Annie Malleron

Click-mediated labeling of bacterial membranes through metabolic modification of the lipopolysaccharide inner core.

Metabolic glycan labeling has recently emerged as a very powerful method for studying cell-surface glycans, which has applications that range from imaging glycans in living multicellular organisms, such as zebrafish or mice, to the identification of metastasis-associated cell-surface sialoglycoproteins. This strategy relies on the cellular biosynthetic machinery assimilating a modified monosaccharide that contains a bioorthogonal chemical reporter. The metabolic incorporation of this reporter into glycans can be further visualized by chemical ligation with a label, such as a fluorescent probe. Somewhat surprisingly, previous studies have mainly focused on the labeling of vertebrate glycans by using derivatives of common monosaccharides, such as Nacetyl neuraminic acid (or its N-acetylmannosamine precursor), N-acetylglucosamine, N-acetylgalactosamine, and fucose. In spite of a much higher degree of diversity in their monosaccharide building blocks as well as an essential role in bacterium–host interactions and bacterial virulence, bacterial polysaccharides have been poorly explored in terms of in vivo structural modifications. Bacteria are divided into Grampositive and Gram-negative bacteria. Whereas Gram-positive bacteria are surrounded by a peptidoglycan cell wall, Gramnegative bacteria are covered by a dense layer of lipopolysaccharides that are embedded in their outer membrane. These lipopolysaccharides are involved in the structural integrity of the cell and are often considered as determinants of pathogenicity. Although lipopolysaccharides appear to be an interesting target for specific and well-defined glycan metabolic labeling in Gram-negative bacteria, attempts to achieve this goal have been limited to the introduction of modified l-fucose derivatives into a customized, genetically engineered strain of Escherichia coli. Although it is a very interesting proof of concept, this l-fucose-based approach has some limitations as l-fucose is not generally present within the lipopolysaccharides of all Gram-negative bacteria, but is found in the O-antigens of specific strains. Secondly, free lfucose is not an intermediate in the normal E. coli “de novo” pathway and, therefore, should not be directly activable into a nucleotide-sugar donor without the introduction of an alternative pathway, known as the “salvage pathway”, into the organism of interest by genetic engineering (metabolic pathway engineering). Furthermore, once activated in the form of a modified guanosine-5’-diphosphate–fucose (GDP– Fuc), the l-fucose analogue might be transformed into a correspondingly modified GDP–mannose (GDP–Man) by the reverse de novo pathway, and potentially further metabolized into various other compounds, a process which could result in the chemical reporter being spread through other pathways of sugar metabolism or beyond. As a result of all of these limitations, and as our goal was labeling the lipopolysaccharides of bacteria with no genetic modification, we investigated whether another sugar could be used as a target for the metabolic modification of glycans. From all of the potential targets, 3-deoxy-d-mannooctulosonic acid (KDO) appears to be a very attractive candidate. Indeed, KDO is a specific and essential component of the inner core of lipopolysaccharides, and has long been considered as being present in the lipopolysaccharides of almost all Gram-negative species (as well as higher plants and algae), in which at least one residue is directly connected to lipid A (Scheme 1a). Because of its vital importance, KDO has been considered as a determinant for the characterization of Gram-negative bacteria, and the KDO pathway as a potential target for the development of new antibacterial compounds. In the KDO pathway (Scheme 1b), arabinose5-phosphate (arabinose-5-P) is condensed with phosphoenolpyruvate (PEP) to give KDO-8-phosphate (KDO-8-P), which is then transformed into free KDO, and further activated to form the cytidine monophosphate (CMP)–KDO donor prior to lipopolysaccharide elaboration. For all of these reasons, we hypothesized that the KDO pathway, as a lipopolysaccharidespecific pathway, may be tolerant enough to incorporate a modified analogue of KDO, such as 8-azido-8-deoxy-KDO (1, Scheme 2), into the core of E. coli lipopolysaccharides, and potentially other Gram-negative bacteria. Given the presence of free KDO as an intermediate in the pathway, we postulated that if the cell penetration of this analogue of KDO was sufficient, it could then be directly activated, partially replace endogenous KDO in lipopolysaccharides, and be detected on the cell surface by azide–alkyne click chemistry (Figure S1 in the Supporting Information). Moreover, modification of the C8-position of KDOwith a bioorthogonal azido group should prevent reverse metabolism by KDO-8-P [*] Dr. A. Dumont, Dr. S. Dukan Aix Marseille Universit , Laboratoire de Chimie Bact rienne (UMR 7283), Institut de Microbiologie de la M diterran e (IMM), CNRS, 31 Chemin Joseph Aiguier 13402 Marseille (France) E-mail: sdukan@imm.cnrs.fr

