Polysaccharides O-specific

A structural study on lipid A and the O-specific polysaccharide of the lipopoly-saccharide from a clinical isolate of Bacteroides vulgatus from a patient with Crohn s disease was conducted by Hashimoto and coworkers [39]. They separated two potent virulence factors, capsular polysaccharide (CPS) and lipopolysaccharide (LPS), from a clinical isolate of B. vulgatus and characterized the structure of CPS. Next, they elucidated the strucmres of O-antigen polysaccharide (OPS) and lipid A in the LPS. LPS was subjected to weak acid hydrolysis to produce the lipid A fraction and polysaccharide fraction. Lipid A was isolated by PLC, and its structure was determined by MS and NMR. 

Fig. 3. Top Resolution-enhanced 600 MHz H NMR spectrum of the octasaccharide repeating unit from Hafnia alvei strain 2 O-specific polysaccharide, (a)-(g) 500 MHz ID TOCSY subspectra with selective excitation of the respective anomeric proton resonances, (h) ID TOCSY subspectrum with the H3eq resonance of the neuraminic acid residue selectively excited. (Reproduced (adapted) with permission from Gamian et al. [25J. Copyright 1991...

Three monosaccharides of the group were identified which contain an amino group at both C-2 and C-3. These include acylated 2,3-diamino-2,3-dideoxy-D-glucose, which is a component of the lipid A region of lipopoly-saccharides from Rhodopseudomonas,144 and 2,3-di(acetamido)-2,3-dideoxy-D-glucuronic145 and -D-mannuronic146 acids, identified as constituents of P. aeruginosa O-specific polysaccharides. The pathway of their biosynthesis, and their activated forms, remain unknown. 

Gram-negative bacterium.160,163 D-Rhamnose is a constituent of O-specific polysaccharides from Xanthomonas campestris164 and Pseudomonas cepacia.165... 

An analogous reaction of UDP-2-acetamido-2,6-dideoxy-D-xy/o-hexos-4-ulose (7d) results in formation of the derivatives of 2-acetamido-2,6-dideoxy-D-glucose and -D-galactose.166 These amino sugars were identified as components of several O-specific polysaccharides (for reviews, see Refs. 95 and 167). 

CDP-6-deoxy-D-xy/o-hexos-4-ulose (7b) serves as a precursor in the biosynthesis of CDP-3,6-dideoxyhexoses28,168 having the d-ribo (paratose, 3,6-dideoxy- D-glucose ), o-xylo (abequose, 3,6-dideoxy- D-galactose ), and d-arabino (tyvelose, 3,6-dideoxy- D-mannose ) configurations. These monosaccharides are characteristic components of O-specific polysaccharides from Salmonella and Yersinia pseudotuberculosis. 

Such a situation is rather common for D-galactose and the structurally related monosaccharides L-arabinose218 and D-fucose thus far, the latter monosaccharide has been identified only as the furanose form in the O-specific polysaccharide of Eubacterium saburreum.219 6-Deoxy-L-altrose is present as the pyranose in the lipopolysaccharide of Yersinia enteroco-litica,20 but as the furanose in a similar polymer220 of Y. pseudotuberculosis type VB. Paratose, which is usually present in polysaccharides as the a-pyranose, was identified as the /3-furanose in the O-specific polysaccharide221,222 of Y. pseudotuberculosis serotype IB. 

Deoxy-L-g/ycero-pentulosonic acid was found to be present in Klebsiella K38 polysaccharide249 and 3-deoxy-D-threo-hexulosonic acid was identified as a component of Azotobacter capsular polysaccharide.2S0 O-Specific polysaccharides of Pseudomonas aeruginosa and Shigella boydii were shown2SI... 

Approximately 400 different glycosyltransferases are necessary in order to ensure the synthesis of those bacterial polysaccharides whose structures have thus far been elucidated. This estimate is based on the results of an analysis of the structures, made in order to ascertain how many different disaccharide fragments are present. An example of such an analysis is shown in Table V for the disaccharide sequences L-rhamnopyranosyl-D-galacto-pyranose, D-mannopyranosyl-L-rhamnopyranose, and D-galactopyranosyl-D-mannopyranose that are characteristic for the O-specific polysaccharides of Salmonella serogroups A, B, D, and E, the objects of many biosynthetic studies. Full details of similar analyses for other disaccharide sequences will be published elsewhere, as the resulting Tables are too voluminous for inclusion in this Chapter, but the most interesting results are summarized in Tables VI and VII. 

The structures of polyprenyl diphosphate-linked intermediates of Salmonella O-specific-polysaccharide biosynthesis were confirmed by chemical synthesis of their analogs derived from the plant polyprenols ficaprenol and moraprenol (structurally related to bacterial polyprenol57) with the following study of their behavior as substrates of enzymic reactions. Synthetic polyprenyl a-D-galactopyranosyl diphosphate291,292 was found to serve as an effective acceptor for the transfer of L-rhamnosyl groups.293"295 Two synthetic, isomeric disaccharide derivatives,292 13 and296 14, were tested as acceptors for enzymic D-mannosyl transfer from GDP-Man, but only the former was found to be an efficient substrate.294... 

