Application of spectroscopic methods for structural analysis of chitin and chitosan.

Chitin, the second most important natural polymer in the world, and its N-deacetylated derivative chitosan, have been identified as versatile biopolymers for a broad range of applications in medicine, agriculture and the food industry. Two of the main reasons for this are firstly the unique chemical, physicochemical and biological properties of chitin and chitosan, and secondly the unlimited supply of raw materials for their production. These polymers exhibit widely differing physicochemical properties depending on the chitin source and the conditions of chitosan production. The presence of reactive functional groups as well as the polysaccharide nature of these biopolymers enables them to undergo diverse chemical modifications. A complete chemical and physicochemical characterization of chitin, chitosan and their derivatives is not possible without using spectroscopic techniques. This review focuses on the application of spectroscopic methods for the structural analysis of these compounds.

Chemical structure of the O-polysaccharides isolated from Cronobacter turicensis sequence type 5 strains 57, 564, and 566.

The Cronobacter spp. are Gram-negative bacterial pathogens that can cause infections in all age groups, and have a high mortality rate in neonates due to necrotizing enterocolitis and meningitis. Recent genotyping studies have revealed a strong clonal lineage in the genus, but this has not been compared with physiological traits. The O-polysaccharides (OPS) were isolated from three C. turicensis sequence type 5 strains (57, 564, and 566) and structurally characterized using (1)H and (13)C NMR spectroscopy, including two-dimensional DQF-COSY, TOCSY, ROESY, and HSQC analysis. Further compositional determination was undertaken using classical chemical methods followed by GLC, and GLC-MS analysis. The repeating unit of the isolated O-polysaccharides consists of GlcNAc, Rha, Glc, and had the structure shown below and therefore complemented the sequence type. [structure: see text].

Chemical structure of wall teichoic acid isolated from Enterococcus faecium strain U0317.

Wall teichoic acid (WTA) was isolated from Enterococcus faecium strain U0317 and structurally characterized using (1)H, (13)C, and (31)P NMR spectroscopy, including two-dimensional COSY, TOCSY, ROESY, HMQC, and HMBC experiments. Further compositional determination was undertaken using classical chemical methods and HF treatment followed by GLC and GLC-MS analyses. The repeating unit of WTA consisted of two residues of 2-acetamido-2-deoxy-D-galactose, glycerol (Gro), and phosphate, and has the structure shown below: [See formula in text].

Chemical structure of the O-polysaccharide isolated from Pectobacterium atrosepticum SCRI 1039.

The lipopolysaccharide (LPS) of the bacterium Pectobacterium atrosepticum SCRI 1039 was hydrolyzed and the products were separated. A study of the obtained O-polysaccharide by means of chemical methods, GLC, GLC-MS, and NMR spectroscopy allowed us to identify a branched polymer with a pentasaccharide repeating unit of the structure shown below, in which the fucose residue was partially O-acetylated at C-2, C-3 or C-4.

Serodiversity of opsonic antibodies against Enterococcus faecalis--glycans of the cell wall revisited.

In a typing system based on opsonic antibodies against carbohydrate antigens of the cell envelope, 60% of Enterococcus faecalis strains can be assigned to one of four serotypes (CPS-A to CPS-D). The structural basis for enterococcal serotypes, however, is still incompletely understood. Here we demonstrate that antibodies raised against lipoteichoic acid (LTA) from a CPS-A strain are opsonic to both CPS-A and CPS-B strains. LTA-specific antibodies also bind to LTA of CPS-C and CPS-D strains, but fail to opsonize them. From CPS-C and CPS-D strains resistant to opsonization by anti-LTA, we purified a novel diheteroglycan with a repeating unit of →6)-ß-Galf-(1→3)- ß-D-Glcp-(1→ with O-acetylation in position 5 and lactic acid substitution at position 3 of the Galf residue. The purified diheteroglycan, but not LTA absorbed opsonic antibodies from whole cell antiserum against E. faecalis type 2 (a CPS-C strain) and type 5 (CPS-D). Rabbit antiserum raised against purified diheteroglycan opsonized CPS-C and CPS-D strains and passive protection with diheteroglycan-specific antiserum reduced bacterial counts by 1.4-3.4 logs in mice infected with E. faecalis strains of the CPS-C and CPS-D serotype. Diheteroglycan-specific opsonic antibodies were absorbed by whole bacterial cells of E. faecalis FA2-2 (CPS-C) but not by its isogenic acapsular cpsI-mutant and on native PAGE purified diheteroglycan co-migrated with the gene product of the cps-locus, suggesting that it is synthesized by this locus. In summary, two polysaccharide antigens, LTA and a novel diheteroglycan, are targets of opsonic antibodies against typeable E. faecalis strains. These cell-wall associated polymers are promising candidates for active and passive vaccination and add to our armamentarium to fight this important nosocomial pathogen.

Structure of the O-polysaccharide isolated from Cronobacter sakazakii 767.

The Cronobacter spp., previously known as Enterobacter sakazakii, are Gram-negative enterobacterial pathogens that can cause necrotizing enterocolitis, meningitis, and septicemia with a high mortality rate in neonates. The O-specific polysaccharide (O-PS) was isolated from Cronobacter sakazakii strain 767 and structurally characterized using (1)H and (13)C NMR spectroscopy, including two-dimensional DQF-COSY, TOCSY, ROESY, HSQC, and HMBC experiments. Further compositional determination was undertaken using classical chemical methods followed by GLC, and GLC-MS analysis. The repeating unit of O-PS isolated from C. sakazakii 767 was a branched heptasaccharide composed of l-Rha, d-Glc, d-GlcNAc, and d-GalA, and had the structure shown below. One of the Rha residues was partially O-acetylated at C-4. C. sakazakii 767 was originally isolated from a fatal neonatal meningitic case, and the structure of its O-PS significantly differs from the O-PS structures previously described for Cronobacter spp.