Complex O-acetylation in non-typeable Haemophilus influenzae lipopolysaccharide: evidence for a novel site of O-acetylation

The structure of the lipopolysaccharide (LPS) of non-typeable Haemophilus influenzae strain 723 has been elucidated using NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS) on O-deacylated LPS and core oligosaccharide material (OS), as well as ESI-MSn on permethylated dephosphorylated OS. It was found that the LPS contains the common structural element of H. influenzae, l-alpha-D-Hepp-(1-->2)-[PEtn-->6]-l-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-l-alpha-D-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdo-(2-->6)-Lipid A, in which the beta-D-Glcp residue (GlcI) is substituted by phosphocholine at O-6 and the distal heptose residue (HepIII) by PEtn at O-3, respectively. In a subpopulation of glycoforms O-2 of HepIII was substituted by beta-D-Galp-(1-->4)-beta-D-Glcp-(1--> or beta-D-Glcp-(1-->. Considerable heterogeneity of the LPS was due to the extent of substitution by O-acetyl groups (Ac) and ester-linked glycine of the core oligosaccharide. The location for glycine was found to be at Kdo. Prominent acetylation sites were found to be at GlcI, HepIII, and the proximal heptose (HepI) residue of the triheptosyl moiety. Moreover, GlcI was acetylated at O-3 and/or O-4 and HepI was acetylated at O-2 as evidenced by capillary electrophoresis ESI-MSn in combination with NMR analyses. This is the first study to show that an acetyl group can substitute HepI of the inner-core region of H. influenzae LPS.     

Structural investigation of lipopolysaccharides from nontypeable Haemophilus influenzae: investigation of inner-core phosphoethanolamine addition in NTHi strain 981

LPS of NTHi comprises a conserved tri-l-glycero-D-manno-heptosyl inner-core moiety (l-alpha-D-Hepp-(1-->2)-[PEtn-->6]-l-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-l-alpha-D-Hepp-(1-->5)-alpha-Kdop) in which addition of PEtn to the central heptose (HepII) in strain Rd is controlled by the gene lpt6. It was recently shown that NTHi strain 981 contains an additional PEtn linked to O-3 of the terminal heptose of the inner-core moiety (HepIII). In order to establish whether lpt6 is also involved in adding PEtn to HepIII, lpt6 in strain 981 was inactivated. The structure of the LPS of the resulting mutant strain 98llpt6 was investigated by MS and NMR techniques by which it was confirmed that the lpt6 gene product is responsible for addition of PEtn to O-6 of HepII in strain 981. However, it is not responsible for adding PEtn to O-3 of HepIII since the 981lpt6 mutant still had full substitution with PEtn at HepIII.     

Structure of a highly pyruvylated galactan sulfate from the Pacific green alga Codium yezoense (Bryopsidales, Chlorophyta)

A polysaccharide fraction consisting of d-galactose, sulfate, and pyruvate in a molar proportion of 4:2:1 was isolated from the green seaweed Codium yezoense by water extraction followed by ion-exchange chromatography. To elucidate its structure, modified polysaccharides were prepared by desulfation, depyruvylation, and by total removal of non-carbohydrate substituents. Structures of the native polysaccharide and of the products of its chemical modifications were investigated by methylation analysis as well as by 1D and 2D (1)H and (13)C NMR spectroscopy. The polysaccharide devoid of sulfate and pyruvate was subjected to two subsequent Smith degradations to afford a rather low-molecular and essentially linear (1-->3)-beta-d-galactan. A highly ramified structure was suggested for the native polysaccharide, which contains linear backbone segments of 3-linked beta-d-galactopyranose residues connected by (1-->6) linkages, about 40% of 3-linked residues being additionally substituted at C-6, probably by short oligosaccharide residues also containing (1-->3) and (1-->6) linkages. Sulfate groups were found mainly at C-4 and in minor amounts at C-6. Pyruvate was found to form mainly five-membered cyclic ketals with O-3 and O-4 of the non-reducing terminal galactose residues. The minor part of pyruvate forms six-membered cyclic ketals with O-4 and O-6. The absolute configurations of ketals (R for six-membered ketals and S for five-membered ones) were established using NMR spectral data.     