The factors that determine the degree of polymerization of the polysaccharides remain unclear. It seems that the process is not under strict control, as significant heterogeneity in chain length is observed for Salmonella O-specific polysaccharides.302 304 In some cases, the degree of polymerization was found to depend on the conditions of cultivation of the micro-... 

Incorporation of abequosyl side-chains in O-specific polysaccharides (12) of Salmonella serogroup B occurs at the stage of repeating-unit assembly. The polyprenyl trisaccharide diphosphate 16a serves as an acceptor for abequosyl transfer from its CDP derivative.83,307 A similar reaction was demonstrated with an enzyme preparation from Citrobacter.290 Polymerases from S. typhimurium83,308 and S. bredeney286 were able to act on the derivatives of linear trisaccharide 16a, but, in the former case, the efficiency of... 

Another example of O-specific polysaccharides that are assembled through the block mechanism are the polymers from Salmonella serogroups C2 and C3. Their polysaccharide chains (18) are composed97 of linear, tetrasaccharide repeating-units having abequosyl and D-glucosyl branches,... 

The biosynthesis of other O-specific polysaccharides has still not been investigated. The only additional example of demonstration of such a process is in the formation of (l->3)-a-D-galactan, the Ol antigen of Klebsiella.333 UDP-Gal was identified as the glycosyl donor in the reaction, but further details of the mechanism remain unclear. 

Biosynthetic type of the main chain 1 O-Specific polysaccharides Exopoly- saccharides Gram-positive cell-wall Mechanisms of the chain... 

The block mechanism of chain assembly is characteristic for polymeric chains of the UGT type (see Salmonella O-specific polysaccharides 10-12 and 18) and the UG type (see capsular polysaccharides 25, 27, and 33), with UDP-activated sugars serving as initiators of chain growth. It seems rather safe to suggest that the biosynthesis of other polymers of these types occurs through a block mechanism as well. 

Preparation of modified, bacterial polysaccharides having monosaccharide analogs inserted into the polymeric chain is of interest for study of the structure-properties relationship in these biopolymers. Incorporation of chemically prepared, modified, biosynthetic precursors of the polymers in enzymic reactions seems a promising approach for achieving this aim. Such an approach, which may be termed chemical-enzymic synthesis, has now been studied by our group,439-441 using O-specific polysaccharides (10-12) of Salmonella serogroups B and E as an example. 

The chemical-enzymic approach to the synthesis of modified polysaccharides presents a good prospect for the preparation of small quantities of these polymers, which may prove very useful for immunochemical studies. The approach is certainly not limited by the specific case of Salmonella polysaccharides 10-12, and may well be extended to other polymers. The first results from this group322 show that several analogs of O-specific polysaccharides (18) of Salmonella serogroups C2 and C3 may be prepared through this approach. 

Figure 2. Specific immunoabsorbents for Salmonella O and R form antibodies. Key PS, O-specific polysaccharide, core, core fragment and GlcNHr glucosamine and fatty acids. Specific immunoabsorbents for lipid A antibodies have also been prepared (C. Galanos and D. Nerkar, unpublished results Luderitz et a ., 1982).

Synthesis of f -D-(l,6) linked disaccharides of N-fatty acylated 2-amino-2-deoxy-D-glucose an approach to the lipid A component of the bacterial lipopolysaccharide. Carbohydr. Res., 88, C10-C13 Kochetkov, N.K., Dmitriev, B.A., Malysheva, N.N., Chernyak, A.Ya., Klimov, E.M., Bayramova, N.E., and Torgov, V.I. (1975). Synthesis of O-p-D-mannopyranosy l-( 1 - 0-0-ot -L-rhamnopyranosy l-( 1 -3)-D-galacto-pyranose, the trisaccharide repeating-unit of the O-specific polysaccharide from Salmonella anatum. Carbohydr. Res. 15, 283-290... 

Synthesis of the tetrasaccharide repeating-unit of the O-specific polysaccharide from Salmonella senftenberg. Carbohydr. Res. 5 1, 269-27 1... 

B. A. Dmitriev, Y. A. Knirel, N. K. Kochetkov, and I. L. Hofman, Somatic antigens of Shigella. Structural investigation on the O-specific polysaccharide chain of Shigella dysenteriae type 1 lipopolysaccharide, Eur. J. Biochem., 66 (1976) 559-566. 

V. N. Shibaev, L. L. Danilov, T. N. Druzhinina, L. M. Gogilashvili, S. D. Maltsev, and N. K. Kochetkov, Enzymatic synthesis of Salmonella O-specific polysaccharide analogs from modified polyprenyl pyrophosphate sugar acceptors, FEBS Lett., 139 (1982) 177-180. 

E. V. Vinogradov, Y. A. Knirel, A. S. Shashkov, and N. K. Kochetkov, Determination of the degree of amidation of 2-deoxy-2-formamido-D-galacturonic acid in O-specific polysaccharides of Pseudomonas aeruginosa 04 and related strains, Carbohydr. Res., 170 (1987) cl-c4. 

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