Characterization of Glycosaminoglycan (GAG) Sulfatases from the Human Gut Symbiont Bacteroides thetaiotaomicron Reveals the First GAG-specific Bacterial Endosulfatase

Background: Sulfatases are emerging as key adaptive tools of commensal bacteria to their host. Results: The first bacterial endo-O-sulfatase and three exo-O-sulfatases from the human commensal Bacteroides thetaiotaomicron, specific for glycosaminoglycans, have been discovered and characterized. Conclusion: Commensal bacteria possess a unique array of highly specific sulfatases to metabolize host glycans. Significance: Bacterial sulfatases are much more diverse than anticipated. Despite the importance of the microbiota in human physiology, the molecular bases that govern the interactions between these commensal bacteria and their host remain poorly understood. We recently reported that sulfatases play a key role in the adaptation of a major human commensal bacterium, Bacteroides thetaiotaomicron, to its host (Benjdia, A., Martens, E. C., Gordon, J. I., and Berteau, O. (2011) J. Biol. Chem. 286, 25973–25982). We hypothesized that sulfatases are instrumental for this bacterium, and related Bacteroides species, to metabolize highly sulfated glycans (i.e. mucins and glycosaminoglycans (GAGs)) and to colonize the intestinal mucosal layer. Based on our previous study, we investigated 10 sulfatase genes induced in the presence of host glycans. Biochemical characterization of these potential sulfatases allowed the identification of GAG-specific sulfatases selective for the type of saccharide residue and the attachment position of the sulfate group. Although some GAG-specific bacterial sulfatase activities have been described in the literature, we report here for the first time the identity and the biochemical characterization of four GAG-specific sulfatases. Furthermore, contrary to the current paradigm, we discovered that B. thetaiotaomicron possesses an authentic GAG endosulfatase that is active at the polymer level. This type of sulfatase is the first one to be identified in a bacterium. Our study thus demonstrates that bacteria have evolved more sophisticated and diverse GAG sulfatases than anticipated and establishes how B. thetaiotaomicron, and other major human commensal bacteria, can metabolize and potentially tailor complex host glycans.

Stannous triplate mediated glycosidations. A sterroselective synthesis of β -d-glucosides.

Abstract Various 1,2 trans,β- d -linked disaccharides with glucose as non-reducing unit have been prepared with complete stereoselectivity from acetobromoglucose and suitably protected sugar derivatives using stannous triflate as promoter.

Chemo-enzymatic synthesis of oligosaccharides using a dendritic soluble support

A new soluble support with high loading capacity is described. This support was used for chemical sulfatation and enzymatic synthesis of the trisaccharide Lewis X .

Stannous triflate mediated glycosidations. A stereoselective synthesis of 2-amino 2-deoxy-β-D-glucopyranosides directly with the natural N-acetyl protecting group.

Abstract Various 2-acetamido 2-deoxy β-D-glucopyranosides have been prepared in good yield from the crystalline 2-acetamido 3,4,6-tri-O-acetyl 2-deoxy α-D-glucopyranosyl chloride using stannous triflate as promoter.

Outstanding stability of immobilized recombinant α(1→3/4)-fucosyltransferases exploited in the synthesis of Lewis a and Lewis x trisaccharides

Recombinant human α(1→3/4)-fucosyltransferases (FucT-III) expressed in CHO cells or baculovirus-infected insect cells, immobilized on Ni2+-agarose through a 6His tag, exhibit a marked stability, which was exploited in the synthesis of Lewis a and Lewis x trisaccharides.

Synthesis of chiral β-alkoxy-α, β-unsaturated ketones

Abstract The title compounds are synthesized in one-pot from the easily available sodium salts of unsymmetrical 1,3-dicarbonyl derivatives and chiral alkoxides.