Structural determination of the O-antigenic polysaccharide from the Shiga toxin-producing Escherichia coli O171

The structure of the O-antigenic part of the lipopolysaccharide (LPS) obtained from the verotoxin-producing Escherichia coli O171 has been determined. (1)H and (13)C NMR spectroscopy techniques in combination with component analysis were used to elucidate the O-antigen structure of O-deacylated LPS. Subsequent NMR analysis of the native LPS revealed acetylation at O-7/O-9 of the sialic acid residue. The sequence of sugars was determined by inter-residue correlations in (1)H,(1)H-NOESY and (1)H,(13)C-heteronuclear multiple-bond correlation spectra. The O-antigen is composed of pentasaccharide repeating units with one equivalent of O-acetyl groups distributed over two positions: -->4)-alpha-Neu5Ac7,9Ac-(2-->6)-beta-D-Galp-(1-->6)-beta-DGlcp-->(1-->3)-beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1--> Based on biosynthetic considerations, this should also be the biological repeating unit.     

The structure of the carbohydrate backbone of the LPS from Myxococcus xanthus strain DK1622

Gram-negative rod shaped bacterium Myxococcus xanthus DK1622 produces a smooth-type LPS. The structure of the polysaccharide O-chain and the core-lipid A region of the LPS has been determined by chemical and spectroscopic methods. The O-chain was built up of disaccharide repeating units having the following structure: -->6)-alpha-D-Glcp-(1-->4)-alpha-D-GalpNAc6oMe*-(1--> with partially methylated GalNAc residue. The core region consisted of a phosphorylated hexasaccharide, containing one Kdo residue, unsubstituted at O-4, and no heptose residues. The lipid A component consisted of beta-GlcN-(1-->6)-alpha-GlcN1P disaccharide, N-acylated with 13-methyl-C14-3OH (iso-C15-3OH), C16-3OH, and 15-methyl-C16-3OH (iso-C17-3OH) acids. The lipid portion contained O-linked iso-C16 acid.     

The structure of the carbohydrate backbone of the lipopolysaccharide of Pectinatus frisingensis strain VTT E-79104

The structure of the carbohydrate backbone of the lipopolysaccharide from Pectinatus frisingensis strain VTT E-79104 was analyzed using chemical degradations, NMR spectroscopy, mass spectrometry, and chemical methods. The LPS contains two major structural variants, differing in the presence or absence of an octasaccharide fragment. The largest structure of the carbohydrate backbone of the LPS, that could be deduced from experimental results, consists of 20 monosaccharides arranged in a nonrepetitive sequence: [carbohydrate structure: see text] where R is H or 4-O-Me-alpha-L-Fuc-(1-2)-4-O-Me-beta-Hep-(1-3)-alpha-GlcNAc-(1-2)-beta-Man-(1-3)-beta-ManNAc-(1-4)-alpha-Gal-(1-4)-beta-Hep-(1-3)-beta-GalNAc-(1- where Hep is a residue of D-glycero-D-galacto-heptose; all monosaccharides have the D-configuration except for 4-O-Me-L-Fuc and L-Ara4N. This structure is architecturally similar to the oligosaccharide system reported previously in P. frisingensis VTT E-82164 LPS, but differs from the latter in composition and also in the size of the outer region.     

Structural analysis of the lipopolysaccharide from nontypeable Haemophilus influenzae strain 1003.

