to antibiotherapy and host defenses. Herein, we have focused on the tetrameric soluble lectin which is involved in bacterium adherence to host cells, biofilm formation, and cytotoxicity. It binds to l-fucose, d-mannose and glycan exposing terminal fucose or mannose. Using a competitive assay on microarray, 156 oligosaccharides and polysaccharides issued from fermentation or from the biomass were screened toward their affinity to LecB. Next, the five best ligands (Lewisa, Lewisb, Lewisx, siayl-Lewisx and 3-fucosyllactose) were derivatized with a propargyl aglycon allowing the synthesis of 25 trivalent, 25 tetravalent and 5 monovalent constructions thanks to copper catalyzed azide alkyne cycloaddition. The 55 clusters were immobilized by DNA Directed immobilization leading to the fabrication of a glycocluster microarray. Their binding to LecB was studied. Multivalency improved the binding to LecB. The binding structure relationship of the clusters is mainly influenced by the carbohydrate residues. Molecular simulations indicated that the simultaneous contact of both binding sites of monomer A and D seems to be energetically possible.