Structural analysis of the lipopolysaccharide (LPS) of nontypeable Haemophilus influenzae strain 1003 has been achieved by the application of high-field NMR techniques, ESI-MS, capillary electrophoresis coupled to ESI-MS, composition and linkage analyses on O-deacylated LPS and core oligosaccharide material. It was found that the LPS contains the common structural element of H. influenzae, l-alpha-D-Hepp-(1-->2)-[PEtn-->6]-l-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-l-alpha-D-Hepp-(1-->5)-[PP Etn-->4]-alpha-Kdop-(2-->6)-Lipid A, in which the beta-D-Glcp residue is substituted by phosphocholine at O-6 and an acetyl group at O-4. A second acetyl group is located at O-3 of the distal heptose residue (HepIII). HepIII is chain elongated at O-2 by either a beta-D-Glcp residue (major), lactose or sialyllactose (minor, i.e. alpha-Neu5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp), where a third minor acetylation site was identified at the glucose residue. Disialylated species were also detected. In addition, a minor substitution of ester-linked glycine at HepIII and Kdo was observed.     

Structural analysis of lipopolysaccharide oligosaccharide epitopes expressed by non-typeable Haemophilus influenzae strain 176

The structure of the lipopolysaccharide (LPS) from non-typeable Haemophilus influenzae strain 176 has been investigated. Electrospray ionization-mass spectrometry (ESIMS) on O-deacylated LPS (LPS-OH) and core oligosaccharide (OS) samples obtained after mild-acid hydrolysis of LPS provided information on the composition and relative abundance of the glycoforms. ESIMS tandem-mass spectrometry on LPS-OH confirmed the presence of minor sialylated and disialylated glycoforms. Oligosaccharide samples were studied in detail using high-field NMR techniques. It was found that the LPS contains the common inner-core element of H. influenzae, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-L-alpha-D-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdop-(2-->6)-Lipid A having glycosyl substitution at the O-3 position of the terminal heptose as recently observed for non-typeable H. influenzae strain 486 [M?nsson, M.; Bauer, S. H. J.; Hood, D. W.; Richards, J. C.; Moxon, E. R.; Schweda, E. K. H., Eur. J. Biochem. 2001, 268, 2148--2159]. The following LPS structures were identified as the major glycoforms, the most significant being indicated with an asterisk (*) (glycoforms are partly substituted with Gly at the terminal Hep):     

Structure of a highly substituted beta-xylan of the gum exudate of the palm Livistona chinensis (Chinese fan)

Structural features of the acidic, highly substituted glycanoxylan (LCP; 87% yield) from the gum exudate of the palm, Livistona chinensis, family Arecaceae, were determined. It had [alpha]D -30 degrees, Mw 1.9x10(5) and a polydispersity ratio Mw/Mn of approximately 1.0. Acid hydrolysis gave rise to Rha, Fuc, Ara, Xyl, and Gal, in a 1:6:46:44:3 molar ratio, and 12% of uronic acid was present. LCP had a highly branched structure with side-chains containing nonreducing end-units (% values are approximate) of Araf (15%), Fucp (4%), Xylp (7%), GlcpA, and 4-Me-GlcpA, and internal 2-O- (5%) and 3-O-substituted Araf (8%), and 2-O-substituted Xylp (14%) units. The (1-->4)-linked beta-Xylp main-chain units of LCP were substituted at O-3 (4%), O-2 (17%), and O-2,3 (16%). Partial acid hydrolysis gave 4-Me-alpha-GlcpA-(1-->2)-[beta-Xylp-(1-->4)](0-2)-Xyl, identified by showing that the uronic acids were single-unit side-chain substituents on O-2. Milder hydrolysis conditions removed from O-3 other side-chains containing Fucp and Araf nonreducing end-units and internal Arap, and 2-O- and 3-O-substituted Araf units. Carboxyl-reduced LCP contained 4-O-methylglucose and glucose in a 3.2:1 molar ratio, arising from GlcpA and 4-OMe-GlcpA nonreducing end-units, respectively. The gum contained small amounts of free alpha-Fucp-(1-->2)-Ara, which corresponds to structures in the polysaccharide. Free myo- and D- or L-chiro-inositol were present in a 9:1 ratio.     

Structure of the O-polysaccharide leads to classification of Proteus penneri 31 in Proteus serogroup O19

O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide (LPS) of Proteus penneri strain 31. Sugar and methylation analyses along with NMR spectroscopic studies, including 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C and 1H,31P HMQC experiments, demonstrated the following structure of the polysaccharide: [carbohydrate structure: see text] where FucNAc is 2-acetamido-2,6-dideoxygalactose and EtnP is 2-aminoethyl phosphate. The polysaccharide studied has the same carbohydrate backbone as the O-polysaccharide of Proteus vulgaris O19. Based on this finding and close serological relatedness of the LPS of the two strains, it is proposed to classify P. penneri 31 in Proteus serogroup O19 as an additional subgroup. In contrast, D-GlcNAc6PEtn and alpha-L-FucNAc-(1-->3)-D-GlcNAc shared with a number of other Proteus O-polysaccharides could not provide any significant cross-reactivity of the corresponding LPS with rabbit polyclonal O-antiserum against P. penneri 31.     

Structure of extended lipopolysaccharide glycoforms containing two globotriose units in Haemophilus influenzae serotype b strain RM7004

Lipopolysaccharide (LPS) is a major virulence determinant of the human bacterial pathogen Haemophilus influenzae. Structural elucidation of the LPS from H. influenzae type b strain RM7004 was achieved by using electrospray ionization mass spectrometry (ESI-MS) and high-field NMR techniques on delipidated LPS and core oligosaccharide samples of LPS. It was found that the organism elaborates a series of related LPS glycoforms having a common inner-core structure, but differing in the number and position of attached hexose residues. LPS glycoforms containing between four and nine hexose residues were structurally characterized. The inner-core element was determined to be L-alpha-D-Hepp-(1-->2)-[PEA-->6]-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-L-alpha-D-Hepp-(1-->5)-[P-->4]-alpha-KDOp-(2-->, a structural feature which has been identified in every H. influenzae strain investigated to date. Two major groups of isomeric glycoforms were characterized in which the terminal Hepp residue of the inner-core element was either substituted at the O-2 position with a beta-D-Galp residue or not. The structures of the major LPS glycoforms were found to have oligosaccharide chain extensions from O-3 of the middle Hepp residue. Glycoforms containing five and six hexose residues were most abundant and were shown to carry the tetrasaccharide unit alpha-D-Galp-(1-->4)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->4)-alpha-D-Glcp at the O-3 position of the middle heptose. This tetrasaccharide displays the globoside trisaccharide (globotriose) as a terminal epitope, a structure that is found on many human cells (P(k) blood group antigen) and which is thought to be an important virulence determinant for H. influenzae. LPS glycoforms were characterized that had further chain extension from the beta-D-Glcp-(1--> residue of the proximal Hepp. In the fully extended LPS (Hex9/Hex8' glycoforms), both the proximal and middle heptose residues carried tetrasaccharide chains displaying terminal globotriose epitopes. In addition, the LPS was found to carry phosphorylcholine and O-acetyl groups.     

Characterization of the exopolysaccharide produced by Streptococcus thermophilus 8S containing an open chain nononic acid.

The exopolysaccharide produced by Streptococcus thermophilus 8S in reconstituted skimmed milk is a heteropolysaccharide containing d-galactose, d-glucose, d-ribose, and N-acetyl-d-galactosamine in a molar ratio of 2 : 1 : 1 : 1. Furthermore, the polysaccharide contains one equivalent of a novel open chain nononic acid constituent, 3,9-dideoxy-d-threo-d-altro-nononic acid, ether-linked via C-2 to C-6 of an additional d-glucose per repeating unit. Methylation analysis and 1D/2D NMR studies (1H and 13C) performed on the native polysaccharide, and mass spectrometric and NMR analyses of the oligosaccharide obtained from the polysaccharide by de-N-acetylation followed by deamination and reduction demonstrated the 'hepta'saccharide repeating unit to be: -->4)-alpha-D-Galp-(1-->2)-beta-D-Ribf-(1-->4)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1--7')-Sub-(1-->4)-beta-D-GalpNAc-(1--> in which Sug is 6-O-(3',9'-dideoxy-d-threo-d-altro-nononic acid-2'-yl)-alpha-d-glucopyranose.     

Structure of the O-specific polysaccharide of Proteus vulgaris O15 containing a novel regioisomer of N-acetylmuramic acid, 2-acetamido-4-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucose

An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus vulgaris O15 and studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY, and H-detected 1H,(13)C HMQC experiments. The polysaccharide was found to contain an ether of GlcNAc with lactic acid, and the following structure of the repeating unit was established:-->3)-alpha-D-GlcpNAc4(R-Lac)6Ac-(1-->2)-beta-D-GlcpA-(1-->3)-alpha-L-6dTalp2Ac-(1-->3)-beta-D-GlcpNAc-(1-->where L-6dTal and D-GlcNAc4(R-Lac) are 6-deoxy-L-talose and 2-acetamido-4-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucose, respectively. The latter sugar, which to our knowledge has not been hitherto found in nature, was isolated from the polysaccharide by solvolysis with anhydrous triflic acid and identified by comparison with the authentic synthetic compound. Serological studies with the Smith-degraded polysaccharide showed an importance of 2-substituted GlcA for manifesting of the immunospecificity of P. vulgaris O15.     

Structural elucidation of the O-antigenic polysaccharide from the enteroaggregative Escherichia coli strain 180/C3 and its immunochemical relationship with E. coli O5 and O65

The structure of the O-antigen polysaccharide (PS) from the enteroaggregative Escherichia coli strain 180/C3 has been determined. Sugar and methylation analysis together with (1)H and (13)C NMR spectroscopy were the main methods used. The PS is composed of tetrasaccharide repeating units with the following structure: -->2)beta-D-Quip3NAc-(1-->3)beta-D-RIBf-(1-->4)beta-D-Galp-(1-->3)alpha-D-GalpNAc-(1-->. Analysis of NMR data indicates that the presented sequence of sugar residues also represents the biological repeating unit of the O-chain. The structure is closely related to that of O-antigen polysaccharide from E. coli O5 and partially to that of E. coli O65. The difference between the O-antigen from the 180/C3 strain and that of E. coli O5 is the linkage to the D-Quip3NAc residue, which in the latter strain is 4-O-substituted. The E. coli O65 O-antigen contains as part of its linear pentasaccharide repeating unit a similar structural element, namely -->4)-beta-d-GalpA-(1-->3)-alpha-D-GlcpNAc-(1-->2)-beta-D-Quip3NAc-(1-->, thereby indicating that a common epitope could be present for the two polysaccharides. Monospecific anti-E. coli O5 rabbit serum did not distinguish between the two positional isomeric structures neither in slide agglutination nor in an indirect enzyme immunoassay. The anti-O65 serum did react with both the 180/C3 and O5 LPS showing a partial cross-reactivity.     

Structure of a heteroxylan of gum exudate of the palm Scheelea phalerata (uricuri)

The polysaccharide isolated from the gum exudate of palm Scheelea phalerata (SPN) was water-insoluble and composed of Fuc, Ara, Xyl, and uronic acid moieties in a 5:34:54:7 molar ratio: 12% of phenolics were also present. A soluble polysaccharide (SPNa) was obtained after alkaline treatment, which contained Fuc, Ara, Xyl and uronic acid in a 7:44:42:7 molar ratio, with only 2% phenolics. SPNa had an M(W) approximately 1.04 x 10(5) g mol(-1) and was almost monodisperse (M(W)/M(N) : 1.25 +/-0.22). It had a branched structure with side chains of 2-O-substituted Xylp (approximately 8%) and 3-O-substituted Araf (12%) units, and a large proportion of nonreducing end-units of Araf (15%), Fucp (10%), Xylp (4%), and Arap (6%). The (1 --> 4)-linked beta-Xylp main-chain units were 3-O- (9%), 2-O- (13%), and 2,3-di-O- (13%) substituted. Its (13)C NMR spectrum contained at least 9 C-1 signals, those at delta 108.6 and 107.7 arising from alpha-Araf units. Others were present at delta 175.4 from C-6 of alpha-GlcpA and delta 15.6 from C-6 of Fucp units. The main chain of SPNa was confirmed by analysis of a Smith-degraded polysaccharide (SPDS): methylation analysis provided a 2,3-Me(2)-Xyl (65%) derivative and its (13)C NMR spectrum showed five main signals typical of a (1 --> 4)-linked beta-Xylp units. Methylation analysis of a carboxy-reduced polysaccharide (SPN-CR) revealed a 2,3,4,6-Me(4)-Glc derivative (4%) arising from nonreducing end-units of GlcpA. Alpha-GlcpA-(1 --> 2)-alphabeta-Xy1p and alpha-GlcpA-(1 --> 2)-beta-Xylp-(1 --> 4)-alphabeta-Xylp were obtained via partial acid hydrolysis of SPN, showing the structure of side-chain substituents on O-2 of the main-chain units.     

Cassia grandis Linn. f. seed galactomannan: structural and crystallographical studies

Cassia grandis is a small or medium sized tree, found in abundance throughout India. The seeds contain about 50% endosperm gum and possess the characteristics of becoming a potential source of seed gum. The purified polysaccharide has been characterized as a pure galactomannan having a mannose-galactose ratio of 3.15; molecular weight (Mw) 80,200; polydispersity (Mw/Mn), 1.35 and intrinsic viscosity [eta], 848 mL/g. Methylation, periodate oxidation, Smith degradation and 13C NMR studies confirm that the polysaccharide has the basic structure of legume galactomannans consisting of a beta-(1-->4)-linked main mannan backbone to which galactose units are attached at O-6. The orthorhombic lattice constants of the hydrated gum are as follows: a=9.00, b=24.81, c=10.30 A. The crystallographic data establish that the probable space group symmetry of the unit cell is P2(1)2(1)2. The results are in contradiction to earlier reports (Indian J. Chem. 16B (1978) 966; J. Indian Chem. Soc. 55 (1978) 1216) in which a non-galactomannan polysaccharide structure has been assigned having a main chain of (1-->4)-linked galactose and mannose units in the molar ratio 6:3, where 50% of the galactose units branched with two galactose and one mannose through 1-->3 linkage.     

Structure of the O-polysaccharide of Providencia stuartii O49

The O-specific polysaccharide chain (O-antigen) of the lipopolysaccharide (LPS) of Providencia stuartii O49 was studied using sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, ROESY, H-detected 1H, 13C HSQC and HMBC experiments. The polysaccharide was found to have the trisaccharide repeating unit with the following structure: -->6)-beta-D-Galp(1-->3)-beta-D-GalpNAc(1-->4)-alpha-D-Galp(1-->     

Structural analysis of the lipopolysaccharide from nontypeable Haemophilus influenzae strain R2846

We here report the lipopolysaccharide (LPS) structures expressed by nontypeable Haemophilus influenzae R2846, a strain whose complete genome sequence has recently been obtained. Results were obtained by using NMR techniques and ESI-MS on O-deacylated LPS and core oligosaccharide material (OS) as well as ESI-MS (n) on permethylated dephosphorylated OS. A beta- d-Glc p-(1-->4)- d-alpha- d-Hep p-(1-->6)-beta- d-Glc p-(1-->4) unit was found linked to the proximal heptose (HepI) of the conserved triheptosyl inner-core moiety, l-alpha- d-Hep p-(1-->2)-[ PEtn-->6]- l-alpha- d-Hep p-(1-->3)- l-alpha- d-Hep p-(1-->5)-[ PPEtn-->4]-alpha-Kdo-(2-->6)-lipid A. The beta- d-Glc p (GlcI) linked to HepI was also branched with oligosaccharide extensions from O-4 and O-6. O-4 of GlcI was substituted with sialyllacto- N-neotetraose [alpha-Neu5Ac-(2-->3)-beta- d-Gal p-(1-->4)-beta- d-Glc pNAc-(1-->3)-beta- d-Gal p-(1-->4)-beta- d-Glc p-(1-->] and the related structure [( PEtn-->6)-alpha- d-Gal pNAc-(1-->6)-beta- d-Gal p-(1-->4)-beta- d-Glc pNAc-(1-->3)-beta- d-Gal p-(1-->4)-beta- d-Glc p-(1-->]. The distal heptose (HepIII) was substituted at O-2 by beta- d-Gal. Phosphate, phosphoethanolamine, phosphocholine, acetate, and glycine were found to substitute the core oligosaccharide. Two heptosyltransferase genes, losB1 and losB2, have been identified from the R2846 genome sequence and are candidates to add the noncore heptose to the LPS. Mutant strain R2846 losB1 did not show dd-heptose in the extension from HepI but still contained minor quantities of ld-heptose at the same position, indicating that the losB1 gene is required to add dd-heptose to GlcI. The LPS from strain R2846 losB1/ losB2 expressed no noncore heptose, consistent with losB2 directing the addition of ld-heptose.     

Bis(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)amine obtained by a fusion reaction

A unique, alkali-soluble polysaccharide has been isolated from the cell walls of the basidiomycete Coprinus macrorhizus microsporus. The polysaccharide, which is primarily a glucan, contains a large proportion of α-(1→4)-linked d-glucose residues and a smaller amount of β-(1→3) and (1→6) linkages, as suggested by methylation, partial acid hydrolysis, periodate oxidation, and enzymic studies. Hydrolysis of the methylated polysaccharide gave equimolar amounts of 2,4-di- and 2,3-di-O-methyl-d-glucose; no 2,6-di-O-methyl-d-glucose was identified, indicating the absence of branch points joined through O-1, O-3, and O-4. The isolation and identification of 2-O-α- glucopyranosylerythritol from the periodate-oxidized polysaccharide suggests that segments of the a-(1→4)-linked d-glucose residues are joined by single (1→3)-linkages. An extracellular enzyme-preparation from Sporotrichum dimorphosporum (QM 806) containing both β-(1→3)- and α-(1→4)-d-glucanohydrolase activity released 76% of the reducing groups from the polysaccharide. The polysaccharide also contains minor proportions of xylose, mannose, 2-amino-2-deoxyglucose, and amino acids.     

The structural basis for the serospecificity of Actinobacillus suis serogroup O:2.

Actinobacillus suis is an important bacterial pathogen of healthly pigs. An O-antigen (lipopolysaccharide; LPS) serotyping system is being developed to study the prevalence and distribution of representative isolates from both healthy and diseased pigs. In a previous study, we reported that A. suis serogroup O:1 strains express LPS with a (1-->6)-beta-D-glucan O-antigen chain polysaccharide that is similar in structure to a key cell-wall component in yeasts, such as Saccharomyces cerevisiae and Candida albicans. This study describes the O-antigen polysaccharide chemical structure of an O:2 serogroup strain, A. suis H91-0380, which possesses a tetrasaccharide repeating block with the structure: -->3)-beta-D-Galp-(1-->4)-[alpha-D-Galp-(1-->6)]-beta-D-Glcp-(1-->6)-beta-D-GlcpNAc-(1-->. Studies have shown that A. suis serogroup O:2 strains are associated with severely diseased animals; therefore, work on the synthesis of a glycoconjugate vaccine employing O:2 O-antigen polysaccharide to vaccinate pigs against A. suis serogroup O:2 strains is currently underway.